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doi:10.3390/brainsci10110868. The Sleeping Brain: Harnessing the Power of the Glymphatic System through Lifestyle Choices. Oliver Cameron Reddy and Ysbrand D van der Werf. Department of Anatomy and Neurosciences, Amsterdam UMC, Vrije Universiteit Amsterdam. Brain Science, 2020, volume 10, page 868. Abstract: The glymphatic system is a “pseudo-lymphatic” perivascular network distributed throughout the brain, responsible for replenishing as well as cleansing the brain. Glymphatic clearance is the macroscopic process of convective fluid transport in which harmful interstitial metabolic waste products are removed from the brain intima. This paper addresses the glymphatic system, its dysfunction and the major consequences of impaired clearance in order to link neurodegeneration and glymphatic activity with lifestyle choices. Glymphatic clearance can be manipulated by sleep deprivation, cisterna magna puncture, acetazolamide or genetic deletion of AQP4 channels, but how lifestyle choices affect this brain-wide clearance system remains to be resolved. This paper will synthesize existing literature on glymphatic clearance, sleep, Alzheimer’s disease and lifestyle choices, in order to harness the power of this mass transport system, promote healthy brain ageing and possibly prevent neurodegenerative processes. This paper concludes that: 1. Glymphatic clearance plays a major role in Alzheimer’s pathology. 2. The vast majority of waste clearance occurs during sleep. 3. Dementias are associated with sleep disruption, alongside an age-related decline in AQP4 polarization and, 4. Lifestyle choices such as sleep position, alcohol intake, exercise, omega-3 consumption, intermittent fasting and chronic stress all modulate glymphatic clearance. Lifestyle choices could therefore alter Alzheimer’s disease risk through improved glymphatic clearance, and could be used as a preventative lifestyle intervention for both healthy brain ageing and Alzheimer’s disease. Keywords: glymphatic system, protein aggregates, Alzheimer’s disease, amyloid-beta, sleep, disease prevention. Section 1. Introduction. Discovered in 2012, the glymphatic system, which stands for glial-dependent lymphatic transport, has been categorized as a macroscopic waste clearance system. Due to the similarities in function, the glymphatic system has been described as the central nervous system’s analogue to the lymphatic system. The transportation of the central nervous system’s interstitial fluid (ISF) has long been thought to move via diffusion, but recently ISF was observed moving at a much faster rate than that possible through diffusion. This suggests the involvement of a mass transport system. This glial cell-dependent paravascular network removes soluble proteins and metabolites from the central nervous system, but in addition supplies the brain with glucose, lipids and neuromodulators, utilizing paravascular tunnels formed by astroglial cells. Since this is a relatively new discovery, the amount of scientific literature surrounding the glymphatic system is rapidly increasing, and therefore its definition is continuously being renewed. This has caused controversy surrounding both the directionality and the anatomical space in which this system resides. For instance, the movement of ISF along paravascular spaces of veins remains disputed, and some claim that a distinct route exists for this clearance pathway. These discrepancies can, however, be partially explained by the limited amount of literature and methodological differences between studies. The glymphatic system is constantly filtering toxins from the brain, but during wakefulness, this system is mainly disengaged. During natural sleep, levels of norepinephrine decline, leading to an expansion of the brain’s extracellular space, which results in decreased resistance to fluid flow. This is reflected by improved cerebrospinal fluid (CSF) infiltration along the perivascular spaces, and therefore increased interstitial solute clearance. The increase in clearance happens specifically during non-rapid eye movement sleep (N), also known as quiescent sleep. The third N stage, N3 or slow-wave sleep, is categorized by slow oscillatory brain waves, that create a flux of CSF within the interstitial cavities, leading to an increase in glymphatic clearance. The role of sleep in glymphatic clearance has been conclusively demonstrated, and since the vast majority of clearance occurs during sleep, the glymphatic system can simply not be investigated without examining the basic aspects of sleep. Impaired glymphatic clearance has been linked to neurodegenerative diseases. Alzheimer’s disease is a chronic neurodegenerative disease and the most common dementia, typically beginning with disorientation and then proceeding to a gradual deterioration of memory, language and physical independence, among others. Amyloid-beta and tau protein aggregations are heavily associated with Alzheimer’s disease, creating plaques and neurofibrillary tangles in the brain that lead to brain degradation. Glymphatic clearance moves tau proteins and amyloid-beta aggregates out of the brain. This suggests that the glymphatic system is involved in modulating, or possibly protective against, Alzheimer’s disease. This paper will focus on Alzheimer’s disease, since it is the most frequent dementia, but will hopefully remain applicable to other neurodegenerative diseases, since several dementias are thought to be caused by protein aggregation. The need for an intervention is gaining urgency. Benveniste and colleagues recently used MRI scans in combination with contrast agents to monitor CSF flow through the brain in real time, yet a method for manipulating glymphatic activity in humans still remains to be developed. Regulating glymphatic clearance could increase waste removal of aggregates in diseases associated with protein deposition, slowing or even reversing neurodegeneration. Sleep is a primary driver of glymphatic clearance. However, research on a wealth of other lifestyle choices such as sleep quality, quantity, physical exercise, changes in body posture, omega 3, chronic stress, intermittent fasting and low doses of alcohol has begun to emerge. Despite these advances, scholars in this field have not yet adequately harnessed the power of lifestyle-regulated glymphatic clearance. Lifestyle choices remain to be evaluated and compared. No guides or literature reviews exist on how to use preventative measures to bolster glymphatic activity. With the incidence of neurodegenerative disease increasing and evidence of the glymphatic systems’ involvement growing, there is an urgent need to capitalize on the uses of this mass transport system. Lifestyle changes decelerating disease progression could be an important discovery, opening a therapeutic avenue and the potential for improvements in quality of life. In order to infer the causal relationships of lifestyle choices in reducing brain ageing and Alzheimer’s disease, this paper will first investigate why glymphatic clearance primarily occurs during sleep, and which underlying mechanisms drive glymphatic clearance. Next, this paper will inspect the implications of a dysfunctional glymphatic pathway and establish the relationship between glymphatic clearance and neurodegenerative disease. Finally, this paper will investigate how lifestyle choices affect this mass transport system and how they can be used as a protective and preventive measure in the context of aging and Alzheimer’s disease. Section 2. Materials and Methods. This literature review synthesizes research on the glymphatic system, neurodegenerative disease and various lifestyle choices. By critically analyzing this literature, we aim to work towards a preventive guide for Alzheimer’s disease, and hopefully also other neurodegenerative diseases. We have gathered papers from the scientific database “PubMed”, as well as additional sources from the reference lists of some of the papers found through the database searches. Using the keywords “glymphatic” and “system”, the search yielded 389 results. Of these 389 papers, those including lifestyle choices and those related to Alzheimer’s disease were selected, based on title, abstract and applicability to the research question. A PRISMA flow chart documents the precise selection process and assessment of eligibility, see Figure 1 in the original text. Section 3. Results. 3 point 1. The Glymphatic System of the Brain. 3 point 1 point 1. Fluid Movement in the Brain. The brain consists of four aqueous compartments: CSF, ISF, intracellular fluid and blood, all separated by two main barriers regulating their ionic and biochemical composition: the blood–brain barrier and the blood, CSF barrier, see Figure 2. The blood–brain barrier is located throughout the brain along the vasculature. Tight junctions on endothelial cells block the movement of macromolecules but allow fluids and solutes to diffuse into the brain from the perivascular space between endothelial cells and astrocytic endfeet. The blood–CSF barrier, on the other hand, has fenestrated endothelial cells allowing macromolecules into the interstitial space. This barrier is located within the choroid plexus of the two lateral, the third and the fourth ventricles. Its epithelial cells have an abundance of tight junctions in order to regulate CSF composition. These do allow the movement of macromolecules and principally transport Sodium, Potassium, Chlorine and Bicarbonate ions through primary active transport using a Sodium, Potassium ATPase. The constant production of CSF by the choroid plexus drives the flow direction of CSF through the brain. Collectively, this results in a CSF production of around 500 milliliters each day, flowing from the lateral ventricles to the third and then fourth ventricle, entering the subarachnoid space, bathing the brain. It exits predominantly through the perineural spaces of the cranial nerves along the internal carotid artery, or into the olfactory-nasal submucosa pathway, ultimately draining into deep cervical lymph nodes. Figure 2. The four fluid compartments of the brain, separated by the blood–brain barrier or the blood–CSF barrier. The blood–brain barrier is situated wherever the vasculature reaches. The blood–CSF barrier is situated only in the choroid plexus and allows the passage of macromolecules. 3 point 1 point 2. Paravascular Spaces. Within the interstitial spaces of the brain, CSF travels towards perivascular and perineural spaces, and in doing so clears solutes from the neuropil into meninges. These then exit the brain and drain into cervical lymphatic vessels and are ultimately degraded in the liver. CSF enters the brain via para-arterial channels and exchanges with ISF, which in turn is cleared by paravenous pathways, depicted in Figure 3. CSF from the subarachnoid spaces enters the Virchow–Robin spaces along para-arterial channels and exchanges with ISF, contrary to the classical model of CSF secretion and absorption. CSF enters the brain exclusively via periarterial spaces and ISF drains exclusively into perivenous spaces. The CSF influx is balanced by the perivenous eflux of ISF ridding the neuropil of proteinaceous metabolites. Patients suffering from central oedema show a significant decrease in CSF entering perivascular spaces, suggesting that a CSF movement is driven not only by a pressure difference, but also by pulsations of arterial smooth muscle. Figure 3. This figure depicts the circulation of cerebrospinal fluid, CSF, and its interchange with interstitial fluid, ISF, CSF entering the perivascular space of penetrating arteries, then through convective flow clearing waste products into the perivenous spaces, ultimately leaving the brain through paravenous efflux routes. 3 point 1 point 3. Fluid Movement within the Interstitial Space. Once deeper within the brain, CSF movement is facilitated by aquaporin 4, AQP4, water channels on the endfeet of astrocytes, which ensheathe the blood vasculature. CSF then enters the parenchyma and mixes with the ISF, where both continuously interchange. The separation here has been proven by AQP4 knockout mice, which have significantly less CSF to ISF exchange than wild type mice, suggesting that AQP4 channels are responsible for CSF, ISF exchange, but the influx of CSF into the periarterial spaces was not affected. Although convection occurs within the perivascular spaces, within the extracellular space, diffusion is responsible for the movement of ISF. Overlapping astrocytic endfeet which completely ensheathe the cerebral microvasculature inhibit the access of molecules with larger molecular weight from entering the interstitium. 3 point 2. Sleeping the Brain Clean. “Innocent sleep. Sleep that soothes away all our worries. Sleep that puts each day to rest. Sleep that relieves the weary labourer and heals hurt minds. Sleep, the main course in life’s feast, and the most nourishing.” William Shakespeare, Macbeth. In 1606, William Shakespeare was already aware that sleep has vital and specific roles: to repair both the body and mind. Four hundred years later, sleep still largely remains an enigma and is one of the last physiological processes with a lack of scientific consensus. What we do know is that sleep is a quiescent behavioral state, associated with reduced responsiveness to weak stimuli and rapid reversibility in response to strong stimuli, and is required for memory formation, brain plasticity and immune function, among others. Sleep comes in two metabolic and electrophysiological varieties, namely rapid eye movement sleep (R) and non-rapid eye movement sleep (N). In order to correctly classify these stages, this paper will use the American Academy of Sleep Medicine, AASM, scoring, classifying sleep stages as N1, N2, N3 and R sleep, replacing the previous Rechtschaffen and Kales 1968 scoring of sleep stages: NREM 1, 2, 3, 4 and REM sleep. Regardless of the classification, the N sleep stages are not considered distinct entities, rather gradual transitions in wave form densities detected by electroencephalography. 3 point 2 point 1. The Glymphatic System and Sleep. The glymphatic system is constantly filtering toxins from the brain, but during wakefulness, this system remains mainly disengaged. Although sleep is often associated with rest, glymphatic activity is dramatically boosted during sleep. Photoimaging of in vivo mice demonstrated a 90 percent reduction in glymphatic clearance during wakefulness, and twice the amount of protein clearance from the brain intima during sleep. Sleep-induced enhancement of glymphatic function appears to arise from the expansion of the ISF space. In a human in vivo study, blood oxygen level-dependent functional magnetic resonance imaging (Bold fMRI) was used in combination with electroencephalograph and CSF measurements in order to detect in which sleep state most brain activity occurred. They found that during wakefulness, CSF flow had a small-amplitude rhythm, peaking at around 0.25 Hertz, one cycle per four seconds, whereas during sleep, large oscillations occurred every 20 seconds, peaking at around 0.05 Hertz, or one cycle per twenty seconds, resulting in a significantly greater inflow of CSF than during the day. As well as cleansing the brain, the replenishing role of the glymphatic system was observed. Glymphatic-induced reoxygenation of the brain occurs during large pulsations of CSF. The pulsating fashion in which these sleep oscillations occur suggests that the majority of glymphatic activity occurs during N3 sleep. During this stage of sleep, slow oscillatory brain waves were shown to increase the amount of CSF within the interstitial cavities, leading to an 80 to 90 percent increase in glymphatic clearance relative to the waking state, and demonstrate the importance of slow-wave sleep. 3 point 2 point 2. Slow-Wave Sleep. Glymphatic clearance mainly occurs in slow-wave sleep, which is synonymous with the N3 sleep stage. It is characterized by high-voltage synchronized electroencephalograph waveforms: delta oscillations and slow oscillations. Slow-wave sleep has numerous functions including learning, memory and metabolite clearance. In young adults, slow-wave sleep makes up between 10 and 25 percent of total sleep time, but this kind of sleep is not evenly distributed throughout the night, mostly occurring in the first half, with more R sleep occurring in the second half. These slow waves usually lie between 0.5 and 4.5 hertz on electroencephalography and have been linked to sleep pressure, occurring in abundance early in the night and then decreasing. The phenomenon of large bundles of neurons coordinating their electrical activity, rhythmically and repetitively depolarizing, is termed slow oscillatory neuronal activity. These pulsations range from 20 to 30 seconds and reflect important physiological restoration of the brain and blood oxygenation, precisely matching the time, rhythm and electrical activity of the N3 stage, confirming that this waste clearance system is primarily active during slow-wave sleep. 3 point 2 point 3. Sleep and Alzheimer’s Disease. The most common dementia and a chronic age-related neurodegenerative disease, Alzheimer’s is associated with a deterioration of memory, language and the ability to self-care, among others. The complex cascade of neurotransmitters and hormones involved in sleep regulation of the brainstem and hypothalamus is the same as that responsible for Alzheimer’s disease. Sleep abnormalities such as insomnia and sleep apnoea are highly prevalent in patients with neurodegenerative disease, often predating the onset of cognitive or neurological impairment. Although this finding suggests that sleep contributes to the onset of Alzheimer’s disease, the direction of causality is not clear. Sleep disorders may be connected to Alzheimer’s disease itself or associated factors such as pain, depression or drug therapy. Major sleep disturbances include insomnia, sleep apnoea syndrome (SAS) and circadian rhythm sleep disorder. Sleep disturbances occur early in disease progression, with minor cases already demonstrating impaired sleep. Alzheimer’s patients have a shorter total sleep time, increased awakening and worse sleep efficiency compared to controls; specifically, slow-wave sleep was found to be impaired. In Alzheimer’s disease, sleep-related issues appear to be associated with the suprachiasmatic nucleus, which regulates the circadian rhythm and naturally deteriorates with age. Sleep disturbances are present in 25 to 35 percent of Alzheimer’s patients, often resulting in impaired slow-wave sleep, a shorter total sleep time and sleep fragmentation. Despite this, however, only one third of Alzheimer’s patients suffer from clinically diagnosed sleep disturbances, placing some question over the causal interrelationship of sleep and neurodegenerative disease. Although only associations have been made so far, there are genetic links emerging between sleep and Alzheimer’s disease. One of the major genetic risk factors for Alzheimer’s disease is apolipoprotein E, APOE, of which reduced function is associated with sleep apnoea, the progression of sleep disturbances, cognitive performance and sleep deterioration. APOE therefore provides a genetic link between sleep and neurodegenerative disease, adding to the validity of this relationship. We therefore suggest that sleep and sleep impairments likely play a compelling role in neurodegenerative disease in particular in regard to waste removal from the central nervous system. It remains to be demonstrated whether other mechanisms aside from sleep universally impair this process. 3 point 2 point 4. Sleep and Toxic Waste Products in Animals. Using position emission tomography (PET) scans and the tracer F-florbetaben, amyloid-beta levels in 20 mice were assessed during normal sleep and sleep deprivation; a one night comparison showed that there was a significant increase in amyloid-beta levels in the hippocampus and the thalamus in 19 out of 20 mice, demonstrating in vivo evidence of the effects of sleep deprivation on recognized neurodegenerative processes. This relationship could be bidirectional, since amyloid-beta has also been linked to decreasing sleep quality. This can be attributed to the increase in interstitial space volume during sleep. There is a doubling of amyloid-beta clearance in the sleep state, and conversely sleep deprivation shows a reduction in the clearance of CSF metabolites. This indicates the usefulness of sleep monitoring as a non-invasive prognostic marker for neurodegenerative disease. The accumulation of amyloid-beta peptides within the brain parenchyma can lead to neuroinflammation and cognitive decline. 3 point 2 point 5. Slow-Wave Sleep and Age. Sleep varies greatly across our lifespan, starting as polyphasic sleep during early life, becoming monophasic during childhood, then slowly decreasing in duration until the age of 60, when the amount of sleep either remains constant or increases. During this time span, many micro and macro changes occur, the largest of which is the amount of slow-wave sleep, peaking during puberty and then declining with age. The origin of the decrease in slow-wave sleep is still unknown, but a suggested mechanism is the neuronal loss occurring with age, as ageing is often accompanied by a significant loss in cortical grey matter, most of which occurs in the prefrontal cortex, where slow oscillations originate, according to the global hypothesis. Alzheimer’s disease is often regarded as accelerated ageing. The gradual deterioration of slow-wave sleep over time is a possible explanation and would therefore result in less glymphatic clearance and an increased risk of neurodegeneration. 3 point 2 point 6. Governors of Sleep. Although the timing and structure of sleep are controlled by both circadian rhythm and homeostatic processes, slow-wave sleep, R sleep, cortisol and melatonin levels are not affected by circadian rhythm and are mainly driven by homeostatic forces. For example, the amount of slow-wave sleep increases with the number of waking hours. Alongside these slow-wave oscillations, the neuromodulator norepinephrine has been found to regulate glymphatic clearance. 3 point 2 point 7. The Chief Modulator: Norepinephrine. The level of arousal also plays an important role in the movement of CSF and ISF. As we sleep, the central levels of norepinephrine decline, due to lowering locus coeruleus-derived noradrenergic tone, leading to the expansion of the extracellular space, decreasing resistance and therefore increasing CSF influx and ISF efflux. Natural sleep is therefore associated with improved tracer penetration along the periarterial spaces and increased interstitial solute clearance, such as amyloid-beta. These findings were recreated in anesthetized mice, with the volume fraction of the interstitial space during wakefulness being 13 to 15 percent and 22 to 24 percent during both sleep and anaesthesia, again suggesting that sleep eases convective fluid flow. Additionally, norepinephrine receptor antagonists induced glymphatic clearance, suggesting norepinephrine release during the daytime could be suppressing clearance, by decreasing the amount of interstitial space. This blockade of adrenergic signaling expanded the ISF volume, accelerated glymphatic clearance and was associated with slow-wave electroencephalograph activity. Norepinephrine also suppresses choroid plexus CSF production. These expansions and increases in CSF production decrease resistance and increase perfusion, leading to a further boost in the removal of metabolic waste products from the brain. These findings indicate glymphatic clearance increase, its specificity to sleep and the link to levels of CSF flow, which can be modulated in a clinical setting. 3 point 2 point 8. Heartbeat or Breathing Rate. During wakefulness, CSF exhibits a small-amplitude rhythm synchronized to the respiratory signal, whereas during sleep, within the N3 stage, large oscillations occur every 20 seconds, driven by ventricular movement, resulting in significantly greater inflow of CSF. The physical forces propelling CSF in glymphatic clearance are intracranial pulsations. Intracranial pulsations have an established relationship with oscillations of blood pressure, which coincide with heart rate. As well as heart rate, lower-frequency events of respiration, such as vasomotion, were also demonstrated to contribute to glymphatic pulsations. As opposed to systemic arterial pulsations dissipating at an arteriolar and venous level, in the brain, the rigid skull promotes propagation of arterial pulsations, which are still measurable in the microvasculature and venous outflow. Even acute decreases in arterial pulsations impair glymphatic clearance. Paradoxically, the N3 sleep stage which has the highest levels of CSF influx and amyloid-beta removal also has the lowest rates of arterial pulsations, suggesting that other factors are at play. Only recently, lower-frequency intracranial pressure oscillations produced by respiration were shown to complement cardiac pulsations, which could alternatively drive clearance. Ultrafast magnetic resonance imaging demonstrated that forced inspiration was a main driver of CSF flow in both the lateral ventricles and the subarachnoid space. A combination of both heartbeat and respiratory rate appears to drive these pulsations. 3 point 3. Impaired Glymphatic Clearance. 3 point 3 point 1. Alzheimer’s Disease and Glymphatic Clearance. All prevalent neurodegenerative diseases are characterized by the accumulation of aggregated proteins. Accumulations of amyloid-beta plaques and neurofibrillary tangles of hyperphosphorylated tau are implicated in the cognitive decline in Alzheimer’s disease. Perivascular drainage pathways function as a sink for interstitial amyloid-beta and perivascular spaces are also associated with amyloid deposition and Alzheimer’s pathology. Additionally, an abnormal perivascular space has been linked to impaired glymphatic clearance. Amyloid-beta plays a role in synaptic regulation and neuronal survival. Interstitial bulk flow and amyloid-beta accumulation both occur in the perivascular space, but the predominant site of amyloid-beta accumulation is the cerebral arteries. Alongside this, abnormal enlargement of the perivascular space is a frequently observed difference between Alzheimer’s patients and healthy controls. Since glymphatic clearance is responsible for the movement of tau and amyloid-beta aggregates out of the brain, glymphatic clearance is of utmost importance to neurodegenerative disease, but remains understudied. 3 point 3 point 2. Alzheimer’s, Endfeet and AQP4. Glymphatic clearance is impaired in a rodent animal model of Alzheimer’s disease, due to changes in the number of AQP4 water channels responsible for the movement of CSF and ISF, expressed on astrocytic endfeet. AQP4 is usually on the endfeet of astrocytes rather than the soma, with abnormal AQP4 localization associated with perturbed glymphatic clearance. Since AQP4 polarity is crucial for CSF inflow and the clearance of amyloid-beta, the loss of AQP4 polarization in the brain contributes to the impairment of glymphatic function. In the brain, AQP4 mainly exists in two isoforms: a long isoform, AQP4-M1, and a short isoform, AQP4-M23, which both form hetero-tetramers in the plasma membrane of astrocytic endfeet. Furthermore, mouse models using AQP4 deletion showed a decreased clearance of amyloid-beta, confirming their involvement in neurodegeneration. 3 point 3 point 3. Glymphatic Clearance and Age. Glymphatic clearance seems to clear toxic aggregates efficiently until the end of the reproductive lifespan, then the system seems to fail. In old mice, a decrease in AQP4 expression, mis-localization of AQP4 away from the astroglial endfeet and reduced pulsations of the arterial wall led to a 40 percent reduction in amyloid-beta clearance from the brain, depicted in Figure 4. Glymphatic activity in old mice was observed to be reduced by 80 to 90 percent. This could explain the increase in frequency of amyloid-beta in aged brains. Amyloid-beta accumulation is increased due to impaired glymphatic clearance, but high levels of amyloid-beta in the interstitial space also impair fluid movement, creating a positive feedback loop, further reducing amyloid-beta deposition. This means patients with Alzheimer’s disease will mostly have impaired glymphatic clearance, which gradually gets worse. Although the loss of AQP4 polarization favors AD pathology, the cause and effect are not yet clarified. In aged brains, the AQP4 channels on astrocytic endfeet relocate to the astrocytes’ soma due to astrogliosis, slowing the rate of CSF–ISF exchange. Impaired glymphatic clearance was also observed in aged transgenic mice with amyloid plaques, but also in younger mice without plaques. Interestingly, injection of amyloid-beta into CSF reduced glymphatic clearance. Higher amyloid-beta levels resulted in lower clearance of tau tangles. In addition, breathing rates during sleep increase with age due to decreasing lung efficiency. These shallower breaths will decrease intracranial pressure and weaken glymphatic clearance. The strength of penetrating arterial pulsations also decreases with age. Figure 4. Model of glymphatic function in Young, Old and Alzheimer’s disease. In young people, CSF travels along periarterial routes, entering the brain parenchyma, and washes solutes and waste products into the veins. In older people, the loss of AQP4 water channels will result in reduced glymphatic clearance. In those with Alzheimer’s disease, the accumulation of amyloid-beta impairs fluid movement within the interstitial space, decreasing glymphatic clearance. 3 point 4. Lifestyle Choices. No suitable drug has yet been developed for Alzheimer’s disease. Research has begun to emerge on individual lifestyle choices and their modulation of glymphatic activity. Behavioral interventions can be both preventative and curative and are frequently preferred over medication. Since we have established the link between glymphatic activity and the absence of suitable treatment, we next investgate an overview of lifestyle choices and their effects on glymphatic clearance, that may in turn exert an effect on neurodegenerative processes. 3 point 4 point 1. Omega-3 Consumption. Marine-based fish oils known as omega-3 polyunsaturated fatty acids, n3-PUFA’s, have been found to modulate glymphatic activity. Epidemiological studies associate increased levels of n3-PUFA’s with lower incidence of neurodegenerative disease, and n3-PUFA supplementation has been suggested to delay or prevent the onset of Alzheimer’s disease. The central nervous system is highly enriched with n3-PUFA’s that exhibit potent anti-inflammatory properties. High endogenous levels of n3-PUFA’s improve impairment of spatial learning and memory induced by amyloid-beta. Both endogenous and exogenous n3-PUFA’s promote amyloid-beta clearance and reduce aggregate formation by inhibiting the activation of astrocytes, protecting against the loss of AQP4 polarization, thus reducing the chance of amyloid-related injury. They also exhibit anti-amyloidogenic activity, modulate aggregation and decrease downstream toxicity. Dietary intake of n3-PUFAs improved cognitive decline in mild Alzheimer’s disease. Supplementation demonstrates higher CSF influx and clearance, with AQP4 remaining polarized at the astrocytic endfeet, increasing the speed of glymphatic clearance. AQP4 knockout mice exhibited no difference in glymphatic activity even with dietary n3-PUFA supplementation, indicating that AQP4 water channels are essential in n3-PUFAs’ improvement of glymphatic function. n3-PUFA supplementation has therefore been suggested to delay or prevent the onset of AD, by improving glymphatic transport and decreasing amyloid aggregation. 3 point 4 point 2. Intermittent Fasting. Intermittent fasting consists of cycles of fasting and then eating; a specific variation of this is alternate-day fasting, consisting of a day of eating followed by a fasting day for a number of days consecutively. In the brain, AQP4 mainly exists in two isoforms: a long isoform, AQP4-M1, and a short isoform, AQP4-M23, which both form hetero-tetramers in the plasma membrane of astrocytic endfeet. Intermittent fasting ultimately downregulates the expression of AQP4-M1, decreasing the AQP4-M1 to AQP4-M23 ratio, and therefore increases AQP4 polarization along the paravenous space, boosting glymphatic clearance. Intermittent-day fasting, meaning, alternatingly fasting on one day and then eating ad libitum the next, lowered the amount of amyloid-beta deposition. This fasting causes the liver to switch to fatty acid oxidation which increases the amount of beta hydroxybutyrate in the blood after 12 hours. Beta hydroxybutyrate crosses the blood–brain barrier and acts as an endogenous histone deacetylase three (HDAC) inhibitor, which has been shown to exert a protective effect in Alzheimer’s disease progression. HDAC inhibitors prevent histone acetylation, which regulates the expression of microRNA-130a, which represses the expression of AQP4-M1, changing the ratio between isoforms. This endogenous HDAC inhibitor also increases the polarity of AQP4 expression on astrocytic endfeet, further increasing glymphatic clearance. Interestingly, the Alzheimer brain has significantly higher amounts of histone deacetylase 3, consistent with its involvement in neurodegeneration. Importantly, cells exposed to amyloid-beta show a decrease in microRNA-130a expression and an increase in HDAC expression, creating a positive feedback loop that will antagonize the positive effect of intermittent fasting on the neurodegenerative process. 3 point 4 point 3. Sleeping Position. Gravity affects the movement of blood and CSF through the brain, and therefore sleep position will likely play a role in the clearance of waste products from the brain. Both intracranial pressure and cerebral hemodynamics are influenced by body posture, and patients with dementia were found to spend a much larger percentage of time in the supine position compared to controls, establishing an association between time in supine position and dementia. An important factor in this clearance pathway is the stretch of nerves and veins in each position. Glymphatic transport is most efficient in the right lateral sleeping position, with more CSF clearance occurring compared to supine and prone. The average person changes sleeping position 11 times per night, but there was no difference in the number of position changes between neurodegenerative and control groups, making the percentage of time spent in supine position the risk factor, not the number of position changes. The suggested mechanisms behind the effects of posture on clearance would appear to result from gravity and a restriction of venous drainage of the carotid veins. Unfortunately, detecting which position you spend most time in is only possible in a sleep laboratory, since self-reported sleep positions are often false. 3 point 4 point 4. Alcohol Consumption. Alcohol can either boost or hinder glymphatic clearance, depending on dosage and whether consumption is chronic or acute. Alcohol’s effect on glymphatic clearance is independent of the wake or sleep state. Prolonged amounts of excessive alcohol consumption were shown to have adverse effects on the central nervous system, with acute and chronic exposure to high doses of alcohol, 1 gram per kilogram, dramatically reducing glymphatic transport in awake mice. Chronic exposure to high levels of alcohol increases GFAP expression, inducing the depolarization of AQP4 channels, but conversely decreasing the levels of inflammatory cytokines. AQP4 polarity is crucial for CSF inflow and the clearance of amyloid-beta, potentially leading to alcohol-induced changes in these water channels to hinder glymphatic clearance. Heavy alcohol consumption for prolonged periods of time greatly increases the risk of developing Alzheimer’s disease. Intermediate alcohol consumption was also found to decrease glymphatic clearance for both acute and chronic usage. Both intermediate and heavy dosage induced non-permanent changes in glymphatic activity, as after 24 hours of sobriety, glymphatic function was fully restored. In contrast, both acute and chronic exposure to low doses of alcohol, 0.5 grams per kilogram increased glymphatic clearance, due to decreased GFAP expression, reducing the risk of Alzheimer’s disease. For the scale reference of a 75 kilogram person, half a gram per kilogram alcohol consumption is equivalent to 37.5 grams of alcohol, which is contained in 94 milliliters of 40 percent vodka, 300 milliliters of 12 percent wine, or 750 milli-liters of 5 percent beer. 3 point 4 point 5. Exercise. Bulk glymphatic flow is accelerated by physical training and notably improves both memory and cognition in neurodegenerative disease. Voluntary running over a six-week duration restored protein homeostasis in the brain, reduced inflammation by decreasing the activation of microglia and astrocytes, improved cognition and reduced the deposition of amyloid-beta through an increase in glymphatic clearance, but showed no effect on the blood–brain barrier permeability. In addition to this, six weeks of physical exercise accelerated glymphatic clearance and reduced amyloid-beta accumulation by increasing the movement of ISF. AQP4 expression in the cortex was also found to be higher along the paravascular space in the exercise group. This improved AQP4 polarization and led to a decrease in both amyloid plaques and neuroinflammation. These findings are consistent with the benefits of exercise on brain health and cognition in the elderly and demonstrates the usefulness of exercise as a neuroprotective lifestyle choice for brain aging and neurodegeneration. According to the WHO, beneficial amounts of exercise consist of at least 150 minutes of moderate or 75 minutes of vigorous exercise a week. 3 point 4 point 6. Chronic Stress. Chronic psychological stress is a common risk factor for Alzheimer’s disease. Short-term stress is crucial for adaptation and survival, but long-term stress can be detrimental to both body and mind. Chronic stress accelerates the accumulation and deposition of amyloid-beta. Mice exposed to stress exhibited decreased glymphatic influx and efflux, loss of AQP4 polarization and a reduction in AQP4-bearing astrocytes. Stress triggers the hypothalamic–pituitary–adrenal (HPA) axis to release glucocorticoids. Alzheimer’s disease is associated with a dysfunctional HPA axis, demonstrated by high levels of cortisol in the blood. Glucocorticoids act by binding to glucocorticoid receptors (GR) or mineralocorticoid receptors (MR) and decrease astrocyte numbers downregulating the number of AQP4 channels. Stress increases the levels of glucocorticoids and therefore GR activation, which also trigger the amyloid precursor protein to form amyloid-beta. Mifepristone, a GR antagonist, significantly improves impaired glymphatic clearance impaired by stress reversing AQP4 expression. Mifepristone might therefore be a useful treatment for Alzheimer’s disease mediated through the glymphatic system. Section 4. Discussion. This paper provides a synthesis of currently tested lifestyle choices which could aid in preventing or slowing the progression of Alzheimer’s disease through increased glymphatic activity. The incidence of Alzheimer’s disease is rising, but there is currently no effective disease-modifying treatment. Similar to other neurodegenerative diseases, Alzheimer’s disease is characterized by the accumulation of aggregated proteins. The accumulation of amyloid-beta peptides and tau within the brain parenchyma causes neuroinflammation, amyloid-beta plaques and tau tangles. This deposition occurs along perivascular spaces. Glymphatic clearance acts within these spaces, moving tau and amyloid-beta aggregates out of the brain and thus reducing neurodegenerative processes. Glymphatic clearance also offers an explanation for why dementias are generally age-related. In mice, clearance of misfolded proteins and other cellular debris is generally efficient but reduces in capacity over time and begins to fail at the end of the reproductive lifespan. This was demonstrated by glymphatic clearance in old mice being reduced by 80 to 90 percent, and may at least partly explain the increased concentration of amyloid-beta in aged brains. One suggested mechanism behind this is the loss of polarization of AQP4 water channels. AQP4 channels are usually polarized along astrocytic endfeet, but can lose polarization, becoming more evenly distributed around the soma and thus slowing the rate of CSF to ISF exchange. Since AQP4 polarity is crucial for CSF inflow and the clearance of amyloid-beta, the loss of AQP4 polarization in the brain contributes to the impairment of glymphatic function. AQP4 deletion results in decreased clearance of amyloid-beta, supporting its involvement in neurodegenerative processes. The vast majority of glymphatic clearance occurs during sleep. There is a 90 percent reduction in glymphatic clearance during wakefulness and twice the amount of protein clearance from the brain intima during sleep. During slow-wave sleep, delta oscillations are nested in high-voltage slow oscillatory neuronal activity, causing large bundles of neurons to harmonize, rhythmically and repetitively depolarizing over 20 to 30 seconds. This increases CSF inflow within the interstitial cavities and boosts glymphatic activity, increasing interstitial solute clearance. Sleep is a primary driver of bulk flow and is crucial in its modulation. These slow oscillations have been linked to sleep pressure, occurring in abundance early in the night and then decreasing over time. Slow-wave sleep is linked to time spent awake, with an increase in waking hours increasing the amount of slow-wave sleep. As well as within each night, sleep changes greatly across our lifespan. The percentage of slow-wave sleep is highest during puberty, and then declines with age, exacerbated in Alzheimer’s disease. Age-related neuronal and cortical grey matter loss is thought to be responsible for the decrease in slow-wave sleep, particularly in the prefrontal cortex where slow oscillations are believed to originate. The neuromodulator norepinephrine regulates sleep, but also glymphatic clearance. During sleep, the decrease in norepinephrine levels causes the expansion of the extracellular space, decreasing resistance and therefore increasing the rate of glymphatic clearance. Norepinephrine also suppresses choroid plexus CSF production. These expansions, together with the increase in CSF production, decrease resistance and boost perfusion, leading to a further increase in the removal of metabolic waste products from the brain. Norepinephrine controls the overall quantity of solute clearance, but intracranial pulsations are the physical force that propel CSF along the parenchyma. Intracranial pulsations have an established relationship with oscillations of blood pressure, which coincide with heart rate. These disperse throughout the brain, aiding metabolism, and at the same time eliminate toxic waste products. Alongside heart rate, lower-frequency events of respiration, and vasomotion contribute to glymphatic pulsations. Slow-wave sleep is linked to glymphatic clearance, but also dementia. A third of Alzheimer’s patients suffer from clinically diagnosed sleep disturbances, and the vast majority Alzheimer’s patients have a shorter total sleep time and impaired slow-wave sleep, with both these deteriorations of sleep often predating its onset. The complex cascade of neurotransmitters and hormones involved in sleep regulation is affected in Alzheimer’s disease. Additionally, in healthy mice, a single night of sleep deprivation was sufficient to increase amyloid-beta deposition. Sleep impairment therefore appears as an influential risk factor for neurodegenerative disease that should ideally be recognized by general practitioners and medical specialists alike. In this manuscript, we have described the results of lifestyle choices on glymphatic clearance; we here provide a summary of the findings. Sleep position, alcohol intake, exercise, omega-3 consumption, intermittent fasting and chronic stress all modulate glymphatic clearance, thereby potentially altering the risk for Alzheimer’s disease. 1. Gravity affects the movement of blood and CSF throughout the brain, and sleep position will therefore play a role in the clearance of waste products from the brain. Neurodegenerative patients spend a much larger percentage of time in the supine position, which suggests a connection between time in supine position and dementia. Glymphatic transport is most efficient in the right lateral sleeping position, with more CSF clearance occurring compared to supine and prone. 2. High levels of both endogenous and exogenous marine-based fish oils known as omega-3 polyunsaturated fatty acids, n3-PUFA’s, are associated with lower incidence of neurodegenerative disease, and n3-PUFA supplementation has been suggested to delay or prevent the onset of Alzheimer’s disease. n3-PUFA’s promote amyloid-beta clearance and reduce aggregate formation by inhibiting the activation of astrocytes, protecting against the loss of AQP4 polarization, and therefore reduce the chance of amyloid-related injury. They exhibit anti-amyloidogenic activity, decrease amyloid-beta production, modulate aggregation and decrease downstream toxicity. 3. Alcohol consumption can either boost or hinder glymphatic clearance, depending on dosage and chronic or acute consumption. Acute and chronic exposure to high doses of alcohol, 1 grams per kilograms, dramatically reduces glymphatic transport in awake mice. This suggests that heavy alcohol consumption for prolonged periods of time greatly increases the risk of developing Alzheimer’s disease. On the other hand, both acute and chronic exposure to low doses of alcohol, a half of a gram per kilogram, increased glymphatic clearance. Low doses of alcohol improved glymphatic function, due to decreased GFAP expression, and avoided the loss of AQP4. Physical training in mice showed a notable improvement in both memory and cognition impairments, associated with neurodegenerative disease. 4. Physical exercise decreased astrocyte and microglia activation, leading to reduced inflammation, and increased the movement of ISF. The increase in ISF movement accelerated glymphatic clearance and reduced amyloid-beta accumulation. The increase in ISF movement is due to improved polarization of AQP4, resulting in a decline in amyloid plaques and neuroinflammation. This confirms the benefits of exercise on brain health and cognition in the elderly and demonstrates the usefulness for exercise as a neuroprotective lifestyle choice for brain aging and neurodegeneration. 5. Chronic stress is a common risk factor for Alzheimer’s disease. Short-term stress is crucial for adaptation and survival, but long-term stress can be detrimental to both body and mind. Chronic stress accelerates the accumulation and deposition of amyloid-beta. Mice exposed to stress exhibited decreased glymphatic influx and efflux, decreased expression and loss of the polarization of AQP4 and a reduction in AQP4-bearing astrocytes. 6. Intermittent fasting ultimately downregulates the expression of AQP4-M1, decreasing the AQP4-M1 to AQP4-M23 ratio, and therefore increases AQP4 polarization along the paravenous space, increasing glymphatic clearance. Intermittent fasting therefore improves cognitive function and decreases amyloid-beta deposition, by increasing polarity mediated through, and by the upregulation of, the AQP4-M1 to AQP4-M23 ratio. Intermittent-day fasting decreases the amount of amyloid-beta deposition. These lifestyle choices in various ways modulate the levels of glymphatic clearance, lowering the risk of, or possibly even preventing, Alzheimer’s disease. Each lifestyle choice has a different mechanistic route, but all seem to function by changing the number or polarity of AQP4 channels. These can be split into two categories, with differing degrees of recommendations. Easily modifiable lifestyle choices include alcohol intake, omega-3 consumption, sleep position and exercise. Alcohol should only be consumed in low doses of half a gram per kilogram, if at all, and avoided in moderate or high quantities. Omega-3 supplementation is recommended. Self-reported sleep position is often unreliable, however with the use of sleep positional therapies, an individual can be trained to alter their sleep position. Each individual should exercise moderately for 150 minutes a week or vigorously for 75 minutes. The less clear-cut lifestyle choices include intermittent fasting and chronic stress reduction. Intermittent fasting has only been investigated thus far in animal models, and can have harmful effects on humans, thus it requires further investigation. Chronic stress is treatable, but this is not as simple as taking supplementation and may require other therapeutic means. Although medication for chronic stress exists, this is a sensitive case-specific option that requires detailed and precise clinical assessment and is beyond the scope of this paper. Easily modifiable or not, lifestyle choices undoubtedly impact glymphatic clearance and should be harnessed to avoid brain ageing and neurodegeneration. A limitation within this paper is the lack of direct information on Alzheimer’s disease sufferers. Although this literature review clearly highlights the effectiveness of lifestyle choices as a prevention of neurodegeneration, most of these findings come from murine studies, as there is only little in vivo human evidence for lifestyle choices altering neurodegeneration. Firstly, these findings need to be replicated in humans. Secondly, since this is emerging research and little literature exists, especially concerning lifestyle choices, each recommendation should be examined further before application. Therefore our suggestion that these lifestyle choices are causally linked is provided with the caveat that it is based on a small number of studies that need to replicated. Thirdly, these findings only demonstrate impaired glymphatic clearance, but the precise causal relationships still remain to be elucidated. Fourth and finally, these lifestyle choices need to be assessed in relation to each other, since we simply do not know whether these effects will summate, synergize or cancel each other out. There seems a compelling need to capitalize on the glymphatic system to harness the potential for reducing dementia rates. Although AQP4 channels have been identified as a potential drug target, a suitable drug is yet to be developed; lifestyle choices therefore remain the best available option for regulating AQP4 numbers and polarization. Future studies should amongst others, see Table 1, empirically confirm the causality between lifestyle choices and improved glymphatic clearance, to quickly develop effective lifestyle interventions. Since existing glymphatic research consists mostly of animal studies, these findings need to be replicated in humans. Other avenues of research may include the role of exosomes, small extracellular vesicles, in transporting protein aggregates. It is as yet unknown if disregulation of the glymphatic system may contribute to neurodegenerative processes by reducing exosome removal. Table 1. This table summarizes the future challenges arising from this paper. Future Studies Surrounding the Glymphatic System and Lifestyle Choices should Aim to: 1. Confirm the link between the glymphatic system and lifestyle choices. 2. Replicate all glymphatic-related murine research in humans. 3. Test easily modifiable lifestyle choices affecting glymphatic clearance in a lifestyle-based intervention in humans. 4. Investigate whether the effects of multiple lifestyle choices on glymphatic activity cumulate or cancel each other out. 5. Confirm that AQP4 channels are the causal pathway behind glymphatic activity. 6. Conduct a randomized controlled trial to confirm whether lifestyle choices have an effect on glymphatic activity. 7. Investigate the effect of Mifepristone on glymphatic activity 5. Conclusions. The glymphatic system is a brain-wide bulk flow waste removal network. Impaired glymphatic clearance contributes to the risk of developing Alzheimer’s disease, due to reduced removal of aggregated proteins such as amyloid-beta and tau. There is no effective treatment for Alzheimer’s disease, and with an increasing incidence and burden on society, the need for an intervention is well recognized. Lifestyle choices may be our current best option for reducing or even stopping the neurodegenerative process, as they constitute non-invasive, readily available, relatively inexpensive and accessible interventions Author contributions, funding and twenty-one references. https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Index of Sciencey stuff:

Biological Functions of Autophagy Genes: A Disease Perspective. Beth Levine and Guido Kroemer. Cell. 2019 January 10. Volume 176 (1 to 2). Pages 11 to 42. doi:10.1016/j.cell.2018.09.048. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347410/ Index of other science articles: https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Abstract. The lysosomal degradation pathway of autophagy plays a fundamental role in cellular, tissue and organismal homeostasis and is mediated by evolutionarily conserved autophagy-related (ATG) genes. Definitive etiological links exist between mutations in genes that control autophagy and human disease, especially neurodegenerative, inflammatory disorders and cancer. Autophagy selectively targets dysfunctional organelles, intracellular microbes and pathogenic proteins, and deficiencies in these processes may lead to disease. Moreover, ATG genes have diverse physiologically important roles in other membrane trafficking and signalling pathways. This review discusses the biological functions of autophagy genes from the perspective of understanding, and potentially reversing, the pathophysiology of human disease and aging. Introduction. A decade has elapsed since our review in 2008 in Cell on “Autophagy in the Pathogenesis of Disease”. During this period, more than 33,000 new articles related to autophagy were published, a Nobel prize was awarded for the discovery of the molecular mechanisms of autophagy to Levine and Klionsky in 2017, and to Mizushima in 2018. Considerable interest has also emerged in autophagy modulation as a potential target in clinical medicine. The fundamental concepts discussed in our 2008 review remain unchanged. The lysosomal degradation pathway of macro autophagy, herein referred to as autophagy, plays a crucial role in cellular physiology, including adaptation to metabolic stress, the removal of dangerous cargo, for example, protein aggregates, damaged organelles, intracellular pathogens, the renovation during differentiation and development, and the prevention of genomic damage. Generally, these and other functions protect against numerous diseases, including infections, cancer, neurodegeneration, cardiovascular disorders, and aging. Under certain circumstances, autophagy may be detrimental either via its pro-survival effects, such as in cancer progression, or via possible cell death-promoting effects. Over the past ten years, significant progress has been made in understanding the molecular mechanisms of autophagy, the regulation of autophagy, and the effects of autophagy on physiology and pathophysiology. New major conceptual advances underscore the plurality of functions of the autophagic core machinery in various membrane trafficking and signaling events and delineate the exquisite specificity with which autophagy targets selected cargo for degradation. These advances, together with discoveries in human genetics linking ATG gene mutations to specific diseases, provide a multidimensional perspective of mechanisms by which ATG gene-dependent pathways protect against mammalian disease. Herein we review selected highlights of the past decade of research on the biological functions of autophagy genes, primarily from a perspective of understanding and treating human disease. Autophagy and other Autophagy Gene-Dependent Pathways. The original scientific definition of autophagy, from the Greek for “self-eating”, is the delivery of cytoplasmic cargo to the lysosome for degradation. There are at least three distinct forms of autophagy, chaperone mediated autophagy, micro autophagy and macro autophagy, which differ in terms of mode of cargo delivery to the lysosome. Macro autophagy is the major catabolic mechanism used by eukaryotic cells to maintain nutrient homeostasis and organelle quality control. It is mediated by a set of evolutionarily conserved genes, the autophagy-related (ATG) genes, originally discovered in yeast genetic screens. With a few exceptions, all ATG genes are required for the efficient formation of sealed autophagosomes that proceed to fuse with lysosomes. In higher eukaryotes, many ATG genes are functionally diversified to facilitate delivery of extracellular cargo to the lysosome, to promote the plasma membrane localization or extracellular release of intracellular cargo, and to coordinate intracellular communication with various cell signaling pathways, Figure 1. These other functions are not, sensu stricto, autophagy and accordingly, will be referred to as ATG gene-dependent pathways. There are broad implications of ATG gene functions in different membrane trafficking and signaling pathways for mammalian cell biology, physiology and disease. Degradative Autophagy: The reason for the existence of Autophagy Genes. The function of ATG genes as originally discovered is to orchestrate and mediate the formation of double-membraned structures that deliver intracytoplasmic contents to the lysosome for degradation. This process is conserved in all eukaryotic organisms, occurs at basal levels in nearly all cell types, and is increased by diverse intracellular and extracellular cues. It is essential for cellular homeostasis, cellular protein and organelle quality control, and organismal adaptation to environmental stress. These principles are firmly supported by nearly two decades of studies involving genetic ablation of the autophagy machinery in diverse eukaryotic species. This lysosomal degradation pathway is usually described as involving a set of around 16 to 20 core conserved ATG genes. The ATG proteins encoded by these genes are traditionally classified into distinct biochemical and functional groups that act at specific stages of autophagosome initiation or formation. In this scheme, see other recent reviews for details, the ULK1 serine threonine kinase complex, involving ULK1, FIP200, ATG13 and ATG101, plays a major role in autophagy initiation, phosphorylating multiple downstream factors. Two distinct Beclin 1, class 83 phosphatidylinositol 3-kinase (PI3KC3) complexes generate phosphatidylinositol 3-phosphate (PI3P) to act in auto phagosome nucleation, or endolysosomal and auto phago lysosomal maturation. Vesicles containing ATG9A, the only transmembrane core ATG protein, supply membrane to auto phagosomes. WIPI, WD repeat domain phosphoinositide-interacting, proteins and their binding partners, ATG2A or ATG2B, function in early stages of membrane elongation at the site of PI3P generation. Autophagosome membrane expansion and completion involves two ubiquitin-like protein conjugation systems: the Ub-like ATG12 conjugates with ATG5 and ATL16L1 and the Ub-like LC3 subfamily, ATG8 in yeast, conjugates with membrane-resident phosphatidylethanoloamine, PE. Unlike in yeast, the ubiquitin-like protein conjugation systems are not essential for auto phagosomal membrane completion in mammalian cells, although they determine the efficiency of the process. This classification of the ATG proteins has provided a useful framework for studying and understanding autophagy. However, its apparent simplicity is at variance with extensive data indicating a highly complex level of interconnectivity among the ATG proteins and newly described functions of ATG proteins at different stages of autophagy. Based on unbiased proteomic analyses, most ATG proteins interact with other ATG proteins that reside outside of their “classic” functional complex. Experimentally, some of these interactions are known to be important for autophagosome formation. For example, FIP200, a member of the ULK1 kinase complex, interacts with ATG 16L1 to properly target it to the isolation membrane, also known as the phagophore, of the nascent autophagosome. ATG14, a component of the autophagy-specific PI3KC3–C1 complex, also functions in SNARE-driven membrane fusion. Similarly, Atg13, a component of the yeast Atg1, mammalian ULK1 kinase complex, interacts with Atg9 to recruit Atg9 vesicles to the pre-autophagosomal structure. The broader interconnectivity and functional multiplicity of core autophagy proteins in autophagosomal biogenesis requires further elucidation. Moreover, as indicated by a recent conditional genetic interactions study using diverse yeast omics datasets, new systems biology approaches will likely identify additional genes required for autophagy, especially those that may function in a stimulus-dependent, cell type-dependent or species-specific manner. The core ATG proteins, conserved from yeast to humans, are necessary but not sufficient for degradative autophagy. The degradation of autophagosomal cargo cannot proceed without successful fusion to an available and functional lysosome. Research in the past decade has unmasked some of the key factors required for lysosomal biogenesis, autophagolysosomal fusion, lysosomal function during autophagy and autophagic lysosome reformation. Adenoviral-mediated gene delivery of TFEB, a master transcriptional regulator of lysosomal biogenesis, improves outcomes in various rodent disease models, including Parkinson’s disease, lysosomal storage disorders, tauopathies, alpha 1-antitrypsin deficiency, and hepatic hyper-ammonemia. Auto phago lysosomal fusion requires changes in lysosomal pH, certain cytoskeleton motor proteins (dynein), tethering factors, the HOPS complex, the Rab GTPase, RAB7, SNARE proteins, the Q-SNARE, syntaxin 17 on autophagosomes which interacts with R-SNARE proteins, SNAP29 and VAMP8 on endosomes, lysosomes, phospholipids, and members of the LC3 Gabarap family that are bridged to tethering factors or SNARES by adaptor proteins. Screens in C elegans identified novel metazoan-specific genes required for fusion steps in degradative autophagy. One example relevant to human disease is EPG5, which encodes a RAB7 effector. Autosomal recessive mutation of EPG5 results in Vici syndrome, a neurodevelopmental and multisystem disorders, see Table 1. Mutations in genes that regulate lysosomal acidification such as ATP6AP2 and presenilin 1 are associated with X-linked Parkinsonism and Alzheimer’s disease, see Table 1. Thus, we must consider regulators of lysosomal biogenesis, the fusion machinery, and determinants of lysosomal function in our efforts to decipher how deficient autophagy leads to disease and how autophagy can be regulated to prevent or treat disease. Beyond Self-Eating: Autophagy Genes Function in Phagocytosis. Several core ATG genes function in a process that shares some similarities with autophagy but involves digestion of unwanted extracellular, rather than intracellular, material. During this process, termed LC3-associated phagocytosis (LAP), single-membraned macroendocytic vacuoles, macropinosomes, phagosomes and entotic vacuoles, engulf extracellular cargo (such as bacteria, dead cells or live cells), become decorated by lipidated LC3, and are directed to the lysosome for degradation. LAP is distinguished from autophagy by four main features: (1) The origin of the vacuolar contents, extracellular versus intracellular, (2) The requirement of cargo engagement of an extracellular receptor for activation, (3) The type of membrane that fuses with the lysosome, single membrane versus double membrane, and (4) The utilization of a subset versus all of the core ATG proteins. LAP requires NADPH-oxidase (NOX2) to generate reactive oxygen species (ROS), certain components of the Beclin 1, VPS34 complexes, PI3P generation, LC3-conjugation to the single membrane of the phagosome, and all components of the LC3 conjugation machinery. However, it does not require other core ATG proteins, such as components of the ULK1 complex or the autophagy-specific Beclin 1, VPS34 complex component, ATG14. Somewhat enigmatically, LAP requires Rubicon, an inhibitory component of the autophagy-specific Beclin 1, VPS34 complex. The precise effects of LC3 decoration of phagosomes on their fusion with lysosomes and on lysosomal function are unknown. The presence of LC3 on phagosomes may enhance efficiency of phagolysosomal maturation, perhaps through a mechanism similar to that of LC3, GABARAP family members in autophagolysosomal maturation. LAP was originally described in murine macrophages during phagocytosis of particles that engage Toll-like receptors (TLRs) and is involved in type I interferon (IFN) secretion in response to DNA immune complexes and other TLR9 ligands. Physiologically important functions of LAP have been identified by comparing phenotypes of mice with myeloid-specific deletion of LAP-specific genes, for example Rubicon or NOX2, and autophagy-specific ATG genes, for example FIP200 or Atg14. LAP is required for degradation of photoreceptor outer segments by retinal pigment epithelium (RPE), a process necessary for intact vision. LAP is induced by the fungus, A fumigatus, and the intracellular bacterium, L monocytogenes, and enhances host defense against these pathogens. Mice lacking several components of the LAP pathway develop an autoimmune systemic lupus erythematosus (SLE)-like disease, perhaps due to a defect in the clearance of dying cells that triggers enhanced pro inflammatory signaling and autoantibody production. Given the crucial roles of receptor-activated phagocytosis in human physiology, it is likely that LAP, like classical autophagy, will emerge as an important pathway in human disease. While the two pathways utilize overlapping genetic machinery, a critical distinction renders them antagonistic. Specifically, Rubicon is required for LAP but suppresses autophagy, and recent studies confirm a mutually inhibitory relationship between LAP and autophagy in photoreceptor degradation in RPE cells. It is not clear why these two pathways are counter-regulated, possibly, cells may need to shut off the alternative pathway during stress to avoid competition for overlapping resources. At a mechanistic level, it is uncertain how Rubicon functions to promote Beclin 1, VPS34 kinase activity at the phagosome, but inhibit it at other organellar sites. Interestingly, the WD repeat-containing C-terminal domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes in LAP, but dispensable for canonical autophagy, illustrating another difference in the molecular roles of an ATG protein in autophagy and LAP. The genetic overlap and mutual antagonism of LAP and autophagy have practical implications for autophagy-targeted therapies. Theoretically, specificity in autophagy induction might be enhanced by activating the ULK1 complex rather than downstream shared nodes in autophagy and LAP (although the ULK1 kinase complex may have substrates outside of autophagy). The appeal of targeting Rubicon, a negative regulator of autophagy whose knockout in mice has beneficial effects, for example improved high-fat diet-induced hepatic steatosis and increased cardiac protection during lipopolysaccharide-induced sepsis, may be tempered by potential adverse effects of LAP inhibition, such as increased susceptibility to fungal diseases and autoimmunity. Furthermore, treatments that target shared ATG proteins may result in unpredictable effects on each pathway, assuming these proteins are rate-limiting and the two pathways compete for access to these shared core ATG proteins. The identification of LAP as a lysosomal degradation pathway that utilizes certain core ATG genes requires us to adopt a wider interpretative lens for studies of mice with deletions of these genes. Does deficient LAP versus deficient autophagy contribute to pathological phenotypes in mice with whole body or tissue-specific deficiency of genes such as beclin 1, ATG5, ATG7, or ATG16L1? To what extent does increased autophagy versus decreased LAP contribute to Rubicon knockout phenotypes? Do the GWAS associations between polymorphisms in some of these genes and diseases that involve disordered immune regulation, such as asthma, SLE, and inflammatory bowel disease, see Table 1, suggest a role for altered LAP in their pathogenesis? The observation that a deficiency of LAP-associated, but not of non-LAP-associated, ATG genes results in a SLE-like syndrome in mice, underscores the importance of this question. Specific molecular markers to distinguish LAP from autophagy in both animal models and human disease are needed. Beyond Lysosomal Degradation: Autophagy Genes Function in Secretion and Exocytosis. ATG genes are used not only for targeting intracellular cargo to the lysosome for degradation, but also for pathways that involve the targeting of intracellular cargo to either the plasma membrane or extracellular environment, Figure 1. Generally, these pathways have been grouped under the umbrella term “secretory autophagy”, however, as the “phagy” part is missing from the process, we prefer the more linguistically precise term of ATG gene-dependent secretion. There are many different types of ATG gene-dependent secretion, reviewed elsewhere from a cell biology perspective, but the mechanisms governing most of these processes are not well understood. Here, we focus on these pathways as they may relate to mammalian physiology and disease. Unconventional secretion involves the extracellular release of proteins that lack amino-terminal signal peptide leader sequences and bypass “conventional” transit through the endoplasmic-reticulum, ER, Golgi apparatus to reach the plasma membrane. A role for ATG proteins in this process was first discovered in yeast secretion of the acyl-CoA-binding protein, Acb1. In mammalian cells, unconventional secretion of leaderless proteins, such as the pro-inflammatory cytokines processed by the inflammasome, IL-1 beta and IL-18, also require the autophagy protein, ATG5. The precise mechanisms underlying ATG gene-dependent unconventional secretion remain unclear. It is not certain whether targets are captured in an autophagosomal lumen and, or the intermembrane space between the double membrane of the autophagosome, nor is it certain how targets are delivered to and released from the plasma membrane. Autophagosome-like vesicles containing IL-1 beta bypass syntaxin 17-dependent fusion with lysosomes and instead use specific SNAREs and syntaxins involved in vesicle fusion with the plasma membrane for cargo secretion. A function of ATG genes in secretion of pro-inflammatory mediators, and more broadly, other leaderless proteins, could have vast importance for inflammatory disorders and a wide range of other diseases. However, it is currently difficult to assess the physiological importance of ATG gene-dependent secretion of IL-1 beta and IL-18 in vivo, as macrophage, or hematopoietic cell specific deletion of Atg5, Atg16l1, and Atg7 in mice is associated with increased, rather than decreased, levels of IL-1 beta and IL-18 production. These findings may reflect basal functions of ATG genes in the negative control of inflammasome activation, whereas the ATG gene-dependent secretion of pro-inflammatory mediators may be unmasked during certain stress conditions, such as inflammasome activation triggered by lysosomal membrane damage. The possibility of an autophagy-dependent secretome in vivo warrants further investigation and may lead to the identification of proteomic signatures of autophagy activation as clinically useful serum biomarkers. Theoretically, autophagy-inducing therapies might lead to untoward effects via the unconventional secretion of pro-inflammatory mediators or other pathogenic proteins. Perhaps the best-established link between ATG gene-dependent secretion and mammalian physiology and disease relates to the exocytosis of secretory granules and lysosomes. Notably, human genome wide association studies (GWAS) that revealed a polymorphism in a core ATG gene, ATG 16 L1 T300A, as a major risk allele for Crohn’s disease spurred the discovery of a fundamental role for the ATG protein conjugation machinery in secretory granule exocytosis. In mice, the hypomorphic expression of Atg 16 L1, referred to as the Atg 16 L1 T300A knock-in mutation, Paneth cell-specific deletion of Atg16L1, Atg5, or Atg7, or the whole-body deletion of Atg4b results in abnormal granule morphology and a defect in granule exocytosis and lysozyme secretion by Paneth cells. Paneth cells are a specialized ileal epithelial cell type that controls the intestinal microbiota by secreting lysozyme and antimicrobial peptides. Similar defects in Paneth cell morphology are observed in patients with, but not those without, the ATG 16 L1 T300A Crohn’s disease risk allele. The precise membrane trafficking mechanisms by which ATG proteins facilitate secretory granule exocytosis in Paneth cells or other cell types remain unknown. However, a recent study indicates that lysozyme is localized in large LC3-positive vesicles in Paneth cells from wild-type but not Atg 16 L1 T300A mice. Thus, in a manner similar to autophagosome-like vesicles involved in unconventional protein secretion, secretory granules may be earmarked for exocytosis by the presence of LC3 on their surface. A related, but topologically distinct, link between autophagy and secretory lysosome exocytosis was uncovered in another specialized type of secretory cell, the osteoclast. Osteoclasts resorb bone by a mechanism that involves secretory lysosome fusion with bone-apposed plasma membrane composed of ruffled borders, with the discharge of matrix-degrading molecules into the site of osteal degradation. In mice, the ATG protein conjugation machinery and the Rab GTPase, Rab7, are essential for generating an LC3-labeled ruffled border, cathepsin K release and normal bone resorption, thus indicating a role for ATG genes in mediating polarized secretion of lysosomal contents to the extracellular space. In this scenario, the plasma membrane, not the secretory lysosome, is labeled by LC3. Thus, during secretion, the ATG protein conjugation machinery and resulting lipidated LC3 can function either in the formation of normal secretory granules that properly fuse with the plasma membrane or in the creation of a specialized plasma membrane that fuses with secretory lysosomes. The predicted clinical outcome of defects in ATG gene-dependent osteoclast functions would be osteopetrosis, a disease marked by abnormally dense bone. Consistent with this prediction, mutations in PLEKHM1, a Rab7 effector, and the v-ATPase alpha 3 subunit involved in lysosomal acidification, are each associated with osteopetrosis in patients. In contrast, aging, which is associated with reduced autophagy in most cell types, is accompanied by osteopenia and osteoporosis in mice and humans. This may reflect the roles of ATG genes in other cell types in the bone that favor bone growth and normal bone density, including protection against endoplasmic reticulum (ER) and oxidative stress in osteoblasts and osteocytes and maintenance of the proper function of bone mesenchymal stem cells. Thus, studies in bone represent an elegant example of how the autophagic machinery can exert different specialized functions in distinct cell types within a given organ, functions that may have opposite effects, such as bone resorption and bone formation, to orchestrate overall tissue homeostasis. As osteopenia, osteoporosis and associated skeletal fractures are a major cause of morbidity and mortality in aging humans, this area warrants further investigation as a potential clinical target for autophagy upregulation. Numerous other defects in protein secretion in ATG gene knockout mice have been described, although it is unclear whether they reflect a direct role for ATG genes in autophagy-independent trafficking or more indirect consequences of autophagy deficiency on secretory processes. These include defects in the assembly and secretion of octogonial core proteins which leads to abnormalities in vestibular development and defects in pancreatic beta cell insulin granule morphology and secretion, melanogenesis and pigmentation, and mucus secretion of airway epithelial cells and intestinal goblet cells. Accumulating evidence suggests that ATG proteins also have pleiotropic effects on the cellular release of exosomes, a process that is mediated by fusion of the multivesicular body (MVB) with the plasma membrane. Autophagy induction can prevent, whereas ATG gene silencing or pharmacological inhibition can increase, extracellular release of exosomes, including those containing pathogenic protein cargoes, such as alpha-synuclein, prions and amyloid precursor protein. This regulatory mechanism is presumed to involve MVB fusion with auto phagosomes, thereby diverting MVB transport away from the plasma membrane. Increased exosome release in the setting of impaired autophagy may function as an alternative quality control pathway to maintain cellular homeostasis and prevent cell death due to proteotoxicity. However, there are also examples in which ATG genes stimulate exosome production. Atg5, but not Atg7, has been shown to decrease late endosome acidification by disrupting the v-ATPase, thereby promoting the production of exosomes that enhance tumor metastasis. Similarly, the ATG3-ATG12 conjugate which is required for LC3 lipidation during basal, but not starvation, conditions interacts with the ESCRT protein, Alix, and positively controls Alix-dependent exosome biogenesis. Given the expanding repertoire of exosome-dependent processes, including neurodegeneration, immune signaling, metabolism, tumor metastasis and viral infection, the effects of the autophagic machinery on the fate of the MVB lysosomal degradation or exocytosis, may partly underlie the pathophysiological effects of ATG gene mutation. The ATG machinery modulates retromer function to control the endosome-to-cell-surface recycling pathway. During metabolic stress, LC3 on autophagic structures binds to the RabGAP protein TBC1D5 to sequester it away from an inhibitory interaction with the retromer complex. This sequestration allows retromers to associate with endosomal membranes and mediate plasma membrane translocation of the glucose transporter, GLUT1, a facilitator of glucose uptake. GLUT1 is required for the low levels of basal glucose uptake required to sustain cellular respiration, and its plasma membrane localization normally increases when cells are exposed to low glucose. Perturbation in ATG protein conjugation may significantly cripple this metabolic homeostatic mechanism involving GLUT1 trafficking and, in addition, affect the cell surface localization of other as-of-yet-unidentified receptors. Autophagy Genes in Other Dynamic Membrane Events. Non-autophagic functions of ATG genes in membrane trafficking modulate the infection of host cells by viruses, bacteria and other pathogens. These include processes described above such as LAP, which may be partially antagonized by virulent micro-organisms that enter professional phagocytes, and LC3-regulated exocytosis, which is involved in the egress of viruses that either reside inside autophagosomes or whose envelopes become decorated with LC3. Many additional ATG gene-dependent membrane reorganization events, or interference with such events, also regulate infection. For example, several ATG genes are required for the formation of intracytoplasmic membrane-associated replication factories of certain medically important RNA viruses, such as hepatitis C viru. Similarly, the formation of multi-membranous vacuoles that support replication of the bacterium, B. abortus, involves ATG genes required for class 3 PI3K activity but not those required for LC3 conjugation. In contrast, IFN gamma inhibits T gondii replication by a process involving LC3, Gabarap lipidation and recruitment of IFN gamma inducible GTPases to the parasitophorous vacuole, where they disrupt the membrane and destroy the parasite’s replicative niche. Similarly, IFN gamma mediated control of murine norovirus, a model for human epidemics of gastroenteritis, involves labeling membrane-associated viral replication complexes with lipidated LC3 and recruitment of IFN gamma inducible GTPases. Thus, marking replication-associated membrane structures by LC3 conjugation may represent a conserved mechanism underlying IFN gamma mediated control of intracellular pathogen replication. Further understanding of the precise processes by which different subsets of ATG proteins provide or destroy host membranes necessary for different stages of pathogen replication may lead to the development of new anti-infective strategies. Beyond Membrane Trafficking: Autophagy Proteins Have Other Functions. The autophagy proteins not only help orchestrate the cross-talk of diverse vesicular trafficking pathways, but also interface with multiple other cellular pathways, including, but not limited to, cell death pathways, cell cycle regulation, and innate immune signaling. The interaction of FIP200 with Atg13 is essential for autophagy in vivo and neonatal survival in mice, but the non-autophagic function is sufficient to maintain embryogenesis through a mechanism involving protection against TNF alpha induced apoptosis. Atg7, independently of its E1-like enzymatic activity and function in autophagy, regulates p53-dependent cell cycle arrest and apoptosis, and the neonatal lethality of Atg7 knockout mice is partially rescued by inhibition of the DNA damage response through deletion of the protein kinase Chk2. In mice, deletion of Atg9a, but not Atg5, results in a defect in necrosis at the bone surface during developmental morphogenesis. The precise mechanisms underlying these, and additional, functions of individual ATG proteins in cell death and cell cycle regulation are not well understood. ATG proteins regulate inflammatory and immune signaling both through autophagy-dependent mechanisms, such as by the autophagic removal of damaged mitochondria that produce ROS and activate RIG One signaling and the NLRP3 inflammasome, and autophagy-independent mechanisms that generally involve ATG protein interactions with immune signaling molecules. For example, the ATG5-ATG12 conjugate inhibits type One IFN signaling in response to viral infection by binding to the CARD’s, caspase activation and recruitment domains, of RNA recognition molecules such as RIG one and MAV’s. Similarly, the cytosolic DNA sensing innate immunity pathway mediated by cGAS, cyclic GMP-AMP, cGAMP, synthase and STING, Stimulator of interferon genes, is regulated by autophagy proteins. The generation of cGAMP by cGAS activates ULK1, which phosphorylates and inhibits STING-dependent cytokine production. As unrestrained STING signaling, either via inherited mutations in the ADAR and ribonuclease H2 complex or gain-of-function mutations in STING, causes human auto inflammatory diseases, ULK1 activating agents have been proposed as potential treatments for such disorders. Beclin 1 binds cGAS to suppress cGAMP synthesis and halt interferon production. Atg9a may also function as a negative regulator of innate immune signaling by decreasing the assembly of STING and TBK1 in the presence of double-stranded DNA. Of note, these same RNA-sensing and cytosolic DNA-sensing signaling pathways are activators of autophagy, which is itself an important innate immune effector pathway. Thus, ATG proteins play a crucial role in both mediating innate immunity and in providing feedback inhibition to fine-tune inflammatory signaling so as to avoid deleterious consequences. The Selectivity of Autophagy: a Guardian of Cellular Homeostasis. For nearly half a century, the process of macro autophagy was believed to lack cargo specificity. In fact, the morphological identification of an auto phagosome required visualizing the simultaneous presence of diverse cytoplasmic contents, such as ER, ribosomes and mitochondria inside a double-membraned vacuole. However, a transformative body of work over the past decade has fully dispelled this notion. We now know that there can be extreme specificity in governing the choice of cargo that is degraded by the autophagosome and an intricate system for earmarking and capturing such cargo. This process, termed selective autophagy, may be more crucial in protection against most mammalian diseases than “bulk autophagy”, which is primarily a homeostatic mechanism during nutrient stress. Many parts of the cell can be “selected” for degradation by autophagy, Figure 2 and Table 2. Numerous studies have reported the selective autophagy of various organelles, including mitochondria, ER, peroxisomes, lipid droplets, ribosomes, midbody rings and the nucleus. Autophagy selectively degrades aggregation-prone misfolded proteins such as those involved in the pathogenesis of certain neurodegenerative, skeletal and cardiac muscle, and liver diseases. In addition, it degrades the individual proteins that serve as adaptors to bridge cargo with the nascent phagophore as well as specific inflammatory and immune signaling molecules. Moreover, selective autophagy can target pathogens that reside inside vacuoles or directly inside the cytosol for lysosomal degradation. Once captured, cargo degradation proceeds through a route that involves the same molecular machinery as bulk autophagy. Different forms of selective autophagy are often named by a term comprising a prefix derived from the cargo. For example, mito, ER, ribo, nucleo, pexo, lipo, and the suffix “phagy”. For selective autophagy of microbial invaders, the term xenophagy is commonly used. Major advances have been made in understanding certain aspects of selective autophagy, particularly how cargo binds to the forming phagophore, Figure 2. In most known instances, the cargo either contains an identifiable LC3-interacting region or LIR motif that directly binds LC3, or it must be labeled with a tag such as ubiquitin, which then binds adaptor proteins that contain both a ubiquitin-binding domain and a LIR motif, thus serving as a bridge between the cargo and the LC3, or Gabarap’s, conjugated to the phagophore membrane. Alternatively, specific proteins (particularly those involved in the inflammasome or IFN signaling) can bind to TRIM (tripartite motif) family members, which serve as adaptors that interact with Gabarap’s to target such proteins for autophagic degradation. Of note, the proteins we refer to as “adaptors” are often called autophagy “receptors”. However, as these are bridging molecules that are not integral parts of cellular membranes that undergo ligand-dependent activation, the designation as “receptors” can be confusing. Several layers of control are needed to properly dictate the targeting of cargo for autophagy. In theory, cargo should be disposed of when it is constitutively harmful, for example, intracellular pathogens, potentially dangerous to the cell, for example, mitochondrial damage, or obsolete as a result of cellular differentiation, for example, organelles during erythrocyte maturation. In the scenario where LC3 directly binds to a protein on an organelle containing a LIR motif, there must exist ways to hide or expose the LIR motif in a regulated-fashion. Two mechanisms identified thus far include: (1) Stimulatory and inhibitory phosphorylations of residues near or in the LIR motif (such as occurs for the mitochondrial outer membrane protein, FUNDC1, that mediates hypoxia-stimulated mitophagy and (2) The exposure of a normally hidden LIR motif, such as occurs when proteasomal-dependent rupture of the outer mitochondrial membrane exposes the inner mitochondrial membrane LC3-binding protein, prohibitin 2. Under circumstances where LC3 binds to an adaptor protein, there must exist ways to recruit the adaptor to the cargo destined for degradation. This process generally involves the concerted action of E3 ligases that ubiquitinate targets, for example, Parkin, SMURF1, kinases that recruit E3 ligases, for example, PINK1, or that phosphorylate LIR domains of adaptors, for example, TBK1, deubiquitinating enzymes, DUB’s that counter E3 ligase activity, for example, USP30, USP15, and acetylation, deacetylation of mitochondrial and ER target proteins. All mechanisms for earmarking cargo must be tightly coordinated with the formation of autophagosomes to ensure final cargo disposal. Some potential nodes of coordination have recently been described. Certain autophagy adaptors, most notably the TRIM family proteins, bind not only substrate proteins and LC3, Gabarap family members but also assemble the ULK1 and Class 3 PI3K complexes to initiate autophagosome formation. ULK1, a “master kinase” that phosphorylates multiple sites on downstream core autophagy proteins, is recruited to and phosphorylates proteins involved in selective autophagy, such as the LIR domain of the mitochondrial membrane protein, FUNDC1. An organelle-specific LC3, Gabarap binding protein, the ER membrane protein, CCPG1, interacts with a key component of the autophagy-initiating ULK1 complex, FIP200. Thus, the machinery involved in selective autophagy substrate recognition may play an active role in autophagy initiation. For selective autophagy targeting events that involve substrate ubiquitination, the precise mechanisms that dictate the choice between autophagic versus proteasomal degradation are uncertain. In yeast, substrate aggregation and oligmerization of the ubiquitin-binding proteins may favor autophagic degradation. The lysine residues used for linkage and the length and nature of the ubiquitin chains have also been proposed to contribute to pathway selection, but definitive evidence is lacking. Moreover, despite elegant studies characterizing the ubiquitylome of selective autophagy cargo, such as mitochondria and intracellular pathogens, the ubiquitin substrates required for autophagic targeting remain largely undefined. Selective autophagy seems to involve multiple concurrent targeting mechanisms that act in a cooperative, potentially hierarchical and, or partially redundant manner to ensure proper removal of cargo. This “combinatorial design” may allow specific cell types to more precisely regulate when and how selective autophagy occurs for a given cargo. The partial redundancy also renders it more feasible to study loss-of-function phenotypes of genes required for selective autophagy but dispensable for bulk autophagy, as compared to those required for bulk autophagy, as they are less likely to be lethal to the cell or organism. As selective autophagy genes are partially redundant, this may explain why their loss-of-function mutation seems to be better tolerated in the human population than loss-of-function mutation of core ATG genes. Indeed, mutations in many of the known molecules involved in selective autophagy are associated with susceptibility to a variety of human diseases, Table 1. Mutations in the genes encoding the adaptor proteins p62, SQSTM and optineurin, the E3 ligase Parkin, and the kinases PINK1 and TBK1, are among the most common causes of familial and early onset neurodegenerative diseases, including Parkinson’s disease, frontotemporal dementia and amyotrophic lateral sclerosis. Hereditary sensory and autonomic neuropathy type II is caused by mutations in an ER-specific LC3 binding protein, FAM134B, required for ER-phagy. Mutations in TRIM20, also known as pyrin, that impair its ability to target inflammasome components for autophagic degradation result in an inherited autoinflammatory disorder, familial Mediterranean fever. Inflammatory bowel disease-associated genes encode proteins that function in multiple steps of autophagy, including the selective targeting of bacteria by the adaptor, Calcoco2, NDP52, and the E3 ligase, SMURF1. The numerous links between mutations in selective autophagy genes and human diseases underscore the likely physiological importance of different forms of selective autophagy, Table 2. However, the precise mechanisms that connect genotype to phenotype remain largely undefined. For example, it is not known why mutations in Parkin and PINK1 are associated with Parkinson’s disease, whereas mutations in optineurin, TBK1, and p62, SQSTM1 are associated with amyotrophic lateral sclerosis and frontotemporal dementia, Table 1. In addition to potential cell non-autonomous effects of mutations in these genes in tissues outside of the brain, cell type-specific differences in various populations of neurons and glia may exist with respect to: (1) Dependency on subsets of selective autophagy genes. (2) Expression and activity of DUBs and other negative feedback mechanisms that regulate selective autophagy, and, or (3) levels and types of stress that mandate different types of selective autophagy responses to maintain homeostasis, such as mitophagy or other forms of selective autophagy such as aggrephagy that are relevant to neurodegenerative diseases. Parkin knockout mice, unlike flies lacking Parkin, do not develop spontaneous neurodegeneration, but they do develop dopaminergic neuronal degeneration, resembling that observed in human Parkinson’s disease, when crossed with “mutator” mice with a proof-reading-defective mitochondrial DNA polymerase (PolG) that accumulate mitochondrial mutations. The localization of disease in dopaminergic neurons may be related to increased mitochondrial stress in these cells as compared to other neuronal populations in the brain. Intriguingly, the motor defect and neurodegeneration in Parkin-null, mutator mice can be rescued by deletion of STING, a regulator of Type One IFN responses to cytosolic DNA. Thus, aberrant inflammatory signaling as a result of defects in mitophagy may contribute to the pathogenesis of neurodegenerative disease in patients with Parkin or PINK1 mutations. While most, if not all, forms of selective autophagy are likely to contribute to normal physiology and protection against disease, mitophagy has been the most extensively studied. Mitophagy is an essential component of mammalian developmental and differentiation processes, including elimination of paternal mitochondria from the fertilized egg, removal of mitochondria during red blood cell maturation and beige-to-white adipocyte differentiation. In addition to Parkinson’s and other neurodegenerative diseases, defective mitophagy is thought to contribute to organ-specific and systemic inflammatory diseases, cancer development and, or progression, and potentially aging. The removal of damaged mitochondria by mitophagy maintains normal cellular metabolism, reduces mitochondrial generation of ROS that trigger inflammation and genotoxic stress, and prevents mitochondrial release of pro-apoptotic factors. Thus, maintenance of proper mitochondrial function by mitophagy is crucial for cellular and organismal health. Other forms of selective autophagy, including xenophagy, likely operate in an analogous manner to mitophagy, in that the mechanisms by which they regulate physiology and disease are a function of the normal “duties” of their substrate and the ensuing pathological consequences of abnormal substrate accumulation, see Table 2. Our expanding knowledge of the mechanisms and physiological functions of selective autophagy may open up new, albeit unchartered, pathways for drug discovery. Knockdown of the mitochondrial deubiquitinase, USP30, rescues mitophagy defects and disease in flies with pathogenic mutations in Parkin, suggesting a potential role for the inhibition of DUBs that target selective autophagy E3 ligases in the treatment of Parkinson’s and other diseases. Indeed, novel highly selective inhibitors of USP30 that accelerate mitophagy have recently been reported. As phosphorylation of substrates is also a common mechanism involved in selective autophagic targeting, it may be possible to activate specific kinases to enhance selective autophagy. Potentially, it may also be possible to develop novel strategies to attach high-affinity LIR domains selectively to harmful cargo so that they can be more efficiently captured by an LC3-decorated nascent autophagosome. Autophagy Regulation: A Nexus for Therapeutics? Autophagy was originally studied in yeast and mammalian cells as a nutrient stress response pathway. During the past decade, we have dramatically expanded our knowledge of autophagy regulation, particularly the spectrum of physiological and pathophysiological stimuli that control autophagy, the mechanisms that regulate the activity of the core autophagy proteins, and the interconnectivity of autophagy with other cellular stress response pathways. These concepts have been reviewed elsewhere, here, we highlight selected aspects relevant to physiology and disease. Post-translational protein modifications such as phosphorylation, ubiquitination, and acetylation play a central role in coordinating the activity of ATG proteins. In most cases, the upstream kinases, phosphatases, ubiquitin ligases, DUB’s, and acetyltransferases simultaneously modify both ATG proteins and proteins involved in other cellular stress-response pathways that are co-regulated with autophagy. As a result, pharmacological targeting of these enzymes will elicit broad-based modulation of multiple intertwined stress-response pathways. Depending on the enzyme and its substrates, such nonspecific targeting may be harmful in some instances, and useful in others. One important example is the stimulation of AMPK, a low energy-sensing kinase activated by ATP depletion, which phosphorylates multiple proteins to both stimulate catabolic pathways, including autophagy, and restrain anabolic pathways, including mTORC1 signaling, thereby ensuring limitation of ATP consumption and generation of new ATP via breakdown of metabolic products. In recent years, AMPK has been shown to not only activate autophagy through inhibition of mTORC1, but also directly phosphorylate several ATG proteins, including ULK1, ATG9A, Beclin 1, and VPS34. In addition, AMPK promotes mitophagy through effects on ULK1 and stimulates TFEB-dependent activation of the CLEAR, Coordinated Lysosomal Expression and Regulation, network of genes required for autophagy. This pro-autophagic activity of AMPK occurs concurrently with its effects on mitochondrial homeostasis and on lipid and glucose metabolism. AMPK activation may underlie the beneficial effects of metformin, a drug widely prescribed for the treatment of diabetes. Metformin activates AMPK indirectly through mitochondrial depletion of ATP, and direct AMPK activators that yield effects similar to metformin are in pre-clinical development. The extent to which autophagy stimulation contributes to beneficial effects of AMPK activation in mice or patients is not known, but it seems likely that autophagy represents a critical part of an AMPK-activated hub that protects against various metabolic diseases, including diabetes, obesity, and non-alcoholic fatty liver disorders, as well as certain cancers and aging-related phenotypes. In Drosophila, deficiency of the Beclin 1 orthologue (ATG6) impairs the ability of metformin to prevent intestinal stem cell aging, and lifespan extension by neuronal AMPK expression requires the fly ULK1 orthologue, ATG1. In mice, AMPK upregulation of autophagy is correlated with improved function of aging muscle stem cells, additionally, muscle-specific AMPK deficiency results in defective autophagy, fasting-induced hypoglycemia, and aging-associated myopathy. In yeast, core ATG genes are required for AMPK-mediated lipid droplet degradation and survival during acute glucose deprivation. The lysine acetylation, deacetylation of ATG proteins has emerged as a central node of autophagic control regulated by metabolic sensors involved in lipid, glucose and protein metabolism. Moreover, this control center may function independently from, but intertwined with, AMPK and mTORC1. During acute nutrient depletion, cells undergo a rapid decrease in levels of cytosolic acetyl coenzyme A (AcCoA), which leads to the deacetylation of cellular proteins. Sirtuin 1, which is downstream of AMPK, deacetylates multiple ATGs, for example, ATG5, ATG7, ATG12, Beclin 1, VPS34, LC3) and thereby promotes autophagy, as does reduced activity of the acetyl transferase EP300. Hence, endogenous activators of sirtuin-1, for example, nicotine adenine dinucleotide NAD plus, endogenous inhibitors of EP300, for example, spermidine, a dietary polyamine), and reduced availability of AcCoA (a rate-limiting step for EP300 function) all stimulate autophagy. Compounds that act on these pathways, thereby mimicking the effects of caloric restriction, so-called “caloric restriction mimetics”, are an active area of investigation, and genetic evidence suggests that autophagy is essential for their beneficial effects in vivo. Reservatrol, an indirect sirtuin activator, requires the autophagy machinery for its favorable effects on longevity in nematodes. Spermidine-induced autophagy is required for several of its beneficial health effects in model organisms, including lifespan extension in flies, worms, and mice, prevention of cardiac aging in mice, improvement in neuronal function in aging flies, and preservation of myocyte stemness in mice. Moreover, caloric restriction mimetics improve anti-tumor immune surveillance and enhance chemotherapy responses in autophagy-competent, but not autophagy-incompetent, mouse tumor allografts. Over the past decade, the lysosome, an organelle traditionally viewed as the downstream “workhorse” for autophagosomal cargo degradation, has been shown to also play a crucial role in the upstream regulation of autophagy. The nutrient-sensing kinase complex, mTORC1, detects both cytosolic and intra-lysosomal amino acids through distinct mechanisms to inhibit autophagy. Amino acids, such as arginine, inside the lysosomal lumen are sensed by the amino acid transporter SLC38A9, which interacts with the lysosomal V ATPase, Rag, Ragulator complex to activate mTORC1. This both restrains autophagy during baseline conditions and provides feedback inhibition to terminate autophagic responses to acute nutrient depletion. mTORC1 activation in the fed state and, or hyperactivation, as a result of mutations in regulatory signals, switches the cell to a state of anabolic growth and energy storage. Although essential for cell growth and proper metabolic regulation, sustained mTORC1 activation at the organismal level is associated with a variety of pathophysiological consequences, including impaired neonatal gluceoneogenesis and survival, accelerated age-related decline in pancreatic beta-cell function, late-onset muscle atrophy, altered lipogenesis and adipogenesis, immune suppression, epileptic seizures and autistic traits, tumorigenesis, and aging. While in some cases, impaired induction of autophagy has been documented in mice with hyperactive mTORC1 signaling and is postulated to contribute to pathological phenotypes, for example, impaired neonatal gluconeogenesis, late-onset muscle atrophy, the precise role of autophagy inhibition in most diseases associated with mTORC1 signaling remains unknown. There has been some interest in using FDA-approved mTOR inhibitors for the treatment of neurodegenerative disorders that may benefit from autophagy induction. However, the safety and efficacy of using mTOR inhibitors to induce therapeutic autophagy is uncertain, given the broad range of essential catabolic functions regulated by mTORC1 along with the lack of full specificity of existing agents to target mTORC1 rather than mTORC2, which functions primarily as an effector of insulin, PI3K signaling. Both AMPK and mTORC1 participate, in opposite directions, in a signaling axis that links autophagy, the lysosome, and the transcription factor EB (TFEB) and related family members. During the acute response to autophagic stimuli, transcriptional activation is not required, as evidenced by the observation that enucleated cells (cytoplasts) undergo autophagy. However, sustained autophagy requires TFEB, a transcription factor that (when inactive) binds to Ragulator at the lysosomal membrane, is phosphorylated by mTORC1, and retained in the cytoplasm by 14 3 3 proteins. Following mTORC1 inhibition, TFEB dephosphorylation releases it from the cytoplasm, allowing its nuclear translocation and subsequent activation of the CLEAR gene network, which includes genes encoding lysosomal hydrolases, lysosomal v-ATPase pumps, lysosomal regulators and autophagy regulators. As noted above, AMPK also activates TFEB-dependent gene expression, this occurs through multiple different mechanisms. In addition, a recent study showed that phosphorylation of acetyl-CoA synthetase 2 (ACSS2) promotes its transport into the nucleus, where it binds to TFEB and favors the acetylation of histone H3 residues within the promoters of TFEB target genes. In addition to TFEB, other transcription factors from the same family, such as micropthalmia-associated transcription factor, MITF, and TFE3, or from other families, for example, FOXO3A, HSF1 or TP53, stimulate autophagy. Bromodomain 4, BRD4, a transcription factor that represses autophagy and lysosomal genes, is displaced from chromatin in response to starvation by a signaling cascade involving an AMPK-SIRT1 axis. Thus, multiple known, and probably yet-to-be-identified, transcription factors regulate the synthesis of genes required for autophagy, including both the formation of the autophagosome and degradation of its contents by lysosomes. Not surprisingly, the activity of these transcription factors is tightly regulated by numerous signaling factors that also regulate core ATG protein function by post-translational modifications. Modulation of the activity of TFEB, a master regulator of both lysosomal biogenesis and autophagy, has emerged as a potential therapeutic strategy. Conceptually, this approach is attractive, since limitations in lysosomal numbers and function either occur intrinsically as part of many rare, but devastating, difficult-to-treat, primary diseases, such as lysosomal storage disorders, LSD’s, or are acquired during the progression of diseases associated with the clearance of toxic aggregates progress, such as Huntington’s, Parkinson’s, Alzheimer’s disease and tauopathies. In mice, TFEB overexpression ameliorates several LSDs, neurodegenerative diseases, and alpha 1-antitrypsin deficiency, and it also promotes lipophagy, thereby reducing obesity and associated metabolic syndrome. The mechanisms by which TFEB overexpression partially corrects lysosomal malfunction in LSDs are not fully understood, but may involve induction of lysosomal exocytosis for the secretion of undigested material. One potential obstacle to strategies for enhancing TFEB family activity is the risk of tumorigenesis associated with constitutive activation, for example, renal clear cell carcinoma with TFEB and pancreatic cancer with MITF, TFE3, and TFEB. While MITF, TFE3, TFEB-dependent autophagy-lysosomal activation is thought to sustain metabolic reprogramming in pancreatic cancer cells by maintaining intracellular amino acid pools, further genetic investigations are warranted to confirm that ATG genes are involved in these effects. Moreover, enhanced activity of BRD4, a transcriptional repressor of autophagy, drives another type of cancer, NUT midline carcinoma, suggesting the effects of transcriptional regulators of autophagy on tumorigenesis may be cell-type specific. It is unclear whether specific subsets of the TFEB-regulated gene network can be induced to avoid genes that contribute to tumorigenesis without losing beneficial effects on the autophagy-lysosomal pathway. An alternative strategy is to activate TFEB on an intermittent basis and, or for limited periods to avoid potential oncogenic effects. Intriguingly, one of the most widely used medications in the world, aspirin, has been reported to upregulate TFEB in brain cells, via activation of PPARC alpha, induce lysosomal biogenesis, and decrease amyloid plaque pathology in a mouse model of Alzheimer’s-like disease. Aspirin (and its active metabolite salicylate) also induces autophagy via inhibition of the acetyltransferase EP300 and via AMPK activation, mTORC1 inactivation. However, there is as-of-yet no direct genetic evidence that autophagy contributes to the health benefits of aspirin. Highly specific activation of autophagy may be possible through strategies that enhance the activity of the upstream components in the core autophagy pathway, meaning the ULK1 serine, threonine kinase complex and, or Beclin 1, VPS34 lipid kinase complexes. As a key allosteric regulator of VPS34 lipid kinase activity, Beclin 1 activity is tightly regulated by multiple post-translational modifications, ubiquitination, acetylation, phosphorylation, which govern its stability, heterodimeric binding to ATG14 or UVRAG, homodimerization in an inactive form, and, or binding to negative regulators. Diverse stress kinases, including AMPK, as well as the upstream ATG protein, ULK1, mediate stimulatory phosphorylations of Beclin 1. The oncogenic kinases, Akt and EGFR, and the EP300 acetylase inhibit the autophagic activity of Beclin 1, mutation of their target post-translational sites in Beclin 1 demonstrates that suppression of Beclin 1-dependent autophagy promotes tumor growth in mouse xenograft models. Enhanced proteasome-mediated degradation of Beclin 1 due to decreased binding of the deubiquitinase, ataxin 3, may contribute to dysregulated autophagy in cells of patients with polyglutamine expansion protein-related diseases, such as Huntington’s and spinocerebellar ataxia type 3.. Disruption of Bcl-2 binding to Beclin 1 represents a central mechanism by which autophagy is activated in response to stress stimuli, such as starvation, exercise, and immune signaling. This disruption can be triggered by phosphorylation of the BH3 domain of Beclin 1 by DAPK, ubiquitination of the Beclin 1 BH3 domain by the E3 ligase TRAF6, phosphorylation of Bcl-2 by JNK1, or competition by BH3-only proteins. Mice containing knock-in non-phosphorylatable mutations in Bcl-2 that prevent disruption of its binding to Beclin 1 are deficient in starvation and exercise-induced autophagy, have decreased exercise endurance, and fail to manifest the beneficial effects of exercise on glucose metabolism. Conversely, mice with a knock-in mutation in Beclin 1 that decreases Bcl-2 binding exhibit increased autophagy and extended lifespan and healthspan, including protection against Alzheimer’s-like disease and HER2-mediated breast cancer. Thus, disruption of Beclin 1, Bcl-2 binding may be a safe and effective approach to induce autophagy in vivo, preclinical studies are in progress to develop agents that act through this mechanism. Cell-penetrating peptides, Tat-Beclin 1, derived from a flexible hinge region of Beclin 1 important for VPS34 membrane association and lipid kinase activity are sufficient to induce autophagy in vitro and in vivo. In mice, Tat-Beclin 1 protects against West Nile virus, chikungunya virus and E. coli bacterial infections, lipopolysaccharide-induced cardiac dysfunction, pressure overload-induced heart failure, hyperammonemia in liver failure and urea cycle disorders, and bone loss in LSDs and in FGF-deficiency. It also enhances chemotherapeutic effects of murine cancers in immune competent mice, reduces the growth of human HER2-positive breast cancer xenografts in immune-deficient mice, and acts synergistically with erastin to increase animal survival in an orthotopic pancreatic cancer model. In rats, intrahippocampal injection of Tat-Beclin 1 improves long-term spatial memory. In a zebrafish model of human polycystic kidney disease, Tat-Beclin 1 ameliorates renal cyst formation. Further studies are needed to examine whether Tat-Beclin 1 induces these effects through autophagy, autophagy-independent effects of Beclin 1, or alternative mechanisms. Moreover, precise definition of its mechanism of action may lead to the development of novel small drug-like molecules that mimic its activity. Recent structural advances elucidating the atomic details of the Beclin 1, VPS34 complexes may provide a basis for rational drug design to selectively activate autophagy-specific Beclin 1-associated VP34 lipid kinase activity. Autophagy in Tissue and Whole-Body Homeostasis. The health of multicellular organisms requires the coordinated regulation of cellular life and death decisions, cell fate determinations, preservation of genomic integrity, immune responses, and metabolic circuitries. The autophagy machinery, via its diverse functions described above, and yet-to-be-discovered mechanisms, plays a crucial role in these processes. Herein, we highlight some recent advances related to the role of autophagy in cell death, preservation of stem cells, tumor suppression, longevity and defense against metabolic diseases. Autophagy as a Homeostat During both routine “housekeeping” and responses to acute stress, cells must find ways to maintain adaptive cytoprotective levels of autophagy while simultaneously avoiding potentially maladaptive levels and, or detrimental effects of autophagy. This balance involves self-control of the levels of autophagy, mechanisms of preventing degradation products from becoming toxic to cells, avoidance of degrading essential cargo, and suppression of unwarranted cell death, which likely is a combined function of the aforementioned processes. Cellular self-titration of levels of autophagy involves multiple different inhibitory feedback loops, including feedback regulation of nutrient sensing signals by the generation of amino acids, acetyl-CoA and respiratory substrates, cytosolic retention of pro-autophagic transcription factors by ATG7, and TFEB-mediated activation of mTORC1. Cellular toxicity by degradation products may be avoided during autophagy, as evidenced by the observation that the generation of lipid droplets generated by autophagy-dependent dismantling of lipid membranes during starvation-induced autophagy sequesters fatty acids, thereby protecting mitochondria against lipotoxicity and preserving cellular viability. It is not known whether starvation-induced autophagy preferentially induces removal of certain, perhaps aged, structures, and if so, by what mechanisms, or whether it is non-specific. Mitochondria elongate during starvation, which spares them from the autophagic capture that generally occurs after fission. Numerous yet-to-be-discovered mechanisms likely protect mitochondria and other organelles from excessive autophagic capture during stress-induced autophagy. The aforementioned negative feedback loops restrain autophagy to adaptive (rather than maladaptive) levels, allowing this homeostatic pathway to exert cytoprotective effects during stress, and thereby, prevent apoptotic and necroptotic cell death. In addition, ATG gene-dependent processes, such as increased plasma membrane localization of the GLUT1 glucose transporter, may increase the threshold of damage required to kill cells and thereby promote successful organismal adaptation to stress. Promotion of cell survival in vivo during stress-induced autophagy may depend on concurrent antagonism of the sodium, potassium ion, ATPase pump, which normally consumes a large fraction of the ATP available to the cell. Interestingly, during acute bouts of exercise or nutrient limitation, potent physiological stimuli of autophagy that generally do not result in cell death, endogenous cardiac glycosides that target sodium, potassium ion ATPase are upregulated 50 to 500 fold, resulting in decreased cellular ATP consumption. However, when organisms are pushed beyond physiological limits of energy deprivation, adaptive mechanisms are insufficient to keep cells alive and to prevent tissue damage. In the liver of patients with anorexia nervosa or in neonatal rodents subjected to severe cerebral ischemic injury, a morphologically and genetically distinct form of cell death occurs called autosis, which requires both ATG genes and sodium, potassium ion, ATPase activity. It is not clear how the cell’s major consumer of ATP, meaning the sodium, potassium ion, ATPase pump) and the cell’s major mobilizer of ATP-generating substrates during stress conditions, meaning autophagy, interact to regulate life and death decisions of the cell. However, this interaction may represent a fundamental energy homeostatic mechanism that becomes pathologic during different types of ischemic conditions. Another recently identified bona fide form of autophagic cell death, meaning demonstrating a genetic requirement for ATG genes, involves GBA1, the gene encoding the lysosomal enzyme, glucocerebrosidase (GCase, which metabolizes glucosylceramide (GlcCer) to ceramide and glucose. GB

Index of Science videos: https://rumble.com/v406mdz-index-of-robert-heinlein-audiobooks..html Open Voice: Versatile Instant Voice Cloning. Zengyi Qin, MIT, and others. We introduce OpenVoice, a versatile instant voice cloning approach that requires only a short audio clip from the reference speaker to replicate their voice and generate speech in multiple languages. OpenVoice represents a significant advancement in addressing the following open challenges in the field: 1) Flexible Voice Style Control. OpenVoice enables granular control over voice styles, including emotion, accent, rhythm, pauses, and intonation, in addition to replicating the tone color of the reference speaker. The voice styles are not directly copied from and constrained by the style of the reference speaker. Previous approaches lacked the ability to flexibly manipulate voice styles after cloning. 2) Zero-Shot Cross-Lingual Voice Cloning. OpenVoice achieves zero-shot cross-lingual voice cloning for languages not included in the massive-speaker training set. Unlike previous approaches, which typically require an extensive massive-speaker multi-lingual (MSML) dataset for all languages, OpenVoice can clone voices into a new language without any massive-speaker training data for that language. OpenVoice is also computationally efficient, costing tens of times less than commercially available API’s that offer even inferior performance. To foster further research in the field, we have made the source code and trained model publicly accessible. We also provide qualitative results in our demo website. Prior to its public release, our internal version of OpenVoice was used tens of millions of times by users worldwide between May and October 2023, serving as the backend of MyShell.ai. One. Introduction. Instant voice cloning (IVC) in text-to-speech (TTS) synthesis means the TTS model can clone the voice of any reference speaker given a short audio sample without additional training on the reference speaker. It is also referred to as Zero-shot TTS. IVC enables the users to flexibly customize the generated voice and exhibits tremendous value in a wide variety of real-world applications, such as media content creation, customized chatbots, and multi-modal interaction between humans and computers or large language models. An abundant of previous work has been done in IVC. Examples of auto-regressive approaches include VALLE and XTTS, which extract the acoustic tokens or speaker embedding from the reference audio as a condition for the auto-regressive model. Then the auto-regressive model sequentially generate acoustic tokens, which are then decoded to raw audio waveform. While these methods can clone the tone color, they do not allow users to flexibly manipulate other important style parameters such as emotion, accent, rhythm, pauses and intonation. Also, auto-regressive models are relatively computationally expensive and has relatively slow inference speed. Examples of non-autoregressive approach include YourTTS and the recently developed Voicebox, which demonstrate significantly faster inference speed but are still unable to provide flexible control over style parameters besides tone color. Another common disadvantage of the existing methods is that they typically require a huge MSML dataset in order to achieve cross-lingual voice clone. Such combinatorial data requirement can limit their flexibility to include new languages. In addition, since the voice cloning research by tech giants are mostly closed-source, there is not a convenient way for the research community to step on their shoulders and push the field forward. We present OpenVoice, a flexible instant voice cloning approach targeted at the following key problems in the field: In addition to cloning the tone color, we ask, how can we have flexible control of other important style parameters such as emotion, accent, rhythm, pauses and intonation? These features are crucial for generating in-context natural speech and conversations, rather than monotonously narrating the input text. Previous approaches can only clone the monotonous tone color and style from the reference speaker but do not allow flexible manipulation of styles. How to enable zero-shot cross-lingual voice cloning in a simple way. We put forward two aspects of zero-shot capabilities that are important but not solved by previous studies: One, if the language of the reference speaker is not presented in the MSML dataset, can the model clone their voice? Two, if the language of the generated speech is not presented in the MSML dataset, can the model clone the reference voice and generate speech in that language? In previous studies, the language of the reference speaker and the generated language by the model should both exist in great quantity in the MSML dataset. But what if neither of them exist? The challenge is how to realize a super-fast speed real-time inference, without downgrading the quality, which is crucial in a massive commercial production environment. To address the first two problems, OpenVoice is designed to decouple the components of a voice as much as possible. The generation of language, tone color, and other important voice features are made independent of each other, enabling flexible manipulation over individual voice styles and language types. This is achieved without labeling any voice style in the MSML training set. We would like to clarify that the zero-shot cross-lingual task in this study is different from that in VALLE-X. In VALLE-X, data for all languages need to be included in the MSML training set, and the model cannot generalize to an unseen language outside the MSML training set. By comparison, OpenVoice is designed to generalize to completely unseen languages outside the MSML training set. The third problem is addressed by default, since the decoupled structure reduces requirement on model size and computational complexity. We do not require a large model to learn everything. Also, we avoid the use of auto-regressive or diffusion components to speed up the inference. Our internal version of OpenVoice before this public release has been used tens of millions of times by users worldwide between May and October 2023. It powers the instant voice cloning backend of MyShell.ai and has witnessed a user growth of several hundredfold on this platform. To facilitate the research progress in the field, we explain the technology in great details and make the source code with model weights publicly available. Two. Approach. The technical approach is simple to implement but surprisingly effective. We first present the intuition behind OpenVoice, then elaborate on the model structure and training. Two point 1. Intuition. The Hard Part. It is obvious that simultaneously cloning the tone color for any speaker, enabling flexible control of all other styles, and adding new language with little effort could be very challenging. It requires a huge amount of combinatorial datasets where the controlled parameters intersect, and pairs of data that only differ in one attribute, and are well-labeled, as well as a relatively large-capacity model to fit the dataset. The Easy Part. We also notice that in regular single-speaker TTS, as long as voice cloning is not required, it is relatively easy to add control over other style parameters and add a new language. For example, recording a single-speaker dataset with 10K short audio samples with labeled emotions and intonation is sufficient to train a single-speaker TTS model that provides control over emotion and intonation. Adding a new language or accent is also straightforward by including another speaker in the dataset. The intuition behind OpenVoice is to decouple the IVC task into separate subtasks where every subtask is much easier to achieve compared to the coupled task. The cloning of tone color is fully decoupled from the control over all remaining style parameters and languages. We propose to use a base speaker TTS model to control the style parameters and languages, and use a tone color converter to embody the reference tone color into the generated voice. Two point 2. Model Structure. We illustrate the model structure in Figure 1. The two main components of OpenVoice are the base speaker TTS model and the tone color converter. The base speaker TTS model is a single-speaker or multi-speaker model, which allows control over the style parameters, for example, emotion, accent, rhythm, pauses and intonation, accent and language. The voice generated by this model is then passed to the tone color converter, which changes the tone color of the base speaker into that of the reference speaker. Base Speaker TTS Model. The choice of the base speaker TTS model is very flexible. For example, the VITS model can be modified to accept style and language embedding in its text encoder and duration predictor. Other choices such as InstructTTS can also accept style prompts. It is also possible to use commercially available (and cheap) models such as Microsoft TTS, which accepts speech synthesis markup language (SSML) that specifies the emotion, pauses and articulation. One can even skip the base speaker TTS model, and read the text by themselves in whatever styles and languages they desire. In our OpenVoice implementation, we used the VITS model by default, but other choices are completely feasible. We denote the outputs of the base model as X (Of LI, SI, and CI), where the three parameters represent the language, styles and tone color respectively. Similarly, the speech audio from the reference speaker is denoted as X (Of LO, SO, and CO). Tone Color Converter. The tone color converter is an encoder-decoder structure with an invertible normalizing flow in the middle. The encoder is a 1D convolutional neural network that takes the short-time Fourier transformed spectrum of X (Of LI, SI, and CI) as input. All convolutions are single strided. The feature maps outputted by the encoder are denoted as Y (Of LI, SI, and CI). The tone color extractor is a simple 2D convolutional neural network that operates on the mel-spectrogram of the input voice and outputs a single feature vector that encodes the tone color information. We apply it on X(Of LI, SI, and CI) to obtain vector v (Of CI), then apply it on X (Of LO, SO, and CO) to obtain vector v(CO). The normalizing flow layers take Y (Of LI, SI, and CI) and v(CI ) as input and outputs a feature representation Z (Of LI, and SI ) that eliminates the tone color information but preserves all remaining style properties. The feature Z( Of LI, and SI) is aligned with International Phonetic Alphabet (IPA) along the time dimension. Details about how such feature representation is learned will be explained in the next section. Then we apply the normalizing flow layers in the inverse direction, which takes Z(Of LI, and SI ) and v(Of CO) as input and outputs Y (Of LI, SI, and CO). This is a critical step where the tone color CO from the reference speaker is embodied into the feature maps. Then the Y (Of LI, SI, and CO) is decoded into raw waveforms X (Of LI, SI, and CO) by HiFi-Gan that contains a stack of transposed 1D convolutions. The entire model in our OpenVoice implementation is feed-forward without any auto-regressive component. The tone color converter is conceptually similar to voice conversion, but with different emphasis on its functionality, inductive bias on its model structure and training objectives. The flow layers in the tone color converter are structurally similar to the flow-based TTS methods but with different functionalities and training objectives. Alternative Ways and Drawbacks. Although there are alternative ways to extract Z (Of LI, and SI), we empirically found that the proposed approach achieves the best audio quality. One can use HuBERT to extract discrete or continuous acoustic units to eliminate tone color information, but we found that such method also eliminates emotion and accent from the input speech. When the input is an unseen language, this type of method also has issues preserving the natural pronunciation of the phonemes. We also studied another approach that carefully constructs information bottleneck to only preserve speech content, but we observed that this method is unable to completely eliminate the tone color. A Remark on Novelty. OpenVoice does not intend to invent the submodules in the model structure. Both the base speaker TTS model and the tone color converter borrow the model structure from existing work. The contribution of OpenVoice is the decoupled framework that separates the voice style and language control from the tone color cloning. This is very simple, but very effective, especially when one wants to control styles, accents or generalize to new languages. If one wanted to have the same control on a coupled framework such as XTTS, it could require a tremendous amount of data and computing, and it is relatively hard to fluently speak every language. In OpenVoice, as long as the single-speaker TTS speaks fluently, the cloned voice will be fluent. Decoupling the generation of voice styles and language from the generation of tone color is the core philosophy of OpenVoice. We also provided our insights of using flow layers in the tone color converter, and the importance of choosing a universal phoneme system in language generalization in our experiment section. Two point 3. Training. In order to train the base speaker TTS model, we collected audio samples from two English speakers, with American and British accents, one Chinese speaker and one Japanese speaker. There are 30K sentences in total, and the average sentence length is 7 seconds. The English and Chinese data has emotion classification labels. We modified the VITS model and input the emotion categorical embedding, language categorical embedding and speaker id into the text encoder, duration predictor and flow layers. The training follows the standard procedure provided by the authors of VITS. The trained model is able to change the accent and language by switching between different base speakers, and read the input text in different emotions. We also experimented with additional training data and confirmed that rhythm, pauses and intonation can be learned in exactly the same way as emotions. In order to train the tone color converter, we collected 300K audio samples from 20K individuals. Around 180K samples are English, 60K samples are Chinese and 60K samples are Japanese. This is what we called the MSML dataset. The training objectives of the tone color converter is two-fold. First, we require the encoder-decoder to produce natural sound. During training, we feed the encoder output directly to the decoder, and supervised the generated waveform using the original waveform with mel-spectrogram loss and HiFi-GAN loss. We will not present details here, as it has been well explained by previous literature. Second, we require flow layers to eliminate as much tone color information as possible from the audio features. During training, for each audio sample, its text is converted to a sequence of phonemes in IPA, and each phoneme is represented by a learnable vector embedding. The sequence of vector embedding is passed to a transformer encoder to produce the feature representation of the text content. Denote this feature as L is an element of Real c times l, where c is the number of feature channels and l is the number of phonemes in the input text. The audio waveform is processed by the encoder and flow layers to produce the feature representation Z is an element of Real c times t, where t is the length of the features along the time dimension. Then we align L with Z along the time dimension using dynamic time warping, an alternative is monotonic alignment, to produce L bar is an element of Real c times t, and minimize the KL-divergence between L bar and Z. Since L bar does not contain any tone color information, the minimization objective would encourage the flow layers to remove tone color information from their output Z. The flow layers are conditioned on the tone color information from the tone color encoder, which further helps the flow layers to identify what information needs to be eliminated. In addition, we do not provide any style or language information for the flow layers to be conditioned upon, which prevents the flow layers from eliminating information other than tone color. Since the flow layers are invertible, conditioning them on a new piece of tone color information and running its inverse process can add the new tone color back to the feature representations, which are then decoded to the raw waveform with the new tone color embodied. Three. Experiment. It is hard to be objective in the evaluation of voice cloning for several reasons. First, different research studies usually have different training and test sets. The numerical comparison could be intrinsically unfair. Even though their metrics such as Mean Opinion Score can be evaluated by crowdsourcing, the diversity and difficulty of the test set would significantly influence the results. For example, if many samples in the test set are neural voices that concentrate on the mean of human voice distributions, then it is relatively easy for most methods to achieve good voice cloning results. Second, different studies usually have different training sets, where the scale and diversity would have considerable influence of the results. Third, different studies can have a different focus on their core functionalities. OpenVoice mainly aims at tone color cloning, flexible control over style parameters, and making cross-lingual voice clone easy even without massive-speaker data for a new language. These are different from the objectives of previous work on voice cloning or zero-shot TTS. Therefore, instead of comparing numerical scores with existing methods, we mainly focus on analyzing the qualitative performance of OpenVoice itself, and make the audio samples publicly available for relevant researchers to freely evaluate. Accurate Tone Color Cloning. We built a test set of reference speakers selected from celebrities, game characters and anonymous individuals. The test set covers a wide voice distributions including both expressive unique voices and neutral samples in human voice distribution. With any of the 4 base speakers and any of the reference speakers, OpenVoice is able to accurately clone the reference tone color and generate speech in multiple languages and accents. We invite the readers to this website for qualitative results. Flexible Control on Voice Styles. A premise for the proposed framework to flexibly control the speech styles is that the tone color converter is able to only modify the tone color and preserves all other styles and voice properties. In order to confirm this, we used both our base speaker model and the Microsoft TTS with SSML to generate a speech corpus of 1K samples with diverse styles, emotion, accent, rhythm, pauses and intonation, as the base voices. After converting to the reference tone color, we observed that all styles are well-preserved. In rare cases, the emotion will be slightly neutralized, and one way that we found to solve this problem is to replace the tone color embedding vector of this particular sentence with the average vector of multiple sentences with different emotions from the same base speaker. This gives less emotion information to the flow layers so that they do not eliminate the emotion. Since the tone color converter is able to preserve all the styles from the base voice, controlling the voice styles becomes very straightforward by simply manipulating the base speaker TTS model. The qualitative results are publicly available on this website. Cross-Lingual Voice Clone with Ease. OpenVoice achieves near zero-shot cross-lingual voice cloning without using any massive-speaker data for an unseen language. It does require a base speaker of the language, which can be achieved with minimum difficulty with the off-the-shelf models and datasets. On our website, we provide an abundance of samples that demonstrate the cross-lingual voice clone capabilities of the proposed approach. The cross-lingual capabilities are two-fold: When the language of the reference speaker is unseen in the MSML dataset, the model is able to accurately clone the tone color of the reference speaker. When the language of the generated speech is unseen in the MSML dataset, the model is able to clone the reference voice and speak in that language, as long as the base speaker TTS supports that language. Fast Inference with Low Cost. Since OpenVoice is a feed-forward structure without any autoregressive component, it achieves very high inference speed. Our experiment shows that a slightly optimized version of OpenVoice, including the base speaker model and the tone converter, is able to achieve 12 times real-time performance on a single A10G GPU, which means it only takes 85 milliseconds to generate one second of speech. Through detailed GPU usage analysis, we estimate that the upper bound is around 40 times real-time, but we will leave this improvement as future work. Importance of IPA. We found that using IPA as the phoneme dictionary is crucial for the tone color converter to perform cross-lingual voice cloning. As we detailed in Section 2 point 3, in training the tone color converter, the text is first converted into a sequence of phonemes in IPA, then each phoneme is represented by a learnable vector embedding. The sequence of embedding is encoded with transformer layers and compute loss against the output of the flow layers, aiming to eliminate the tone color information. IPA itself is a cross-lingual unified phoneme dictionary, which enables the flow layers to produce a language-neutral representation. Even if we input a speech audio with unseen language to the tone color converter, it is still able to smoothly process the audio. We also experimented with other types of phoneme dictionaries but the resulting tone color converter tended to mispronounce some phonemes in unseen languages. Although the input audio can be correct, there is a high likelihood that the output audio will be problematic and sounds non-native. Four. Discussion. OpenVoice demonstrates remarkable instance voice cloning capabilities and is more flexible than previous approaches in terms of voice styles and languages. The intuition behind the approach is that it is relatively easy to train a base speaker TTS model to control the voice styles and languages, as long as we do not require the model to have the ability to clone the tone color of the reference speaker. Therefore, we proposed to decouple the tone color cloning from the remaining voice styles and the language, which we believe is the foundational design principle of OpenVoice. In order to facilitate future research, we have made the source code and model weights publicly available. References to eighteen other publications in text.

https://www.nature.com/articles/srep12115 https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Fasting activates macro-autophagy in neurons of an Alzheimer’s disease mouse model, but it is insufficient to degrade amyloid-beta. Xigui Chen, Department of Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, Japan, and others. We developed a new technique to observe macro autophagy in the brain in vivo, and examined whether fasting induced macro-autophagy in neurons, and how the induction was different between Alzheimer’s disease (AD) model and control mice. Lentivirus for EGFP-LC3 injected into the brain successfully visualized auto-phagosome in living neurons by two-photon microscopy. The time-lapse imaging revealed that fasting increased the number, size and signal intensity of auto-phagosome in neurons. In AD model mice, these parameters of auto-phagosome were higher at the basal levels before starvation, and increased more rapidly by fasting than in control mice. However, the metabolism of exogenous labeled A Beta evaluated by the new technique suggested that the activated macro-autophagy was insufficient to degrade the intracellular A Beta increased by enhanced uptake from extracellular space after fasting. Ordinary immunohistochemistry also revealed that fasting increased intracellular accumulation of endogenous A Beta, triggered cell dysfunction but mostly did not decrease extracellular A Beta accumulation. Moreover, we unexpectedly discovered a circadian rhythm of basal level of macro autophagy. These results revealed new aspects of neuronal autophagy in normal, AD states and indicated usefulness of our method for evaluating autophagy functions in vivo. Autophagy, especially macro-autophagy mediated by auto phagosomes, has been implicated in various neurodegenerative diseases including AD. Ultrastructural analysis of postmortem human AD brains revealed increased auto phagosomes in dystrophic neurites. Macro autophagy was also suggested to be a pathway of generating amyloid beta (A Beta) in the cytoplasm. Meanwhile autophagy-related genes were induced in autopsy brains of AD patients and auto phagosomes were co-localized not only with A Beta in AD but also with a-synuclein and tau aggregation in autopsy brains of Parkinson’s disease and frontotemporal lobar degeneration, suggesting that misfolded disease proteins might generally induce autophagy. Some neurodegenerative diseases have more direct relationships to autophagy. Familial Parkinson’s disease causative proteins, PARK2, Parkin and PARK6, PINK1 act as indicators of functionally abnormal mitochondria to induce mitophagy. Hereditary spastic paraparesis type 15 are linked to mutations of the SPG15 gene that promotes autophagosome maturation. Mutations of an adaptor protein for selective autophagy, p62 are associated with amyotrophic lateral sclerosis, ALS. In vivo analysis of autophagy after nutritional starvation was performed in a pioneering work by Mizushima, Ohsumi and their colleagues with LC3-GFP transgenic mice, but induction of macro-autophagy was not detected in the brain tissues after fixation. Meanwhile, it was reported thereafter that inhibition of m TOR induced autophagy and ameliorated poly-glutamine disease pathology. Moreover, autophagic response of neurons might be conditional. In contrast to inducible autophagy, constitutive autophagy is established to protect neurons in vivo from neurodegeneration through clearance of ubiquitinated proteins. The discrepancy awaits further analysis with a new technique to observe living neurons to settle the issue of in vivo. Results. A new method of in vivo imaging of macro autophagy in the brain of living animals based on two-photon microscopy. To visualize auto phagic vacuoles in living neurons in the brain, we generated lentiviral vector expressing EGFP-LC3. We injected 5 micro liters of lentiviral vector, titer 5 million vector genomes per milliliter, into retrosplenial dysgranular cortex (RSD) or cerebellar cortex. Twenty days after injection the mice were investigated by two-photon microscopy, FV1000 MPE2, Olympus, Japan, with the thin skull method as described in Methods. In both areas, clustered EGFP-positive vesicles and dispersed fine EGFP-positive dots were observed, Figure 1a. Especially in the cerebellar cortex, the vesicles with high intensities were clustered in a narrow area of 10 to 20 micron diameter, suggesting that they correspond to the cell body of Purkinje cells aligned in a single layer, Figure 1a, Supplementary Figure 1. In reconstructed images of the cerebellum, EGFP-LC3 vesicles were also aligned in the main dendrite of Purkinje cells, Supplementary Figure 1. To verify that such clusters of EGFP-LC3 vesicles actually corresponded to the cell body of Purkinje cells, we used double transgenic mice, loxP-flanked STOP cassette Td Tomato by Ptf1a-promoter-Cre, that express red fluorescent protein in GABAergic Purkinje cells in the cerebellum. Infected lentiviral vector actually expressed EGFP-LC3 protein in Purkinje cells, a part of granule cells, but not in Td Tomato positive GABAergic neurons in molecular cell layer, see Supplementary Figure 2. To verify the expression of EGFP-LC3 in cortical neurons, we performed immunohistochemistry with anti-NeuN or GFAP antibody and examined co-localization of non-stained native EGFP-LC3 with a cell-specific marker, Figure 1b. The result revealed EGFP-LC3 vesicles, dots were distributed in NeuN-positive neurons, Figure 1b. GFAP positive astrocytes might also possess EGFP-LC3-positive dots, while the signals were weak in comparison to neuronal EGFP-LC3 vesicles, Figure 1b. On the other hand, infection of AAV-EGFP generated diffuse intracellular signals of EGFP, Figure 1c, supporting that the EGFP-LC3 vesicles were not the artificial self-aggregates of EGFP as reported. Moreover, we found by confocal microscopy that a part of the EGFP-LC3 vesicles was co-stained with a lysosome marker, LAMP2A in brain tissues, indicating that these EGFP-LC3 vesicles were actually fused with lysosomes, Figure 1d. Starvation-dependent induction and circadian rhythm of macro autophagy in neurons. Since these results supported usefulness of two-photon microscopic observation of EGFP-LC3 for evaluation of macro autophagy, we applied the technique to answer the questions: 1) Whether fasting treatment induces macro autophagy in neurons and 2) How the auto phagic response is different between 5XFAD mice, one of the severest mouse AD models that firstly shows A Beta deposition at 3 months of age, and the background mice, C57BL, 6 times SJL, Figure 2. We firstly examined the effect of fasting treatment on body weight and blood glucose, and confirmed that 5XFAD and background mice showed similar responses to fasting in these parameters, Supplementary Figure 3. In this experiment, 20 days after injection of EGFP-LC3 lentivirus, the mice were fasted and supplied only with water for 48 hours, Figure 2a. Two-photon microscopic observation was performed at 0, 6, 12, 24 and 48 hour time points during fasting, Figure 2a. Using the vessels as markers, the position of observation was strictly controlled, Supplementary Figure 4. Before analyzing the effect of fasting, we needed to test whether auto phagosome formation possesses a circadian rhythm, Supplementary Figure 5, because it had not been investigated previously. Unexpectedly, our live imaging of the brain revealed that the number, volume, and signal intensity per cell of the EGFP-LC3 vesicles changed in a circadian rhythm pattern, Supplementary Figure 5. All the parameters increased during daytime (light) and decreased in nighttime, dark. Interestingly, however, the parameters started to decrease around 4 PM when mice do not eat much, Supplementary Figure 5, suggesting that the circadian rhythm genes might affect auto phagosome formation independently of feeding behavior. However, the question whether circadian rhythm genes affect auto phagosome formation through or not through feeding behavior is an open question requiring further investigation. Therefore we started the observation strictly at the same time points to evaluate the response of autophagosome to the fasting treatment, Figure 2a, b. Two photon microscopy images, 100 micron by 100 micron by 100 micron volume, were obtained from four groups of mice. The number, signal intensity and volume of EGFP-LC3 vesicles were quantified and their mean and SD were calculated, Figure 2c. More than five mice were analyzed in each group of 5XFAD fasting, 5XFAD non-fasting, Wt fasting and Wt non-fasting mice, respectively, Figure 2c. Detailed methods for acquiring these parameters are described in the Methods section. The time-lapse live imaging revealed that basal levels of EGFP-LC3 vesicles were higher in 5XFAD mice at the number, intensity, and total volume of vesicles per cell but not the average size of puncta, Figure 2c. In addition, when these values were corrected by the basal values in each mouse group, the increasing ratio was also higher in 5XFAD mice, Figure 2c. We also employed an ordinary immune-histo-chemistry method with postmortem brains of 5XFAD mice after fasting treatment to detect endogenous LC3 vesicles. In this analysis, sensitivity of the endogenous LC3 detection was far lower than that of AAV-EGFP-LC3 by our new method, and it was hard to evaluate the number of macro auto phagosome strictly. However, the result of LC3 signals still suggested induction of macro auto phagosome after fasting, which was generally observed across multiple brain regions, Figure 2d. Figure 1. In vivo imaging of macro autophagy in neurons. (a) EGFP-LC3 lentivirus was injected into the cerebellar cortex (left panel) and retrosplenial dysgranular cortex (RSD, right panel) of wild type mice (C57BL, 6 by SJL) at 3 months of age. Twenty days later, EGFP-LC3 signals were directly observed by two photon microscopy. The EGFP-positive vesicles were distributed in a group as if they were auto phagosomes in a cell. (b) The brain tissues of wild type mice injected with EGFP-LC3 lentivirus were immunostained with anti-NeuN and GFAP antibodies. EGFP-LC3 vesicles surrounded NeuN-positive neuronal nuclei, yellow arrows, indicating that they were auto phagosomes in neurons. Such distributions of EGFP-LC3 were not found in GFAP-positive astrocytes, white arrowhead, suggesting that most EGFP-LC3 vesicles were located in neurons. EGFP-LC3 was directly observed without immunostaining in these experiments. (c) Two-photon microscopic observation of the RSD region of AAV-EGFP-injected wild type and 5XFAD mice at 3 months. Both genotypes of mice showed homogeneous signals of EGFP in cells, supporting the specificity of EGFP-LC3 signals. (d) Colocalization of a part of EGFP-LC3 vesicles with LAMP2A in RSD region of 5XFAD mice at 3 months. Figure 2. In vivo imaging of auto phagosome in AD model and control mice. (a) Experimental protocol of the in vivo time-lapse imaging to test the effect of fasting on auto phagosome formation. (b) Time-lapse imaging of EGFP-LC3 vesicles was performed at a similar region of AD model and control mice at 3 months with or without fasting. The signal intensities were higher in 5XFAD mice than control mice before fasting. Fasting induced the increase of signal intensities in both genotypes, while the induction was more prominent in 5XFAD mice. (c) Quantitative analyses of the chronological changes of EGFP-LC3 vesicles with or without fasting. In mean signal intensity per cell, mean vesicle number per cell, and mean vesicle volume per cell, the values were higher in 5XFAD mice than control mice. Some of these values were also increased more remarkably in 5XFAD mice than control mice. The mean volume of the vesicles was not changed so remarkably. More information is included in the text of the paper. Effect of starvation-induced macroautophagy on extracellular and intracellular A Beta accumulation. Finally, we tested whether induced auto phagosome was really effective for degradation of A Beta because it was reported previously that AD-asscociated mutation of presenilin-1 impairs autolysosome acidification and cathepsin activation to inhibit proceeding of autophagy processes. Such a dysfunction in macro autophagy might occur in 5XFAD mice and might prevent degradation of the substrates within auto phagosomes after fusion with lysosomes. For this purpose, we injected A Beta labelled with TAMRA into the retrosplenial dysgranular cortex (RSD), the brain area that corresponds to human precuneus, and observed dynamics of A Beta by time-lapse imaging for 2 days from 24 hours after injection, Figure 3a. The experiment might mimic over-secretion of A Beta from hyperactivated neurons in brain regions composing the default mode neural network. First we found that injected A Beta was taken up into neurons within 24 hours after injection, Figure 3b. Importantly, the amount of A Beta (red vesicle) in neurons was obviously higher in 5XFAD mice, Figure 3b,c. The increase of intracellular A Beta could be explained by increased uptake of extracellular A Beta by endocytosis. Analysis of the yellow vesicle volume per cell that reflects secondary lysosome, the fused vesicle of endosome and autophagosome, revealed that the amount of secondary lysosome increased in neurons during the time of fasting whereas in which A Beta remained undegraded, Figure 3c. The increase of such “residual body” containing A Beta was observed both in wild type and 5XFAD mice, while the extent of increase was more remarkable in 5XFAD mice, Figure 3c. Figure 3. Endocytosis and auto phagosome-dependent degradation of A Beta by cortical neurons. (a) Experimental protocol of the chronological in vivo imaging to test A Beta uptake and degradation in the cortical neurons is shown. (b) Time lapse imaging of TAMRA Beta amyloid (upper panels) and TAMRA Beta amyloid plus EGFP-LC3 vesicles (lower panels) at 24, 0, 48, 24, 60, 36, and 72, 48, hours after injection of TAMRA Beta amyloid. Interaction between endosomes (red) and auto phagosomes (green) were observed chronologically. (c) Quantitative analyses of the chronological changes of endosomes, TAMRA Beta amyloidpositive vesicles, left graph, and secondary lysosomes, TAMRA Beta amyloid and EGFP-LC3 double positive vesicles, right graph, with or without fasting. More information in the text of the paper. Figure 4. Fasting treatment does not affect extracellular A Beta accumulation. (a) Images of A Beta at various brain regions of 5XFAD mice with or without fasting at 3 months. DAB satin was used to visualize the antibody reaction. No obvious difference was detected in intracellular and extracellular A Beta accumulation between fasting (minus) and fasting (plus) groups at all brain regions. Abbreviations. RSD, retrosplenial dysgranular cortex. FC, frontal cortex. LSV, ventral part of lateral septal nucleus. VM, ventromedial thalamic nucleus. PnC, caudal pontine reticular nucleus. OB, olfactory bulb. Sub, subiculum. CA1, hippocampus CA1. CA2, hippocampus CA2. CA3, hippocampus CA3. DG, dentate gyrus. Cbm, cerebellum. (b) Images of A Beta visualized by fluorescent secondary antibody at various brain regions of 5XFAD mice with or without fasting at 3 months. No obvious difference was detected in intracellular and extracellular A Beta accumulation between fasting (minus) and fasting (plus) groups at all brain regions. (c) DAB staining of sagittal sections revealed that extracellular A Beta accumulation was decreased in visual cortex, arrows. For the comparison of the effect of fasting treatment on A Beta accumulation across different regions of the brain, we employed a method using the post-fixed brain samples of 5XFAD mice at 3 months taking the advantage of the pathological stage. In this case we observed the effect of fasting on accumulation of endogenous A Beta of 5XFAD mice instead of exogenous A Beta TAMRA. The DAB and fluorescence immunostainings with anti-A Beta antibody revealed no significant difference of extracellular A Beta accumulation between non-fasting and fasting groups of 5XFAD mice in most brain regions, Figure 4a,b. However, we detected a tendency that extracellular A Beta accumulation was decreased in visual cortex in fasting groups of 5XFAD mice, Figure 4c. Intracellular A Beta accumulation was also confirmed across different brain regions as we reported previously. Also unexpectedly, we observed that intracellular A Beta accumulation accompanies blurring or fading-out of DAPI or NeuN stains of the nuclei of neurons, Figure 5a,b. This suggested that a certain type of cell death was induced by intracellular A Beta accumulation and that extracellular plaque formation might be triggered by the seed of A Beta foci of ghost neurons, the residual intracellular A Beta accumulation of dead neurons. Indeed, we often observed various images supporting the progression from intracellular to extracellular A Beta aggregates, Figure 5c, and queer ballooning’s of the cytoplasm and apoptoic changes of the nuclei in intracellular A Beta positive cells, Figure 5d. Hence, we categorized such types of cells with intracellular A Beta accumulation into three groups, vital cells with clear nuclear margin with DAPI stains (Group A); dying cells with blurred or faint DAPI nuclear stains (Group B); and dead cells with the defect of DAPI nuclear shape (Group C), Figure 5a. Quantitative analysis revealed that the total cell number of neurons with intracellular A Beta accumulation was increased by fasting treatment generally in all brain regions, Figure 5e. Quantification of each group revealed that fasting treatment induced a shift from A to C generally in all regions of the brain, Figure 5e. These results were consistent with the hypothesis that intracellular A Beta accumulation triggers the cell death and that plaque formation is seeded by the ghost of intracellular A Beta accumulation, whereas this hypothesis should be examined more extensively by employing additional methods in the future. Figure 5. Intracellular A Beta accumulation was affected by fasting. (a) Various forms of intracellular A Beta accumulation observed in the cortex of 5XFAD mice at 3 months. Accumulation of intracellular A Beta often accompanied with blurring or fading out of the nuclei, thus the were aligned with hypothetical progression of the cell pathology. Intracellular A Beta positive cells with intact nuclei, with abnormal nuclei, and with no nuclei are classified to group A, B and C, respectively. (b) Co-staining of NeuN and A Beta revealed that neurons are the cells possessing intracellular A Beta accumulation. (c) Hypothetical progression of extracellular A Beta aggregates from the seed of intracellular A Beta accumulation is shown with representative images in RSD of 5XFAD mice at 3 months. White arrows suggest the defect of dead neurons. Yellow arrows suggested an enlarged nucleus with abnormal DAPI stains of the nucleus. (d) Abnormal ballooning of cells with intracellular A Beta accumulation suggesting atypical cell death. (e) Quantitative analysis of the number of group A, B and C cells at various brain regions in fasting and non-fasting 5XFAD mice at 3 months. P-values for comparison in each group or total cell numbers between fasting and non-fasting were calculated by Welch’s test. Discussion. Our study firstly proved that macro autophagy was actually induced by starvation in mouse neurons in vivo. This finding was consistent with a previous result with post-mortem analysis of GFP-LC3 transgenic mice after fasting. The GFP-LC3 transgenic mice were starved for 24 or 48 hours, perfused and fixed. After killing the mice, the authors of the previous study sampled the brain tissues and observed the GFP-LC3 fluorescence. Compared with their method, our technique has an advantage to directly observe the change of macro autophagy in the brain of living animals but not of dead animals. On the other hand, the protocol used in this study was limited to observation at one or several restricted regions of the brain. However, since our AAV vector is also applicable for systemic delivery by intravenous injection, it might be possible to overcome the limitation of our method in the future. The finding in this study that fasting induces macro autophagy basically supports previous results showing that activation of macro autophagy by chemicals or vectors ameliorated the pathology of neurodegenerative diseases in various animal models. In addition we unexpectedly discovered circadian rhythm of neuronal macro autophagy in vivo. The circadian rhythm might be interesting if we consider it with the previous finding that A Beta secretion is reduced during the sleep. These results would collectively contribute to understanding of the significance of autophagy in the brain. Our results also suggested even though autophagy was activated in 5XFAD mice under starvation, the intracellular degradation of A Beta was still insufficient to compensate the increased uptake of A Beta from extracellular space, Figure 3. This idea was further supported by immunohistochemistry of 5XFAD mice, showing that fasting treatment enhanced intracellular accumulation of endogenous A Beta, Figure 5. Moreover, fasting did not largely affect extracellular accumulation of endogenous A Beta in 5XFAD mice, Figure 4a,b. If this is the case in human AD pathology, such enhanced uptake of A Beta by calorie restriction could be harmful for neuronal function if the intracellular A Beta triggers abnormal signaling cascades. Supporting this hypothesis, we observed an increased number of neurons with intracellular A Beta that lost viability after fasting treatment, Figure 5. However, it is also of note that we had injected a high amount, concentration of A Beta to visualize the metabolism in vivo and that the expression level of A Beta in the 5XFAD mice was significantly higher than in human AD patients. Therefore, starvation–activated macro autophagy might still ameliorate the A Beta pathology if A Beta concentration in extracellular space is not so high. In that case, induction of A Beta uptake by starvation might reduce the extracellular A Beta and the intracellular A Beta could be degraded at a relatively low level by activated macro autophagy. Then, the resultant decrease of extracellular A Beta might also rescue the synaptic transmission. The critical point of concentration of the two hypotheses needs further investigation. Collectively, the balance among secretion, endocytosis and degradation of A Beta should play a pivotal role in initiation and progression of human AD pathology. Therefore nutritional condition and circadian rhythm, which may be influenced by our life style, are considered to be intriguing factors for AD. In conclusion, this study revealed that nutritional starvation induces macro autophagy in neurons but the induction is insufficient to degrade a high amount of A Beta in AD-associated pathological condition. Technical sections, including Methods, AD model mice and the Generation of lentiviral vectors. The Titration of viral vectors, Injection of viral vectors and fluorescence of beta amyloid. In vivo imaging with two photon microscopy, the fasting treatment of mice and Immunohistochemistry. The Positioning of the image fields, Statistics, Ethics, and 25 References. Acknowledgments, Author Contributions, and Additional Information.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8749320/pdf/jcp-26-4-224.pdf https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Calorie Restriction for Cancer Prevention and Therapy: Mechanisms, Expectations, and Efficacy. An average quality review by Chiara Vidoni, Laboratory of Molecular Pathology, University oriental pasta, Novara, Italy. Cancer is one of the most frequently diagnosed diseases, and despite the continuous efforts in searching for new and more effective treatments, its morbidity and mortality remain a significant health problem worldwide. Calorie restriction, a dietary manipulation that consists in a reduction of calorie intake, is gaining attention as a potential adjuvant intervention for preventing and, or fighting cancer. Several forms of energy reduction intake, which include caloric restriction tout-court, dietary restrictions, and intermittent fasting, are being explored for their ability to prevent or slow cancer progression. Additional anti-cancer approaches under investigation rely on the use of nutraceuticals known as “Caloric Restriction Mimetics” that can provide caloric restriction-mediated benefits without subjecting the patients to a strict diet. Preclinical in vitro and in vivo studies consistently show that diet modifiers reducing caloric intake have impact on tumor microenvironment and cancer metabolism, resulting in reduced growth and progression of cancer. Preliminary clinical studies show that patients subjected to a reduced nutrient, energy intake experience improved outcomes from chemo and radiotherapy while better tolerating the side effects. Here, we review the state of the art on the therapeutic potential of calorie restriction and of caloric restriction mimetics in preventing or retarding tumor development by modulating a subset of cellular processes. The most recent clinical progresses with caloric restriction mimetics in the clinical practice are also discussed. Key Words. Ketogenic diet, Warburg effect, Caloric restriction mimetics, Tumor microenvironment, Drug therapy. Introduction. Calorie restriction (CR) consists of a 20 to 40 percent reduction of the average daily caloric intake without incurring malnutrition or deprivation of essential nutrients. It encompasses the restriction of specific hyper caloric nutrients, which can be substituted with others, that are metabolized with less production of energy. Mammals undergo a metabolic adaptation in response to food restriction. The circulating glucose concentration is the first biomarker to decline under CR condition, which results in the utilization of stored glycogen as a main energy source. Once the glycogen stocks are depleted, the organism utilizes glycerol and fatty acids mobilized from the adipose tissue, and thus ketone bodies become the main fuel. CR has a huge impact on promoting longevity by delaying the severity and the onset of inflammatory and several age-related diseases including obesity, cardiovascular, neurodegenerative and ophthalmic disorders, and cancer. Such pleiotropic effects rely on several mechanisms, although the principal common denominator is the ability of CR to dampen the oxidative stress and inflammation. Aging is one of the major risk factors favoring the development of many cancer types. Epidemiological studies reveal that five out of six cancer-related deaths occur in patients aged 60 years and older. The continuous exposure to carcinogenic factors, which leads to the accumulation of mutations and epimutations in cancer-sensitive genes surely accounts for the increased risk of cancer development with aging. Additional contributors are: The progressive decline of the immune surveillance, the efficiency of DNA repair and of autophagy. The dysregulation of the inflammatory process. The increased production of reactive oxygen species (ROS). Increased levels of circulating insulin and Many other hormones promoting cell growth, and other factors. CR may arrest and slow-down age-related decline of cellular protective systems, especially by improving autophagy and dampening inflammation and ROS production, as well as reducing circulating growth hormones, and this could result in reduced risk of cancer. Preclinical and preliminary clinical data support the view that reducing calorie intake as well as periodic fasting or dietary restriction, in which intake of macronutrients is limited with no reduction in total calories, has the potential to prevent and treat cancer. Typically, in a CR or dietary restriction regimen, carbohydrates, the main source of energy in the regular diet, are reduced and replaced partially or nearly completely, as in the ketogenic diet, by fat. Indeed, reducing sugar intake seems to be a good strategy to fight cancer, given that cancer cells use glucose as the main fuel. Likewise providing ketones as an alternative energy source may limit cancer growth because cancer cells do not efficiently harness ketones for their anabolism. Here, we describe the cellular and molecular mechanisms underlying the pathophysiological effects of calorie and nutrient restrictions and review the scientific proofs of their beneficial effects in preventing cancer onset and progression as well as in improving the anti-cancer therapeutic effects. We also discuss the anti-cancer effects of drugs and nutraceuticals with proven caloric restriction mimetic (CRM) activity. Finally, we present the clinical trials currently investigating the efficacy of caloric restriction dietary regimens as an adjuvant therapy in anti-tumor treatment. Molecular and cellular effects of calorie restriction at a glance. For a long time, the beneficial impact of CR was regarded merely as the result of the passive effect of nutrient limitation and of a slow metabolism. It is now recognized that the organismal effects of CR are actively regulated processes aiming to reduce oxidative stress, and that CR triggers a robust defense program involving multiple metabolic pathways in which nutrient sensors are centrally positioned in such regulation. However, the effects of CR depend on multiple factors such as individual characteristics and the dose and timing of CR. The metabolic adaptations to CR include: (1) A decrease in growth factors and production of anabolic hormones. (2) An upregulation of anti-oxidant systems, which in turn decreases free radical-induced DNA damage. (3) A downregulation of pro-inflammatory cytokines and an increase in circulating levels of corticosteroids, ghrelin and adiponectin, collectively resulting in the reduction of inflammation, and (4) A delay of aging-associated deterioration of host immune surveillance. More in detail, many of the benefits exerted by CR are associated with the upregulation of genes promoting DNA repair, for example, genes belonging to the base excision repair pathway, the removal of damaged cells through apoptosis, autophagy, stress response and anti-oxidant defense, in parallel with the downregulation of pro-inflammatory genes and of energy metabolism pathways. Particularly, autophagy represents the primary stress response to calorie and nutrient restrictions. This process is in fact regulated mainly by two pathways that sense the lack of energy sources and ATP production in the cell, via the AMP-activated kinase (AMPK) and hexokinase 2 (HK2) mTOR complex 1 (mTORC1) pathway, and the lack of growth factors and of amino acids, via the protein kinase B (AKT)-mTORC1 pathway, Figure 1. Autophagy, herewith referring to macro autophagy, consists in the p62, SQSTM1-mediated entrapment of cellular components, such as protein aggregates, membranes, and mitochondria (mitophagy) along with portions of cytoplasm, within a double-membrane organelle named the auto phagosome that upon fusion with the lysosome determines the degradation of those components. This process is regulated by several signaling pathways and autophagy-related (ATG) proteins that also include oncogene products and tumor suppressors, which explains why this process is dysregulated in cancer. Under metabolic stress conditions such as those determined by the lack of nutrients, amino acids, glucose, and of hormones and growth factors, autophagy is upregulated to provide energy and substrates from degradation of redundant cell components. As illustrated in Figure 1: (1) Amino acids, especially, methionine, leucine and arginine, directly activate mTORC1, the mechanistic target of rapamycin complex 1, which then inhibits the axis Unc-51 like autophagy activating kinase 1 complex 1 (ULKC1)-phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3KC3)-BECLIN-1 that positively triggers autophagy. (2) The presence of growth factors and hormones elicits the activation of mTORC1 via the PI3KC1-AKT pathway thus resulting also in inhibition of autophagy. (3) Soon after entry, glucose is phosphorylated to glucose-6-phosphate (G6P) by HK2, and this prevents HK2 from interacting and inhibiting mTORC1, and this results in inhibition of autophagy as well. Therefore, autophagy is maximally induced when all these nutrients and growth factors are absent in the tumor microenvironment (TME), as for instance that occurs during starvation. Upregulation of autophagy in cancer cells may have several beneficial outcomes in terms of improved DNA repair efficiency, improved TME, reduced growth and migration, invasive ability. Calorie restriction and cancer progression. From a molecular point of view, several signaling pathways collaborate and cross-talk to control carcinogenesis under CR conditions. To date, the major effectors known to be responsible for the CR-mediated anti-cancer activity include insulin-like growth factor-1 (IGF-1), phosphatidylinositol-3-kinase, PI3K, AKT, mTOR, the Sirtuin family proteins, Aldolase A (ALDOA), DNA-dependent protein kinase, DNA-PK, p53, NF-kappa B and AMPK signaling pathways. However, further studies aiming to characterize the molecular mechanisms by which CR mediates its cancer inhibitory effects are essential for development of new drugs and therapeutic regimens to prevent tumor initiation and, or interrupt tumor promotion and progression. CR can also modulate epigenetic changes, particularly DNA methylation, histone modifications, chromatin remodeling and generation of microRNA, which regulate the expression of genes involved in those processes responsible for CR anti-cancer activity. Notably, CR has been shown to have a wide impact not only on cancer cells but even on TME by allowing enhanced drug delivery, by decreasing the availability of substrate and growth factors for cancer cells, and by reducing inflammation. Tumor vascularization represents one of the most crucial steps in cancer progression by ensuring nutrients, soluble factors and oxygen to reach the tumor mass. CR has been capable of counteracting this aspect by hampering the secretion of pro-angiogenic factors such as VEGF, factor eight, interleukin-6, IL-6, TNF-alpha, plasminogen activator inhibitor-1, PAI-1, and others. Consequently, tumor neo-vascularization was delayed or even arrested as demonstrated by the reduction in the size, number and density of blood vessels in the CR-fed mice in comparison with the trends observed in ad libitum-fed ones. Additionally, CR can shape the tumor immune microenvironment by specifically decreasing the number of tumor associated macrophages, increasing the formation of a reservoir of CD8 plus cytotoxic T cells and memory T cells while negatively modulating immunosuppressive Treg cells’ activity and immunosuppressive cytokine levels. Other pivotal players in the TME are the cancer-associated fibroblasts. CAF’s, that by releasing onco metabolites, growth factors, inflammatory cytokines and proteolytic enzymes cooperate in the establishment of a malignant liaison between the stroma and cancer parenchymal cells. The evolution of tumor fibrosis, that originates from cancerous lesions, causes an excessive deposition of extracellular matrix and, as a consequence, damaged epithelial cells produce a large amount of pro-inflammatory and pro-fibrotic cytokines, leading to more and more aggravated deposition of collagen and fibrotic tissue. In this context, CR can elicit an anti-fibrotic effect by downregulating TGF-Beta signaling, that normally promotes the phenotypic conversion of normal fibroblasts in CAFs. In this respect, a highly dense and viscous stroma prevents the cells of the immune system to target the tumor, thus making it much more resistant. By preventing fibrosis, CR may facilitate the interaction of immune cells with cancer. The remodeling of the TME mediated by CR is schematically represented in Figure 2. Benefits of caloric restriction in anti-cancer therapy. To date, chemotherapy is one of the main therapeutic strategies for the treatment of several malignancies. However, this approach causes many side effects, such as cardio, neuro, haematological toxicity, nausea, gastrointestinal symptoms, fatigue, weakness, hair loss and stomatitis, that can negatively affect the cancer patients’ quality of life and cause discontinuity of the therapy. Disappointingly, most of the drugs used to manage the symptoms of toxicities may themselves have significant adverse effects. Although most of the available studies regarding CR in anti-cancer therapy are still in the pre-clinical phase, CR appears to be a promising approach to modulate the chemotherapy induced side effects while enhancing the efficacy of the treatment. Reduction of adverse effects would improve the quality of life and potentially reduce the costs of hospitalization as well as the use of drugs, for example anti-emetics, antibiotics. In detail, CR can induce healthy cells to invest their energy in reparation and maintenance pathways rather than cell proliferation. This effect promotes an increased resistance of normal cells to chemotherapeutic drugs known as “differential stress resistance”. On the other hand, cancer cells bearing mutations in oncogenes, for example, IGF-1R, Ras, AKT and and the mTOR pathways, that cause constitutive activation of proliferation pathways in external growth factor-independent manner, and onco suppressor genes, for example, p53, p16 and Rb, that cause insensitivity to growth-inhibitory signals, are not prone to adapt to fasting conditions and continue to proliferate at a high rate. This results in an enhanced sensitization of cancer cells to chemotherapy-induced apoptosis while protecting normal cells from such effect, leading to the so called “differential stress sensitization”. Several reports indicate that fasting potently triggers autophagy, both in normal cells and cancer cells, to recycle critical components and produce energy. The upregulation induction of autophagy before chemotherapy may protect benign cells by providing an alternative mechanism to remove damaged macromolecules and organelles, particularly when the proteasomal degradation pathway is saturated. However, autophagy may also play a pro-survival role in some cancer cells. On the other hand, over activation of autophagy may lead to what is referred to as autophagy-associated cell death. Given the complex role of autophagy in tumor biology, which is strictly dependent on the context and the stage of malignancy, further studies are needed to dissect the balance between benefits and side effects related to CR-induced upregulation of autophagy. Even though CR displays numerous benefits in anti-cancer therapy, the real applicability of fasting regimens in the clinical practice could be limited to a small subset of cancer patients, as some potential risks may be associated with this approach, such as malnutrition, cachexia and sarcopenia, that are strongly associated with chemotherapy-related toxicity, reduced response to cancer treatment, low quality of life and a worse overall prognosis. Another concern is related to the anti-inflammatory effect of CR that could be disadvantageous for those patients that experience immunodeficiency due to cancer progression and, or as a consequence of repeated chemotherapy treatments. Therefore, more tolerable adjuvant regimens should be developed. In this perspective, fasting-mimicking dietary interventions as well as CRM’s, that will be discussed more in detail in the next section, may represent a more feasible therapeutic approach to circumvent these limitations. Overall, the global impact of CR and CRM’s on the anti-cancer therapy is illustrated in Figure 3. Caloric restriction Mimetics. An alternative therapeutic strategy that extends life expectancy and improves health markers, while reducing the development of several age-related diseases, including cancer, involves the use of the pharmacological group of compounds known as CRM’s. These compounds act, either through direct interaction with signaling molecules or via epigenetic mechanisms, those pathways that are triggered when energy intake is reduced, yet in the presence of adequate nutrition. Many CRM’s are bioactive food components able to elicit anti-proliferative, pro-apoptotic and anti-metastatic effects, avoiding a fasting regimen that could not be tolerated by the cancer patient. The family of polyphenol substances are all a good source of potential CRM’s, since they have a wide range of biological activities, including anti-oxidant, anti inflammatory, anti-carcinogenic and epigenetic modulation activities. They include phenolic acids and derivatives, flavonoids, stilbenes, and coumarins. CRM’s modulate energy and nutrient-sensing pathway impinging on many biological mechanisms, including activation of autophagy, enhancement of insulin sensitivity, inhibition of oxidative stress and inflammation, and modulation of glucose metabolism. The molecular targets of CR involve sirtuins, acetyl-CoA, activated AMP protein kinase, insulin, and mTOR. CRM’s in clinical practice. We will focus on the beneficial effects of the most relevant and promising CRM’s, summarized in Table 1, both FDA approved and not yet approved, and will illustrate their potential clinical applications as new effective anti-cancer strategies. Resveratrol. Resveratrol, 3, 5, 4 tri hydroxyl stilbene, RV, is a natural stilbene compound present in vegetables and fruits in general, but especially abundant in grapes. RV acts as a CRM as well as a protein restriction mimetic. RV has pleiotropic beneficial effects not limited to cancer, but even to metabolic syndromes and neurodegenerative diseases. The tumor suppressive effects of RV on manifestation of malignant phenotype of cancer cells involve the repression of the drug resistance and metastatic ability, counteracting hypoxia, inhibition of inflammation and oxidative stress, and so on. In details, RV reverts cell invasion, which is promoted by high generation of ROS through activation of the Hedgehog pathway. Cumulative studies have illustrated the impressive anti-inflammatory properties of RV. In vivo experiments showed that mice treated with RV exhibit low levels of pro-inflammatory cytokines like TNF-alpha, IL-6, IL-1 and IL-8, typical biomarkers of the inflammation. Further, RV increases the number of T cells, specifically natural killer and CD8+ T cytotoxic cells, implementing anti-cancer immune surveillance. Another anti-inflammatory property mediated by RV is the suppression of the NF-kappa B pathway and of TNF alpha induced cancer cell migration and invasion. Additionally, RV can block tumor development by targeting cytochrome p-450 enzymes able to activate pro-carcinogenesis factors. Furthermore, RV positively impacts to expand lifespan as an epigenetic modulator, specifically through the activation of sirtuin deacetylases (SIRT1) and autophagy mediated via AMPK pathway. Besides limiting glucose uptake and reverting the inflammatory phenotype of CAF’s, RV is a potent autophagy inducer. Many preclinical and clinical trials in different types of cancer, for example, breast, colon, and prostate, support its anti-cancer effects. Although RV has many anti-carcinogenic properties, its poor bioavailability limits its clinical use. Nevertheless, there is evidence that RV, either alone or in combination with other agents, is active. Therefore, an alternative strategy is to modify the RV structure for improving its bioavailability and reducing its toxicity. Nowadays, it is clear that RV is a fascinating adjunctive cancer treatment when associated with standard chemotherapeutic agents, but there is still the necessity to define the optimal conditions to ameliorate the delivery and the efficiency. Table 1. Overview of the ongoing clinical trials with caloric restriction mimetics, CRM’s. The table reports the CRM’s that are employed in ongoing clinical trials in cancer patients. The table elaborated with data extracted from the clinical trials dot gov site. Curcumin. Curcumin is a polyphenol compound, FDA-approved, for CRM properties that has caught the attention of many researchers. It is the main bioactive compound isolated from the rhizomes of Curcuma longa (Turmeric). Several investigations have revealed the multitude of biochemical and biological activities of curcumin with therapeutic potential, including anti-inflammatory, anti-oxidant, anti-cancer and anti androgenic effects. Particularly remarkable is its anti-cancer activity exerted through induction of apoptosis, inhibition of cell proliferation and of tumor invasion, and downregulation of NF-kappa B, COX-2, and STAT3. Furthermore, curcumin counteracts the Warburg effect, meaning, the aerobic glycolysis occurring in cancer cells) via the suppression of pyruvate kinase M2 (PKM2). Additionally, curcumin suppresses the PI3K, Akt, mTOR pathway (by decreasing Akt and mTOR phosphorylation in parallel with PTEN upregulation) thus promoting cell death in cancer cells. Of note, curcumin also abrogates CAF-induced aggressiveness of cancer cells through the inhibition of the mTOR, HIF-1 alpha signaling. The anti-carcinogenic property of curcumin is well-documented in several types of cancer, which makes it a promising co-adjuvant agent in cancer therapy. Metformin. Metformin (dimethylbiguanide hydrochloride) is a derivative of natural biguanidines isolated from the French lilac, Galega officinalis, a plant used for the treatment of type 2 diabetes and metabolic syndrome since the nineteen sixties. Metformin administration is not yet certified as adjuvant of anti-cancer therapy. Mechanistically, it suppresses hepatic gluconeogenesis and decreases insulin levels thus acting as a hypoglycemic drug. This effect is attributed to the activation of energy sensor AMPK via the repression of the mitochondrial electron transport chain complex One, thus leading to the inhibition of mTORC1. For this reason, this molecule is associated with prolonged lifespan, promotion of autophagy, and suppression of oxidative stress and inflammation. As epigenetic modulator, metformin inhibits class two HDAC’s, while stimulates class three HDAC SIRT1 activity. Another important physiological action of metformin involves the immune system. This compound can modulate lymphocyte differentiation during the aging process, promoting CD8 plus memory T cell differentiation, and simultaneously reducing the expression of several pro-inflammatory cytokines. The latter aspect could represent a relevant opportunity to counteract the development of immune evasion within the TME. Taken together, metformin has crucial functions in modulating energy metabolism, while its capacity in retarding or contrasting cancer progression is less addressed. In addition, recent clinical trials are also testing its anti-cancer activity, especially in colon, breast, ovarian, prostate and lung tumors, however, further investigations are needed. Spermidine. Spermidine is a polyamine naturally found in a variety of foods, including wheat germ, soybean, mushrooms, and mature cheese. Further, it is produced by the intestinal microbiota. The effects of this polyamine include extending the lifespan in many model organisms, an effect correlated to induction of autophagy and inhibition of acetyltransferase activity. Moreover, spermidine stimulates AMPK, while it limits the mTORC1 activity. Predominantly, spermidine is able to stimulate mitophagy in both in vitro and in vivo assays, sustaining its capability to slow down the aging process and to sustain tissue renewal. Another molecular mechanism underlying the cancer preventive action of spermidine involves the competition of spermidine with acetyl-CoA for EP300 binding which may contribute to a reduced cancer-related mortality in patients. The inhibition of acetyl transferase EP300 triggers autophagy by the deacetylation of many ATG genes. Furthermore, spermidine, through autophagy activation, can also improve anti-cancer immune surveillance. To explore and support the spermidine administration as adjuvant anti cancer treatment, more clinical trials are needed. Hydroxycitrate Hydroxycitrate (HC) or hydroxycitric acid (HCA) is a CRM present in tropical plants as Garcinia cambogia and Hibiscus sabdariffa. It is widely used as a weight-loss drug in obese patients, but it also possesses anti-cancer activity. A HC’s peculiarity is its ability to block acetyl-CoA synthesis by inhibiting the enzyme ATP citrate lyase, thus representing an innovative approach to target cancer metabolism. This compound enhances autophagy flux, since it reduces lysine acetylation of cellular proteins. It has been found that the treatment of HC promotes the depletion of regulatory T cells from the tumor, improving immunosuppressive ability and counteracting lung cancer progression. Based on these premises, further synthetic agents, namely acetyl-CoA inhibitors, have been proposed as CRM’s: perhexiline maleate is now used in the clinical practice as an anti-anginal agent with cardioprotective and anti-tumor effects. Halofuginone. Halofuginone (HF) is a synthetic derivative of febrifugine, a natural quinazolinone alkaloid found in the plant Dichroa febrifuga Lour. It is known for its anti-protozoal activity and it is used as an anti-malarial agent in traditional Chinese medicine. Its activity includes inducing amino acid starvation response (AAR) in cancer cells in parallel with the concomitant activation of autophagy. Accordingly, the molecular explanation for its action is that HF inactivates mTORC1 by causing its detachment from the lysosomes and its degradation in proteasome, while promoting the nuclear translocation of the ATG transcription factor TFEB. Furthermore, HF shows its anti-inflammatory propriety by inhibiting the differentiation of inflammatory Th17 cells, an effect clearly linked to induction of AAR. More significantly, HF is a well-known inhibitor of collagen type I synthesis due to the repression of the TGF-Beta pathway. HF also prevents keloid fibrosis by reducing the deposition of ECM and decreasing the proliferation and migration of TGF-Beta activated myofibroblasts. In agreement with this, HF has found clinical application as a therapeutic agent in fibrotic disease and in some types of malignancies, such as lung and bladder cancer. In this respect, further clinical trials are needed to validate the anti-fibrotic property of HF in a wide range of tumors. Rapamycin. Rapamycin, also known as sirolimus, is a macrolide compound that was first isolated in 1975 from the bacterium Streptomyces hygroscopicus, found in the soil of Easter Island. Rapamycin is the most promising CRM with an anti-cancer activity, and its efficacy has been addressed in various clinical trials. Its molecular mechanism entails the inhibition of mTOR, a major regulator of cell proliferation and protein synthesis, by binding the protein FKBP12. Since rapamycin is an inhibitor of mTOR, this CRM promotes autophagy. Consequently, sirolimus provokes the deregulation of mTOR downstream effectors resulting in a prolonged lifespan and in a healthier metabolism. Additionally, this macrolide mediates immunosuppressive effects by controlling survival and proliferation of regulatory T-cells. Because of its side effects, including risk of cataracts, insulin-resistance and increased infections, the search for analogues of rapamycin, called rapalogs, occurred, which resulted for example, in NVP-BEZ235, OSI-027, and RapaLink 1. Everolimus, which belongs to the first-generation of rapalogs, was certified for the treatment of hormone receptor-positive, HER2, neu-negative advanced breast cancer, whereas temsirolimus, first-generation drug, was identified as a therapeutic agent in metastatic renal cell carcinoma. Currently the anti-cancer activity of rapamycin and rapalogs is under investigation in several clinical trials, opening several possibilities for innovative anti-cancer treatments. To sum up, see Table 1, it is well established that CRM’s can mimic the actions of CR, or rather delay aging and extend the patients longevity in parallel with improvement of physiological function and the reduction of the risk of many chronic diseases. This results in the avoidance of many side effects that occur with CR, together with better patient compliance. Nevertheless, even CRM’s-based therapeutic approaches show some limitations. For instance, many of them have not been investigated in a sufficient number of clinical trials, for example HF, HC, spermidine, to guarantee the safety and the feasibility of their applications. Moreover, some CRM’s fail to extend lifespan to the same degree as CR, suggesting that CR might suppress distinct mechanisms that are partially targeted by CRM’s. Accordingly, innovative clinical protocols for the employment of CRM’s are under investigation. Recently, the anti-tumor effects of everolimus combined with metformin have been examined. This combination results in an improvement of clonogenicity suppression, cancer cell death and inhibition of mTOR signaling. Therefore, combining different CRM’s could synergize their anti-cancer activities and achieve health benefits. Additionally, to escalate CRM’s effectiveness, it is possible to combine these substances with non-CRM compounds or with nutritional approaches, as CR, intermittent fasting and physical exercise. In this respect, HC, rapamycin and metformin, in association with standard chemotherapeutic drugs, are already being applied as anti-cancer therapies. Finally, there is a relation between CRM’s and the “personalized medicine”, which is the targeting of specific molecular pathways and cancer types with these compounds. The employment of HC and spermidine in the fight against lung metastases through the use of aerosolization is an innovative, efficient and non-invasive way to deliver CRM’s to the lungs. This method possesses several advantages. First of all, it guarantees a higher local concentration of CRM in a particular tissue and, secondly, it limits the arise of systemic adverse effects. Hence, many researchers have pointed out the existence of specificity of certain CRM’s for a precise cancer type. For example, RV is commonly used in breast cancer therapy in conjunction with chemotherapy. Altogether, new clinical trials need to be undertaken to define how these compounds could become a real “personalized target therapy”, making these mimetics into an effective and adjunctive weapon to fight the battle against cancer. Concluding remarks and perspectives. Cancer cells have a high affinity for glucose and amino acids, glutamine, methionine, leucine, arginine, and others, and need growth factors for cell proliferation and cell motility. Thus, starving cancer cells is an appealing strategy to halt cancer growth and metastatic spread. It has been hypothesized that a low energy diet could influence tumor progression and prognosis. Indeed, preclinical and preliminary clinical studies have confirmed that fasting has potential benefits by improving the effectiveness of chemotherapy while attenuating the toxic side effects, by protecting normal tissues from DNA damage, by reducing the inflammation in the TME, by restoring anti-tumor autophagy and apoptosis, and by favoring the immune response. All in all, available data suggest that a regimen with very-low-carbohydrate and low-protein intake, substituted by a relatively high-fat intake, may benefit cancer patients in terms of overall survival and, or progression free survival. However, patients may not tolerate such a CR diet for prolonged time. Therefore, an intermittent fasting regimen has been proposed as alternative, whose beneficial effects also appear promising though controversial in preclinical settings. Intermittent Fasting will require further elucidation in controlled clinical trials. An interesting alternative is represented by compounds known as CRM’s that can mimic the caloric, energic restriction condition while allowing an adequate supplementation of nutrients. These CRM’s elicit their action by triggering anti-cancer biochemical pathways through direct interaction with targeted signaling molecules and, or via epigenetic regulation of the expression of relevant regulators. It is likely that CRM’s activity is influenced by the genetic background and the TME context of the tumor. Therefore, understanding the molecular mechanisms underpinning the effects of such CRMs is mandatory for harnessing their adjuvant benefits in the frame of personalized cancer therapy. Acknowledgments, conflicts of interest, orcid and 125 references.

doi:10.1038/s43587-020-00013-3 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8932957/ https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Intermittent and periodic fasting, longevity and disease. Valter Longo, Longevity Institute, Leonard Davis School of Gerontology and Department of Biological Sciences, University of Southern California, and others. Published in Nature Aging, January 2021, pages 47 to 59. Abstract. Intermittent and periodic fasting, I-F and P-F, respectively, are emerging as safe strategies to affect longevity and healthspan by acting on cellular aging and disease risk factors, while causing no or minor side effects. I-F lasting from 12 to 48 hours and repeated every 1 to 7 days and PF lasting 2 to 7 days and repeated once per month or less have the potential to prevent and treat disease, but their effect on cellular aging and the molecular mechanisms involved are only beginning to be unraveled. Here, we describe the different fasting methods and their effect on longevity in organisms ranging from yeast to humans, linking them to the major nutrient-sensing signaling pathways and focusing on the benefits of the fasting and the refeeding periods. We also discuss both the therapeutic potential and side effects of I-F and PF with a focus on cancer, autoimmunity, neurodegeneration and metabolic and cardiovascular disease. Dietary restriction (D-R) refers to regimens including the reduction of the intake of either calories or of specific components of the diet, such as protein or certain amino acids, and to intermittent and periodic fasting, I-F and PF, respectively, which may or may not require an overall reduction in calorie intake. Instead, calorie restriction (CR), which in most cases involves a chronic reduction in calorie intake by 20 to 40 percent below the standard, extends lifespan and health span in yeast, invertebrates, laboratory rodents and non-human primates. In humans, 15 percent CR reduces markers or risk factors for a range of age-related diseases, including diabetes, cancer and cardiovascular disease, but it also causes side effects, which include a low or very low body mass index (BMI) when applied chronically. Moreover, a chronic CR of 20 to 60 percent in mice can have positive effects on aging and immune function, but can increase susceptibility to certain pathogens, such as the influenza virus and intestinal parasites. Protein restriction (PR) independently of calorie intake also extends lifespan in mice and improves the health of young and middle-age mice and humans, but moderate to severe PR can lead to frailty and,or disease in old mice or individuals over the age of 65. Severe PR, in which calories from proteins are below 5 percent of the total, could also have detrimental effects, including weight loss in younger organisms, as shown in mice. Signaling pathways by which CR and PR extend lifespan include those activated by growth hormone, insulin-like growth factor-1 (IGF-1) and insulin, and involve downstream factors, including phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin complex 1 (mTORC1), protein-kinase A (PKA), AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator 1 alpha, PGC-1 alpha, sirtuins and forkhead transcription factors (FOXOs), that are well established to regulate or affect aging and longevity. Thus, both CR and PR can have strong effects on nutrient signaling pathways and aging, but there is a need to identify novel interventions that optimize health span while minimizing side effects and the burden imposed by chronic dietary interventions. These studies could allow the identification of specific dietary regimens effective in delaying, and even partially reversing, aging and age-related diseases. In this Review, we discuss the role of dietary restriction in aging and disease, with a focus on I-F and PF regimens (Table 1 and Box 1). The use of generic terms like I-F, fasting and even dietary restriction, which include interventions lasting from a few hours to months and represent many dietary compositions and severities of caloric restriction, needs to be limited and replaced by specific terminology referring to a clearly defined intervention so that use can be standardized for the laboratory, the clinic and eventually the public. These intermittent or periodic dietary interventions can promote cell protection and repair as well as the clearance of damaged cells and intracellular components, in part through the modulation of conserved stress-response or nutrient-sensing pathways. Whereas the dietary-restriction field has mostly focused on the benefits of continuous caloric or macronutrient restriction, here we focus both on the importance of much shorter restriction periods and the refeeding and post-refeeding phases, which is accompanied by a regenerative process that is not observed or is much less active during chronic restrictions. We summarize the metabolic and cellular responses triggered by these feeding regimens, their impact on nutrient signaling pathways and their link to age-related diseases. Fasting methods and effects. Common fasting methods. Different fasting regimens (Table 1) can affect metabolism, aging, disease and mortality in simple organisms and mammals. I-F includes various eating patterns; water-only fasting or severely restricted (over 50 percent) calorie intake lasts between 12 and 48 hours, and in most cases is repeated in cycles occurring every day to once per week. There are several types of I-F diets commonly adopted in rodents and human clinical studies: complete water-only fasting that occurs every other day, also called alternate-day fasting, ADF; 70 percent energy restriction every other day; The 5 to 2 diet, which provides between 500 and 700 calories for 2 days per week; And time-restricted feeding (TRF), in which food intake is in most cases restricted to 6 to 12 hours per day. Thus, I-F regimens usually encompass a period in which only water is consumed or calorie intake is very low, which is followed by a normal feeding period that in most cases lasts between 12 and 48 hours. In contrast, PF refers to a prolonged and severely calorie-restricted or water-only fasting period lasting in most cases between 48 hours and 1 week, although several studies have investigated longer fasting periods. Unlike I-F, it can occur at specific intervals or on an as needed basis, but it is usually carried out less than once every 2 weeks and in most cases only a limited number of times per year for periods lasting 2 days or longer in mice or 4 days or longer in humans. There are two major methods of PF: (1) water-only PF25 and (2) a fasting-mimicking diet (FMD), which is a plant-based caloric-restricted diet containing low proteins, low sugars and high unsaturated fats that are able to mimic the effects that water-only fasting has on IGF-1, IGF binding protein 1 (IGFBP-1), ketone bodies and glucose. Metabolic effects of fasting. In most mammals, the liver serves as the main reservoir of glucose, which is stored in the form of glycogen. In humans, depending upon their level of physical activity, 12 to 24 hours of fasting typically results in a decrease in serum glucose and depletion of hepatic glycogen, accompanied by a switch to a metabolic mode in which glucose, fat-derived ketone bodies and free fatty acids are used as energy sources. After hepatic glycogen depletion, lactate, pyruvate, fat-derived glycerol and amino acids account for the gluconeogenesis-dependent generation of approximately 80 grams per day of glucose, which is mostly utilized by the brain. Depending on the severity and length of the restriction, fatty acids are mobilized, leading to an increase in circulating ketone bodies and adiponectin and a lowering of circulating leptin. In humans, the fed-state blood levels of ketone bodies are at or below the limit of detection and reach levels of 0.2 to 0.5 milli Molar within 8 to 12 hours after the onset of fasting, but reach levels between 1 and 2 milli Molar by 48 hours. This metabolic switch occurs more rapidly in rodents, as plasma ketone levels are elevated within 4 to 8 hours after the onset of fasting and reach millimolar levels by 24 hours. Intermittent fasting, metabolism and aging. Intermittent fasting in simple organisms and rodents. I-F affects longevity in multiple organisms. In worms, I-F activates mitochondrial-network plasticity as they are switched between fasted and fed states, which may contribute to longevity extension. Moreover, recent studies in mice show that cycles of fasting lasting from 12 to 72 hours followed by a refeeding period have beneficial effects on longevity, markers for health, stress, metabolic response, age-related diseases and tissue regeneration. Cycles of fasting lasting 48 hours or longer will be covered in more detail in the Effects of periodic fasting and fasting-mimicking diet section. In flies, chronic I-F has failed to extend lifespan, suggesting that flies can be sensitive to a shorter starvation period. In mice, however, I-F can have both neutral or positive effects on lifespan (see following section) and the beneficial effects of D-R on longevity appear to be due, at least in part, to the time-restricted access to food, and therefore to extended daily fasting periods. New studies in flies investigating short-term I-F regimens for only a part of the lifespan followed by a switch to ad libitum feeding at later stages indicate stronger effects on the aging process. Again, more marked effects are observed when the flies are switched back to an ad libitum diet after 30 days of I-F, in agreement with a series of studies in mice, indicating that the refeeding period is as important as the fasting or restricted stage. A number of studies have investigated the effect of I-F on the health and lifespan of rodents. In one of the earliest studies, Goodrick et al, reported an increase of up to 80 percent in the average lifespan of rats maintained on a regimen of ADF, started at 5 weeks of age. Later studies show much smaller effects of ADF, but several of them confirm an extension of median, as well as maximal, lifespan. In addition, longevity in male C57BL, 6J mice subjected to ADF is associated with a delayed onset of lethal neoplastic disorders that typically limit natural lifespan in many mouse strains, but this was achieved without a delay of the aging process, in agreement with earlier studies. Thus, the magnitude of the effects of ADF on longevity in rodents depends upon the species, mouse versus rat, strain and age at regimen initiation, and can range from a small negative effect to as much as an 80 percent lifespan extension. Similarly, it has emerged from a genetic screening that CR can exert positive or even detrimental effects on longevity, depending on genetic variations in different mouse backgrounds. For example, in two different mouse genetic backgrounds A, J and C57BL, 6J, I-F did not extend mean lifespan, and even reduced lifespan, when initiated at 10 months. When initiated at 1.5 months, I-F either increased longevity or had no effect. However, in rodents, I-F enhances cognitive performance, which may be caused in part by its stimulatory effect on synaptic plasticity, improves insulin sensitivity and reduces blood pressure and heart rate. Moreover, 2-month-old male C57BL, 6J mice fed a time-restricted high-fat diet (16-hour daily fasting period), show protection against obesity, hyperinsulinemia, hepatic steatosis and inflammation with improved motor coordination, despite a caloric intake equivalent to that of the group with ad libitum access to a high-fat diet. At the molecular level, TRF in mice affects the energy signaling mediated by c-AMP responsive element binding protein (CREB) and AMPK, the pro growth mTOR pathways and the expression of circadian clock gene. A deep understanding of the type and length of fasting and mechanisms that can maximize longevity effects, as well as of the detrimental effects that may be counterbalancing the positive ones, is required. Fasting may be consistently protective in young and middle-aged laboratory rodents that are affected by cellular damage and aging, which lead to insulin resistance, inflammation, genomic instability and so on, but may have at least some detrimental effects in old or very old animals after they begin to lose weight or become frail. Human studies on intermittent fasting, aging and disease risk factors. A rapidly increasing number of studies have investigated the effects of different I-F regimens produces mild caloric restriction and weight loss in obese adults. A number of trials testing I-F in humans show positive effects on metabolic markers but, together with epidemiological studies, also point to potential side effects, particularly after long-term use. Varady and colleagues showed that ADF in overweight or obese adults with insulin resistance produces a greater reduction in insulin levels and insulin resistance than CR does, despite achieving a similar decrease in body weight. On the other hand, Madeo and colleagues found that either short-term (4 weeks) or long-term (6 months) ADF carried out in healthy middle-aged humans has beneficial effects on metabolic and cardiovascular markers alongside reduced levels of soluble intracellular adhesion molecule-1 (sICAM-1), an age-associated inflammatory marker, low-density lipoprotein (LDL) and the metabolic regulator triiodothyronine. Panda and colleagues, who had shown the beneficial effects of TRF for preventing and treating obesity and metabolic disorders in mice, showed similar cardiometabolic benefits in people with metabolic syndrome who consumed food for only 10 hours daily. TRF prevents excessive body-weight gain, improves sleep, attenuates age and diet-induced deterioration in cardiac performance and improves blood pressure and accumulation of atherogenic lipids. Cienfuegos and colleagues also reported that 4 and 6 hour TRF reduces body weight, insulin resistance and oxidative stress compared with results from non-time-restricted controls, supporting further studies on TRF as a promising intervention for weight loss and cardiometabolic protection. The health effects of I-F are accompanied by weight loss, but how much reduced adiposity affects disease risk factors remains poorly understood. In one trial, 16 healthy participants, aged between 23 and 53 years with a BMI between 20 and 30, assigned to a regimen of ADF for 22 days lost 2.5 percent of their initial weight and 4 percent of their fat mass, with a 57 percent decrease in fasting insulin levels. In two other trials, overweight women, with approximately 100 women in each trial, were assigned to either a 5 to 2 I-F regimen or a 25 percent reduction in daily caloric intake. The women in the two groups lost the same amount of weight during the 6-month period, but those in the group assigned to the 5 to 2 I-F had a greater increase in insulin sensitivity and a larger reduction in waist circumference. However, there are also a number of studies indicating that frequent fasting cycles may not only be difficult to carry out for long periods, but also increase side effects and even mortality. For example, the risk of gallstone disease nearly doubles between women who fast for 8 hours per day and those who fast for over 14 hours per day. Furthermore, skipping breakfast, which is perhaps the most common method adopted to reach a daily 14 to 18 hour daily fasting period, is associated with an increased risk of mortality from cardiovascular and all-cause mortality in the US population. Clearly, epidemiological data are not easy to interpret and the association between long daily fasting periods and increased incidence of disease or mortality could be explained by factors other than the fasting itself. However, until further studies, including randomized clinical studies and additional epidemiological studies, are conducted, the use of this type of daily fasting intervention should be limited to short-term periods and applied to only people with disease for which regular I-F has been demonstrated to be effective. It is also important to gain an understanding of the effect of long daily fasting periods in which dinner is skipped instead of breakfast. In contrast, daily fasting, TRF periods of approximately 12 hours appear to be associated with benefits without known negative effects. Effects of periodic fasting on aging Periodic fasting and fasting-mimicking diet. In contrast to the short and very frequent fasting periods of I-F, PF or FMD last in most cases between 2 and 7 days, 2 to 3 days in mice and 4 to 7 days in humans, and are followed by a high-nourishment refeeding period of at least 1 week. Another major difference from I-F is that PF can be periodic and does not have to be carried out at a specific interval, but can be applied for one or several cycles either as a preventive measure or to treat a specific disease or condition. PF was traditionally carried out in specialized clinics with water-only or very-low-calorie methods, but outside of a clinic, such a regimen can be difficult to maintain and unsafe because it can cause side effects, including malnourishment, rapid weight loss, reduced blood pressure and hypoglycemia, as well as the exacerbation of existing micronutrient deficiencies. These safety concerns and the scarcity of preclinical and clinical data may explain why historically the potential benefits of PF have emerged multiple times within the medical community, but have eventually disappeared and have not been integrated into standard-of-care practices. Thus, FMDs were developed to promote the effects of fasting while standardizing dietary composition, providing nourishment and minimizing the burden and side effects associated with water-only fasting. Notably, these steps are necessary for PF and possibly I-F to move toward approval from the US Food and Drug Administration and standard-of-care applications. The FMD composition, which includes low protein, low sugar and high unsaturated fat, achieves a reduction in IGF-1 and glucose, and an increase in ketone bodies, and IGFBP-1, similar to that caused by water-only fasting in mice. Various versions of the rodent FMD have been utilized, but in most cases, they provide between 10 percent and 50 percent of the normal caloric intake for periods ranging from 2 to 5 days, with the most severe restrictions lasting from 2 to 3 days. A longer regimen with less caloric restriction is also used, which consists of 5 days with a caloric intake ranging from 50 percent on the first day to 30 percent for the rest of the days. Mice undergoing FMD cycles lose about 15 to 20 percent of their body weight, which is recovered upon refeeding. In fact, the severe caloric restriction is compensated by overeating during the refeeding period, resulting in the same or similar caloric intake in the FMD and control groups. Rodent studies on prolonged fasting or fasting-mimicking diet and longevity. Sixteen-month-old female C57BL, 6 mice placed on a periodic 4-day FMD twice per month, alternating with a normal diet, display an 11 percent increase in their median lifespan, in addition to significant weight and visceral-fat loss, without loss of muscle mass. Moreover, FMD cycles reduce tumor incidence by 45 percent and delay tumor development, with most being detected after 26 months of age. FMD cycles that are started at middle age reduce skin inflammation including dermatitis by 50 percent and improve motor coordination along with long and short-term memory. Notably, the FMD cycles also promote changes leading to an immune-system profile in 20.5-month-old mice more similar to that of younger mice, 4 months old, in agreement with the effect of PF on hematopoietic stem cell (HSC)-dependent regeneration of immune cells. In addition, FMD cycles selectively reduce visceral fat without an overall reduction in per-month calorie intake, indicating a potential acceleration in metabolism during the refeeding period. In summary, similarly to the well-established effects of CR, FMD cycles delay the onset and reduce the incidence of age-related diseases, but achieve this with minimal or no long-term reduction in calorie intake and with positive effects on immunity and a targeted reduction in visceral fat. Thus, PF, FMD but potentially also certain D-R, including I-F, may achieve many beneficial effects by mechanisms that are independent of reduced calorie intake. In fact, chronic protein restriction, without calorie restriction, is well established to extend longevity and healthspan. Notably, I-F and PF, similarly to chronic CR, delay disease incidence but also reduce the lifelong portion of animals that develop any type of disease, and particularly cancer. One limitation of studies with PF and I-F is that they have been conducted on only a few mouse strains, and mostly on C57BL6 mice, so we do not yet know whether the health benefits and lifespan increase is not dependent on rodent strain. On the other hand, several of these studies were conducted in both female19 and male mice, and some of them began when the mice were young and others when the mice were already middle aged (16 months), indicating that the health and lifespan benefits of I-F or PF can be achieved when started at middle age. A recent study has shown that there is memory of CR done during early mouse life, and that age-dependent mortality can depend on the nutrition earlier in life. This is another effect of CR, I-F and PF that is poorly understood and could help achieve the goal of maximizing healthspan and longevity with minimal burden. Human studies on prolonged fasting, fasting-mimicking diet and longevity. A study assessed longer periods of fasting in large cohorts that included non-obese participants. In a 1 year observational study, 1,422 non-obese participants aged between 18 and 99 participated in a fasting program consisting of fasting periods of between 4 and 21 days in which they fasted with a daily caloric intake of 200 to 250 kcal accompanied by a moderate-intensity lifestyle program. Significant reductions in weight, abdominal circumference and blood pressure were observed, along with a reduction in blood glucose levels and increase in ketone bodies, proving the fasting-related metabolic switch. However, these effects are detected during the fasting period. Also, a single period of extended fasting with concomitant weight reduction leads to significant, rapid improvement of fatty-liver index in people with or without type 2 diabetes, age greater than or equal to 18 and BMI greater than or equal to 19 kilograms per meter squared by the end of the fasting cycle. However, the long-term effect of this regimen on fatty liver after return to the normal diet is not known. The major limitation of these very severe and prolonged fasting periods is that they must be carried out in a specialized clinic to avoid adverse events. Furthermore, we do not know the long-term effects of the long periods (weeks) of water-only or very-low-calorie fasting on health. In fact, long periods of CR have been associated with a reduction in metabolic rates, which could actually promote, rather than reduce, fat accumulation after the end of the fasting or CR period, which would result in the regain of weight as established in studies involving severe restrictions or fasting lasting 10 days and longer. In addition, multiple cycles of weight loss (ranging between 7 percent and 10 percent) and regain (yo-yo diets) are associated with increased mortality, indicating that long and severe fasting periods could have short-term benefits followed by long-term beneficial as well as detrimental effects. The periodic use of FMD and refeeding cycles was studied to identify interventions to maximize effects against aging and age-related diseases, while minimizing side effects and the burden of frequent restrictions. In a randomized clinical trial with 100 relatively healthy volunteers, FMD cycles lasting 5 days, carried out once per month for 3 months, reduce multiple risk factors for age-related diseases, including diabetes, cancer and cardiovascular disease. These effects include reduced body weight and trunk fat, lowered blood pressure and decreased IGF-1, along with decreased BMI, glucose, triglycerides, cholesterol and C-reactive protein 5 to 7 days after returning to a normal diet in people that displayed elevated levels of these markers at baseline. Notably, the beneficial effects of the FMD on several of these markers, risk factors were maintained for months after subjects returned to a normal diet. These studies underline the potential of PF, FMDs and other types of fasting for extension of not only health span but also youth span, the range of time in which an organism remains youthful, healthy and fully functional. Thus, PF and FMD cycles that alternate with normal refeeding periods appear to be safe in both rodents and humans and to have beneficial effects on disease or disease risk factors when started at middle to old age, 16 months in rodents. Although FMDs appear to be safe, their use should be limited to three times per year in healthy people with normal levels of disease risk factors, until additional and long-term clinical studies demonstrating the safety of more frequent use are carried out. In contrast, the more severe and longer forms of fasting should only be done in a specialized clinic in the presence of medical personnel. How much of the benefit of PF is due to effects on cellular aging and dysfunction versus the effect of weight loss remains unclear and is addressed in the following sections. Because there is a concern that all types of fasting methods may in the long term cause side effects, including those listed earlier, when done too frequently, the beneficial effects of I-F and PF must be weighed against their potential side effects, particularly in healthy or relatively healthy individuals. Both basic and clinical research should focus more on interventions that maximize efficacy against aging and age-related diseases while minimizing side effects and the burden of the intervention, which is usually inversely correlated with long-term compliance. Periodic fasting, intermittent fasting and nutrient signaling pathways. Reduced activity of the nutrient-sensing pathways that regulate aging in yeast, Figure 1), worms and flies can also extend longevity in rodents, Figure 2. Thus, inhibition of the mTOR pathway either pharmacologically with rapamycin or genetically by deletion of the ribosomal S6 kinase 1 (S6K1) extends longevity in mice. In addition, S6K1-mutant mice show a delayed onset of age-related phenotypes, such as bone-matrix loss, immune and motor dysfunction and insulin resistance. The GH IGF-1 axis, acting upstream of mTOR PI3K AKT-1 and PKA signaling, has been intensively studied in mammals because of its effects on the incidence of age-related diseases and lifespan. Indeed, mice lacking the GH receptor binding protein (GHR-BP) display a 40 percent longer average lifespan than that of controls, with reduced tumor incidence. A number of additional studies have shown similar extended lifespans for mice with defects leading to both GH and IGF-1 deficiencies, which are also associated with downregulation of TOR signaling in multiple cell types. Whereas the mTORC1 inhibitor rapamycin increases longevity in wild-type mice, in mice with knocked out growth hormone receptor that have constitutively suppressed mTORC1 and upregulated mTORC2 signaling, it leads to a drastic reduction in mTORC2 in liver, muscle and subcutaneous fat, which in turn causes an elevation of the inflammation marker interleukin-6, reduced numbers of functional immune cells and a shortened lifespan. These results together, with a series of previous studies, indicate that mTORC2 is not as clearly linked to aging as mTORC1 and that its inhibition may even be deleterious. Because carbohydrates and proteins play a central role in growth, it is not surprising that the protein and sugar restriction associated with PF, I-F and CR causes conserved changes in growth factors and nutrient signaling. It is well established that higher protein levels increase IGF-1 levels and that several amino acids are sufficient to promote an increase in the levels of this growth factor in the serum and also in TOR-S6K signaling. For example, methionine regulates the growth hormone (GH)-IGF-1 axis, whereas amino acids including leucine and arginine can activate TOR-S6K signaling. Moreover, the balance of macronutrients, not just their levels, and particularly of protein and carbohydrate can affect lifespan. In the mouse liver, when protein is replaced with carbohydrate, compensatory mechanisms are inhibited and protein uptake is suppressed, leading to mTOR inhibition. Furthermore, the quality of macronutrient, for instance animal versus plant-derived proteins, can influence aging and disease. In fact, high protein intake in adult life (up to age 65) is associated with increased risk of overall mortality and cancer-related death, however this association is attenuated or eliminated when the higher intake of proteins is from plant-derived sources. It has also emerged that amino-acid quality can have an important effect on aging, and in fact an imbalance of branched-chain amino acids compared with other amino acids (high BCAA:non-BCAA ratio) leads to reduced longevity through a mechanism independent of mTOR activation and caused by hyperphagia. Sugars can also activate various pathways including Ras or PKA signaling either through insulin action or by an insulin-independent mechanism of insulin and resulting in reduced antioxidant protection and cellular stress sensitivity. Thus, it is not surprising that GH or GHR-deficient mice and those undergoing I-F or PF, all of which reduce common proaging pathways, share an extended longevity and a major reduction in disease incidence. For example, CR (from 10 percent to 50 percent) reduces IGF-1 and insulin, as well as Tor-S6K signaling, in rodent models. In agreement with mouse studies, in humans CR reduces IGF-1 levels only when protein intake is also restricted, underlining the need to focus both on calorie intake and dietary composition and their effects on nutrient signaling pathways. I-F, including ADF and TRF (8-week fast, 16 hours per day, 16 to 8, also decrease IGF-1, blood glucose and insulin levels while increasing insulin sensitivity and adiponectin levels. PF and FMDs also affect the levels TOR-S6K, IGF-1, insulin and glucose in mice, but these effects are reversed when animals return to the normal diet. Thus, it is likely that a long-lasting reduction of factors including glucose, insulin and IGF-1 caused by CR is not necessary for at least part of the lifespan and healthspan effects. However, in humans, FMD cycles can have long-lasting effects on IGF-1, insulin and glucose, raising the possibility that at least some of the effects of dietary restriction and FMDs on longevity may involve long-term effects on the levels of these factors. Another mechanism that may explain the role of the temporary reduction of these factors on the longevity extension caused by PF is the activation of HSC-based and other regenerative processes discussed later in this Review. Notably, these effects depend on the activation of regenerative processes that begin during fasting periods but are completed after mice return to a normal diet. I-F lasting between 12 and 24 hours can also have effects on IGF-1, IGFBP-1, glucose and ketone bodies, but most of the changes are smaller than those obtained by the longer PF or the chronic CR. For example, in mice, 4 weeks of every-other-day I-F did not elevate ketone bodies but instead caused a reduction in beta-hydroxybutyrate levels and beta-hydroxybutyrate dehydrogenase activity in the mouse liver but not in the cerebral cortex, where levels remained unchanged or enhanced, supporting the importance of further investigating the mechanisms of ketone-body production, release and delivery. However, circulating IGF-1, insulin and glucose were decreased after resistance-trained men (aged 29 to 33 and weighing 85 to 92 kilograms) were placed on a 16 to 8 fast, indicating that I-F can have a positive effect on these factors. Notably, because this I-F period also caused loss of fat mass, it is difficult to establish whether the effects of I-F on insulin and glucose and possibly IGF-1 are mediated by effects on adipose tissue. Importantly, these positive effects in men undergoing the 16 to 8 TRF were also accompanied by negative ones, including an over 20 percent decrease in total testosterone. These results point to the importance of continuing to study both the short-term and long-term molecular changes caused by I-F, PF and CR and to connect them to the positive as well as negative effects on metabolic markers, but also diseases and other conditions. Fasting-refeeding and regeneration. Fasting and refeeding regimens are powerful promoters of stem-cell self-renewal mechanisms and activators of tissue regeneration, in part through inhibition and reactivation of the IGF-1, PKA and mTOR pathways (Figure 3). The PF and FMD regimens can promote a rejuvenation process in tissues, organs and cells through the activation of cell death and autophagy followed by the activation of stem or progenitor cells. Notably, the refeeding period appears to be responsible for a major component of the regeneration process leading to the replacement of senescent or damaged cells with new cells arising from tissue-specific stem cells. Not surprisingly, the effects of short-term fasting and FMD on stem-cell function and regeneration depend on the content of the diet, its effects on different pathways and the timing and duration of the regimen. For example, lifelong CR does not prevent the age-related functional decline of HSCs in mice, whereas short-term fasting periods, as well as FMD regimens followed by refeeding periods, do promote regenerative and rejuvenating effects in the hematopoietic and immune systems. This is a fundamental distinction between cycles of fasting and refeeding compared with chronic dietary interventions, which may have smaller regenerative effects compared to fasting, refeeding by preventing or limiting the regenerative phase which requires high levels of macro and micronutrients and possibly higher calories to support macromolecular synthesis, cellular division and growth and tissue and organ expansion. Stem-cell-based regeneration is stimulated by I-F and PF through nutrient-sensing pathways. The positive effects of fasting have been described in multiple stem-cell types, including muscle stem cells, HSC’s, intestinal stem cells, ISC’s, and neuronal stem cells, NSC’s. The common mechanisms through which fasting affects stem-cell function involve modulation of the amino-acid and glucose-sensing pathways, including IGF-1 TOR PKA, also implicated in longevity regulation. However, the fasting refeeding cycles also affect inflammation and can cause long-lasting epigenetic changes or changes in the stem-cell niche, which could contribute to the biological age of cells and organs. Whether changes in stem-cell function in response to nutrient availability are mediated by epigenetic changes is still unclear, but a few studies have indicated this possibility. For instance, the regulation of NSC proliferation in response to decreased glucose availability is governed by the nutrient sensors CREB and SIRT1, and the effects of CR are accompanied by an increase in histone H3 acetylated at Lys 9 (H3K9Ac). Changes in dietary intake are accompanied by massive changes in chromatin states in several species. On the basis of these studies and on previous results of PF and FMD on tissue regeneration, we speculate that FMD and, or refeeding periods may cause long-lasting epigenetic changes, although further studies are required to test this hypothesis and to understand its possible role in stem-cell maintenance during aging. During aging, a skewing in the lineage of differentiation of HSCs occurs with relatively more myeloid cells being produced than lymphoid cells. This skewing contributes to an age-related impairment of adaptive immunity. PF reduces IGF-1-PKA signaling and promotes HSC self-renewal and long-term repopulation capacity upon serial transplantations and lineage-balanced regeneration of the immune system. Also, four periodic cycles of PF or FMD started at 16 months of age rejuvenate the hematopoietic system, leading to an increased number of white blood cells 1 week after refeeding, and to a partial reversion of the age-dependent lineage skewing. Moreover, FMD cycles can partially revert the age-dependent decline of mesenchymal stem and progenitor cells, thus indicating that they promote proliferation of new stem and progenitor cells in various tissues. Quiescent satellite cells express the transcription factor paired box 7 (Pax-7), and when activated, coexpress Pax-7 and MyoD, the myoblast determination protein, which promotes transcription of muscle-specific target genes and plays a role in muscle differentiation. When they proliferate, they downregulate Pax-7 and differentiate. Interestingly, mice undergoing FMD cycles show increases in Pax-7 and MyoD in muscle tissue, with associated reversal of markers of impaired autophagy. Of note, the boost in protein expression of the regenerative markers was detected during the refeeding period, while no major changes were measured at the end of the fasting period. These results indicate that different systems may activate quiescent stem or progenitor cells at different stages during the fasting and refeeding periods. Notably, in old mice, short-term administration of spermidine, a known CR mimetic, reverses the age-associated defect of autophagy in muscle cells, normally characterized by loss of proteostasis, increased mitochondrial dysfunction and oxidative stress. The molecular mechanism of spermidine action involves the increase of protein deacetylation and autophagy activation through induction of arginine and NO (nitric oxide) metabolism, as well as downregulation of pro inflammatory cytokines in muscle stem cells (satellite cells). In the mouse pancreas, PF and FMD promote a decrease in the numbers of differentiated or committed pancreatic cells, followed by the induction of transitional alpha-to-beta or beta-to-alpha cells that coexpress both alpha (that is, glucagon) and beta (that is, PDX-1 or insulin) cell markers, and finally a major increase in the proliferation and number of insulin-generating beta cells. The metabolic reprogramming caused by the FMD also affects lineage determination in pancreatic islets with increased pluripotency and beta cell reprogramming markers, especially one day after refeeding. Results in human pancreatic islets from healthy donors and people with type 1 diabetes indicate that FMD induces the expression of SOX-2, NGN3 and insulin in part by reducing IGF-1 and inhibiting both mTOR and PKA signaling. Recent work confirms the effect of FMD on pancreatic regeneration and diabetes in a genetic mouse model of type 2 diabetes and also confirms that a FMD promotes reduced blood glucose, increased insulin sensitivity, beta cell proliferation and NGN3 expression. The protein content of this specific FMD formulation was higher (17 percent versus 6 percent), whereas fat was lower (14 percent versus 65 percent), than the content of the Cheng et al, formulation, but the diet was administered for nearly twice as long and was much more calorically restricted (7 days at 30 percent of daily calorie intake). Thus, in the latter study, changes in the key signaling pathways, similar to those caused by the shorter FMD, were likely to be achieved by a longer and more severely restricted period with a less-fasting-mimicking macronutrient ratio. Thus, the beneficial regenerative effects achieved are affected by dietary composition, severity of the calorie restriction and length of the PF or FMD period. In another study, one day of fasting was sufficient to increase ISCs and progenitor activity both in young and old mice by inducing a fatty-acid oxidation program. Interestingly, a recent study carried out in a mouse model of intestinal bowel disease shows that FMD cycles stimulate protective gut microbiota, reduce intestinal inflammation, increase stem-cell number and reverse intestinal pathology. Notably, water-only fasting increases regenerative and reduces inflammatory markers without reversing pathology, supporting the possibility that specific nutrients contained in the FMD, and possibly prebiotic ingredients contained in plant-based foods, can have a crucial impact on microbiota and consequently on the course of the disease. A recent study highlights that dietary macronutrients, in particular carbohydrate and protein, can also be major drivers of microbial response and can reshape microbiome by dictating nitrogen availability to bacteria. Previous studies also indicate that long-term dietary restriction can increase the regenerative capacity of ISCs via an extrinsic mechanism involving reduction of mTORC1 in the niche surrounding Paneth cells, suggesting that the refeeding period is not necessary for ISC activation, although it may enhance or maximize regeneration as indicated in other studies. Taken together, these studies suggest that the mechanisms of regeneration may vary depending on the timing, duration, composition and severity of both the fasting and refeeding diet. However, mechanisms involving IGF-1, TOR and PKA are likely to represent common denominators in the protective and regenerative effects of I-F, PF and CR. Notably, both the inhibition of these signaling proteins and enzymes during the fasting and their activation during the refeeding are likely to be important for the regenerative process. In fact, their role in regeneration during the refeeding period is poorly understood and should be investigated further in multiple tissues. Intermittent fasting, periodic fasting and aging-related diseases. Advancing age is the major risk factor for most major diseases, including cancer, diabetes and neurodegenerative, cardiovascular and immunological diseases. Because I-F, PF and FMD cycles have been shown to slow down and partially reverse cellular aging in rodent models, a number of studies have investigated their potential application to the prevention and treatment of age-related diseases (Table 2). Neurodegeneration. Fasting has been shown to protect neurons and ameliorate cognitive impairment in animal models. A triple transgenic mouse model of Alzheimer’s disease (AD), which expresses familial AD mutations in the beta-amyloid precursor protein (APP), presenilin 1 and Tau, fed either a 40 percent CR or ADF dietary regimen for 1 year starting at 5 months of age, developed reduced cognitive impairment, compared with that in ad libitum-fed control mice. Interestingly, 40 percent CR, but not ADF, reduces the levels of beta amyloid (A Beta) and Tau accumulation in the brains of the AD mice. The intermittent restriction of essential amino acids also protects mice from pathology and cognitive decline in triple transgenic AD mice, suggesting that the protein-restriction component of the fasting plays a key role in its protective effects. The latter study did not detect reduced levels of Abeta after restriction of essential amino acids, although it did observe reduced accumulation of phosphorylated Tau in the hippocampus. This suggests that fasting and protein restriction can protect the nervous system even in the presence of high levels of Abeta, although their effect on the fibrillar versus soluble forms of Abeta remain poorly understood. The mechanism by which fasting protects neurons from degeneration has been linked to increased expression of neurotrophic factors important for neuronal cell growth and stress resistance, including BDNF and FGF2. Studies also suggest important roles for the ketone body beta-hydroxybutyrate and the mitochondrial sirtuin SIRT3 in the neuroprotective mechanism of IF. Ketone bodies may be protective against GABAergic interneurons degeneration through a mechanism dependent on SIRT3, which was shown to reduce anxiety-like behavior and to improve hippocampus-dependent memory in mouse models of AD. A common genetic mouse model for Parkinson’s disease (PD) that overexpresses human alpha-synuclein exhibits progressive accumulation in neurons and shows motor dysfunction and premature death. When these mice were treated with ADF, the autonomic nervous system deficit was reversed, while it was exacerbated even more in mice on a high-fat diet. Bai et al, in a recent study showed that, in response to rapamycin treatment and the consequent inhibition of mTOR, these PD model mice displayed reduced oxidative stress and synaptic damage and an overall improvement of motor function. Hence, ADF cycles have the potential to improve the overall pathology progression. Although it has not been tested with AD mouse models yet, PF or FMD has been shown to increase neural stem cells and increase cognitive performance in normal, old mice. Major issues remaining to be addressed when considering human trials are the burden of fasting every other day and the side effects described earlier, particularly in people with PD or AD, who in most cases will be over age 70. The identification of the specific dietary compositions responsible for neuroprotective and regenerative effects and the description of the mechanisms involved should eventually allow the design and development of fasting-like interventions that are able to protect against neurodegeneration with minimal side effects. Diseases of the immune system. Aging is associated with progressive immune senescence, which is caused in part by the age-dependent impairment of HSC function. This results in a higher ratio of myeloid cells relative to lymphoid cells, accompanied by a decline in common lymphoid progenitors, and ultimately reduced T and B-cell lymphogenesis as well as stem-cell exhaustion and reduced regenerative capacity. Dysfunctional lymphocytes can consequently give rise to immunosuppression or immune senescence, but may also contribute to autoimmune disorders such as asthma, systemic lupus erythematosus, multiple sclerosis (MS) and rheumatoid arthritis. Certain dietary restriction regimens have the potential to prevent and, or reverse age-dependent immune dysfunction by killing or altering autoimmune cells and activating HSC-dependent regeneration. Notably, severe CR (66 percent CR) and 8 weeks of ADF regimen prevent autoimmune encephalomyelitis (EAE) in mice. Although the molecular mechanisms of autoimmune disease suppression are not yet known, these diet regimens have been shown to decrease the amount of circulating T cell and inflammatory cytokines and chemokines. In contrast to the prevention of autoimmunity described above, for the treatment of already-established MS symptoms and pathology, FMD refeeding cycles were shown to attenuate EAE by modulating immune cells and promoting regeneration of oligodendrocyte precursor cells. The FMD cycle increased apoptosis in autoreactive T cells, which are replaced in part by newly generated naive T cells during the refeeding period. Notably, several studies show that fasting and CR can cause both the death and relocalization of different immune-cell populations. In agreement with the rodent study, a clinical trial in humans also reported a reduction in lymphocytes upon FMD intervention and an improvement in quality of life in people with MS. However, larger studies are necessary to determine whether the FMD can reduce multiple sclerosis pathology and progression in people. Cancer. Recently, a series of studies in animal models has shown that PF and FMD lasting two or more days can be as effective as chemotherapy at delaying the progression of a wide range of cancers but, more importantly, can protect normal cells from the toxic effects of chemotherapy while sensitizing cancer cells to the treatment. PF and FMD cycles appear to increase the killing of cancer cells by causing system-wide changes that affect the ability of malignant cells to survive or adapt, which includes a reduction in IGF-1, insulin, glucose, leptin and cytokines, but likely also changes in hundreds of enzymes or pathways. Notably, fasting and FMD are most effective against cancer cells when combined with chemotherapy, radiotherapy, kinase inhibitors, metabolic drugs or hormone therapy. Another important mode of action of cycles of PF or FMDs and refeeding is the activation of the immune surveillance to promote T-cell-dependent killing of cancer cells. These effects of the fasting, FMD on immunity-dependent attack of cancer cells was confirmed by a recent study from Collins et al, showing that mice that underwent severe 50 percent CR for few weeks accumulated memory T cells in the bone marrow, and that caused enhanced protection against infections and tumors. The broad effect of fasting and FMD in decreasing circulating levels of glucose, and IGF-1 and increasing ketone bodies, IGFBP-1 and so on together with the targeted toxicities of standard cancer therapies have the potential to promote major improvements in therapeutic index and cancer or progression-free survival. Diabetes and cardiovascular disease. Both I-F and PF or FMDs can be effective in reducing not only weight, abdominal circumference and body fat, but also risk factors for metabolic syndrome, diabetes and cardiovascular disease. ADF and chronic CR caused similar effects on weight loss, reducing body and abdominal fat and lipids and improving insulin sensitivity in humans. Notably, ADF had stronger effects on high-density lipoprotein (HDL) and LDL than did CR. Another study on people with one or more risk factors for metabolic syndrome compared a 6-month ADF against chronic CR. Both groups experienced a similar decrease in body weight, 8 to 9 kilograms, as well as improvements in blood pressure, triglycerides and HDL (Table 2). In another study, 8 weeks of a 10-hour time-restricted eating decreased body weight (4 kilograms), waist circumference (4 cm), systolic BP (13 millimeters of Mercury) and glucose in people with metabolic syndrome. No significant changes were found in LDL cholesterol, HDL and insulin or insulin resistance (Table 2). For the PF interventions, three monthly cycles of a FMD were effective at reducing risk factors for diabetes and CVD in higher risk people. Independently of initial weight, people underwent a reduction in BMI, although individuals with BMI greater than 30 displayed a greater decrease (Table 2). Significant improvements in total cholesterol, LDL, fasting glucose, C-reactive protein and triglycerides were also observed in people with high levels of these risk factors at baseline (Table 2). In summary, both I-F and PF or FMD’s can cause a wide range of improvements in cardio metabolic risk factors, which are likely to lead to a reduction in diabetes and CVD’s. Additional and larger studies will be important to determine which of these interventions can become standard of care in disease prevention and treatment. Conclusion and future perspectives. I-F and PF or FMD activate ancient programs that promote entry into alternative metabolic modes focused on conserving energy and on protecting the organism while enduring extended periods of food deprivation to optimize survival and reproduction once food becomes available. In fact, it is the refeeding period that has more recently emerged as an equally important process involved in the regeneration, and possibly rejuvenation, of systems, including organs, cells and organelles. In humans, the alternation of fasting and refeeding periods is accompanied by positive effects on risk factors for aging, diabetes, autoimmunity, cardiovascular disease, neurodegeneration and cancer. But not all fasting interventions are equal, and some are associated with smaller beneficial effects as well as side effects, including, in some cases, reduced longevity. The I-F and PF field should expand the investigation of the effects of dietary macronutrient composition, ratio and quality on health span. In fact, lifespan and health span extension can be reached by a specific macronutrient balance, independently of caloric intake. It is also important to identify the mediators of the effects of I-F and PF not only on different types of mammalian cells and organs and on disease, but also on the microorganisms of the digestive system, with a focus on the molecular mechanisms mediating key effects on cellular protection, aging and regeneration. It will not only be important to separate the positive effects of I-F and PF from the adverse effects, but also to match the type and length of I-F and PF with goals including health span extension, the prevention and treatment of specific diseases and weight management. Acknowledgements and 150 References, and three figures. Figure One. Fasting, nutrient signaling and longevity in yeast. Starvation conditions in yeast cause a major lifespan extension mediated in large part by the lack of amino acids and sugars. On one hand, amino-acid restriction causes the inactivation of TOR Pkh S6k signaling; on the other hand, low glucose levels promote reduced activity of the Ras-adenylate cyclase (Cyr1) PKA pathway. Both the amino-acid and the sugar pathways converge on and inactivate the serine threonine kinase Rim 15. This, in turn, contributes to the activation of stress-resistance transcription factors Gis1, which binds to post diauxic shift (PDS) motif, and Msn 2 and Msn 4, orthologs of mammalian early growth response protein 1 (EGR1), which bind to stress-responsive element (STRE) motif. Figure Two. Conserved nutrient-sensing response pathways in worms, flies and mammals. This model summarizes the conserved nutrient-sensing pathways that regulate longevity and stress-response mechanisms in different model organisms. Fasting or calorie restriction reduces the activity of amino acids and glucose signaling pathways through membrane receptors by reducing circulating ligands such as growth factors like mammalian IGF-1. The fasting-inhibited TOR-S6K pathway (labeled in blue) promotes the expression of nuclear transcription factors such as the hypoxia-inducible factor-1 (HIF-1), the FOXA ortholog PHA-4, the nuclear hormone receptors NHR-62 and NHR-49 and the TFEB ortholog HLH-30 (worms), the FOXA nuclear factor (flies) and the increase of the FOXO nuclear factor (mammals). These transcription factors commonly activate antiaging systems and processes, including autophagy and ribosomal biogenesis, stress response and cellular-protection genes, including antioxidant SODs. The RAS-AC-PKA pathway (labeled in green) is also partially conserved between species. Similar to what was observed in yeast, glucose in certain mammalian cells can signal through the PKA pathway and the transcription factor EGR1, the mammalian ortholog of Msn 2, Msn 4 in yeast. In worms, flies and mice, downregulation of TOR S6K signaling has conserved proaging effects. The fasting-dependent effects on longevity in different organisms may also involve sirtuin pathway activation (labeled in orange), the increase in mitochondria respiration and the activation of autophagy. Also, fasting in flies delays the disruption of tri cellular junctions (TCJs), which is linked to improved intestinal barrier integrity and therefore longevity (labeled in gray). Figure Three. Periodic fasting and tissue regeneration and rejuvenation in mice. Periodic fasting or FMD can affect tissue regeneration in multiple systems and organs in mice. a, In bone marrow, PF or FMD drives self-renewal of HSCs and lineage-balance regeneration of the immune system, leading to a lymphoid-biased phenotype. b, PF and FMD increase mesenchymal stem and progenitor cells (MSPCs) in bone marrow. c, PF and FMD increase neurogenesis in brain tissue, represented by increase in doublecortin (DCX) levels in newly generated bromo deoxyuridine (BrdU plus) neurons. d, In muscle tissue, PF or FMD modulates the expression of the pair box protein Pax-7, a stem-cell marker mainly expressed by muscle satellite stem cells, and MyoD, a marker of early muscle differentiation. e, In the pancreas, PF and FMD drive increased expression of early developmental markers, including SOX-17, and of the downstream NGN3 transcription factor, leading to the regeneration of insulin-producing beta cells. f, In intestinal tissue acute one-day-only fasting, PF and FMD increase levels of ISCs and progenitors in part by inducing a fatty-acid oxidation (FAO) program or by modulating gut microbiota.

The Key Role of Mitochondrial Function in Health and Disease. Inigo San-Millan, Department of Human Physiology and Nutrition, University of Colorado, Colorado Springs, and others. https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Source: https://pubmed.ncbi.nlm.nih.gov/37107158/ Abstract. The role of mitochondrial function in health and disease has become increasingly recognized, particularly in the last two decades. Mitochondrial dysfunction as well as disruptions of cellular bioenergetics have been shown to be ubiquitous in some of the most prevalent diseases in our society, such as type 2 diabetes, cardiovascular disease, metabolic syndrome, cancer, and Alzheimer’s disease. However, the etiology and pathogenesis of mitochondrial dysfunction in multiple diseases have yet to be elucidated, making it one of the most significant medical challenges in our history. However, the rapid advances in our knowledge of cellular metabolism coupled with the novel understanding at the molecular and genetic levels show tremendous promise to one day elucidate the mysteries of this ancient organelle in order to treat it therapeutically when needed. Mitochondrial DNA mutations, infections, aging, and a lack of physical activity have been identified to be major players in mitochondrial dysfunction in multiple diseases. This review examines the complexities of mitochondrial function, whose ancient incorporation into eukaryotic cells for energy purposes was key for the survival and creation of new species. Among these complexities, the tightly intertwined bioenergetics derived from the combustion of alimentary substrates and oxygen are necessary for cellular homeostasis, including the production of reactive oxygen species. This review discusses different etiological mechanisms by which mitochondria could become dysregulated, determining the fate of multiple tissues and organs and being a protagonist in the pathogenesis of many non-communicable diseases. Finally, physical activity is a canonical evolutionary characteristic of humans that remains embedded in our genes. The normalization of a lack of physical activity in our modern society has led to the perception that exercise is an “intervention”. However, physical activity remains the modus vivendi engrained in our genes and being sedentary has been the real intervention and collateral effect of modern societies. It is well known that a lack of physical activity leads to mitochondrial dysfunction and, hence, it probably becomes a major etiological factor of many non-communicable diseases affecting modern societies. Since physical activity remains the only stimulus we know that can improve and maintain mitochondrial function, a significant emphasis on exercise promotion should be imperative in order to prevent multiple diseases. Finally, in populations with chronic diseases where mitochondrial dysfunction is involved, an individualized exercise prescription should be crucial for the “metabolic rehabilitation” of many patients. From lessons learned from elite athletes, the perfect human machines, it is possible to translate and apply multiple concepts to the betterment of populations with chronic diseases. Keywords: mitochondrial dysfunction; cellular bioenergetics; diabetes; cardiovascular disease; cancer; Alzheimer’s disease; metabolic flexibility; exercise. One. Introduction. The role of mitochondrial function in health and disease has become increasingly popular, especially in the past two decades. It is known that the dysregulation of mitochondrial function and cellular bioenergetics are hallmarks of many diseases, such as type 2 diabetes, T2D, cardiovascular disease, CVD, metabolic syndrome, cancer, and Alzheimer’s disease, AD. Although mitochondrial dysfunction is ubiquitous to many non-communicable diseases (NCDs), the etiology and pathogenesis of mitochondrial dysfunction remain elusive and the subject of important biomedical research nowadays as one of the most significant medical challenges in our history. The energy production of an individual is based on the metabolic demand and metabolic efficiency during exercise, resting, and fasting and in a postprandial state. Cellular bioenergetics are quite complex and tightly intertwined with the purpose of producing the necessary energy for cellular survival as well as achieving cellular homeostasis. Mitochondria are the main cellular organelles in charge of energy production and play a pivotal role in the control of cellular hemostasis. Under resting conditions, fatty acids and carbohydrates should be successfully transported into mitochondria and be oxidized to Acetyl-CoA for posterior oxidation in the Kreb cycle (tricarboxylic acid cycle (TCA)) and electron transport chain, E-T-C, through oxidative phosphorylation (OXPHOS) in order to synthesize the key energetic compound in the human body (ATP). Mitochondria are also the production site for reactive oxidative species (ROS), which at physiological levels behave as signaling molecules needed for cellular homeostasis. Hence, mitochondrial malfunctions will impact cellular bioenergetics, cellular function, and cellular homeostasis, making mitochondria a key player in health and disease. There are multiple effectors eliciting mitochondrial dysfunction that have been recognized, including mitochondrial DNA mutations, infections, aging, and a lack of physical activity. However, the etiology of mitochondrial dysfunction in the pathogenesis of multiple diseases has yet to be elucidated. The aim of this review is to assemble multiple components involved in the role of mitochondrial function in health and disease, especially some of the most prevalent NCDs in our society. Furthermore, the development of assessments for mitochondrial function in humans appears imperative in order to detect or diagnose mitochondrial dysfunction or decay. If signs of mitochondrial impairment or decay are detected early in life, there could be a significant window of opportunity to intervene in order to prevent diseases or the further deterioration of existing ones as well as improve multiple diseases through enhancing mitochondrial function through lifestyle interventions such as exercise. It has been known for decades that physical activity is probably the only known intervention that can improve mitochondrial function. The “exercise as medicine” concept continues to grow among health professionals as a necessity to prescribe exercise in a personalized and individualized manner, which seems imperative in the decades to come. However, an individualized exercise prescription should be crucial for the “metabolic rehabilitation” of many patients. This task remains a challenge due to the current lack of vertical and horizontal integration of medical systems, including clinicians, multiple providers, exercise specialists, and health care systems, with the proper means and infrastructures. The scientific and individualized approach to training elite athletes has been shown to be quite successful over the last several decades. Hence, the lessons learned from the work done with elite athletes can be an important “template” to apply to populations with chronic diseases in order to prescribe individualized exercise with the goal of improving mitochondrial function, disease, and overall metabolic health. Two. Mitochondria, the Key Aerobic Microbe for Eukaryotic Cell Evolution. Mitochondria originated about 1.5 billion years ago from a prokaryotic origin linked to archae bacterium, “archae” meaning “ancient bacteria”. According to the endosymbiotic hypothesis proposed by Doctor Lynn Margulis in 1967, eukaryotic species evolved from aerobic prokaryotic microbes (mitochondria) that were engulfed by an eukaryotic cell leading to endosymbiosis. In general, through this symbiotic relationship, mitochondria provided aerobic energy to eukaryotic cells in exchange for protection. The ability of mitochondria to conduct aerobic respiration inside the host eukaryotic cell led to a fundamental change in evolution and the origins of hundreds of new genes and proteins, leading to novel metabolic characteristics of eukaryotic cells providing transformational evolutionary advantages to multiple species including animal life. Mitochondria continued to evolve within eukaryotic cells and both entities improved their symbiotic relationship of energy and protection. On the other hand, the Romanian biologist and chemist Eugene Macovschi developed the biostructural theory. According to this theory, living cells possess a related biological structure conferring on them living features through a so called biostructure by which living matter consists of two distinct and interdependent forms: biostructure matter, which is the living matter itself, and coexistent molecular matter, which is a combination of chemicals in non-living matter. The biostructure in cells forms an inseparable unit such that, according to Macovschi, only one uniform cell could be the origin of all forms of life, contradicting the endosymbiosis hypothesis. Today, the cell nucleus contains genes encoding for about 1200 proteins involved in mitochondrial structure, membrane, and mitochondrial DNA (M-T DNA) repair. Nuclear DNA (nuDNA) is the key to a mitochondrion as its genome only contains 37 genes that encode 13 proteins, all of them involved in OXPHOS; hence, the symbiotic relationship with nuDNA. Mitochondria are referred to as the “powerhouses of cells” since they provide the ATP necessary for cellular functions and life. Mitochondria are found in all cells in the body except for red blood cells, which rely on aerobic glycolysis and lactate for proliferation and survival. Although it is commonly thought that mitochondria are individual organelles, in skeletal muscle they probably evolved to become interconnected in a reticulum or a network, most likely penetrating deep into skeletal muscles for increased bioenergetic efficiency. Two. Mitochondrial Bioenergetics Are Complex and Intertwined. The oxidation of multiple fuels occurs within the matrix of mitochondria through the TCA cycle and OXPHOS. Mitochondria oxidize all major substrates derived from macronutrients: pyruvate derived from carbohydrates, fatty acids derived from fat, and amino acids derived from protein. Lactate, the obligatory byproduct of glycolysis, is also a very important fuel for mitochondria and could even be the fuel preferred by most cells. Other metabolites, such as ketone bodies, are also commonly oxidized by mitochondria, especially under stress and fasting conditions. Skeletal muscle comprises the largest organ in the body and is the largest contributor to aerobic capacity through mitochondrial respiration. Hence, skeletal muscle mitochondrial function is crucial for whole-body metabolic function and health. Under normal, healthy conditions, mitochondrial bioenergetics are complex and tightly regulated for cellular homeostasis. In general, pyruvate, fatty acids, and a few amino acids are linked together upon being converted to Acetyl-CoA, which is the first step in the TCA cycle. The final step in the TCA cycle is the production of reducing equivalents of NADH and FADH2, which deliver electrons and hydrogen ions (H plus) to mitochondrial complexes through the electron transport chain, E-T-C, in the inner mitochondrial membrane. These electrons build up a chemical gradient that drives ATP production. Hydrogen ions are pumped out from the mitochondrial matrix into the intermembrane space through mitochondrial complexes (One to Four). The large gradient of protons that accumulate in the intermediate space will force H plus back to the lower gradient in the mitochondrial matrix to generate ATP. Even before oxidation, mitochondrial transport of multiple substrates is of key importance. In general, medium-chain fatty acids (FAs) can freely enter mitochondria, while long chain FAs need to be transported through palmitoyl transferase 1 and 2 (CPT 1, 2) located on the outer and inner mitochondrial membranes, respectively. Fatty acids are converted to Acyl CoA, which through oxidation is converted to Acetyl-CoA for oxidation in the TCA. Pyruvate, on the other hand, is transported across mitochondria by the mitochondrial pyruvate carrier (MPC) and oxidized to Acetyl CoA by pyruvate dehydrogenase (PDH). Therefore, the dysfunction of any of these elements involved in substrate transport across mitochondria can severely disrupt cellular bioenergetics and function. Substrate kinetics and dynamics are also important in cellular bioenergetics. When there is increased glycolytic flux, such as in the case of high-intensity exercise or high CHO ingestion, pyruvate may accumulate even under fully aerobic conditions and have difficulty being transported across mitochondria and oxidized to Acetyl-CoA, leading to its reduction to lactate. This is a ubiquitous process in exercise bioenergetics where PDH and lactate dehydrogenase (LDH) enzyme kinetics as well as MPC transport kinetics are key players. The LDH-A isoform possess a higher affinity for pyruvate, therefore eliciting a higher rate of pyruvate reduction to lactate. Further, in the oxidation of 2 Glyceraldehyde 3 phosphate (G-3-P) to 1, 3 Diphosphoglycerate, NAD plus is reduced to NADH and, under high glycolytic flux, cytosolic NAD plus may be depleted, leading to halted glycolysis and the disruption of the NAD plus, NADH ratio and intracellular redox state. During this stressful cellular event, NAD plus is “rescued” by lactate through the reduction of pyruvate to lactate through LDH-A and the oxidation of NADH to NAD plus for the continuation of glycolysis and the stabilization of the cellular redox state. Furthermore, increased glycolytic flux can lead to the accumulation of Acetyl-CoA, resulting in inhibition of Malonyl Co-A, which inhibits CPT1 and, therefore, fatty acid transport across the mitochondrial membrane. Lactate is a canonical component of cell biology and at the crossroads of cellular bioenergetics and intermediary metabolism. Lactate is the obligatory end product of glycolysis and behaves as a “lacthormone” by possessing multiple endocrine, paracrine, and autocrine properties. Lactate is mainly oxidized in mitochondria through the mitochondrial lactate oxidation complex (mLOC). Poor mitochondrial lactate oxidation could lead to a significant dysregulation of cellular bioenergetics. Both lactate accumulation in the cytosol and exportation to the blood could have significant effects on the regulation of both fat and carbohydrate metabolism, tightly regulating the intermediary metabolism as well as having an intimate relationship with mitochondrial function. Lactatemia decreases the mRNA expression of GLUT 4 in skeletal muscles, therefore decreasing glucose uptake and oxidation. Furthermore, lactate binds to the G protein coupled receptor (GPR81) on adipocytes, which inhibits lipolysis. Moreover, we have recently shown that lactate decreases the activity of both CPT1 and CPT2 in neonatal rat cardio myocytes, disrupts cardiolipin species, increases reactive oxidative species (ROS), and disrupts cellular bioenergetics by decreasing the rate of ATP production. A decrease in lactate and fat oxidation (FATox) by mitochondria, as in the case of T2D or metabolic syndrome, indicates a direct relationship between lactatemia and FATox. Metabolic flexibility is a term that has emerged in the last two decades and continues to evolve due to its involvement in multiple diseases. Mitochondrial flexibility is defined as the ability to respond or adapt to conditional changes in metabolic demand. However, the work in metabolic flexibility probably dates back over a hundred years to the pioneering work by Harris and Benedict, who studied the metabolism of adult males and females, infants, and patients with diabetes. Especially relevant was Benedict’s work in the nineteen twenties and thirties on the basal metabolism of both humans and animals as well as metabolic responses to exercise. Skeletal muscle substrate utilization and bioenergetics are central to metabolic flexibility. Kelly and Mandarino elegantly demonstrated that skeletal muscle is central to the study of mitochondrial function. They observed that individuals with type 2 diabetes (T2D) and obesity showed metabolic inflexibility in postprandial conditions with altered glucose and fat oxidation. Before the innovative studies by Kelly and Mandarino, De Fronzo and colleagues had shown that under eu-glycemic hyper-sinsulinemic clamp conditions, skeletal muscle uptakes and metabolizes about 85 percent of all glucose. Metabolic flexibility and mitochondrial function are closely intertwined as under resting and postprandial conditions both fat and glucose are oxidized in mitochondria via OXPHOS. In summary, mitochondrial bioenergetics are quite complex and the studies continue to show that the disruption of mitochondrial and, in general, cellular bioenergetics is central to the pathogenesis of multiple diseases. The following decade will be decisive in unveiling further crucial aspects of mitochondrial bioenergetics as well as therapeutic targets. Four. Mitochondria, the Main Producers of Reactive Oxygen Species (ROS). Oxygen consumption and reactive oxidative species (ROS) are ubiquitous to mitochondrial respiration. As electrons flow through the E-T-C, an estimated 0.4 to 4 percent of them leak before reaching Complex Four. Hence, mitochondria are considered to be the main generators of ROS and, within mitochondria, Complex One is the main ROS-generating site. Historically, ROS were previously thought to only cause cellular damage. However, it is well known that at physiological levels ROS generation is necessary and highly involved in the regulation of cellular homeostasis, key signaling pathways, cell proliferation, cell differentiation, cell migration, angiogenesis, and increased lifespan. The process by which physiological ROS are involved in cellular homeostasis and signaling has been named “oxidative eustress” and also mitohormesis. Superoxide anions, (Oh two minus) and hydrogen peroxide (H 2 O 2) are the main ROS and cells have specific mechanisms for counteracting excessive ROS production as well as scavenging free radicals through antioxidants such as superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx), and glutathione reductase (GR). When ROS production exceeds the antioxidant capacity, ROS accumulate and can cause multiple cellular disruptions involved in multiple diseases. Excess ROS production by mitochondria can cause damage to M-T DNA, proteins, and lipids, which in turn can disrupt mitochondrial function and cellular homeostasis. Although ROS production and mitochondrial dysfunction are intimately associated in multiple diseases, the mechanisms by which either primary ROS production leads to mitochondrial dysfunction or vice versa need to be elucidated. Could “faulty” mitochondrial function be responsible for excessive ROS production, or could it be the opposite? ROS generation occurs in the mitochondrial inner membrane, which is proximal to the M-T DNA that depends on nu DNA for maintenance and repair. Therefore, the proximity of M-T DNA to the ROS generation site makes M-T DNA more vulnerable to oxidative damage. A primary mitochondrial dysfunction may lead to a further increase in the generation of ROS, leading to exacerbated oxidative stress, which, in turn, may lead to further mitochondrial dysfunction in a self-perpetuating, feed-forward, and vicious cycle. As an example of the mitochondrial dysfunction and ROS balance for cellular homeostasis, deficiencies in the pyruvate dehydrogenase complex (PDC) lead to the accumulation of pyruvate and lactate, which has been shown to increase ROS levels and decrease the antioxidant capacity. On the other hand, lactate is an important signaling molecule as it stimulates a modest amount of ROS production, which elicits an antioxidant response for pro-survival cellular pathways such as PI3K, AKT and endoplasmic reticulum (ER) chaperones. Moreover, different events and external effectors have been shown to elicit mitochondrial ROS generation. Tumor necrosis factor alpha, TNF alpha, an inflammatory mediator, has been associated with increased ROS generation. Several toxic metals, such as mercury, damage M-T DNA and elicit lipid peroxidation as well as the depletion of glutathione, leading to increased ROS generation and further mitochondrial damage. Iron-deficiency anemia can cause a decrease in the activity of Complex four, eliciting a higher level of oxidative stress. In summary, mitochondria are the main site for ROS generation and for an extended period of time it was thought that ROS production was exclusively detrimental to human cells. However, it is well known that ROS act as key signaling molecules necessary for cellular and mitochondrial homeostasis. Nevertheless, an understanding of the exact balance between homeostatic and pathological ROS production remains elusive and is currently an area in which important research efforts are being made. Five. Etiologies of Mitochondrial Dysfunction. It is important to note that the term “mitochondrial dysfunction” might not be completely appropriate. In most cases where the term “mitochondrial dysfunction” is coined, mitochondria still work but not at full or an appropriate level of potential compared with healthy states, hence the term “mitochondrial dysfunction”. However, in many situations, “decreased mitochondrial capacity” or “mitochondrial impairment” could be more appropriate. From genetic mutations to aging, infections, and a lack of physical activity, the etiology of mitochondrial dysfunction, impairment (Figure 1) is multiple and currently an important field of research due to its implications in health and disease. Five point one. Genetic Mutations. As previously mentioned, the mitochondrial genome contains 37 genes that encode for 13 proteins, all of which are involved in OXPHOS. Therefore, any mutation of those 13 genes could result in a significant disruption of mitochondrial function and cellular bioenergetics. The incidence of inherited mitochondrial mutations is considered to be quite rare, 1 in 5000 individuals . Most mutations occurring in M-T DNA are mainly point mutations and deletions. Other mutations occur in the nucleus, including to autosomal recessive, dominant, or X-linked M-T DNA maintenance genes. Most mitochondrial genetic diseases are involved in neurological disorders, including myopathy, ataxia, and neuropathy. It is important to highlight the importance of the relationship between nuDNA and M-T DNA. As part of the ancient “symbiotic pact” of aerobic energy for protection between eukaryotic and prokaryotic cells, nuDNA encodes all the genes necessary for mitochondrial maintenance, repair, and replication. Hence, inherited or acquired mutations of nuDNA can contribute to mitochondrial instability. Unlike germline mutations, somatic mutations evolve over the life cycle of an individual and exposure to endogenous and exogenous mutagens could lead to potential errors in nuDNA repair and replication. The symbiotic genetic relationship between nuDNA and M-T DNA remains largely unexplored and could confer significant insights into the etiology of multiple diseases. Other mutations at the mitochondrial structural level can also affect cardiolipin (CL), which is a phospholipid in the inner mitochondrial membrane that regulates multiple mitochondrial processes and is also involved in mitochondrial dysfunction. As an example, Barth syndrome (BTSH) is a rare X-linked genetic disease caused by a mutation of the tafazzin gene, encoding for phospholipid transacylase, which is necessary for CL remodeling and characterized by cardiomyopathy, skeletal myopathy, and neutropenia. BTSH is a classic example of a disruption to mitochondrial bioenergetics due to genetic mutations, leading to metabolic reprograming in the heart characterized by a significant decrease in the capacity for mitochondrial oxidation of fatty acids and pyruvate (about 40 to 60 percent). Cancer is another important disease characterized by decreased, impaired mitochondrial function. M-T DNA is more susceptible to DNA damage than nuDNA as it has no introns, histones, or non histone proteins and, therefore, it is continually exposed to endogenous and exogenous mutagens, ROS, and different carcinogens. This vulnerability is substantial and in cancer it has been shown that M-T DNA mutations are significantly higher in number than nuDNA mutations. Vogelstein’s group was the first to decode the mitochondrial genome in tumors, where they found M-T DNA mutations in seven out of ten human colorectal cancer cell lines. In a study by H C Lee and colleagues with 20 different types of cancer in 859 patients, 66 percent of those cancers carried at least one somatic M-T DNA mutation. In cancer, the term “mitochondrial dysfunction” refers to a significant mitochondrial impairment leading to aberrant metabolic reprograming of cellular bioenergetics characterized by accelerated glucose uptake and lactate production. It was discovered by the Nobel Laureate Otto Warburg one hundred years ago. Five point two. Aging. The process of aging has been extensively studied over decades if not centuries. Aging is an inescapable biological process characterized by decreased physiological and cellular function across the body. A decrease in mitochondrial capacity has been observed with aging and is already considered a hallmark. Briefly, damaged and aging mitochondria are controlled by the process of mitophagy, which is the internal cellular autophagy of mitochondria. Mitophagy and mitochondrial biogenesis are indispensable to the regeneration of new mitochondria and achieve a balance for mitochondrial health. Mitochondrial fission and fusion are key processes for the regeneration and maintenance of mitochondrial networks, structure, and function. In general, mitochondrial fission splits mitochondria, where the damaged structures of mitochondria are degraded through mitophagy. The healthy fragments of mitochondria are attached together by the fusion process, allowing for the regeneration of mitochondrial structure and function, including normal metabolic bioenergetics. A decrease in the mitochondrial dynamics between fission and fusion is typical of aging processes, including increased fission and decreased fusion, which can lead to metabolic changes resulting in increased glycolysis and the metabolic reprogramming of multiple cells. As part of the aging process and exogenous mutagens, M-T DNA mutations become more frequent and could disrupt mitochondrial dynamics and bioenergetics over time. One particular mitochondrial deletion, M-T DNA4977, accumulates in multiple organs and is highly correlated with increased O2 consumption, which is a sign of increased glycolysis and metabolic reprograming. The GEHA EU project was an ambitious project whose goal was to compare the M-T DNA variability in 2200 nonagenarian Europeans and the same number of younger individuals as a control. The study showed that the association with longevity was only present when M-T DNA OXPHOS complexes co-occurred. It is also well known that, in aging, ROS and mitochondrial dysfunction are highly Interconnected. According to the free radicals theory of aging first proposed by Harman in 1956, the E-T-C’s inside mitochondria produce intracellular ROS that elicit mitochondrial damage and eventually cellular dysfunction. Mitochondrial ROS production can also interfere with mitophagy by disrupting the balance between fusion and fission, promoting the latter and activating intrinsic apoptotic pathway. However, over the last decade the emphasis on ROS production as the underlying mechanism of the pathogenesis of aging has evolved to mitochondrial bioenergetics and turnover, where ROS generation could be a consequence of aging and mitochondrial dysfunction instead of the primary cause of mitochondrial injury. Moreover, aging elicits an accumulation of damaged mitochondria in the brain, leading to a lower degree of metabolic efficiency, producing less ATP, and increasing the production of ROS, which can result in a disruption of cellular bioenergetics triggering neurogenerative disease. Five point three. Infections. Mitochondria play an important role in regulating the immune response to infections as they trigger multiple modulators involved in the innate immune system, including the transcriptional regulation of cytokines, chemokines, and inflammasomes. Multiple bacteria and viruses modulate cellular bioenergetics in order to increase their survival rate and establish a proliferative niche. One main way by which microbes hijack cellular functions is by targeting mitochondria. Bacteria-like listeria mono cytogenes, helicobacter pylori, shigella flexneri, legionella pneumophila, and chlamydia trachomatis cause mitochondrial fragmentation, mainly by increasing fission. Viruses also target mitochondria through different mechanisms. The Hepatitis C virus targets mitochondria by increasing mitophagy. The HIV virus disrupts the mitochondrial fission-fusion balance in the brain by increasing mitochondrial fusion, causing damage to neurons. PB1-F2, an Influenza A protein, is translocated into the mitochondrial inner membrane, disrupting the membrane potential and leading to mitochondrial fragmentation. Of recent importance is the pandemic caused by the SARS-CoV-2 virus. Although still under investigation, it would seem that SARS-CoV-2 also targets mitochondrial function for survival and replication by downregulating OXPHOS, increasing the elongation and overproduction of ROS. Recently, we observed both metabolic and mitochondrial dysregulation in 50 patients infected with SARS-CoV-2 and affected by post-acute sequelae of COVID-19 (PASC), referring to extreme chronic fatigue. About half of these patients had previous comorbidities, but the other half were healthy and moderately active individuals. We observed significant metabolic dysregulation with an extremely poor capacity to oxidize fatty acids and clear lactate compared with individuals with metabolic syndrome, suggesting mitochondrial dysfunction. In a subsequent study deploying metabolomics, we were able to find robust signatures of mitochondrial dysfunction and impaired fatty acid metabolism in PASC. While mitochondrial function is normally restored when infections cease, patients affected by SARS-CoV-2 and suffering from long lasting effects may have a significant and long-lasting alteration to muscle mitochondrial function, which needs to be studied in more depth. Septicemia (sepsis) due to bacterial infection can also cause metabolic and bioenergetics disruptions in multiple organs that can ultimately lead to multi-organ failure and death. Mitochondrial dysfunction in sepsis has attracted an increasing amount of attention over the last decade in order to explain the bioenergetic dysfunction of organ failure characteristic of patients with sepsis. Impaired perfusion early on in the sepsis process, increased ROS generation, hormonal alterations, and altered transcription of mitochondrial genes can significantly affect mitochondrial function during sepsis. Recently, it has been shown that mitochondrial transcription factor A (TFAM), which is key in mitochondrial biogenesis, is significantly decreased in sepsis. Rahmel and colleagues have shown that intramitochondrial TFAM levels were about 80 percent lower compared with controls and accompanied by decreased M-T DNA copy numbers and cellular ATP content. This finding is relevant as many sepsis survivors suffer from “post-sepsis syndrome”, which includes neuropathies, energetic dysfunction, and muscle weakness and wasting. The metabolic derangements in sepsis survivors also include hyperglycemia, which is a risk factor for the development of T2DM post-sepsis and CVD. In summary, although long-term effects of viral or bacterial infections in general are rare, a decrease in mitochondrial function caused by certain infections can elicit significant metabolic dysregulation through mitochondrial dysfunction increasing the risk of metabolism-related diseases. Five point four. Lack of Physical Activity. Physical inactivity has been associated with multiple diseases, including cardiovascular disease, cancer, Alzheimer’s disease, type 2 diabetes, and Parkinson’s disease. In fact, low cardiorespiratory fitness is considered to be responsible for the highest percentage of all attributable fractions for all cause mortality. The effects of a lack of physical activity on mitochondrial function have been known for decades. In 1979, Houston and colleagues found a 24 percent decrease in a mitochondrial function surrogate, succinate dehydrogenase (SDH), after 15 days of detraining in distance runners. Coyle et al, observed that 56 days of detraining elicited a 40 percent decrease in mitochondrial oxidative enzyme levels and a 22 percent increase in lactagenic enzyme lactate dehydrogenase (LDH) levels with increased blood lactate accumulation during exercise. Fritzen et al, found that 4 weeks of detraining in healthy male subjects elicited a decrease of 32 percent in the activity of another mitochondrial function surrogate, citrate synthase (CS), and a 29 to 36 percent decrease in mitochondrial complexes one to four. Houmard and colleagues showed a decrease in CS activity of 25 percent with just 14 days of detraining. Bed studies have contributed significantly to our understanding of the loss of proper mitochondrial function and metabolic flexibility. Alibegovic and colleagues elegantly showed that 9 days of bed rest altered more than 4500 genes and downregulated 34 metabolic pathways mainly associated with mitochondrial biogenesis, function, and OXPHOS. In this study, the most downregulated pathway was OXPHOS (54 percent of all genes involved in OXPHOS were downregulated). Further, bed rest elicited changes in the DNA methylation of the PPARGC1A gene, which encodes for PGC one alpha, a master regulator of mitochondrial biogenesis. In this same study, upon retraining for four weeks, 82 percent of the genetic expression that was altered with bed rest was restored, showing that physical activity restores major losses in genetic expression in a relatively short period of time. Furthermore, bed rest also induces changes in substrate partitioning favoring glycolysis instead of OXPHOS with a decrease of 37 percent in fat oxidation and an increase of 21 percent in CHO in the post-absorptive state. Moreover, bed rest increases insulin resistance, which primarily occurs in skeletal muscle. Finally, as described in Section 7 (vide infra), there are many studies showing that physical activity can efficiently increase mitochondrial function. Hence, the levels of daily physical activity (or the lack thereof) are significantly involved in mitochondrial function, the prevention of multiple diseases, and decreasing the risk of all-cause mortality. Six. The Role of Mitochondrial Function in Multiple Diseases. Six point one. Type 2 Diabetes. Type 2 diabetes has become an unstoppable epidemic affecting millions around the world and in various countries, regardless of their degree of development and sociocultural characteristics. Currently, in the United States alone, about 52 percent of the adult population has either pre or type 2 diabetes. China is experiencing the largest increase in T2DM in the world and Europe is also experiencing a significant increase in T2DM. Other parts of the world, including developing countries such as Cuba and highly developed countries such as the United Arab Emirates, are also being affected by this epidemic. Insulin resistance is the hallmark of T2D and central to its pathogenesis. As previously mentioned, skeletal muscle is central to the study of mitochondrial function and its relationship to the pathogenesis of T2D. Although the mechanisms remain elusive, multiple studies over the last two decades have implicated skeletal muscle mitochondrial dysfunction in the development of insulin resistance (IR). It is widely known that individuals with T2DM and metabolic syndrome are characterized by decreased mitochondrial content in both intermyofibrillar and subsarcolemmal skeletal muscle regions, mitochondrial oxidative enzymes, mitochondrial DNA, transcriptional factors and genes, and overall mitochondrial function. Furthermore, dysregulated muscle bioenergetics are a prevalent feature in individuals with type 2 diabetes, characterized by a poor capacity to oxidize fats and carbohydrates. The decreased capacity to oxidize both FAs and CHO in mitochondria leads to metabolic inflexibility and metabolic reprogramming with increased reliance on cytosolic glycolysis and lactate production to generate ATP. Furthermore, a lack of mitochondrial capacity for fat oxidation may lead to an accumulation of lipids in skeletal muscle adjacent to mitochondria, which is correlated with increased diacylglycerols, sphingolipids, ceramides, and insulin resistance. In individuals with IR, insulin signaling is disrupted, resulting in a decrease in AKT phosphorylation and the translocation of skeletal muscle glucose transporter (GLUT-4) to the sarcolemma, leading to a decrease in glucose uptake. Fernandez and colleagues developed a transgenic mouse model with the dominant-negative insulin-like growth factor one receptor (KR, IG, IR) in skeletal muscle. Expression of KR, IGF, IR abrogated IGF 1 and insulin receptors, resulting in insulin resistance in skeletal muscle. Since skeletal muscle seems to be the tissue with the highest uptake of glucose, De Fronzo and Tipathy as well as Fernendez and colleagues proposed that T2D debuts in skeletal muscle and that muscle insulin resistance is the primary mechanistic event involved in the development of T2D. Six point two. Cardiovascular Disease. The role of mitochondrial dysfunction in cardiovascular disease has been receiving increasing attention in recent years. The heart can suffer from severe metabolic reprograming and mitochondrial dysfunction with a decrease in oxidative capacity, oxidative phosphorylation, and ATP synthesis and an increase in ROS production. The heart is the most oxidative tissue in the body and about 50-70 percent of ATP is synthesized through the beta oxidation of fatty acids with 30 to 40 percent derived from aerobic glycolysis. Consequently, decreased mitochondrial function of the heart could lead to a disruption of the cellular bioenergetics of cardiomyocytes through increased glycolysis as in the case of cardiac hypertrophy and heart failure. Furthermore, it has been shown that cardiomyocytes of patients with coronary artery disease possess 8 to 2000 more M-T DNA deletions than healthy patients, which can significantly alter mitochondrial function and increase ROS production, leading to cellular damage and a dysregulated cellular metabolism. Moreover, even a small increase in glucose metabolism as a result of mitochondrial dysfunction can lead to cardiomyocytes with metabolic inflexibility. Vascular tissue is also affected by mitochondrial dysfunction. M-T DNA mutations and mitochondrial damage have been correlated with atherosclerosis. Specifically, atherosclerotic plaques are characterized by mitochondrial dysfunction and reduced M-T DNA copy numbers. In the process of angiogenesis, vascular endothelial cells (VECs) possess a high degree of metabolic flexibility in order to adapt to the changing microenvironment of sprouting angiogenesis. Although the mitochondrial composition of VECs is only 2 to 6 percent of the cell volume as opposed to 32 percent in cardiac myocytes, a small percentage of the volume of mitochondria in VECs may be key to maintaining their homeostasis. Because of this specific phenotype, VECs rely on glycolysis and lactate for cell proliferation and angiogenesis. In fact, about 99 percent of the glucose is reduced to lactate in VEC’s as lactate is a major regulator of vascular endothelial vascular growth factor (VEGF) and hypoxia-inducible factor HIF 1, which are both key processes in angiogenesis. The remaining ATP synthesis is derived from fatty acids and glutamine via OXPHOS. Although minor, the role of fatty acid oxidation may be of importance to control and balance VEC proliferation as disruptions in VEC bioenergetics could lead to pathophysiological conditions, including atherosclerosis and hypertension. VECs also suffer from senescence associated with intrinsic mitochondrial impairments involving M-T DNA mutations, E-T-C dysfunctions, changes in the fission-fusion balance, excessive ROS production, and decreases in antioxidant capacity. Six point three. Mitochondrial Dysfunction at the Crossroads of the Connection between Type 2 Diabetes and Cardiovascular Disease. The connection between T2D and CVD has received much attention, especially over the last two decades, as a large number of patients with T2D also develop CVD and vice versa. In many cases, this connection has led to the confluence of both diseases into one emerging disease: cardiometabolic disease. At this point, the connection between these two diseases is mainly epidemiological as the mechanisms behind the relationship remain elusive. As a possible hypothesis, a primary mitochondrial dysfunction in skeletal muscle could be important to understanding the connection between both diseases. A significant histological finding pertaining to skeletal fat metabolism occurs in physically fit individuals as well as in individuals with T2D, where both populations show an accumulation of intramuscular triglycerides. This phenomenon is known as the “skeletal muscle lipid paradox” as both physically fit individuals as well as individuals with T2D are characterized by the presence of a “lipid droplet” adjacent to mitochondria. However, the presence of skeletal muscle lipid or intra myocellular lipid (IMCL) content in highly metabolically fit individuals accounts for a significant source of fat oxidation during exercise. On the other hand, in individuals with T2D, this accumulation of fat possesses different metabolic properties and lipid profiles compared with fit individuals. In the case of individuals with T2D, the composition of intramuscular triglycerides is high in ceramides, which belong to a family of lipids consisting of sphingosines, which are bioactive lipid molecules and are involved in skeletal muscle insulin resistance, and mitochondrial dysfunction. Circulating ceramide levels are already considered to be a biomarker of insulin resistance, T2D, and CVD. Further, in the field of CVD research, it is well known that ceramides are key players in the atherosclerotic process. Historically, circulating ceramides have been thought to primarily originate in the liver, where they are packed in lipoprotein particles and transported to different tissues. However, it could be possible that the decrease in mitochondrial fat transport and oxidation in individuals with T2D could lead to chronic muscle lipid accumulation characterized by an increase in the content of ceramides that could be released into the blood and, consequently, contribute to the atherosclerotic process. Furthermore, as a possible cross-talk and transport mechanism, it has been shown that extracellular vesicles, EV’s, can contain ceramides and that skeletal muscle is very active in secreting EV’s. Could the skeletal muscles of people with T2D secrete EVs containing ceramides to the blood in a way that could influence the atherosclerotic process? Could other components of EV’s, mRNA, microRNA, proteins, enzymes, be implicated in the dysregulation of the metabolic function of the endothelial tissue? Although the relationships between CDV and T2D are quite strong, the mechanisms behind the link between these diseases remain elusive and a significant amount of research is needed. Six point four. Alzheimer’s Disease, Is It the Brain’s Diabetes? Over the past two decades, an increasing number of studies have linked T2D to AD and cognitive impairment. It is known that individuals with T2D have a 1 point 5 to 2 fold higher risk of developing CVD compared with people without T2D. Moreover, a study by Janson and colleagues found that 81 percent of patients with AD had either T2D or impaired fasting glucose. The same study showed that individuals with T2D possessed a higher frequency of islet amyloids and a greater extent of islet amyloids compared with control subjects. The hypothesis of beta amyloid plaque as being the main culprit in the etiology of AD has prevailed since the mid nineteen eighties. However, the therapeutic approaches to treating AD by targeting amyloid plaque have proven to be unsuccessful. Consequently, and due to the necessity of developing novel therapies, innovative pathways and approaches to understanding the pathogenesis of AD have emerged. Consequently, research on brain metabolism and bioenergetics has emerged and attracted a significant amount of attention over the last two decades. Glucose and lactate are the main energy substrates for the brain. A metabolic characteristic of patients with AD is diminished cerebral glucose metabolism characterized by a decreased capacity to uptake and oxidize glucose, signaling dysregulated brain bioenergetics. A traditional methodology for studying cerebral glucose metabolism is the use of an 18-F-Fluorodeoxyglucose (18F-FDG) PET scan. Early studies from the 1990s showed decreased glucose metabolism in AD patients despite normal blood flow. Recently, Hammond and Lin proposed that glucose metabolism is a better marker for predicting AD than amyloid or tau. Further, there has been a recent tendency in clinical practice to incorporate 18F-FDG-PET in the diagnosis and progression assessment of AD. The plethora of studies showing decreased cerebral glucose metabolism in patients with AD have led multiple researchers to inevitably explore the role of IR and mitochondrial dysfunction in the pathogenesis of AD. These pronounced novel interests have shown that, indeed, two main metabolic hallmarks of patients with AD are IR and mitochondrial dysfunction. Since IR and mitochondrial dysfunction are also the main hallmarks of T2D, there seems to be a metabolic connection. Hence, novel terminologies such as “type 3 diabetes”, “brain diabetes”, and “end stage type 2” have emerged in efforts to describe the pathogenesis of AD using a metabolism-centric approach. Furthermore, like skeletal muscle, the brain possesses a lactate shuttle key to brain bioenergetics. Although glucose historically has been thought to be the main fuel for the brain, it is now well known that lactate is a key fuel for neurons, possibly the preferred fuel for the brain, and essential for long term memory. In skeletal muscle, the discovery of the lactate shuttle by Doctor George Brooks was instrumental in understanding skeletal muscle glucose and intermediary metabolism. Briefly, lactate is shuttled from fast to slow-twitch muscle fibers, where lactate is oxidized in the mitochondria of slow twitch muscle fibers via the mitochondrial lactate oxidative complex (mLOC) for fuel purposes. Like skeletal muscle, the brain possesses its own lactate shuttle, which is called the “astrocyte neuron lactate shuttle”. Astrocytes play a key metabolic role in glucose metabolism as they receive glucose from the blood as well as store glycogen and break it down to glucose. Glycolysis is the main metabolic pathway for astrocytes, where most of the pyruvate is reduced to the lactate that is exported to neurons for fuel. From lessons learned from skeletal muscle metabolism, it is possible to observe similarities in brain metabolism through intracellular and extracellular lactate dynamics associated with mitochondrial function. While in skeletal muscle lactate is shuttled from fast twitch muscle fibers to the mitochondria of slow twitch fibers, in the brain, lactate is shuttled from astrocytes to neurons, where lactate is oxidized in the mitochondria of neurons via pyruvate oxidation. As a possible hypothesis, a mitochondrial dysfunction in neurons might lead to reduced astrocyte-derived lactate oxidation resulting in decreased pyruvate oxidation and a disruption of neuronal bioenergetics, as is the case with skeletal muscle, which is ultimately limited not only by glucose transport but by pyruvate oxidation. The etiology of mitochondrial dysfunction in AD patients remains largely unknown. Although the mechanisms behind the pathogenesis of AD remain elusive, novel and exciting advances in our understanding of brain metabolism have been made in the last decade, opening the door towards the generation of novel diagnostic methods and therapeutics. Six point five. Cancer. The lack of progress in targeting genes to cure cancer has led to the development of novel areas of research and clinical applications with exciting therapeutic possibilities. These therapies include immunotherapy and targeted therapies, particularly those that use tyrosine kinase inhibitors, TKI’s. Both immunotherapy and TKIs have helped to extend the lives of and even cure disease in, as in the case of immunotherapy, millions of people. Although they have efficacy in only a relatively small number of tumors and people, we can expect that new and more efficient generations of these therapies will be developed. As a result of the necessity of expanding our understanding of cancer, the field of cancer metabolism has experienced a strong renaissance in the last two decades due to the renewed interest in the Warburg Effect. As previously mentioned, in 1923, the German cell physiologist and Nobel Laureate Otto Warburg discovered that cancer cells show accelerated glycolysis and produce significant amounts of lactate. Although cancer cells increase their glucose uptake, they may not oxidize pyruvate correctly in mitochondria, reducing it to lactate. The observation of the significant amount of lactate that accumulates in cancer cells ledWarburg to posit that cancer is an injury to the cellular respiratory system (mitochondria). However, one century ago, DNA and genetic mutations were not known to exist, as DNA was discovered by Watson and Crick in 1953. It is widely recognized that genetic mutations are ubiquitous to cancer, especially the overexpression of genes such as RAS, MYC, and hypoxia inducible factor 1 alpha (HIF-1-alpha) and the loss of function of the tumor suppressor factor TP53, which confers on cancer cells a selective growth advantage for aberrant cell growth and proliferation. Hence, not all types of cancer necessarily possess a mitochondrial dysfunction. As mentioned above, the Warburg Effect is characterized by accelerated glycolysis and increased lactate production, which was probably what struck Warburg the most. According to the “lactagenesis hypothesis”, the exacerbated lactate production due to cancer cells observed by Warburg one hundred years ago could be the explanation for and purpose of the Warburg Effect. According to this hypothesis, lactate could be a major regulator of the main elements involved carcinogenesis: angiogenesis, immune escape, metastasis, and self-sufficient metabolism. Moreover, it has been shown recently that lactate is an onco metabolite capable of regulating histone acetylation as well as the expression of the main genes involved in ER positive breast cancer cells, including RAS, MYC, and HIF 1 alpha. Since lactate is a canonical element in most cancers, the transcendental question is: why is lactate so ubiquitous to cancer metabolism? As Warburg described a century ago, an injury to mitochondria could be one possible answer. The question of whether this injury is due to a genetic etiology, a metabolic dysregulation, or both will be fundamental to answer and even crucial to finally conquering cancer in the next decade. As previously mentioned, it is known that many cancers have some form of mitochondrial impairment, dysfunction that could be attributable to somatic M-T DNA mutations (as already observed by Vogelstein’s laboratory in the late nineteen ninties as well as M-T DNA depletion. Furthermore, other authors have observed direct disruptions of the mitochondrial structure affecting cristae (cristolysis) in glioblastoma multiforme (GBM), which should have devastating consequences for cellular bioenergetics and homeostasis and could possibly be a reason for the high aggressiveness of GBM. Furthermore, as previously mentioned, mitochondria highly depend on nuDNA as it encodes for about 1200 proteins necessary to mitochondrial repair, maintenance, and biogenesis. Hence, any mutation of any of the mitochondrial nuDNA dependent genes could lead to dysregulation of the mitochondrial bioenergetics resulting in metabolic reprogramming through decreased OXPHOS as well as increased glycolysis and lactate production, leading to carcinogenesis. The crucial symbiotic relationship between the cellular nucleus and the mitochondrion dates back about 1.5 billion years and any disruption of this relationship by either external, internal mutagens or epigenetic effectors could lead to severe consequences for cellular homeostasis. Although the enduring symbiotic relations between the nucleus and the mitochondrion remain largely unexplored in cancer, they may provide us with a better understanding of cancer. In summary, the implications of mitochondrial function in some of the most common NCDs are quite prevalent and central to the pathogenesis of these diseases (Figure 2). Furthermore, there is growing evidence of strong relationships between several diseases, where mitochondrial dysfunction could not only be the etiology behind the pathogenesis of these diseases but also a nexus, which, if true, would elevate medical research to horizons never before reached. Undoubtedly, the research that will be conducted the next decade holds much promise. Seven. Exercise, the Only Known “Medicine” for Maintaining and Improving Mitochondrial Function. It has been known for decades that exercise is the best physiological stimulus for improving mitochondrial function in skeletal muscles and possibly other organs. In this regard, we have learned many lessons from elite athletes that can be translated to multiple populations. Well trained athletes possess the highest mitochondrial function of any humans. The typical characteristic of elite endurance athletes is an increased capacity to oxidize fatty acids as well as carbohydrates, making them highly metabolically flexible. Since the late nineteen sixties and early seventies, multiple studies have demonstrated improvements in mitochondrial biogenesis and function after training. Twelve weeks of endurance training, 5 days a week, increased the number of mitochondrial enzymes by 2 fold and the total amount of protein content by 60 percent . Ten weeks of daily endurance training increased the mitochondrial concentration in the gastrocnemius muscle by about 30 percent and 1 hour of cycling for 4 days a week over five months at an intensity of 70 to 90 percent of the VO2max increased the oxidative capacity and glycolytic capacity by 95 and 117 percent, respectively. Exercise can improve mitochondrial health by increasing mitochondrial content, increasing the transcriptional activity of mitochondrial proteins such as PGC 1 alpha, and decreasing ROS production. A 16 week aerobic exercise program as an intervention in both men and women showed an increase in CS and cytochrome c oxidase of 45 and 76 percent, respectively, as well as an increase in the expression of genes involved in mitochondrial biogenesis, such as PGC1a (55 percent), NRF (15 percent), and TFAM (85 percent). Different studies have also used the model of training followed by detraining in order to measure muscle and mitochondrial plasticity and as a stimulus for physical activity and detraining. Moore and colleagues observed an increase of about 38 percent in CS activity in sedentary subjects after 7 weeks of training followed by a decrease of about 25 percent in CS activity and an increase of about 10 percent in the respiratory exchange ratio (RER) after 3 weeks of detraining, reflecting a decrease in mitochondrial oxidative capacity and flexibility. Klausen et al, observed an increase of 30 to 40 percent in SDH and mitochondrial cytochrome c oxidase (COX) after 8 weeks of training, followed by a decrease to basal levels after 8 weeks of detraining. Wibom et al, found an increase of 70 percent in the mitochondrial ATP production rate after 6 weeks of training followed by a decrease to between 12 and 28 percent after 3 weeks of detraining. Regarding the beneficial effects of exercise on mitochondrial function in multiple diseases, there are multiple studies demonstrating the benefits of exercise on mitochondrial function. For example, a 16-week aerobic training program in sedentary, overweight, obese individuals resulted in a significant increase in mitochondria (76 percent) in the myofiber volume accompanied by improvements in insulin resistance that were highly correlated with mitochondrial size and content (r equals 0.88 and 0.72, respectively, p less than 0.01). Toledo and colleagues also showed that diet and weight loss alone are insufficient to stimulate the mitochondrial capacity in skeletal muscle compared with diet plus exercise. In this study, both groups showed similar improvements in insulin resistance but the exercise group was the only one in which improvements in mitochondrial density, cardiolipin content, and E-T-C were observed. The same group of researchers showed that, in individuals with T2D, a moderate-intensity exercise program for 4 months elicited significant increases in mitochondrial density (67 percent), cardiolipin (55 percent), and mitochondrial oxidative enzymes and improved glycemic and metabolic flexibility. In diabetic mice, eight weeks of aerobic exercise significantly improved the expression of mitofusin-2 (Mf2n), which improves fusion, increases the expression of the mitochondrial transcription factor PGC 1 alpha for mitochondrial biogenesis, increases overall mitochondrial respiration, and decreases IR and ROS production. In patients with mitochondrial myopathies, due to M-T DNA mutations, endurance exercise has been shown to elicit significant improvements in mitochondrial function. Taivassalo and colleagues elegantly showed that 14 weeks of endurance training significantly increased the mitochondrial oxidative capacity, with increases in CS activity, about 50 percent), SDH activity (about 40 percent), and Complex four, about 25 percent, and a decrease in blood lactate accumulation (p less than 0.05). In CVD, exercise is known to improve cardiomyocyte, muscle, and platelet mitochondrial biogenesis, the oxidative capacity, and the antioxidant capacity. In patients with chronic heart failure, a six-month exercise program improved the total volume density of mitochondria by 19 percent and the surface density of mitochondrial cristae by 43 percent. Exercise has also been shown to significantly improve OXPHOS in the platelets of patients with stroke and with peripheral artery disease. Furthermore, exercise was shown to inhibit the pathological mitochondrial remodeling in rats with myocardial infarction (MI) by improving mitochondrial fusion and decreasing mitochondrial fission. Furthermore, eight weeks of exercise post-MI improved the mitochondrial O2 consumption, bioenergetics, and oxidative capacity in mice. Exercise can also improve the mitochondrial function and biogenesis in the brain as well as cognitive function, which opens an exciting door of opportunity to further understand the mechanisms behind the pathogenesis of AD and to improve the therapeutics against this disease. Therefore, we should stress the importance of physical activity not just for the prevention of T2D but possibly for mitigating the severity of and risks associated with AD as has already been shown in. In aging, some studies have obtained promising results. Sustained endurance training over time is also quite effective at maintaining mitochondrial function and flexibility in aging populations. Dube et al, showed that the muscle oxidative capacity, metabolic flexibility, and insulin sensitivity in older endurance-trained master athletes, average age 65 years old, were similar to those of young recreational athletes, average age 28 years old. Another recent study comparing the effects of exercise between elderly, average 80 years old, and young, average age 24 years old, cohorts found that 6 weeks of aerobic exercise increased CS activity by 31 percent in elderly individuals and by 45 percent in younger individuals. Complex one, two three and four increased in both groups by between 51 and 163 percent. The study found that both elderly individuals and younger individuals have the capacity to improve their mitochondrial function after 6 weeks of aerobic training. In summary, there are many studies demonstrating the benefits of exercise on mitochondrial function in many types of populations, including populations with chronic diseases. However, if we consider exercise to be a therapy that we can use to improve mitochondrial and metabolic function, it is essential to optimize and individualize the dose and duration of the exercise that is prescribed. Over the last decade, this is an area where we have gained a wealth of knowledge by working with elite athletes to whom prescribing the right training regime is key to improving athletic performance. Translating this knowledge to populations with chronic diseases is a challenge due to the lack of vertical and horizontal integration of medical systems, including clinicians, multiple providers, exercise specialists, and health care systems, with the proper means and infrastructures. However, all stakeholders should (must) be able to materialize this multidisciplinary partnership in order to achieve proper and individualized exercise prescription programs as exercise continues to be the most important intervention that is known to improve mitochondrial function, metabolic flexibility, and, thus, metabolic health. Eight. Assessment of Mitochondrial and Metabolic Function in the Clinical Setting. Historically, the assessment of mitochondrial respiration and function has focused on the measurement of relevant oxidative enzymes involved in OXPHOS. CS and SDH have been traditionally used in multiple studies as surrogates for mitochondrial function and content. More modern technologies have been developed to measure mitochondrial respiration and substrate utilization in skeletal muscle through two predominant techniques: the Oroboros and Seahorse technologies. These modern techniques, as well as the traditional ones, require muscle biopsies or cell cultures, which are not feasible to obtain on a large scale in humans in order to assess mitochondrial function and respiration. Non invasive techniques based on nuclear magnetic resonance (NMR) and magnetic resonance spectroscopy (MRS) techniques have become popular for research purposes as a valid way to assess mitochondrial respiration in vivo. However, the application of these new techniques to the general population would be very costly and infeasible. Recently, we proposed a novel and simple methodology for indirectly measuring mitochondrial function and metabolic flexibility that can be performed on a large scale in an ambulatory manner. Our methodology is based on the combination of measuring fat oxidation through indirect calorimetry using stoichiometric equations and the measurement of blood lactate levels during exercise, both important mitochondrial substrates. The concept is similar to cardiology stress tests where the heart is stressed through exercise in order to measure its activity and detect pathologies. Through our methodology, we use similar protocols with incremental exercise stages in order to stress the mitochondrial capacity and detect changes in mitochondrial and muscle bioenergetics. As shown in Figure 3, during exercise, both fat oxidation and lactate are oxidized in mitochondria as t

https://doi.org/10.1080/07853890.2020.1770849 Index of Science Articles: https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Unravelling the health effects of fasting: a long road from obesity treatment to healthy life span increase and improved cognition. Annals of medicine 2020, Volume 52, Number 5, pages 147 to 161. Françoise Wilhelmi de Toledo, and others. Buchinger Wilhelmi Clinic, Wilhelm-Beck-Strasse 27, Uberlingen, Germany. In recent years a revival of interest has emerged in the health benefits of intermittent fasting and long-term fasting, as well as of other related nutritional strategies. In addition to meal size and composition a new focus on time and frequency of meals has gained attention. The present review will investigate the effects of the main forms of fasting, activating the metabolic switch from glucose to fat and ketones (G-to-K), starting 12 to 16 hours after cessation or strong reduction of food intake. During fasting the deactivation of m-TOR regulated nutrient signalling pathways and activation of the AMP protein kinase trigger cell repair and inhibit anabolic processes. Clinical and animal studies have clearly indicated that modulating diet and meal frequency, as well as application of fasting patterns, for example, intermittent fasting, periodic fasting, or long-term fasting are part of a new lifestyle approach leading to increased life and health span, enhanced intrinsic defences against oxidative and metabolic stresses, improved cognition, as well as a decrease in cardiovascular risk in both obese and non-obese subjects. Finally, in order to better understand the mechanisms beyond fasting-related changes, human studies as well as non-human models closer to human physiology may offer useful clues. KEY-MESSAGES. 1. Biochemical changes during fasting are characterised by a glucose to ketone switch, leading to a rise of ketones, advantageously used for brain energy, with consequent improved cognition. 2. Ketones reduce appetite and help maintain effective fasting. 3. Application of fasting patterns increases healthy life span and defences against oxidative and metabolic stresses. 4. Today’s strategies for the use of therapeutic fasting are based on different protocols, generally relying on intermittent fasting, of different duration and calorie intake. 5. Long-term fasting, with durations between 5 and 21 days can be successfully repeated in the course of a year. 1. Introduction. In the evolution alternation of food availability and food scarcity has been the consequence of seasonal cycles with variations in sunlight exposure. Before humans could efficiently conserve food, physiological and behavioural adaptations to survive periods of food limitation, have led to the development of the metabolic fasting programmes. Storing food as fat in the adipose tissue instead of carbohydrates or proteins, was a highly efficient solution for humans and animals to cope with the absence of food supply and it permitted to fuel prolonged periods of fasting. In free living animals fasting is coupled with migration, hibernation or huddling to save energy. Bears and ground squirrels reduce energy expenditure by hibernating and undergo prolonged fasting periods of months with no or little food, whereas Emperor penguins fast during moulting and breeding. During fasting, they can stand colder conditions better than any other bird, while maintaining their usual body temperature thanks to the huddling behaviour. Birds can undertake strenuous efforts during long distance migration, while flying hundreds of kilometres without food or drink. Humans also had to adapt to natural cycles of food scarcity for millions of years and only with the onset of agriculture in the Neolithic era, more continuous food supply became available. This culminated in today’s technologies allowing humans in privileged countries to have access to any type of food at any time during the whole year. Extreme situations such as famine, chronic malnutrition, hunger strikes or long course of anorectic denial of food have also provided information about the human capacity to cope with long periods of food shortage. In addition to being a necessity for human survival, the ability to fast has been known in most religions as traditionally ritualised periods lasting from hours to weeks. Religions used fasting empirically for its effects on mind and body, as well as an important factor in community cohesion. In medicine fasting for 2 to 21 days or more was known for its numerous therapeutic effects that led to multidisciplinary fasting programmes, well-known in the public less in the scientific community. Total fasting safely practiced for 31 days has been documented scientifically at the beginning of the twentieth century. Later on, in the nineteen sixties, as obesity started to be a medical issue on a large scale, water fasting to treat morbid obesity and co-morbidities emerged under the names of zero calorie diet or total fasting. The effects of this type of fasting, lasting weeks or months, particularly on obesity, have been extensively documented. Nowadays a revival of the interest for fasting emerges from a different perspective. Instead of focussing only on weight loss in morbid obesity, the new focus is on the effects of the main fasting regimens, that activate the metabolic switch from liver derived glucose to adipose cell-derived ketones (G-to-K) and its reversal K-to-G, on longevity, health span expansion, multi-stress resistance and antioxidant defence stimulation, improved performance and cellular regeneration in animal models and humans. The present review investigates, in animal models and humans, how application of several types of fasting strategies, for example intermittent fasting, periodic fasting, or long-term fasting could be part of a new medical approach leading to improved healthy life span and cognition, as well as to a decrement in cardiovascular risk in both obese and non-obese subjects. 2. Fasting classification. Fasting is defined as the voluntary abstinence or strong limitation of caloric ingestion for a limited period of time, triggering the G-to-K switch and major changes in the activity of signalling pathways. Further effects also take place on refeeding when the K-to-G switch occurs. The big challenge when it comes to classify fasting regimens is that duration has a very different meaning if we consider animals, such as nonobese mice, or humans. One day fasting brings the mouse almost to starvation, ketones decrease and protein catabolism increases, whereas a day in humans is considered as intermittent fasting. Table 1 summarizes the main forms of fasting and other restrictive diets. Calorie restriction (CR) refers to a daily reduction by 15 to 40 percent of calorie intake without malnutrition. Intermittent fasting (IF) refers to fasting lengths between 16 and 48 hours, alternated with usual food intake. The most frequently studied IF procedure is alternate day fasting, ADF, whereby normal food intake occurs on one day and restricted food the next day. Time restricted eating-feeding, TRE-TRF, is characterised by intake of food occurring within a time window of 8 to 12 hours per day or less. Periodic fasting (PF) describes cycles of fasting or calorie restricted diets, meaning 5 to 2 diet (5 to 2), referring to two days per week, consecutive or not, in which food intake is drastically reduced to approximately 600 kcal. In some classifications 5 to 2 is considered as IF. Periods of fasting lasting from many days to weeks have sometimes been referred to as periodic and can be repeated every year. Other Authors referred to them as long-term or prolonged fasting. Considering that these adjectives do not take into consideration differences in the baseline nutritional status and between species, in the present review article we refer to long-term fasting (LF) as a food abstinence from 2 to 21 days or more during which no or minimal amounts of calories, up to 200 to 250 kcal per day are given within appropriate schedules. LF has been well documented in obese and non-obese subjects as well as in animals and during the nineteen sixties in morbidly obese subjects. Other regimens, derived from fasting strategies, are the very-low-calorie diets (VLCD), a hypocaloric formula diet providing 80 to 100 grams of proteins and an average of 1000 kcal per day, designed to treat obesity and to avoid a negative nitrogen balance. Furthermore, the fasting mimicking diet (FMD) is a hypocaloric (800 to 1100 kcal), low protein, ketogenic diet leading to weight loss and to some of the effects of fasting. It is worth mentioning that nutrient restricted normo caloric diets like the ketogenic diet (carbohydrate restriction) and the protein or amino acid (methionine) restricted diet are being also intensively evaluated. 3. Metabolic and cellular responses to fasting. 3 point 1. The metabolic switch. The onset of fasting is characterised by the metabolic switch, that defines the fuel switch occurring when an organism commutes from the eating mode, including generally around 50percent carbohydrates, to the fasting mode. From 12 to 16 hours after interrupting food absorption, glucose levels drop, followed by a decrease of insulin levels and at the same time of circulating amino acid levels. The onset of the metabolic switch depends on the liver glycogen content at the beginning of the fast as well as on the composition of the preceding meal, energy expenditure, and physical activity. Lipids in adipocytes (triacylglycerol and diacylglycerol) are metabolised to free fatty acids (FFAs) whose levels increase in fasting blood, to be partly oxidised in most tissues, such as muscles, kidneys, heart and partly transformed in the liver to ketones (b-hydroxybutyrate, acetoacetate and acetone). Ketones are not only fuels, since they have also signalling effects and regulate expression and activity of transcription factors like the peroxisome proliferator activated receptor c coactivator 1a (PGC-1a), sirtuins (SIRTs), poly-adenosine diphosphate ADP-ribose polymerase 1 (PARP1), and ADP ribosyl cyclase, as well as fibroblast growth factor 21 and nicotinamide adenine dinucleotide, NAD plus. A rise of brain derived neurotrophic factor (BDNF) in the central nervous system (CNS) can enhance brain health. Ketones are effectively oxidised by the fasting brain, more reluctant than other tissues to renounce glucose and metabolise FFA’s. The use of FFA’s and ketones as energy sources reduces the respiratory exchange ratio (RER), compared with the fed state, indicating a greater metabolic efficiency of energy production during weight loss leading to ketosis. The decreased ATP to AMP ratio will activate AMP-activated protein kinase (AMPK) as well as create a mild oxidative eustress leading to the activation of antioxidant and cytoprotective enzymes, meaning superoxide dismutase, catalase, peroxidase, sulfiredoxin 1, thioredoxin reductase 1, haem oxygenase-1, NAD(P)H quinone oxidoreductase 1, glutamate-cysteine ligase, glutathione S-transferases, and uridine 50-diphosphoglucuronosyltransferases. During prolonged periods of fasting, ketosis has been shown to reach a plateau after 4 days, an effect that can last for several hours or days, and to decrease when food is reintroduced, see Figure 1. The G-to-K switch is the key mechanism allowing to spare proteins by reducing protein utilisation, as reflected by the changes in nitrogen balance. It is regulated by inhibition of the m-TOR, mechanistic-mammalian target of Rapamycin, signalling pathway, thus decreasing protein synthesis and enhancing autophagy, that leads to the recycling of endogenous proteins. Relying on ketones for energy during weight loss has the advantage of promoting retention of lean mass; the same may not occur after plain CR. This conclusion was not supported in the most extensive ADF studies to date. At present, the substantial benefit of fasting regimens on lean mass retention needs to be reconsidered. While the adaptation to long-term fasting can promote lean mass retention, this has not been definitely shown to occur preferentially after IF rather than with CR. It should be noted that ketosis can be also obtained with low energy and-or high fat diets. Normocaloric strict high fat ketogenic diets are successfully prescribed to epileptic children who do not respond to drug treatments. Switching from a carbohydrate to a lipid rich diet has been successfully applied for weight reduction, although, based on current evidence, low-CHO and very-low-CHO diets do not appear to be clearly superior to other dietary approaches. Ketones serve as an energy source, being metabolised to acetyl coenzyme A (AcCoA) entering the tricarboxylic acid cycle generating adenosine triphosphate (ATP). They sustain the function of muscle and brain cells during fasting as well as extended periods of physical exertion. Furthermore, ketosis leads to a fully compensated acidosis, believed to be responsible for the characteristic absence of hunger during fasting. Absence of hunger enhances compliance. Blood glucose stabilises at the lower normal level and will remain stable during the whole fasting period, as long as fat reserves can fuel metabolism and the protein pool remains at the physiological limits. Concomitantly, in a well-orchestrated manner, an increase in the hormonal secretion of glucagon, stimulation of glycogenolysis and gluconeogenesis, growth hormone, implicated in lipolysis, cortisol and adrenaline regulate the course of fasting. Furthermore, along with a decrement in the adipokine leptin and a rise in adiponectin, fasting significantly reduces insulin like-growth factor, IGF-1. Only restrictions of 50 percent or more of normal daily energy requirements can reduce IGF-1 levels. Low levels of IGF-1 reduce the intracellular mitogenic signaling pathways, and lead mammalian cells to enter either a non-dividing or a low-dividing state and invest energy resources into cellular protection against various insults (multi-stress resistance). Since during fasting very few or no exogenous proteins or sugars enter the system, nutrient dependent signalling pathways are shut down, m-TOR and Rasadenylate cyclase (AC)-protein kinase A (PKA), along with the de-repression of transcription factors for example SIRT, and forkhead box O1 (FOXO1), leading to the many peculiar features of the fasting mode. The promotion of the multi-stress resistance is characterized by improved antioxidant defences, increased DNA repair and diminished inflammation. Moreover, most cardiovascular risk factors, waist circumference, lipids, blood pressure and insulin resistance, are improved by CR, IF or LF. The fasting mode will be reversed as soon as a mixed diet is reintroduced, triggering the K-to-G switch: glucose and insulin levels increase, ketones drop, m-TOR is reactivated leading to increased protein synthesis and mitochondrial biogenesis, as well as decreased autophagy. Cell regeneration in multiple systems occurs, with increased mesenchymal and progenitor cells. In this refeeding phase, specific effects take place concomitant to cell growth and plasticity, leading to functional tissue remodelling and offering a unique opportunity, in adult life, to boost cellular and tissue regeneration. In some cases, an inappropriate pattern of food reintroduction can lead to relapse of symptoms, as seen in the case of LF prescription to treat polyarthritis. 3.2. Signalling pathways modulated by fasting. In the fasting mode, cellular and metabolic processes are controlled by a complex network of transcriptional regulators. Major regulators are SIRTs, nuclear factor erythroid 2 related factor 2 (NRF2), FOXO1, nuclear factor ‘kappa-light-chain-enhancer’ of activated b-cells (NFkB), hypoxia inducible factor 1 a (HIF-1a), heat shock factor (HSF-1). The decrease of the protein responsive signalling pathway m-TOR and of its downstream effector, the ribosomal protein S6 kinase b-1, leads to global protein synthesis inhibition and recycling of macromolecules by autophagy stimulation. In the brain, in addition to raised neuronal stress resistance through bolstered mitochondrial function there is an improvement in antioxidant defences, DNA repair, and stimulation of BDNF production. BDNF regulates hippocampal neurogenesis, dendrite morphology and synapse plasticity, and increases production of new neurons from neural stem cells. The decrease in glucose levels in parallel to the rise in ketones during fasting is associated with a decrement in the glucose responsive Ras-AC-PKA pathway, implicated in life span extension. Other crucial consequences of fasting are the decrease in insulin-IGF-1 signalling, leading to a reduction in anabolic processes and decrement in the ATP to AMP ratio with consequent activation of AMPK. This last step triggers repair and inhibition of the anabolic processes, Figure 2. Another mechanism associated to the fasting-induced activation of AMPK is autophagy of muscle cells, a process preserving blood glucose levels during LF. As a proof-of-concept, the selective skeletal muscle depletion of AMPK resulted in hypoglycaemia and hyperketosis, an effect not due to an impairment in fatty acid oxidation, but instead to a reduction in autophagy of muscle cells, leading to reduced circulating levels of alanine, an essential amino acid required for gluconeogenesis. Among the many activated transcriptional regulators quoted above, hepatic SIRT1 protein levels are raised during fasting and reduced with refeeding. SIRT1-driven deacetylation of PGC-1a and the increased FOXO1 transcription factor provides a mechanism by which mitochondrial and lipid oxidation genes can be dynamically controlled in response to energy demand. A specific liver knock down of SIRT1 results in a decreased fasting-dependent downregulation of the lipogenic gene expression (SREBP-1c), suggesting this mechanism for the regulation by SIRT1 of multiple SREBP-1 target genes. SIRT1 suppresses glucose production by inhibiting the CREB regulated transcription coactivator 2 (CRTC2)-mediated gluconeogenesis, activated during the G-to-K switch. Similar to SIRT1, the mitochondrial deacetylase SIRT3 regulates metabolic homeostasis during fasting and CR. SIRT3 is critical for fatty acid oxidation and ketogenesis during fasting, by regulating the deacetylation state and the activity of mitochondrial enzymes involved in the metabolic switch. Having thus a major role in liver ketogenesis, mice lacking SIRT3 display lower plasma b-hydroxybutyrate levels during fasting. 4. Evidence from experimental animals. The life and health span extension produced by CR has been an area of interest since the late nineteen seventies. Compared to mice assigned to an ad libitum regimen, 30percent CR and TRF (single-meal feeding) enhanced longevity, regardless of diet composition and showed improvements in morbidity and mortality. Prevention of age-related declines, as evaluated by tests of motor coordination (rotarod) and learning (complex maze) took place in mice exposed to a 50 percent CR regimen. Increased locomotor activity in a runwheel cage, regardless of age was observed, without effects on exploratory activity in a novel arena. This type of functional improvement has been related to anatomical changes, such as a rise in synapse numbers and stimulation of mitochondrial biogenesis, correlated with increased BDNF levels. 3-hydroxybutyrate may also mediate adaptive responses of neurons to fasting, exercise and ketogenic diets. In mice, hippocampus dependent spatial learning and memory deficit improve upon a daily TRF with ameliorated behavioural changes, in particular anxiety-like behaviours. These improvements, found in old mice, may rely on the enhanced cerebral blood flow and blood-brain barrier function, similar to that seen in young mice at 5 to 6 months of age following CR. Relative to TRE, the study of mechanisms leading to changes in the molecular circadian clock genes, activated or deactivated according to light and diet, has opened new horizons. These circadian rhythms, displaying oscillations over a period of 24 hours, are evolutionarily conserved and driven by the need to synchronise biological activity with the ever-changing, but predictable, environment of the rotating Earth. In mammals, feeding behaviour is cyclic with periods of fasting separating feeding bouts, leading the organism to switch from nutrient storage during feeding periods to the use of stored nutrients during fasting. Studies on the effects of a disruption of metabolic circadian cycles, showed that under TRF, in which mice are fed an equivalent amount of food as the ad libitum cohort but for a defined period of 8 hours, mice were protected against obesity, hyperinsulinemia, hepatic steatosis, and inflammation. Compared to rats fed ad libitum, those exposed to ADF present lower resting heart rate and blood pressure (BP), this last remaining lower after exposure to immobilisation and swim stressors, and equal to or greater than that previously obtained with exercise training regimens in rats. Other Authors confirmed that TRF regimens, compared to prolonged ad libitum feeding are associated to an improved hemodynamic profile, meaning reduced heart rate and systolic and diastolic BP. TRF decreases the sympathetic autonomic nervous system activity and raises the parasympathetic or vagal tone. Another TRF regimen of 4 hour feeding per day in mice restored the expression of clock genes, leading to reduced body weight, cholesterolaemia, tumour necrosis factor-a and improved insulin sensitivity. These findings suggest that TRF can normalise the expression of genes involved in fatty acid metabolism, b-oxidation, PPARc and antioxidant defences in the liver. The correlation between clock genes and TRF has recently been documented by a study in mice lacking a circadian clock. When these are provided with access to high energy food ad libitum they gain weight quickly, but when subjected to a TRF regimen, 10 hours, they are protected from the weight gain and metabolic diseases driven by a high-energy diet. Prolonged ADF can upregulate the hemo-oxygenase1 and glucose-regulated protein 7B, both reduced by aging, in the cerebral cortex and striatum. These cytoprotective proteins against metabolic and oxidative stresses rise after ADF and appear to sustain expression, responsivity of genes involved in adaptive neuroplasticity and cognition. Similarly, in the case of animal models of neurologic disorders, including epilepsy, Alzheimer, Parkinson disease and stroke, application of an IF regimen, feeding for 8 hours per day, appears to protect against the neurological damage in ischaemic stroke, with circulating leptin as a possible mediator. Mice maintained on a chronic IF for 11 months exhibited a superior cognitive ability in the Barnes maze test of spatial memory. This type of positive response also occurred after a late-onset short-term IF dietary restriction for three months in old rats, animals fasted overnight. A better cognitive performance was also reported when mice were fed cycles of FMD lasting 4 days, followed by a standard ad libitum diet. Compared to control mice, FMD resulted in a rise in life span, meaning up to 28.3 versus 25.5 months. Bi-monthly application of this dietary approach reduced visceral fat, cancer incidence and skin lesions, rejuvenated the immune system, and retarded bone mineral density loss; when applied later in life, there was improved cognitive performance. 5. Therapeutic fasting: clinical impact in contemporary medicine. Modulating diet and meal frequency as well as several patterns of fasting can represent a new paradigm in today’s medical approaches. As recently highlighted by de Cabo and Mattson, Homo sapiens has adapted to fasting in ways that enable the organism to tolerate or overcome challenges and then restore homeostasis. An increasing number of physiological effects linked to IF can be applied also to LF, at least in the initial phase, leading to: (1) weight loss and metabolic reset, (2) increased insulin sensitivity, (3) reduction in inflammation and oxidative damage to proteins, lipids and DNA, as well as: (4) enhanced immune system function. All these mechanisms are definitely of value in the management of obese as well as nonobese subjects. 5 point 1. LF, a general overview. Whereas the metabolic switch and the changes in signaling pathways above described also apply at the beginning of LF, the question is whether these effects persist, decrease or increase in the course of LF. Evidently, it will depend on the duration of fasting, the individual profile including age, nutritional and health status, as well as personal inclination for the procedure. One of the first detailed scientific observations of a non-obese voluntary subject on total fasting ended safely after 31 days. Other case reports of persons fasting during several weeks were published and brought stupefaction in the medical community and the public. Many Authors have stressed the well-being, the absence of hunger and lifted mood reported by fasting obese and non-obese subjects. The human capacity to live without energy intake for periods of almost 40 days has been often reported in non-obese subjects during hunger strikes. A medically supervised practice of LF has a long tradition in Europe and in particular in Germany. The safety of this programme has been recently documented in large cohort studies. This type of LF, generally lasting from 4 to 21 days, has been studied in various clinical conditions: chronic inflammatory disorders and rheumatoid arthritis, hypertension, irritable bowel syndrome, insulin resistance, type 2 diabetes and metabolic syndrome, fibromyalgia, breast and ovarian cancer, osteoarthritis, obesity, and fatty liver. Furthermore, MRI evaluation documented significant changes in body composition after 14 days of fasting. LF was first documented in morbidly obese subjects in numerous publications from 1959 to 1975, reporting fasting periods from several days up to 249 or, in an extreme case report, to 382 days, called “zero calorie diets” or “total fasting,” sometimes with administration of multi-vitamins and potassium supplements. The main concern at that time was to elucidate how humans could fast that long, having a limited possibility to break down their protein pool and at the same time the need to provide their CNS with energy: the daily calorie needs of the brain in the eating phases are 400 to 570 kcal delivered by 100 to 145 grams glucose. If these needs have to be met by gluconeogenesis, since fatty acids cannot be transformed into glucose, this would mean catabolizing 200 grams of proteins daily. Since the human protein body pool is roughly 6000 to 8000 grams and cannot drop more than a third, this would reduce the ability to fast for long periods. In fasting subjects, after depletion of the glycogen stores, a rapid decrease in carbohydrate oxidation and protein catabolism takes place. Measurement of urinary nitrogen excretion allowed to determine protein utilisation. Whereas at the beginning of fasting 35 grams of nitrogen were excreted per day, these values tended to fall to 2 to 4 grams per day, 12 to 25 grams protein per day after 4 weeks. The brain was increasingly fed with ketone bodies, especially b-hydroxybutyrate, keeping it alert in order to stay able to face challenges. The protein sparing mechanism is induced by the metabolic switch of G-to-K, as described by Longo and Mattson decades later. Cahill demonstrated the fuel change occurring in the brain after 38 to 41 days of fasting. The brain system metabolises only 30 percent of glucose supplied by the glycerol molecule, liberated after cleavage of three fatty acids from triglycerides by lipoprotein lipase. Altogether, during total fasting, up to 41 days, 86 grams per 24 hour glucose is produced: half of this comes from blood cells, producing lactate and pyruvate through glycolysis, then resynthesized to glucose in the liver. The other half comes from glycerol and gluconeogenesis, taking place first in the liver and then in the kidneys from amino acids, respectively alanine and glutamine. This shift produces ammonium to titrate ketone bodies. Three phases have been described in the course of LF in animals, such as the Emperor penguin, that undergo yearly, during 35 years (average life span) fasting periods lasting several weeks until exhaustion of energy reserves. An initial phase 1, when the metabolic switch occurs, is followed by a phase of protein sparing called phase 2, lasting several days to weeks. Phase 3 finally starts when fat and protein reserves have reached a threshold, forcing animals to refeed. Phase 3 is characterised by a high catabolic activity, as well as adrenergic stimulation and underlines the urgent need to replenish energy stores. Nevertheless, the process is fully reversible. In humans these three phases seem to exist in a similar way. Under physiological condition of therapeutic fasting, phase 3 is not reached. Phase 3 has been described in anorexia. The zero-calorie diet, where only non-caloric beverages are permitted, was relatively well tolerated for several weeks or even months, even though obese persons were often without exercise, psychological support or nutritional education to prevent relapses. Pioneers of this type of fasting to treat obesity made even the recommendation, surprising for our contemporaries, that obese subjects should not fast longer than 100 days without nitrogen balance monitoring. The publication of a single case of death caused by acute intractable heart arrest happened on the seventh refeeding day in a 20-year-old woman. She had been on a 30 week zero calorie diet, reducing her weight from 118 to 60 kilograms. By simple calculation this would mean a protein utilisation of 4000 grams, meaning more than 50percent of her protein pool, this being the most plausible death aetiology. At autopsy a decrease of myofibril diameter in the heart with gross fragmentation was diagnosed. Isolated cases of death by very prolonged zero calorie diet periods of several weeks or months in morbidly obese had not always such a clear aetiology; other possible causes of death include factors such as potassium or vitamin deficiencies, imperfect patient selection or compliance, associated medications or pre-existing disease. For several years, zero calorie diets were followed by a medical team in hospital wards. Despite the polymorbidity of the patients, the procedure seemed to be safe for periods of 60 to 100 days. Because of the long duration, the costs for hospitalisation were considered as too elevated and, as a consequence, people started total fasting on their own. Furthermore, obese subjects could also buy protein supplemented diet formulas in supermarkets, without any supervision. One of these products consisting of a liquid based hydrolysate of collagen proteins, the so called “liquid protein diet” (LPD) was linked to deaths from cardiac arrest in 32 out of 44 casualties. Again, the histological diagnosis was myofibrillar gross fragmentation but, unlike the first documented case, the subjects were still obese. The poor protein quality of LPD might have played an accelerating role in protein pool depletion or else in depletion of specific amino acids. After these events, the depletion of the protein pool was incriminated and this led to a strict regulation of what has been called VLCD (very low calorie diet), a hypocaloric formula providing 80 to 100 grams good quality protein per day. The main objective was to avoid a negative nitrogen balance measured by 24 hours urinary excretion. VLCD were prescribed to last not more than 6 weeks. By reflection, this reduction of the fasting period alone could have sufficed to reduce risk drastically. Protein formulas provided a market allowing to design ambulatory multidisciplinary programmes. The need to provide proteins was not questioned any more until today. 5 point 1 point 1. Impact of LF on health and well-being. The practice of LF has reached a wide network of utilization in Europe. Among 1422 subjects who followed fasting periods, daily calorie intake of 200 to 250 kcal accompanied by a multidisciplinary lifestyle programme, lasting between 4 and 21 days, there were significant reductions in body weight, between 3.2 plus or minus 0.0 kilograms after 5 and 8.6 plus or minus 0.3 kilograms after 20 days of fasting, as well as in abdominal circumference, meaning between 4.6 plus or minus 0.1 cm and 8.8 plus or minus 0.8 cm, respectively. BP decreased for the whole group from 131.6 plus or minus 0.7 to 120.7 plus or minus 0.4 for systolic BP and from 83.7 plus or minus 0.4 to 77.9 plus or minus 0.3 for diastolic BP. A reduction of total cholesterol, -0.4 plus or minus 0.0 mmol per Liter, triglycerides, -0.4 plus or minus 0.0 mmol per Liter, glucose, -0.7 plus or minus 0.1 mmol per Liter, and HbA1c, -1.2 plus or minus 0.1 mmol per mol, was reported. The absence of hunger feeling, documented in 93.2 percent of the subjects and an increase of emotional and physical well-being was documented. None of the subjects dropped out of the fasting procedure. Adverse effects, for example cardiac arrhythmia, were reported in less than 1percent and mild symptoms like headache and fatigue occurred rarely and mainly in the first days. In another study on 174 hypertensive patients, who underwent a water-only fasting programme, approximately 10 days, 90percent of the subjects achieved a reduction of BP to below 140 per 90 millimeters of Mercury. An improved fatty liver index (FLI), a surrogate marker of non-alcoholic fatty liver disease (NAFLD), has been recently described after LF in subjects with and without type 2 diabetes. In a series of 697 subjects, out of whom 264 had a baseline FLI greater than 60 (threshold for fatty liver), a LF of 8.5 plus or minus 4.0 days was carried out, providing 250 kcal per day and large quantities of water. FLI decreased in the whole cohort significantly, -14.02 plus or minus 11.67), the largest benefit being noted in diabetics, -19.15 plus or minus 11.0. BMI decreased by 1.51 plus or minus 0.82 kilograms per square meter, 50 percent of the subjects losing more than 5percent body weight. Improvement of FLI was significantly correlated with the number of fasting days and with the magnitude of BMI reduction. 5 point 1 point 2. LF and microbiota. The contribution of gut microbiota to human diseases is being intensively studied, such as in the case of inflammatory bowel disease, gastric ulcers, NAFLD, obesity, metabolic syndrome as well as associated neurologic disorders. This has led to a detailed evaluation of dietary changes since gut microbiota relies, almost entirely, on host diet composition and food processing capacity, to obtain the metabolic substrates needed to cover its energy requirements, 150 to 450 kcal per day, 628 to 1883 kJ per day. Thus, it seems inevitable that periods of fasting may have an impact on gut microbiota. Ten days of fasting in 15 healthy men led to a decrease in abundance of Lachnospiraceae and Ruminococcaceae, as shown by faecal 16S rRNA gene amplicon sequencing, with a concomitant rise in Bacteroidetes and Proteobacteria (Escherichia coli and Bilophila wadsworthia). These changes were associated with an increase in faecal branched-chain amino acids (BCAA) by 18percent, possibly coming from host-derived compounds such as desquamated cells attacked by the microbiota. The effects were reversed three months after fasting. In obese women on a VLCD (800 kcal per day) for 4 weeks major consistent changes in dominant faecal bacterial communities were reported. A potential benefit of microbiomal changes after fasting is the amelioration of the altered gut microbiome in relapsing-remitting multiple sclerosis. Since CR has a clear anti-inflammatory potential and chronic CR has been shown to attenuate autoimmune encephalomyelitis, therapeutic fasting may be of value in the management of CNS autoimmunity. At present, experimental data have shown raised gut bacterial richness after IF, with enhanced anti-oxidative microbial metabolic pathways. In the same report the Authors describe similar microbiomal changes following IF in a series of MS patients. There are at present only plans to test PF. A controlled clinical trial on LF is ongoing in metabolic syndrome, as well as on IF in relapsing-remitting multiple sclerosis. 5 point 2. Impact of CR and IF on cardiometabolic risk factors in non-obese and obese subjects. The CALERIE, Comprehensive Assessment of the Long-Term Effects of Reducing Intake of Energy, study, enrolled participants with BMI between 22 and 28 kilograms per square meter, proposing a 25percent CR throughout a 2 year period with a control group staying on their current diet. CR significantly improved general health and mood. Weight, body fat, fat mass, and fat free mass as well as cardiometabolic risk factors, meaning lipids, BP, C-reactive protein, and insulin sensitivity index were significantly reduced compared to controls. Concerning obese subjects, the TEMPO, Type of Energy Manipulation for Promoting Optimum Metabolic Health and Body Composition in Obesity, trial compared the long-term impact of a 25 to 35percent CR during 12 months versus a 65 to 75 percent CR during 4 months followed by 8 months of a more moderate energy restriction. Both interventions had a prescribed protein intake of 1.0 gram per kilogram of baseline body weight per day. Among the 101 recruited postmenopausal women, those on the 65 to 75 percent CR lost more abdominal subcutaneous and visceral adipose tissue, but they also lost more whole body and thigh muscle lean mass, proportional to the total weight loss. Moreover, participants allocated to the severe group lost more total hip mineral density. Overall, although these changes were not related to differences in muscle strength, prudence should be exerted as in any manipulation of food intake in men or postmenopausal women with sarcopenia or osteoporosis. Relative to the ADF approach, this was proven to be acceptable and effective (weight loss being 2.5percent and fat mass 4percent) in non-obese subjects who underwent fasting every other day for 22 days. Most recently, following a similar approach, Stekovic et al investigated the direct effects of ADF during 4 weeks in a series of normal weight individuals, leading to an average 37 percent reduction of the daily calorie intake. Improved sense of well-being and improvement of cardiovascular risk markers, in particular triglycerides and LDL-cholesterol, with reduced trunk fat, fat-to-lean ratio and of the inflammatory marker soluble inter-cellular adhesion molecule-1 (sICAM) were detected. An increase of b-hydroxybutyrate occurred even in non-fasting days. Interestingly, the pro-aging amino acid methionine was periodically depleted. Another study compared the effects of CR versus ADF enrolling 100 participants. These were randomised to adhere for 1 year, 6-month intervention followed by 6-month weight maintenance, to one of the following regimens: (1) ADF (25 percent of energy needs on fasting days and 125 percent on alternating feast days), and (2) 75 percent CR. Compared to the control group, no differences between interventions were found either in weight loss, 6.0 percent versus 5.3percent, respectively or in systolic and diastolic BPs, heart rate, triglycerides, fasting glucose, C-reactive protein. Overall, the Authors concluded that ADF is not superior to a CR approach and leads to a higher rate of dropouts, meaning 38 percent (13 out of 34) versus 29 percent (10 out of 35), respectively. It should be recalled that, as elsewhere reviewed, IF generally results in significant reductions in body weight and fat mass, the magnitude of weight losses being quite large 5.0 kilograms, except in trials less than 8 weeks in duration. In obese individuals, BMI 30 kilograms per square meter, Catenacci and colleagues compared ADF with CR during an 8 week-intervention. Neither weight loss nor body composition, lipids and insulin sensitivity differed between groups. After 24 weeks of unsupervised follow up aimed at assessing weight regain, there were no significant differences but changes in percent fat mass and lean mass were more favourable in the ADF group. Patients exercising more during the follow-up had a better weight maintenance, an often documented observation. As above described, IF regimens have been shown to reduce global fat mass and visceral fat, both linked to a reduced risk of diabetes development and thus of cardiovascular outcomes. In overweight or obese patients with type 2 diabetes, 12 weeks of the 5 to 2 diet, 400 to 600 Kcal per two days per week, did not differ from continuous energy restriction, 1200 to 1400 kcal per day, in reducing glycated haemoglobin (0.7 percent). The 5 to 2 group showed an increased tendency to hypoglycaemia despite medication reduction. Application of ADF in obese subjects for 10 weeks reduced total cholesterol, LDL-cholesterol and triglycerides with a switch towards a rise of LDL size by 5percent and a reduction of small LDL by 9percent. A similar lipid improvement was found in obese patients following a modified protocol of ADF with an overall 25 percent reduction of energy needs. Furthermore, a decrement in systolic BP as well as in C-reactive protein were also found. In overweight women, daily energy restriction (600 to 650 kcal per day for 2 days per week, versus intermittent energy and carbohydrate restriction was superior in improving body fat reduction and insulin sensitivity. Finally, in a very recent trial, enrolling 19 metabolic syndrome patients, application of TRE, meaning a reduction of daily eating window from 14 hours to a self-selected 10 hour window over 12 weeks, led to healthier body composition, lowered BP, and decreased levels of cardiovascular risk associated lipids. Moreover, TRE led to a modest, but not significant increase in sleep duration with no impact on sleep efficiency and a trend towards a reduction in physical activity. Conclusions. While clinical studies on fasting have generally show positive effects on health and possibly on life span, documenting benefits and challenges of long-term fasting still needs further studies. The question whether repeated cycles of fasting, as it happens spontaneously in animals, can enhance benefits, is still awaiting a definite answer. After having been rated, in the nineteen sixties, as a successful strategy to treat obesity and comorbidities, additional benefits of fasting other than weight loss have been uncovered. Among others are improvements in glucose regulation, BP and heart rate, as well as abdominal fat loss. The key point in the clinical approach of fasting will be dependent upon acceptance and compliance, as well as on safety. These issues are linked to the emotional and physical well-being, the absence of hunger and the presence of professional guidance. TRE and IF can well fit in everyday life and may be possibly adopted as a lifelong eating behaviour. Long-term fasting, in fact, requires specialised settings, possibly away from the usual environment. An emerging aspect, in the planning of the fasting strategy, is the after-fast period, when food is reintroduced. When the fasting process is reversed, symptom relapses can occur in the absence of specific nutritional guidelines. This may partially reduce the numerous health benefits of fasting. The individual limits of this procedure need to be carefully analysed and, similar to any manipulation of food intake, prudence is necessary, particularly in older subjects and in the case of low BMI, sarcopenia or eating disorders. Figure 1. Metabolic switch from carbohydrates to fatty acids and ketones induced by 10 days of fasting, daily energy intake of about 250 kcal and multidisciplinary programme. A regression spline was fitted on individual acetoacetic values to show the variations in ketosis during the course of the study. T: transition to the fasting mode; RF: progressive reintroduction of food. Figure 2. Representation of signalling pathways modulated fasting. The reduced levels of circulating amino acids and of IGF-1 consequent to fasting repress the activity of m-TOR and its downstream effector leading to an inhibition of global protein synthesis and promote recycling of macromolecules by autophagy stimulation. There is a rise in the AMP-to-ATP ratio leading to the activation of AMPK. SIRT1-driven deacetylation of PGC-1a and FOXO1 transcription factors provides a mechanism by which mitochondrial and lipid oxidation genes can be dynamically controlled in response to energy demand. AMPK: AMP-activated protein kinase; FOXO1: forkhead box O1; IGF: insulin-like growth factor; NAD plus : nicotinamide adenine dinucleotide; PGC-1a: peroxisome proliferator activated receptor c coactivator 1a; m-TOR: mammalian target of Rapamycin; SIRT: sirtuin.

Effects of Intermittent Fasting on Health, Aging, and Disease. By Rafael de Cabo, P-H-D, and Mark P Mattson, P-H-D. 2020. Index of Science Articles: https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html According to Weindruch and Sohal in a 1997 article in the Journal, reducing food availability over a lifetime, caloric restriction, has remarkable effects on aging and the life span in animals. The authors proposed that the health benefits of caloric restriction result from a passive reduction in the production of damaging oxygen free radicals. At the time, it was not generally recognized that because rodents on caloric restriction typically consume their entire daily food allotment within a few hours after its provision, they have a daily fasting period of up to 20 hours, during which ketogenesis occurs. Since then, hundreds of studies in animals and scores of clinical studies of controlled intermittent fasting regimens have been conducted in which metabolic switching from liver-derived glucose to adipose cell–derived ketones occurs daily or several days each week. Although the magnitude of the effect of intermittent fasting on life-span extension is variable, influenced by sex, diet, and genetic factors, studies in mice and nonhuman primates show consistent effects of caloric restriction on the health span, see the studies listed in Section S3 in the Supplementary Appendix, available with the full text of this article at NEJM dot org. Studies in animals and humans have shown that many of the health benefits of intermittent fasting are not simply the result of reduced free-radical production or weight loss. Instead, intermittent fasting elicits evolutionarily conserved, adaptive cellular responses that are integrated between and within organs in a manner that improves glucose regulation, increases stress resistance, and suppresses inflammation. During fasting, cells activate pathways that enhance intrinsic defenses against oxidative and metabolic stress and those that remove or repair damaged molecules, see Figure one. During the feeding period, cells engage in tissue specific processes of growth and plasticity. However, most people consume three meals a day plus snacks, so intermittent fasting does not occur. Preclinical studies consistently show the robust disease-modifying efficacy of intermittent fasting in animal models on a wide range of chronic disorders, including obesity, diabetes, cardiovascular disease, cancers, and neurodegenerative brain diseases. Periodic flipping of the metabolic switch not only provides the ketones that are necessary to fuel cells during the fasting period but also elicits highly orchestrated systemic and cellular responses that carry over into the fed state to bolster mental and physical performance, as well as disease resistance. Here, we review studies in animals and humans that have shown how intermittent fasting affects general health indicators and slows or reverses aging and disease processes. First, we describe the most commonly studied intermittent fasting regimens and the metabolic and cellular responses to intermittent fasting. We then present and discuss findings from preclinical studies and more recent clinical studies that tested intermittent-fasting regimens in healthy persons and in patients with metabolic disorders, obesity, insulin resistance, hypertension, or a combination of these disorders. Finally, we provide practical information on how intermittent fasting regimens can be prescribed and implemented. The practice of long-term fasting, from many days to weeks, is not discussed here, and we refer interested readers to the European clinical experience with such fasting protocols. Intermittent Fasting and Metabolic Switching. Glucose and fatty acids are the main sources of energy for cells. After meals, glucose is used for energy, and fat is stored in adipose tissue as triglycerides. During periods of fasting, triglycerides are broken down to fatty acids and glycerol, which are used for energy. The liver converts fatty acids to ketone bodies, which provide a major source of energy for many tissues, especially the brain, during fasting, Figure 2. In the fed state, blood levels of ketone bodies are low, and in humans, they rise within 8 to 12 hours after the onset of fasting, reaching levels of 0.2 to 0.5 milli Molar, which are maintained through 24 hours, with a subsequent increase to 1 to 2 milli Molar by 48 hours. In rodents, an elevation of plasma ketone levels occurs within 4 to 8 hours after the onset of fasting, reaching milli molar levels within 24 hours. The timing of this response gives some indication of the appropriate periods for fasting in intermittent-fasting regimens. In humans, the three most widely studied intermittent-fasting regimens are alternate-day fasting, 5 to 2 intermittent fasting, fasting 2 days each week, and daily time-restricted feeding. Diets that markedly reduce caloric intake on 1 day or more each week, for example, a reduction to 500 to 700 calories per day, result in elevated levels of ketone bodies on those days. The metabolic switch from the use of glucose as a fuel source to the use of fatty acids and ketone bodies results in a reduced respiratory-exchange ratio, the ratio of carbon dioxide produced to oxygen consumed, indicating the greater metabolic flexibility and efficiency of energy production from fatty acids and ketone bodies. Ketone bodies are not just fuel used during periods of fasting; they are potent signaling molecules with major effects on cell and organ functions. Ketone bodies regulate the expression and activity of many proteins and molecules that are known to influence health and aging. These include peroxisome proliferator activated receptor gamma coactivator one alpha, or PGC one alpha, fibroblast growth factor, nicotinamide adenine dinucleotide, or NAD plus, sirtuins, poly adenosine diphosphate ADP ribose polymerase one, or PARP one, and ADP ribosyl cyclase, or CD38. By influencing these major cellular pathways, ketone bodies produced during fasting have profound effects on systemic metabolism. Moreover, ketone bodies stimulate expression of the gene for brain-derived neurotrophic factor, Figure 2, with implications for brain health and psychiatric and neurodegenerative disorders. How much of the benefit of intermittent fasting is due to metabolic switching and how much is due to weight loss? Many studies have indicated that several of the benefits of intermittent fasting are dissociated from its effects on weight loss. These benefits include improvements in glucose regulation, blood pressure, and heart rate; the efficacy of endurance training; and abdominal fat loss, see Supplementary Section S1. Intermittent Fasting and Stress Resistance. In contrast to people today, our human ancestors did not consume three regularly spaced, large meals, plus snacks, every day, nor did they live a sedentary life. Instead, they were occupied with acquiring food in ecologic niches in which food sources were sparsely distributed. Over time, Homo sapiens underwent evolutionary changes that supported adaptation to such environments, including brain changes that allowed creativity, imagination, and language and physical changes that enabled species members to cover large distances on their own muscle power to stalk prey. The research reviewed here, and discussed in more detail elsewhere, shows that most if not all organ systems respond to intermittent fasting in ways that enable the organism to tolerate or overcome the challenge and then restore homeostasis. Repeated exposure to fasting periods results in lasting adaptive responses that confer resistance to subsequent challenges. Cells respond to intermittent fasting by engaging in a coordinated adaptive stress response that leads to increased expression of antioxidant defenses, DNA repair, protein quality control, mitochondrial biogenesis and autophagy, and down-regulation of inflammation, Figure 3. These adaptive responses to fasting and feeding are conserved across taxa. Cells throughout the bodies and brains of animals maintained on intermittent fasting regimens show improved function and robust resistance to a broad range of potentially damaging insults, including those involving metabolic oxidative, ionic, traumatic, and proteotoxic stress. Intermittent fasting stimulates autophagy and mitophagy while inhibiting the mTOR, mammalian target of rapamycin, protein-synthesis pathway. These responses enable cells to remove oxidatively damaged proteins and mitochondria and recycle undamaged molecular constituents while temporarily reducing global protein synthesis to conserve energy and molecular resources, Figure 3. These pathways are untapped or suppressed in persons who overeat and are sedentary. Effects of Intermittent Fasting on Health and Aging. Until recently, studies of caloric restriction and intermittent fasting focused on aging and the life span. After nearly a century of research on caloric restriction in animals, the overall conclusion was that reduced food intake robustly increases the life span. In one of the earliest studies of intermittent fasting, Goodrick and colleagues reported that the average life span of rats is increased by up to 80 percent when they are maintained on a regimen of alternate-day feeding, started when they are young adults. However, the magnitude of the effects of caloric restriction on the health span and life span varies and can be influenced by sex, diet, age, and genetic factors. A meta-analysis of data available from 1934 to 2012 showed that caloric restriction increases the median life span by 14 to 45 percenr in rats but by only 4 to 27 percent in mice. A study of 41 recombinant inbred strains of mice showed wide variation, ranging from a substantially extended life span to a shortened life span, depending on the strain and sex. However, the study used only one caloric restriction regimen, 40 percent restriction, and did not evaluate health indicators, causes of death, or underlying mechanisms. There was an inverse relationship between adiposity reduction and life span suggesting that animals with a shortened life span had a greater reduction in adiposity and transitioned more rapidly to starvation when subjected to such severe caloric restriction, whereas animals with an extended life span had the least reduction in fat. The discrepant results of two landmark studies in monkeys challenged the link between health-span extension and life-span extension with caloric restriction. One of the studies, at the University of Wisconsin, showed a positive effect of caloric restriction on both health and survival, whereas the other study, at the National Institute on Aging, showed no significant reduction in mortality, despite clear improvements in overall health. Differences in the daily caloric intake, onset of the intervention, diet composition, feeding protocols, sex, and genetic background may explain the differential effects of caloric restriction on life span in the two studies. In humans, intermittent-fasting interventions ameliorate obesity, insulin resistance, dyslipidemia, hypertension, and inflammation. Intermittent fasting seems to confer health benefits to a greater extent than can be attributed just to a reduction in caloric intake. In one trial, 16 healthy participants assigned to a regimen of alternate day fasting for 22 days lost 2.5 percent of their initial weight and 4 percent of fat mass, with a 57 percent decrease in fasting insulin levels. In two other trials, overweight women, approximately 100 women in each trial, were assigned to either a 5 to 2 intermittent fasting regimen or a 25 percent reduction in daily caloric intake. The women in the two groups lost the same amount of weight during the 6-month period, but those in the group assigned to 5 to 2 intermittent fasting had a greater increase in insulin sensitivity and a larger reduction in waist circumference. Physical and Cognitive Effects of Intermittent Fasting. In animals and humans, physical function is improved with intermittent fasting. For example, despite having similar body weight, mice maintained on alternate-day fasting have better running endurance than mice that have unlimited access to food. Balance and coordination are also improved in animals on daily time-restricted feeding or alternate-day fasting regimens. Young men who fast daily for 16 hours lose fat while maintaining muscle mass during 2 months of resistance training. Studies in animals show that intermittent fasting enhances cognition in multiple domains, including spatial memory, associative memory, and working memory; alternate-day fasting and daily caloric restriction reverse the adverse effects of obesity, diabetes, and neuro-inflammation on spatial learning and memory, see Section S4. In a clinical trial, older adults on a short-term regimen of caloric restriction had improved verbal memory. In a study involving overweight adults with mild cognitive impairment, 12 months of caloric restriction led to improvements in verbal memory, executive function, and global cognition. More recently, a large, multicenter, randomized clinical trial showed that 2 years of daily caloric restriction led to a significant improvement in working memory. There is certainly a need to undertake further studies of intermittent fasting and cognition in older people, particularly given the absence of any pharmacologic therapies that influence brain aging and progression of neurodegenerative diseases. Clinical Applications. In this section, we briefly review examples of findings from studies of intermittent fasting in preclinical animal models of disease and in patients with various diseases. Additional published studies are listed in Section S5. Obesity and Diabetes Mellitus. In animal models, intermittent feeding improves insulin sensitivity, prevents obesity caused by a high-fat diet, and ameliorates diabetic retinopathy. On the island of Okinawa, the traditional population typically maintains a regimen of intermittent fasting and has low rates of obesity and diabetes mellitus, as well as extreme longevity. Okinawans typically consume a low-calorie diet from energy-poor but nutrient-rich sources, particularly Okinawan sweet potatoes, other vegetables, and legumes. Likewise, members of the Calorie Restriction Society, who follow the CRON, Calorie Restriction with Optimal Nutrition, diet, have low rates of diabetes mellitus, with low levels of insulin-like growth factor 1, growth hormone, and markers of inflammation and oxidative stress. A multicenter study showed that daily caloric restriction improves many cardio-metabolic risk factors in non-obese humans. Furthermore, six short-term studies involving overweight or obese adults have shown that intermittent fasting is as effective for weight loss as standard diets. Two recent studies showed that daily caloric restriction or 4 to 3 intermittent fasting, 24-hour fasting three times a week, reversed insulin resistance in patients with prediabetes or type 2 diabetes. However, in a 12-month study comparing alternate-day fasting, daily caloric restriction, and a control diet, participants in both intervention groups lost weight but did not have any improvements in insulin sensitivity, lipid levels, or blood pressure, as compared with participants in the control group. Cardiovascular Disease. Intermittent fasting improves multiple indicators of cardiovascular health in animals and humans, including blood pressure; resting heart rate; levels of high-density and low-density lipoprotein, HDL and LDL, cholesterol, triglycerides, glucose, and insulin; and insulin resistance. In addition, intermittent fasting reduces markers of systemic inflammation and oxidative stress that are associated with atherosclerosis. Analyses of electrocardiographic recordings show that intermittent fasting increases heart-rate variability by enhancing parasympathetic tone in rats and humans. The CALERIE, Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy, study showed that a 12 percent reduction in daily calorie intake for a period of 2 years improves many cardiovascular risk factors in non-obese persons. Varady et al reported that alternate-day fasting was effective for weight loss and cardio-protection in normal-weight and overweight adults. Improvements in cardiovascular health indicators typically become evident within 2 to 4 weeks after the start of alternate day fasting and then dissipate over a period of several weeks after resumption of a normal diet. Cancer. More than a century ago, Moreschi and Rous described the beneficial effect of fasting and caloric restriction on tumors in animals. Since then, numerous studies in animals have shown that daily caloric restriction or alternate-day fasting reduces the occurrence of spontaneous tumors during normal aging in rodents and suppresses the growth of many types of induced tumors while increasing their sensitivity to chemotherapy and irradiation. Similarly, intermittent fasting is thought to impair energy metabolism in cancer cells, inhibiting their growth and rendering them susceptible to clinical treatments. The underlying mechanisms involve a reduction of signaling through the insulin and growth hormone receptors and an enhancement of the forkhead box O, FOXO, and nuclear factor erythroid 2 related factor 2, NRF2, transcription factors. Genetic deletion of NRF2 or FOXO1 obliterates the protective effects of intermittent fasting against induced carcinogenesis while preserving extension of the life span, and deletion of FOXO3 preserves the anticancer protection but diminishes the longevity effect. Activation of these transcription factors and downstream targets by means of intermittent fasting may provide protection against cancer while bolstering the stress resistance of normal cells, see Figure one. Clinical trials of intermittent fasting in patients with cancer have been completed or are in progress. Most of the initial trials have focused on compliance, side effects, and characterization of biomarkers. For example, a trial of daily caloric restriction in men with prostate cancer showed excellent adherence, 95 percent and no adverse events. Several case studies involving patients with glioblastoma suggest that intermittent fasting can suppress tumor growth and extend survival. Ongoing trials listed on Clinical Trials dot gov, focus on intermittent fasting in patients with breast, ovarian, prostate, endometrial, and colorectal cancers and glioblastoma, see Supplementary Table S1. Specific intermittent-fasting regimens vary among studies, but all involve imposition of intermittent fasting during chemotherapy. No studies have yet determined whether intermittent fasting affects cancer recurrence in humans. Neurodegenerative Disorders. Epidemiologic data suggest that excessive energy intake, particularly in midlife, increases the risks of stroke, Alzheimer’s disease, and Parkinson’s disease. There is strong preclinical evidence that alternate-day fasting can delay the onset and progression of the disease processes in animal models of Alzheimer’s disease and Parkinson’s disease. Intermittent fasting increases neuronal stress resistance through multiple mechanisms, including bolstering mitochondrial function and stimulating autophagy, neurotrophic-factor production, antioxidant defenses, and DNA repair. Moreover, intermittent fasting enhances GABAergic inhibitory neurotransmission, meaning gamma aminobutyric acid related inhibitory neurotransmission, which can prevent seizures and excitotoxicity. Data from controlled trials of intermittent fasting in persons at risk for or affected by a neurodegenerative disorder are lacking. Ideally, an intervention would be initiated early in the disease process and continued long enough to detect a disease-modifying effect of the intervention. For example a 1-year study. Asthma, Multiple Sclerosis, and Arthritis. Weight loss reduces the symptoms of asthma in obese patients. In one study, patients who adhered to the alternate-day fasting regimen had an elevated serum level of ketone bodies on energy restriction days and lost weight over a 2-month period, during which asthma symptoms and airway resistance were mitigated. A reduction in symptoms was associated with significant reductions in serum levels of markers of inflammation and oxidative stress. Multiple sclerosis is an autoimmune disorder characterized by axon demyelination and neuronal degeneration in the central nervous system. Alternate-day fasting and periodic cycles of 3 consecutive days of energy restriction reduce autoimmune demyelination and improve the functional outcome in a mouse model of multiple sclerosis, experimentally induced autoimmune encephalomyelitis. Two recent pilot studies showed that patients with multiple sclerosis who adhere to intermittent-fasting regimens have reduced symptoms in as short a period as 2 months. Because it reduces inflammation, intermittent fasting would also be expected to be beneficial in rheumatoid arthritis, and indeed, there is evidence supporting its use in patients with arthritis. Surgical and Ischemic Tissue Injury. Intermittent-fasting regimens reduce tissue damage and improve functional outcomes of traumatic and ischemic tissue injury in animal models. Preoperative fasting reduces tissue damage and inflammation and improves the outcomes of surgical procedures. In animal models of vascular surgical injury, 3 days of fasting reduced ischemia–reperfusion injury in the liver and kidneys and, before the injury, resulted in a reduction in trauma-induced carotid-artery intimal hyperplasia. A randomized, multicenter study showed that 2 weeks of preoperative daily energy restriction improves outcomes in patients undergoing gastric-bypass surgery. Such findings suggest that preoperative intermittent fasting can be a safe and effective method of improving surgical outcomes. Several studies have shown beneficial effects of intermittent fasting in animal models of traumatic head or spinal cord injury. Intermittent fasting after injury was also effective in ameliorating cognitive deficits in a mouse model of traumatic brain injury. When initiated either before or after cervical or thoracic spinal cord injury, intermittent fasting reduces tissue damage and improves functional outcomes in rats. Emerging evidence suggests that intermittent fasting may enhance athletic performance and may prove to be a practical approach for reducing the morbidity and mortality associated with traumatic brain and spinal cord injuries in athletes. See the section above on the physical effects of intermittent fasting. Studies in animals have shown that intermittent fasting can protect the brain, heart, liver, and kidneys against ischemic injury. However, the potential therapeutic benefits of intermittent fasting in patients with stroke or myocardial infarction remain to be tested. Practical Considerations. Despite the evidence for the health benefits of intermittent fasting and its applicability to many diseases, there are impediments to the widespread adoption of these eating patterns in the community and by patients. First, a diet of three meals with snacks every day is so ingrained in our culture that a change in this eating pattern will rarely be contemplated by patients or doctors. The abundance of food and extensive marketing in developed nations are also major hurdles to be overcome. Second, on switching to an intermittent fasting regimen, many people will experience hunger, irritability, and a reduced ability to concentrate during periods of food restriction. However, these initial side effects usually disappear within 1 month, and patients should be advised of this fact. Third, most physicians are not trained to prescribe specific intermittent-fasting interventions. Physicians can advise patients to gradually, over a period of several months, reduce the time window during which they consume food each day, with the goal of fasting for 16 to 18 hours a day, Figure 4. Alternatively, physicians can recommend the 5 to 2 intermittent-fasting diet, with 900 to 1000 calories consumed 1 day per week for the first month and then 2 days per week for the second month, followed by further reductions to 750 calories 2 days per week for the third month and, ultimately, 500 calories 2 days per week for the fourth month. A dietitian or nutritionist should be consulted to ensure that the nutritional needs of the patient are being met and to provide continued counseling and education. As with all lifestyle interventions, it is important that physicians provide adequate information, ongoing communication and support, and regular positive reinforcement. Conclusions. Preclinical studies and clinical trials have shown that intermittent fasting has broad-spectrum benefits for many health conditions, such as obesity, diabetes mellitus, cardiovascular disease, cancers, and neurologic disorders. Animal models show that intermittent fasting improves health throughout the life span, whereas clinical studies have mainly involved relatively short term interventions, over a period of months. It remains to be determined whether people can maintain intermittent fasting for years and potentially accrue the benefits seen in animal models. Furthermore, clinical studies have focused mainly on overweight young and middle age adults, and we cannot generalize to other age groups the benefits and safety of intermittent fasting that have been observed in these studies. Although we do not fully understand the specific mechanisms, the beneficial effects of intermittent fasting involve metabolic switching and cellular stress resistance. However, some people are unable or unwilling to adhere to an intermittent-fasting regimen. By further understanding the processes that link intermittent fasting with broad health benefits, we may be able to develop targeted pharmacologic therapies that mimic the effects of intermittent fasting without the need to substantially alter feeding habits. Studies of the mechanisms of caloric restriction and intermittent fasting in animal models have led to the development and testing of pharmacologic interventions that mimic the health and disease-modifying benefits of intermittent fasting. Examples include agents that impose a mild metabolic challenge, 2-deoxyglucose, metformin, and mitochondrial-uncoupling agents, bolster mitochondrial bioenergetics, ketone ester or nicotinamide riboside, or inhibit the mTOR pathway, sirolimus. However, the available data from animal models suggest that the safety and efficacy of such pharmacologic approaches are likely to be inferior to those of intermittent fasting. Figure 1. Cellular Responses to Energy Restriction That Integrate Cycles of Feeding and Fasting with Metabolism. Total energy intake, diet composition, and length of fasting between meals contribute to oscillations in the ratios of the levels of the bioenergetic sensors nicotinamide adenine dinucleotide, known as NAD plus, to NADH, ATP to AMP, and acetyl CoA to CoA. These intermediate energy carriers activate downstream proteins that regulate cell function and stress resistance, including transcription factors such as forkhead box O’s, also called FOXO’s, peroxisome proliferator activated receptor gamma coactivator 1 alpha, abbreviated as PGC 1 alpha, and nuclear factor erythroid 2 related factor 2, referred to as NRF2. Kinases such as AMP kinase, or AMPK, and deacetylases such as sirtuins, abbreviated as SIRT’s. Intermittent fasting triggers neuroendocrine responses and adaptations characterized by low levels of amino acids, glucose, and insulin. Down-regulation of the insulin, insulin-like growth factor 1, IGF-1 signaling pathway and reduction of circulating amino acids repress the activity of mammalian target of rapamycin, abbreviated as mTOR, resulting in inhibition of protein synthesis and stimulation of autophagy. During fasting, the ratio of AMP to ATP is increased and AMPK is activated, triggering repair and inhibition of anabolic processes. Acetyl coenzyme A, CoA, and NAD plus serve as cofactors for epigenetic modifiers such as SIRT’s. SIRT’s deacetylate FOXO’s and PGC-1 alpha, resulting in the expression of genes involved in stress resistance and mitochondrial biogenesis. Collectively, the organism responds to intermittent fasting by minimizing anabolic processes, synthesis, growth, and reproduction, favoring maintenance and repair systems, enhancing stress resistance, recycling damaged molecules, stimulating mitochondrial biogenesis, and promoting cell survival, all of which support improvements in health and disease resistance. The abbreviation cAMP denotes cyclic AMP, CHO carbohydrate, PKA protein kinase A, and redox reduction oxidation. Figure 2. Metabolic Adaptations to Intermittent Fasting. Energy restriction for 10 to 14 hours or more results in depletion of liver glycogen stores and hydrolysis of triglycerides, TG’s, to free fatty acids, FFA’s, in adipocytes. FFA’s released into the circulation are transported into hepatocytes, where they produce the ketone bodies acetoacetate and Beta hydroxyl-butyrate, or Beta HB. FFA’s also activate the transcription factors peroxisome proliferator activated receptor alpha, referred to as PPAR alpha, and activating transcription factor 4, ATF4, resulting in the production and release of fibroblast growth factor 21, known as FGF21, a protein with widespread effects on cells throughout the body and brain. Beta HB and acetoacetate are actively transported into cells where they can be metabolized to acetyl CoA, which enters the tricarboxylic acid, TCA, cycle and generates ATP. Beta HB also has signaling functions, including the activation of transcription factors such as cyclic AMP response element–binding protein, abbreviated as CREB, and nuclear factor Kappa B, shortened as NF kappa B, and the expression of brain-derived neurotrophic factor, BDNF, in neurons. Reduced levels of glucose and amino acids during fasting result in reduced activity of the mTOR pathway and up-regulation of autophagy. In addition, energy restriction stimulates mitochondrial biogenesis and mitochondrial uncoupling. Figure 3. Cellular and Molecular Mechanisms Underlying Improved Organ Function and Resistance to Stress and Disease with Intermittent Metabolic Switching. Periods of dietary energy restriction sufficient to cause depletion of liver glycogen stores trigger a metabolic switch toward use of fatty acids and ketones. Cells and organ systems adapt to this bioenergetic challenge by activating signaling pathways that bolster mitochondrial function, stress resistance, and antioxidant defenses while up-regulating autophagy to remove damaged molecules and recycle their components. During the period of energy restriction, cells adopt a stress-resistance mode through reduction in insulin signaling and overall protein synthesis. Exercise enhances these effects of fasting. On recovery from fasting, eating and sleeping, glucose levels increase, ketone levels plummet, and cells increase protein synthesis, undergoing growth and repair. Maintenance of an intermittent fasting regimen, particularly when combined with regular exercise, results in many long-term adaptations that improve mental and physical performance and increase disease resistance. HRV denotes heart-rate variability. Figure 4. Incorporation of Intermittent-Fasting Patterns into Health Care Practice and Lifestyles. As a component of medical school training in disease prevention, students could learn the basics of how intermittent fasting affects metabolism and how cells and organs respond adaptively to intermittent fasting, the major indications for intermittent fasting, obesity, diabetes, cardiovascular disease, and cancers, and how to implement intermittent fasting prescriptions to maximize long-term benefits. Physicians can incorporate intermittent-fasting prescriptions for early intervention in patients with a range of chronic conditions or at risk for such conditions, particularly those conditions associated with overeating and a sedentary lifestyle. One can envision inpatient and outpatient facilities staffed by experts in diet, nutrition, exercise, and psychology that will help patients make the transition to sustainable intermittent-fasting and exercise regimens, covered by basic health insurance policies. As an example of a specific prescription, the patient could choose either a daily time-restricted feeding regimen, an 18-hour fasting period and a 6-hour eating period, or the 5 to 2 intermittent-fasting regimen, fasting, meaning an intake of 500 calories, 2 days per week, with a 4-month transition period to accomplish the goal. To facilitate adherence to the prescription, the physician’s staff should be in frequent contact with the patient during the 4-month period and should closely monitor the patient’s body weight and glucose and ketone levels.

https://doi.org/10.1111/jcmm.15194 Index Of Science Articles: https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Mitochondrial biogenesis: An update. Lucia-Doina Popov. Journal of Cellular and Molecular Medicine 24, pages 4892 to 4899, 2020. Abstract. In response to the energy demand triggered by developmental signals and environmental stressors, cells launch the mitochondrial biogenesis process. This is a self-renewal route, by which new mitochondria are generated from the ones already existing. Recently, considerable progress has been made in deciphering mitochondrial biogenesis-related proteins and genes that function in health and in pathology related circumstances. However, an outlook on the intracellular mechanisms shared by the main players that drive mitochondrial biogenesis machinery is still missing. Here, we provide such a view by focusing on the following issues: (a) The role of mitochondrial biogenesis in homeostasis of the mitochondrial mass and function, (b) The signalling pathways beyond the induction, promotion, stimulation and inhibition of mitochondrial biogenesis and, (c) The therapeutic applications aiming at the repair and regeneration of defective mitochondrial biogenesis, in ageing, metabolic diseases, neurodegeneration and cancer. The review is concluded by the perspectives of mitochondrial medicine and research. One, mitochondrial homeostasis. Mitochondria are the major source of energy for cellular activity, by ATP generation via oxidative phosphorylation. Emerging evidence of the last decade indicates that mitochondria form a highly dynamic intracellular network that executes the “quality control” of the organelle's population in a process that implies their fusion, fission and autophagic degradation, known as “mitophagy”. Mitochondria regulate the operation of intracellular signalling cascades, generate reactive oxygen species (ROS), execute fatty acid beta-oxidation, participate in amino acid metabolism, pyridine synthesis, phospholipid modifications, calcium regulation and cells survival, senescence and death. The homeostasis of any healthy cell implies also a controlled regulation of mitochondrial mass and function, as an adaptive response to safeguard the mitochondrial (mt) DNA and to meet the energy demands vital for cellular function. Mitochondrial homeostasis is preserved by the fine co-ordination between two opposing processes: generation of new mitochondria, by mitochondrial biogenesis, and the removal of damaged mitochondria, by mitophagy. Among the specific molecules involved in this fine-tuning, the recent data highlight the peroxisome proliferator activated receptor gamma coactivator, PGC one alpha, the main regulator of mitochondrial biogenesis, the PTEN-induced putative kinase 1, shortened to PINK 1Pakin, that activates protein synthesis in damaged mitochondria, and the ligand-activated transcription factor aryl hydrocarbon receptor, that functions also as a protector from oxidative stress. In examining mitochondrial homeostasis, one should consider the particular traits of these organelles in eukaryotic cells: (a) They have a prokaryotic origin and were acquired by eukaryoticcells via an endosymbiotic event. (b) Are semi-autonomous organelles: synthesize a rather small number of proteins by transcription and replication of mt DNA-encoded genes, while the larger proportion of mitochondrial proteome, around sixty to seventy percent, or more than ninety-five percent, is nuclear-encoded, synthesized on cytosolic ribosomes, and finally, sorted and imported to the appropriate intra-mitochondrial location. The encoding factors for nuclear genes identified so far are as follows: PGC-1 alpha, the transcription factor A (TFAM), the uncoupling proteins 2 (UCP2) and the uncoupling proteins 3 (UCP3), (c) Mitochondrial biogenesis implies a specific route consisting in the recruitment of the novel proteins by the pre-existing mitochondria, followed by their fragmentation, via fission. Associated with the rapid cell growth and proliferation, these events ensure the constant renewal of the mitochondrial population. Uncovering the complexity of the mitochondrial biogenesis operation is an exciting ongoing topic, and its main features are briefly examined next. Two. Mitochondrial biogenesis machinery, the associated signalling pathways. The process of mitochondrial biogenesis takes place mainly in healthy cells. Interesting, in cancerous cells enhanced oxidative phosphorylation and mitochondrial biogenesis were correlated with invasion and metastasis. It engages co-ordination between the mitochondrial and the nuclear genomes, in a complex and multistep process, Figure 1, that involves: One. mt DNA transcription and translation. Mt DNA transcription is activated by the family of PGC-1 proteins, PGC-1 alpha, PGC-1 beta and PGC-1, from which PGC-1 alpha is considered the master regulator of mitochondrial biogenesis. The pathway is initiated by PGC-1 alpha activation, by either phosphorylation or deacetylation, followed by stimulation of a series of nuclear transcription factors, that is the nuclear respiratory factor-1, NRF-1, NRF-2 and oestrogen-related receptor-alpha, ERR-alpha, and by the increase in expression of TFAM, the final effector of mt DNA transcription and replication. Next, translation of the mt DNA-encoded genes into proteins takes place with the assistance of specific translation factors, encoded by nuclear DNA, n DNA, such as the initiation factor 2 and 3, mtIF2 and mtIF3, the elongation factors Tu, Ts and G1, mtEFTu, mtEFTs and mtEFG1, the translational release factor1-like, mtRF1L, and the recycling factors, mtRRF1 and mtRRF. Furthermore, the levels of mitochondrial proteins are regulated by the translational activator of cytochrome c oxidase 1, TACO1, that binds the mitochondrial RNA, mRNA. Two. Synthesis, import and assembly of mitochondrial proteins encoded by nDNA. These mitochondrial proteins originate from the preproteins synthesized within the cytosol and provided with an amino-terminal cleavable targeting signal. The translocase TIM23 directs the signal of preproteins towards the mitochondrial matrix, where they assemble, and are sorted to a precise intra-mitochondrial location, that is the matrix or the inner mitochondrial membrane, IMM. The energy required for driving this import pathway is provided by the mitochondrial membrane potential and the ATP, by oxidative phosphorylation. The biogenesis of the outer mitochondrial membrane, OMM, proteins has been studied so far in unicellular organisms, such as the yeast Saccharomyces cerevisiae; the OMM functions as an interface with the cytosol and is particularly important for mitochondrial dynamic changes, fission, fusion, and interaction with other intracellular organelles. The mitochondrial biogenesis markers are the mt DNA copy numbers, the elevated mt DNA, n DNA ratio and the level of mitochondrial gene expression. In cancer cells, an augmented expression of PGC-1 alpha, NRF1 and TFAM was reported, although these cells have a reduced number of mitochondria. Moreover, a recent report underlines that the use of TFAM level as a biogenesis marker is questionable, as it does not always match the mt DNA copy number, and the expression of mt DNA-encoded polypeptides. What are the consequences of mitochondrial biogenesis? The current data acknowledge the increase in the oxidative phosphorylation capacity, the diminishment of pathologic oxidative stress and the repair of mitochondrial-associated dysfunctions. How can mitochondrial biogenesis be measured? Reliable strategies are based on the magnitude of mt DNA synthesis and of mitochondrial membrane phospholipids. In this context, two cautions are noted: (a) a change in the number of mitochondria is not indicative of biogenesis, as their amount is not exclusively due to synthesis, and (b) mitochondrial biogenesis may produce detrimental effects, such as the import of misfolded proteins into the organelle, and the silencing of the unfolded protein response in the endoplasmic reticulum. Two point one. Mitochondrial biogenesis inductors, promoters. Mitochondrial biogenesis induction is associated with activation of transcription factors that act on mitochondrial genes and with up-regulation of local translation of mitochondrial proteins. These effects are produced in response to several natural products, such as 6-gingerol, the main active component of the ginger extracts, and Ursolic acid, a natural triterpene. In contradistinction, relatively few synthetic drugs have been identified as mitochondrial biogenesis inductors. Reportedly, the following signalling pathways sustain transcription activation during mitochondrial biogenesis: The AMPK PGC-1 alpha pathway used by C1q, tumour necrosis factor-related protein-3, CTRP3, to promote biogenesis in cardiomyocytes, and by the ginger extract, in both mice and HepG2 cells. Furthermore, AMPK phosphorylates and activates histone acetyltransferase 1, HAT1, creating a more relaxed chromatin-DNA structure that favours transcription. AMPK phosphorylates also the epigenetic factor DNA methyltransferase 1, DNMT1, that limits transcription factors access to promoters. A recent report shows that the diterpene alkaloid benzoylaconine activates AMPK signaling cascade and stimulates mitochondrial biogenesis. The induction of PGC-1 alpha along with its downstream target genes, NRF1 and TFAM. Such signalling cascade was identified in pancreatic MIN6 beta-cells, after the humanin treatment, and in 3T3-L1 pre-adipocytes, after salicylate medication. Activation of PGC-1 alpha signalling pathway is mediated also by the transcription factor cAMP response element-binding protein CREB. It binds to certain DNA sequences, the cAMP response elements, and subsequently increases, decreases gene transcription. In endothelial cells, CREB, PGC-1 alpha pathway promotes mitochondrial biogenesis by activation of G protein-coupled receptor, TGR5, the route operating also after lixisenatide medication, a drug approved by the US Food and Drug Administration for the treatment of type 2 diabetes. Stimulation of the G beta gamma, a component of heterotrimeric G proteins, Akt-eNOS-sGC, soluble guanylatecyclase, pathway by the beta 2 adrenergic receptor agonists, such as formoterol, allowing recovery from acute and chronic degenerative diseases, and carvedilol, employed in heart failure. The return to normal of the Akt, transcription factor FoxO3a axis under the influence of dietary beta-hydroxy-beta-methylbutyrate, HMB, is another condition that improves mitochondrial biogenesis. The sirtuins assistance in transcription: it is known that the silent information regulator-1 (SIRT1) activates the PGC-1 alpha-mediated transcription of nuclear and mitochondrial genes encoding for proteins during mitochondria proliferation, oxidative phosphorylation and energy production, while SIRT3 stimulates the proteins important for oxidative phosphorylation, tricarboxylic acid cycle and fatty-acid oxidation, and indirectly, the PGC-1 alpha and AMPK. If the above data is taken together, it is obvious that up-regulation of transcription factors is a key event in mitochondrial biogenesis. However, depending on ligands specificity, unwanted genes may be equally activated, conducting to detrimental, neurological and hyperproliferative, effects. Up-regulation of mitochondrial proteins translation is associated with exercise-induced mitochondrial biogenesis, in the plantaris muscle. An interesting mechanism implied in biogenesis of healthy mitochondria was deciphered in Drosphila: the MDI protein of the mitochondrial OM recruits the translational stimulator La-related protein (Larp) and promotes the synthesis, on the mitochondrial surface, of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes. 2.2. Mitochondrial biogenesis stimulators and inhibitors. In physiological conditions, the response of cells to energy demands leads to either up or down-regulation of the transcription factors that stimulate and, or inhibit mitochondrial biogenesis, respectively. The pathology-associated disturbances of mitochondrial biogenesis consist in an impeded mitochondrial biogenesis, a condition in which stimulation of the declined process is required, or in abnormal higher levels of this process, when and a diminishment is necessary. Examples of efficient stimulators of mitochondrial biogenesis are the following: formoterol, used for treating podocytopathies, resveratrol, a polyphenol, that prevents rotenone-induced neuronal degeneration, acetylcholine, protector in hypoxia, reoxygenation injury, adiponectin, a cardioprotector in diabetes, and tetrahydrobiopterin, helpful for the cardiac contractility. The cellular mechanism beyond the above stimulatory effects on mitochondrial biogenesis entails the up-regulated expression of the transcriptional regulator PGC-1 alpha. Recently, normalization of Akt, FoxO3 axis was reported to be involved in the protective effects of dietary HMB against lipopolysaccharide (LPS)-induced muscle atrophy. Another regulatory mechanism is based on phosphorylation of GSK-3 beta exerted by arachidonyl-2-chloroethylamide, ACEA, a selective agonist of cannabinoid receptor1, effective at the beginning of cerebral ischemia. Several natural extracts have been found to stimulate mitochondrial biogenesis. Such regulatory effects were recently reported for the Kaempferia parviflora extracts, containing methoxy flavones, that act through the SIRT1, AMPK, PGC-1 alpha, PPAR delta pathway. For tangeretin, a polymethoxylated flavonoid of mandarin fruits, activator of AMPK, PGC-1 alpha pathway, for salidroside, isolated from Rhodiola rosea, that stimulates the miR22, SIRT1 pathway. For the spice saffron, Crocus Sativus, that augmented NRF-1 gene expression in exercised rats, and for the natural precursor of resveratrol, polydatin that enhances SIRT1 expression. The inhibitors of mitochondrial biogenesis. These down-regulate the expression of the associated-transcription factors, such as PGC-1 alpha, TFAM and AMPK. The activity of PGC-1 alpha pathway is reduced by miR-130b-p, 2-methoxyestradiol, cyclosporine A, XCT790, a potent and selective inhibitor of the oestrogen related receptor alpha, and the high glucose high fat environment. The down-regulation of TFAM takes place at the use of the local anaesthetic ropivacaine and at the in vitro treatment of cells with silica nanoparticles. Furthermore, the diminished AMPK expression explains resistin inhibitory effects on mitochondrial biogenesis. It is evident that reduced biogenesis is accompanied by other mitochondrial dysfunctions, such as an impaired ATP synthesis capacity leading to acceleration of mitophagy, critical for cell health. A reduced mt DNA, nuclear ratio has also been reported to be associated with the impairment of biogenesis, altered biogenesis. The examination of the two opposite sides of mitochondrial biogenesis, that is the impairment, such as in ageing, metabolic and neurodegenerative diseases, and the abnormal intensification, in some tumours, conducted in the last decade have led to the identification of several strategies adequate for exploitation in therapy. Examples are discussed next. Three. Dysregulation of mitochondrial biogenesis and repair strategies. 3.1 Ageing. The cells senescence and the consequent ageing is associated with the impairment of mitochondrial biogenesis and bioenergetics potential, the decrease in mitochondrial dynamics, the altered quality control, the failure in mt DNA repair, the accumulation of mt DNA mutations and the decline in mitophagy. The main factors involved in ageing effects on mitochondrial biogenesis are the reduced activity of AMPK alpha and the decreased expression of SIRT1, PGC-1 alpha, TFAM and NRF-1 and 2, along with the regulatory loop that engages PGC-1 alpha and NRF-2 interaction. Strategies to prevent, delay age-associated decline in mitochondrial biogenesis consists in stimulation of PGC-1 alpha signalling with tetrahydrobiopterin or with resveratrol, modulation of TFAM binding to mt DNA, mitophagy regulation, dietary supplementation with acetyl-l-carnitine, ALCAR, cells exposure to gomisin A, a bio-active compound isolated from the fruit of Schisandra chinensis, regular exercise training, and the calorie restriction. The current endeavours aimed to delay, counteract the age-associated decline of mitochondrial biogenesis may have translational relevance for promotion of healthy ageing, for protection against age-related pathologies and for the improvement of the quality of life of the elderly. 3.2 Metabolic diseases. The impairment of mitochondrial biogenesis and function has been linked to metabolic diseases such as type 2 diabetes and obesity. In a diabetic’ kidney, the mechanism beyond the reduced mitochondrial biogenesis implies the decrease of PGC-1 alpha, AMPK, SIRT-1 signalling pathway. In placentae of diabetic mothers, impaired mitochondrial biogenesis engages PGC-1 alpha, TFAM signalling pathway and is mainly present at male offspring; this trait may explain the propensity for development of future metabolic diseases in adult males. In a diabetics’ heart, earlier studies reported that hypoadiponectinemia impaired AMPK-PGC-1 alpha signaling. More recently, in a model for type 2 diabetes, a high glucose, high-fat medium, adiponectin was found to partial rescue mitochondrial biogenesis in cardiomyocytes, via PGC-1 alpha mediated signalling. This pathway participates in cardioprotection and is evaluated as a novel therapeutic target. Mitochondrion is regarded now as a possible target for the prevention and treatment of chronic metabolic disorders; in this context, the endurance exercise it is routinely used to alleviate the reduced mitochondrial biogenesis. Furthermore, the antidiabetic effect of mitochondrial biogenesis enhancers, such as Spirulina platensis and Alogliptin, a dipeptidyl-peptidase-4 inhibitor, were recently reported. Another ongoing topic is the regulation of mitochondrial biogenesis in adipocytes. The obesity associated signalling entails hyperacetylation of PGC-1 alpha, and induction of pAMPK, PGC-1 alpha, NRF-1 and TFAM, after the salicylate treatment of pre-adipocytes. Activation of AMPK along with stimulation of mitochondrial gene expression and mt DNA replication explain the beneficial effects of isorhamnetin, 3-O-methyl quercetin, on adipocyte mitochondrial biogenesis. AMPK activation contributes also to the anti-obesity effects of zeaxanthin, an oxygenated carotenoid, that promotes mitochondrial biogenesis and expression of brown and beige adipogenesis markers. The regulation of mitochondrial biogenesis in beige adipocytes, in the course of browning, involves PGC-1 alpha signalling, associated with miR-494-3p expression. Other stimulatory factors of mitochondrial biogenesis are NRF-1 and the mitochondrial transcription factor A that intervene in the effect of metformin on brown adipocytes. In contradistinction, decreased UCP1 expression explains the reduced mitochondrial biogenesis generated by arsenite in brown adipocytes. The above basic findings may be used as a basis for further clinical approaches in metabolic diseases. 3.3 Neurodegeneration. Mitochondrial biogenesis is a potential novel therapeutic target for neurodegenerative diseases treatment including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). Although this strategy is based on a plethora of basic and (pre)clinical results, in the present overview only the data on the intracellular pathways beyond mitochondrial biogenesis are mentioned. In AD and PD, mitochondrial biogenesis is impaired and augmenting this process is turned into a therapeutic approach. The intracellular mechanism was uncovered in hippocampal neurons, where amyloid beta 25 to 35 inhibits AMPK-SIRT-1, PGC-1 alpha pathway. Recent reports indicate melatonin, as a promoter of mitochondrial biogenesis, along with resveratrol, that induced PGC-1 alpha and mt TFA expression, berberine, a natural AMPK activator, that stimulates PGC-1 alpha and NRF-2 in neuronal cells, and rotenone, an inhibitor of Complex One. Moreover, necdin, a melanoma antigen, prevents mitochondria-associated neurodegeneration by binding to PGC-1 alpha and suppressing its proteolytic degradation in the ubiquitin-proteasomal system. Interestingly, mt DNA replication appears to be an early response to neurodegeneration associated stress and a precursor for mitochondrial biogenesis in axons. Distinctly, the neurotoxic effect of some medicines is accompanied by reduced mitochondrial biogenesis. An example is the local anaesthetic ropivacaine, employed in medical and dental care, that reduces expression of mitochondrial regulators PGC-1 alpha, NRF-1 and TFAM. 3.4 Cancer. It is known that mitochondrial biogenesis targeted therapies are efficient for the prevention and treatment of relapsed and resistant cancers. The pointed intracellular pathways are PGC-1 alpha, important also for the cells adaptive response against chemotherapeutic stress, AMPK, a proximal signalling step for mitochondrial biogenesis, and dynamin-related protein-1 (Drp1) up-regulation, accompanied by augmented levels of PGC-1 alpha, NRF-1 and TFAM. Among the modulators of mitochondrial biogenesis, sulforaphane, a sulphur-rich compound found in cruciferous vegetables, is considered a potential antineoplastic agent; in prostate cancer cells, it stabilizes NRF-2, increases the expression of PGC-1 alpha and decreases the level of hypoxia-inducible factor-1 alpha, HIF-1 alpha. Chemotherapy medication with cisplatin stimulates PGC-1 alpha expression and up-regulates mitochondrial biogenesis. Mitochondrial biogenesis is increased in some invasive cancer cells, such as osteosarcoma; the use of 2-methoxyestradiol inhibits biogenesis, via regulation of PGC-1 alpha, COX1 and SIRT-3. In this circumstance, the strategy to stop the increased propagation of cancer stem cells consists in doxycycline inhibition of mitochondrial biogenesis. Four. Conclusion and perspectives. The mitochondrial biogenesis is a complex biological process, Figure 1, that controls organelle's self-renewal and the maintenance of mt DNA, ensuing cell homeostasis. This topic is under intense investigation at present. The intracellular signalling pathways uncovered so far identified PGC-1 alpha as a master regulator of mitochondrial biogenesis, implicated in the response to several inductors, promoters, stimulators and inhibitors. Dysregulated mitochondrial biogenesis occurs not only in senescence and ageing, but also in metabolic diseases, neurodegeneration and cancer, and is potentially ameliorated by the novel mitochondria-based therapies. However, there are still several issues that require an answer, such as the association between impaired mitochondrial biogenesis and the early stage of myocardial remodeling, the neuron-specific mechanism of mitochondrial biogenesis, and the limitation of osteoarthrosis progression in chondrocytes, among others. Challenging topics are the exploitation of mitochondria based therapies for the treatment of chronic degenerative diseases and for the prevention of cancer. FIGURE 1. Mitochondrial biogenesis in brief: the central role of PGC-1 alpha activation and the contribution of proteins encoded by both nuclear and mitochondrial genomes, n DNA and mt DNA, to enhance the mitochondrial proteins content. AMPK, 5' adenosine monophosphate activated protein kinase; ERR, the oestrogen-related receptor; NRF, the nuclear respiratory factor; PGC-1 alpha, the peroxisome proliferator-activated receptor gamma coactivator-1 alpha; SIRT-1, the silent information regulator-1; TFAM, the transcription factor alpha; TIM 23, translocase.

The radiation of a uniformly accelerated charge is beyond the horizon: A simple derivation. Camila de Almeida. Departamento de Matematica Aplicada, Campinas. Instituto de Fısica, Universidade de Sao Paulo, Brazil. By exploring some elementary consequences of the covariance of Maxwell’s equations under general coordinate transformations, we show that even though inertial observers can detect electromagnetic radiation emitted from a uniformly accelerated charge, co moving observers will see only a static electric field. This analysis can add insight into one of the most celebrated paradoxes of the last century. One. Introduction. Paradoxes provide good opportunities to learn and teach physics. The long-standing paradox about the electromagnetic radiation emitted by a uniformly accelerated charge has received considerable attention. Eminent figures such as Pauli, Born, Sommerfeld, Schott, von Laue, and many others have contributed to this debate with different answers. The relevant questions we consider are: Does a uniformly accelerated charge actually radiate? In a constant gravitational field should free-falling observers detect any radiation emitted by free-falling charges? Is the equivalence principle valid for such situations? If the answer to the first question is affirmative, a free-falling charge will radiate according to an observer at rest, because in a constant gravitational field, any particle should move with uniform acceleration. However, an observer falling freely with the charge would observe it at rest and no radiation at all. How can this answer be compatible with an affirmative answer to the first question? Moreover, if the equivalence principle is assumed to be valid, we would conclude that a charged particle at rest on a table should radiate, because for free-falling inertial observers the particle is accelerating. To explain this puzzle, we need to recognize that the concept of radiation has no absolute meaning, and that it depends both on the radiation field and the state of motion of the observer. This dependence is the main conclusion of a celebrated and long debate, exhaustively presented in the recent series of papers by Eriksen and Gron, where the reader can find relevant references. Inertial observers have no doubts about the answer to the first question. They will answer it affirmatively by using special relativity and Maxwell’s equations, as it is done in classical electrodynamics texts, see, for example Reference 6 whose conventions we adopt. Nevertheless, co-moving observers, that is, accelerated observers with respect to whom the charge is at rest, will not detect any radiation because the radiation field is confined to a space-time region beyond a horizon that they cannot access. As we will see, uniformly accelerated observers are able, in principle, to detect electromagnetic radiation from an inertial charge. These observations are enough to solve the paradoxes posed by the three questions. The last two conclusions are obtained by considering Maxwell’s equations for non-inertial observers. We will show that we can conclude that co moving observers have no access to the radiation field of a uniformly accelerated charge. The concept of a horizon emerges naturally in this context. Our approach is inspired by the recent analysis of this problem by Gupta and Padmanabhan whose result is much stronger. They show how to calculate the radiation field for an arbitrarily moving charge by solving Maxwell’s equations in the, non-inertial, reference frame where the charge is at rest, and then transforming the electromagnetic field to the inertial reference frame by exploiting the tensorial character of Maxwell’s equations. As stressed in Reference 7, this is a remarkable result because the spatial and time dependences of the radiation field in the inertial frame have to be converted into the geometrical properties of the background metric of the reference frame where the charge is static. We take the converse approach here and will show that it is simple to conclude that the electromagnetic field generated by a uniformly accelerated charge is observed by a co moving observer as a purely electrostatic field. Two. The hyperbolic motion. The speed of light c is the maximum speed that a physical body can attain. Thus the uniformly accelerated motion of a particle should have v goes to c as tau goes to plus or minus infinity where is the proper time as measured by a co-moving clock. It is easy to deduce that a particle moving with constant proper acceleration g along the z direction has a hyperbolic world-line given by the curve r alpha (Of tau): Equations one-a to one-d, in the text. For example c t equals c squared over g times hyperbolic sine of g tau over c. There is no loss of generality if the motion is restricted to the z direction. Such a world-line is displayed in Figure one. “A.” The Horizons. The velocity of a particle according to Equation one approaches plus or minus c as tau goes to plus or minus infinity, and its trajectory tends asymptotically to the lines plus or minus c t equals z, with z greater than zero as shown in Figure one. Consider the point Q. Its past light-cone intersects the hyperbolic trajectory. Indeed, a large part of the trajectory, tau is less than tau Q ret, is entirely contained inside its past light-cone, implying that the point Q could be causally influenced by signals emitted by the particle for tau is less than or equal to tau Q ret. No signal emitted for tau greater than tau Q ret will influence Q, because the points of the trajectory with tau greater than tau Q ret are not contained in the past light-cone of Q. Moreover, a signal emitted in the space time Q will affect only the region corresponding to its future light-cone, implying that no signal emitted in Q will reach the particle moving according to Equation one. The line ct equals z acts as a future event horizon for regions One and Four, or, equivalently, a past event horizon for regions Two and Three. No signal emitted in regions Two or Three will reach regions One and Four, although signals emitted in One or Four can cross the line and enter into regions Two and Three. Analogously, the line minus c t equals is a future event horizon for regions three and four, or a past event horizon for one and two. Because the hyperbolic trajectory is entirely contained in region One, the lines ct equals z and minus ct equals z act, respectively, as the future and past horizons for a particle under uniformly accelerated motion. We will see that such structures appear naturally when we consider the radiation emitted by a uniformly accelerated charged particle. B. The Radiation in the inertial frame. The metric of the inertial Minkowski spacetime is given by equation two: D s squared equals eta “A”, “B” d x “A” d X “B” equals c squared d t squared minus d x squared minus d y squared minus d z squared. Where x “A” equals ct, x, y, z. Maxwell’s equations are not only Lorentz invariant, they can also be cast in a generally covariant way, valid for any reference frame with the metric G “A”, “B”. The derivative of the Faraday tensor, equation three. D “A” of F B C plus D “B” of F C “A” plus D “C” of F “A” B equals zero. And equation four, the derivative of the Faraday tensor equals the current density. G equals the determinant of G “A” B, and J B is the external 4-current. Equation three is automatically satisfied if the 4-potential “A” b is introduced: F b c equals d b of “A” c minus d c of “A” b. In the inertial Minkowski frame, the radiation emitted by a uniformly accelerated charge e corresponds to the solution of Equations three and four with G “A” B equals eta “A” b. The Current density J “A” (Of x) equals the integral of a single point charge. Such a solution for the Faraday tensor is given by equation five, for the conditions of equation six. In the inertial frame we can read from the Faraday tensor F “A”, “B” the usual three-dimensional components of the electric and magnetic field as equation eight, and obtain after some algebra the components of the electric and magnetic fields as in equations nine. These are the electromagnetic fields due to a uniformly accelerated charge moving according to Equation one. The radiation content can be extracted by separating the components that drop off as one over R from the usual Coulomb one over r squared fields. As shown in Figure one, only regions One and Two can experience the fields in Equation nine. The main conclusion of Reference four is that, even though radiation components are present in both regions, only observations performed in region Two, and, perhaps, also on the boundary ct equals z between regions One and Two, would allow us to detect unambiguously the radiation emitted by the charge. This conclusion implies that the co-moving observer would not detect any radiation at all, because region Two is inaccessible to uniformly accelerated observers. Although this conclusion is correct, its logical derivation is involved and not intuitive. Two regions of space-time which have qualitatively distinct behavior for the radiation field according to inertial observers are identified and, then, it is shown that the co-moving observers have access only to the region where inertial observers are not able to detect any radiation field. However, this conclusion does not directly imply that the co-moving observers cannot detect the radiation because, as we have discussed, the detection of radiation has no absolute meaning because the detection depends both on the radiation field and the state of motion of the observer. C. No Radiation in the co-moving frame. We can show directly that a co-moving observer will observe the fields in Equation nine as a static electric field. The reference frame of a uniformly accelerated observer corresponds to the Rindler space-time, which in our case is spanned by the co-ordinates x prime a (Of x) equals c tau (Of t, z), x, y, Xi (OF t, z) defined by equation ten. T equals the square root of two xi over g times shine g tau over c. And z equals c times square root two xi over g times gosh g tau over c. With Xi greater than zero. The particle under the hyperbolic motion of equations one in the Rindler reference frame is at rest at xi equals c squared over two g, and its proper time is measured by tau. In these coordinates the spacetime interval is given by: D s squared equals G “A”, “B” d x “A”, d x “B”. Note that the coordinates defined by Equation ten cover only region One of the original Minkowski spacetime. Because static observers, where Xi is a constant, correspond to uniformly accelerating observers in the original Minkowski spacetime, their velocity in the inertial frame will approach c as tau goes to infinity, implying that no signal coming from region Two will ever reach them see Figure one. As mentioned, the line c t equals z behaves as an event horizon, and no signal emitted in regions two or three can escape into regions one and four. The coordinate transformation, ten, can be used to obtain the solution of Maxwell’s equations, three and four for the Rindler spacetime with a charge e at rest in Xi equals c squared over two g. Recall that the electromagnetic field F “A”, “B” is a tensor and hence under a coordinate transformation x “A” goes to X “A” prime it transforms as equation twelve. Because Maxwell’s equations (3) and (4) are covariant under general coordinate transformations, F prime “A”, “B” will be a solution for the coordinate system x prime “A” (Of x) if the Faraday tensor F “A” “B” is a solution of Equations three and four in the coordinate system x “A”. The magnetic components of F prime “A”, “B” are given by equation set thirteen. Strictly speaking, we need to be careful about the interpretation of F prime 1, 3 F prime 2, 3 and F prime 1, 2 as the components of the magnetic field as observed in the Rindler reference frame. A proper definition of electric and magnetic fields for non-inertial reference frames can be obtained from the Lorentz force formula. This issue will not be relevant to our analysis. By using equation nine, the components of the faraday tensor can be written down, as in equation fourteen. From the equations ten, which relate the transformation between proper time tau and rest frame time t, we can calculate. D tau, d t equals z over two xi. D tau, d z equals minus t over two xi. D xi, d t equals minus g t. D xi, d z equals g z over c squared. This leads to F prime 1,3 equals F prime 2,3 equals zero. Therefore the only non-vanishing components of the electromagnetic field experienced by Co-moving observers are F prime 0-1, 0-2 and 0-3. Because the only non-vanishing component of the 4-current J rime “A” is J prime zero for a static charge in the Rindler reference frame, we conclude from Equation four that the remaining non-vanishing components of the electromagnetic field are static, that is, d F prime zero one, with respect to 0, equals d F prime zero two, with respect to 0, equals d F prime zero three, with respect to 0, equals zero. So that there is no radiation field in region one, the Rindler reference frame. This result answers our question. A co-moving observer will not detect any radiation from a uniformly accelerated charge. The co-moving observer can receive signals only from regions one and four. The field emitted by the accelerated charge does not reach region four, and in region one, it is interpreted by the co-moving observer as a static field. We note that essentially the same argument was used by Rohrlich to show that in a static homogeneous gravitational field, static observers do not detect any radiation from static charges. The situation is qualitatively different beyond the horizon in region two. Although uniformly accelerated observers will never receive any information from region two, they can affect this region. The coordinate system ten can be extended to include region two by considering Xi is less than zero and the equation set sixteen. T, and z are expressible using transformations of the hyperbolic cos and sine of the proper time. The metric eleven and the expressions fifteen, valid for region one, also hold in region two, but with a crucial difference due to the change of sign of the metric components: in region two, xi instead of tau plays the role of a time parameter. Thus the metric of equation eleven is not static in region two. The magnetic components of F prime “A”, “B” in region two can be obtained from transformations such as Equation thirteen if we take into account that the components zero, temporal, and three, spatial are, respectively, Xi and c tau in equation seventeen: One over y F prime 1, 3 equals one over x F prime 2, 3 equals the charge e over a function of tau, z and t. The fields in equation seventeen, together with the electric components that can be obtained in an analogous way, are time-dependent solutions of the, vacuum, Maxwell equations in region two, having radiating parts. However, they are inaccessible to a co-moving observer because they are confined beyond his-her future horizon. Three. CONCLUDING REMARKS. The physics of the Rindler space is sufficiently subtle to deserve some extra remarks. Trajectories with constant Xi, see Figure two, correspond to uniformly accelerated trajectories in the inertial frame, but with distinct accelerations. The trajectory one corresponds to the static world-line Xi equals c squared over two g in the Rindler frame. From the inertial frame point of view, the true co-moving observer should correspond also to xi equals c squared over two g, because any other static Rindler observer would be in relative motion according to the inertial frame point of view with respect to the charge following Equation one. Our results show that the electromagnetic field of the uniformly accelerated charge is realized as a purely electrostatic field everywhere in the Rindler frame, implying that even observers with Xi is not equals c squared over two g, for which the charge is indeed accelerating when observed from the inertial point of view, would not detect the emitted radiation. This observation, which anticipates an intriguing quantum result described by Matsas, reinforces the role played by the horizon, the unique property that the trajectories of these distinct observers have in common, see figure two. The discussion can be considerably enriched by the introductions of quantum mechanical concepts. The classical radiation emitted by the accelerated charge in the inertial frame consists of a large number of real photons, which due to some subtle quantum effects cannot be detected by co-moving observers. To illustrate the novelties brought by quantum mechanics, consider in the Minkowski space a uniformly accelerated observer following a trajectory such as that in Equation one and a charge at rest at the origin. The world-line for this charge is the ct axis, and it is restricted to regions two and four. The solution of Maxwell equations in the inertial frame is the static Coulomb field is given in equation eighteen. Which spreads over all four regions of Figure one. In region one, where a uniformly accelerated observer can detect any field coming from the charge, the static Coulomb field will be measured by such observers as a time-depending electromagnetic field with components given in equation nineteen. The fields nineteen are solutions of the, vacuum, Maxwell equations in region one as seen by uniformly accelerated observers. These fields have radiative components, although it is not so easy in this case to identify the terms dropping off as one over R. Note that the accelerated observers are completely unaware of the charge fate in region two. Because they can detect only the contributions coming from regions one and four, they will never discover what eventually happens to the charge in region two if it, for instance, accelerates or even if it vanishes. An analysis of this problem based on quantum field theory, however, demands that the trajectory of the charge be entirely inside region one. Is it possible to conclude something in this case? Astonishing the answer is yes. In the Rindler reference frame static trajectories with Xi goes to infinity, see Figure two 2, correspond to uniformly accelerated trajectories in the inertial frame, restricted by construction to region one, but with proper acceleration g equals c squared over two Xi goes to zero. Thus, they correspond to inertial trajectories! Now we can answer the question of whether uniformly accelerated observers could detect any photon emitted by these specific inertial charges, and the answer is no. The detection of radiation is not the only paradox involving accelerated charges. Another very interesting paradox is related to the radiation reaction force. As we discussed, an inertial observer detects the radiation emitted by a uniformly accelerated charge. He-she can even calculate the, non-vanishing, total radiated power. But we know from classical electrodynamics that the radiation reaction force vanishes for a constant proper acceleration. Hence, what is acting as the source of the radiated power? How is it possible to conserve energy in this case? Interesting questions, but that’s another story. Figure One. The hyperbolic trajectory r “A” (Of tau) given by Equation one. The retarded time tau ret associated with a given point (c t, x, y, z) corresponds to the (unique) intersection of the past light-cone of (c t, x, y, z) with the trajectory r “A” (Of tau). For instance, Q prime equals c squared over g times shine g tau ret Q over C, zero, zero gosh g tau ret Q over C, and P prime equals c squared over g times shine g tau ret P over C, zero, zero gosh g tau ret P over C, define, respectively, the retarded times tau ret Q and tau ret P associated with the points Q and P. The future light-cone is the boundary of the causal future of a given point. Thus, any event occurring, for instance, in the spacetime point R will affect only the region enclosed by its future light-cone, with the light-cone surface reserved only to signals moving with velocity c. Note that only regions one and two are affected by the fields due to a charged particle with a world line given by Equation one. Figure two. The lines of constant Xi and tau according to Equation ten and sixteen, respectively, for the regions one and two. In region one, the Rindler frame where Xi is greater than zero, the identified lines correspond to Xi 1 less than Xi 2 is less than Xi three and tau 1 is less than tau 2 is less than tau three. Lines of constant Xi, the hyperbola, are time-like. On the other hand, for region two, known as the Milne frame where Xi is less than zero, the lines of constant tau are time like. The identified lines in two correspond to the cases tau 1 is less than tau 2 is less than tau and Xi 1 is greater than Xi 2 is greater than Xi 3. The horizon, the boundary c t equals z between one and two, corresponds to one half of the degenerated hyperbola corresponding to Xi equals zero. https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html

arXiv:2205.05696v3 [astro-ph.EP] 26 Jul 2023 https://arxiv.org/abs/2205.05696 https://arxiv.org/pdf/2205.05696.pdf https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Direct Imaging and Spectroscopy of Extrasolar Planets. Thayne Currie, and others, 2023. Abstract. Direct imaging and spectroscopy is the likely means by which we will someday identify, confirm, and characterize an Earth-like planet around a nearby Sun-like star. This Chapter summarizes the current state of knowledge regarding discovering and characterizing exoplanets by direct imaging and spectroscopy. We detail instruments and software needed for direct imaging detections and summarize the current inventory of confirmed and candidate directly-imaged exoplanets. Direct imaging and spectroscopy in the past decade has provided key insights into Jovian planet atmospheres, probed the demographics of the outskirts of planetary systems, and shed light on gas giant planet formation. We forecast the new tools and future facilities on the ground and in space that will enhance our capabilities for exoplanet imaging and will likely image habitable zone rocky planets around the nearest stars. 1. Introduction. Identifying, confirming, and characterizing an Earth-like planet around a nearby Sun-like star is a key goal of exoplanet science. In the coming decades, direct imaging and spectroscopy are the likely means by which this goal will be achieved. Over 5000 extrasolar planets and candidates have been detected through indirect means. In contrast, 20 to 25 exoplanets have been directly imaged, see Figure one. Despite this relatively low yield of discoveries, direct imaging comprises a large fraction of the known exoplanet population amenable to atmospheric characterization, since the method provides photons from the planets themselves instead of just inferring their Presence. Almost all of the directly imaged planets known thus far are young, self-luminous gas giants detected in thermal emission. They represent the extremes of planet formation, planets with masses of 2 Jupiter Masses or more orbiting at moderate to wide separations, around ten to 250 astronomical units. A subset of directly detected planets appear to be in or just finished with the final stages of assembly, protoplanets. The direct imaging field currently also provides key insights into Jovian planet formation and the demographics of the outskirts of planetary systems. Remarkably, the direct imaging method enables the acquisition of hundreds to thousands of spectroscopic data points on exoplanets in a few hours of telescope time. This wealth of information can critically constrain individual planet atmospheric properties, for example temperature, clouds, chemistry, and gravity, as well as the atmospheric evolution of gas giants as a population. The first direct images and spectra of planet candidates relied on facility, general-use adaptive optics (AO) systems to deblur starlight or space telescopes, meaning the Hubble Space Telescope, coupled with simple coronagraphs. The past decade has seen the development, demonstration, and honing of dedicated extreme AO systems coupled with advanced coronagraphs and sophisticated post-processing methods, allowing the detection of planets that are fainter and less massive and, or located at smaller angular separations that probe tighter orbits. In-development extreme AO systems and upgrades to first-generation extreme AO systems will provide images of many exoplanets near the ice line. The Coronagraphic Instrument on NASA’s Roman Space Telescope (Roman-CGI) could provide the first detections of mature planets in reflected light, Figure Two. Planned ground-based extremely large telescopes and proposed space missions promise to make the discovery and confirmation of a habitable, Earth-like exoplanet around a Sun-like star a reality within the next 25 years. These facilities will endeavor to reveal biomarkers, for example water, oxygen, ozone, in individual systems. Their surveys will provide the first assessment of potential habitability around stars of different masses and thus the true context for life on Earth. Technological innovation, atmospheric characterization, and demographic studies of young Jovian planets over the past decade provide first, key steps towards this goal. In this Chapter, we provide an updated description of the state of our knowledge about detecting and characterizing exoplanets by direct imaging. The last dedicated direct imaging review chapter in Protostars and Planets was written in 2007, before the first incontrovertible exoplanet imaging detections: super Jovian planets around HR 8799 and Beta Pictoris. Previous reviews from Traub and Oppenheimer in 2010 and Bowler in 2016 bracket the start and end of the era of direct imaging surveys with facility, conventional AO systems. Now, more than 5 years later, the first generation of extreme AO surveys have ended, bringing new discoveries, fundamentally new insights into atmospheric properties of individual systems and imaged planet demographics, motivating substantially more powerful instrumentation and tangible plans for directly detecting true solar system analogues, including Earths. This Chapter is pedagogical in nature and is organized as follows. In Section 2, we outline key direct imaging instrumentation and methods, describing the challenges in achieving planet detections by direct imaging, and detailing critical, novel hardware and software needed to image planets. Section 3 summarizes our current inventory of directly imaged exoplanets, discusses challenges with interpreting direct imaging detections, and outlines synergies with other detection techniques. Section 4 focuses on atmospheric characterization of exoplanets by direct imaging and spectroscopy, using other substellar objects, meaning brown dwarfs, as anchors to provide empirical constraints on exoplanet atmospheres and employing atmospheric models to infer intrinsic properties, including clouds, chemistry, and gravity. Next we overview the architecture of directly imaged planetary systems gleaned from astrometric monitoring, dynamical models, and planet-disk interactions, Section 5. Section 6 summarizes recent direct imaging surveys, the occurrence rates and demographics derived from them, and what these results mean for models of Jovian planet formation. Finally, in Section 7 we forecast the future of direct imaging, describing how technological innovations and new, vastly more powerful facilities may provide humanity with a glimpse of a habitable world for the first time. 2. Direct Imaging Instrumentation and Methods. Direct imaging detections require separating the halo of bright, highly structured scattered starlight from faint exoplanet light. The key metric used to determine the detectability of extrasolar planets is the planet-to-star contrast ratio at a given off-axis angular separation: the contrast ratio needed for a detection varies with planet properties. Section 2.1. Critical hardware like AO, or, more generally, wavefront control systems consisting of sensors and deformable mirrors, and coronagraphs sharpen and then suppress scattered starlight, Section 2.2 to achieve deep raw contrasts. Novel observing techniques allow post-processing algorithms to further remove residual starlight, increasing achievable contrast ratios and thus improving planet detection capabilities, section 2.3. 2.1. Detectability of Planets by Direct Imaging. Even in the absence of an atmosphere to blur starlight, an astronomical image of a point source, for example a distant star, will not be a single point: its intensity distribution follows from the Fourier transform of the telescope pupil function. For a simple unobscured circular aperture, the image intensity follows an Airy pattern whose values compared to the peak intensity I zero at angular separation rho, effective telescope aperture diameter D, and wavenumber k equals two pi over lambda is the standard airy pater using a Bessel function J1, where J1 is the Bessel function of the first kind of order one. The first null of the Airy function occurs at about 1.22 lambda over D. The full-width at half maximum (FWHM) of this point source on the sky, defining the telescope diffraction limit, is given by equation one: Theta in arc seconds is about zero point two one times wavelength in microns over telescope diameter in meters. Even current telescopes in principal have the angular resolution needed to image planets on solar system-like scales orbiting nearby stars. An Earth twin orbiting at a projected separation of 150 Giga-meters, or 1 A-U from a Sun-like star at 308 Peta-meters, or 10 parsec subtends an angle of a tenth of a second of arc, or a half a micro-radian. Diffraction limited imaging with the Hubble Space Telescope at optical wavelengths or with 8 to 10 meter class telescopes like VLT, Subaru, and Keck at near infrared (IR) wavelengths is sufficient to resolve objects at Earth-Sun projected physical separations out to a distance of three to six hundred Peta-meters, which is 10 to 20 parsec. However, the star’s halo light must be reduced through hardware and software to a level sufficient to make the planet’s light detectable. The starlight suppression level required to detect a planet by direct imaging depends on whether the detection is in reflected light or thermal emission and varies with the planet angular separation, age, size, temperature, and other properties, see Figure two. 2.1.1. Reflected Light. Planets reflect the light of the stars they orbit. At optical wavelengths, the contrast ratio for a planet in reflected light can be approximated as equation two: Contrast Ratio is a function of square of the ratio of planet radius to planet-star separation times an Albedo function, and a phase function. The Albedo and the phase function depend on the planet’s atmospheric properties. For a Lambertian phase function, valid for high albedo atmospheres, the phase function follows a simple trigonometric relation. Other potential scattering phase functions include isotropic scattering and Rayleigh scattering. The value of the phase function is considerably lower for Rayleigh scattering appropriate for Jovian atmospheres at angles of sixty to ninety degrees. For an exo-Jupiter and exo-Earth emitting as Lambertian spheres with measured geometric albedos of 0.52 and 0.367, these contrasts at maximum elongation reduce to equations three and four in the text. Contrast ratios are of the order of ten to the minus nine to ten. At V band, a Jupiter and Earth at around 300 Peta-meters, 10 parsec, will then have apparent magnitudes of slightly greater than 27 and 29, respectively, and require starlight removal at angular separations of one half and one tenth arc second better than one part in ten to the minus nine and ten to the minus ten, respectively. These required contrasts are well beyond the capabilities of current ground and space-based high-contrast imaging instruments. Thus far, exoplanet direct imaging has therefore focused on the detection of thermal emission from self-luminous Jovian planets. 2.1.2. Thermal Emission. Like brown dwarfs, Jovian planets cool and contract with time, releasing gravitational potential energy as thermal emission. At ages of one to ten mega year, models for the luminosity evolution of one to ten mass of Jupiter exoplanets predict temperatures of around 500 to 3000 Kelvin and radii up of r p around 2.5 to 3 Radii of Jupiter. By 1 Giga year, these models predict that these planets cool to temperatures T less than around 500 Kelvin and contract to Jupiter-like radii, r p around one to one point two RJ. Thus, thermal emission from young Jovian exoplanets peaks at near-to-mid IR wavelengths at the youngest ages, moving to well into the mid-IR at older ages, see Figure three. The contrast ratio for planets in thermal emission depends on the radius and effective temperature of the planet and star and other properties, equation five. Which can include a function of temperature and atmosphere composition, the thermal flux from the planet as a function of wavelength, The Flux function is the thermal flux from the star as a function of wavelength, and X depends on the planet’s atmospheric characteristics, such as clouds, chemistry, and gravity. Figure three shows how the contrast ratios vary for various types of exoplanets orbiting a G2 dwarf star. In the near-IR, young super Jovian, ten mass Jupiter exoplanets similar to Beta Pictoris B and HR 8799 cde are roughly a factor of ten to the minus three to ten to the minus five fainter than a Sun-like star. In the red optical, predicted contrasts from these planets are 100 times larger; however, mid-IR contrasts are a factor of 10 smaller. Jovian exoplanets of similar masses around much older stars are far cooler, another factor of 100 to a 1000 fainter in the red optical and near-IR, but remain bright in the mid IR. A true Jupiter analogue emits negligible thermal emission for wavelengths greater than four to five microns. Rocky terrestrial planets also emit thermally. An exo-Earth analogue with an effective temperature of 260 Kelvin has blackbody emission peaking at 10 microns, its contrast ratio is ten to the minus seven, a factor of 1000 shallower than its reflected light contrast. 2.2. Wavefront Control and Coronagraphy. 2.2.1. Atmospheric Wavefront Control for Direct Imaging. Wavefront errors, whether induced by atmospheric turbulence on the ground or intrinsic to an optical imaging system on the ground or in space, substantially limit an image system’s achievable contrast. Left unmitigated, these errors preclude the direct detection of exoplanets. In the past decade, the direct imaging field has made key strides in wavefront control hardware and software to drastically reduce wavefront errors and has developed sophisticated coronagraph designs to further suppress diffracted starlight. For ground-based imaging systems, turbulence arising from many atmospheric layers along the path from the star to the telescope induces changes in the optical path length difference of starlight, blurring images. These aberrations must be corrected by an AO system consisting of wavefront sensors and deformable mirrors (DMs). An AO system splits incoming light from a guide star between the science detector and a wavefront sensor to measure and then correct, with a DM(s), atmospheric turbulence distorting the incoming wavefront, sharpening starlight at the image plane. Key terms driving the wavefront error budget for an AO system include: 1) Measurement error sigma m, which depends on the noise properties of a wavefront sensor and the guide star brightness, 2) Temporal bandwidth error Sigma T, which depends on the AO system time lag tau compared to the atmospheric coherence time tau zero, and, 3) The fitting error sigma f which depends on the coherence length r zero compared to the DM actuator density. The quadrature-added sum of these terms sets the Strehl ratio, a measure of the optical quality of the image, and the raw contrast versus angular separation. At the angular separations relevant for most current planet searches theta around zero point two to one arc second, wavefront measurement error and temporal bandwidth error set the contrast floor. Wavefront chromaticity and non-common path aberrations due to instrument optics can also limit contrast. Facility, conventional AO systems were used for the first direct imaging searches. They typically sample and correct the incoming wavefront at frequencies of p;oint two to one kilo Hertz and use DMs with a few hundred actuators: aberrations are measured by standard Shack-Hartmann wavefront sensors. These systems yielded partial corrections, achieving modest Strehl ratios of zero point one to zero point four at near-IR wavelengths focused on by most exoplanet imaging Searches. The past 5 to 10 years have seen the deployment of numerous extreme AO systems on 5 to 10 meter telescopes, which have wavefront control loop speeds of greater than one kilo Hertz and DMs with more than a 1000 actuators. Examples of extreme AO systems include the Large Binocular Telescope Adaptive Optics system, LBTAO, PALM-3000 on the Hale telescope at Palomar Observatory, the Gemini Planet Imager on Gemini South, and the Spectro-Polarimetric High-contrast Exoplanet REsearch instrument (SPHERE) at the VLT, the Subaru Coronagraphic Extreme Adaptive Optics project (SCExAO) on the Subaru Telescope, and MagAO-X on the Clay telescope at the Magellan Observatory. These systems obtain higher-quality AO corrections, Strehl ratios of pont seven to zero point nine five at 1.6 microns, and yield a factor of 10 to a 100 deeper contrast at sub-arc second separations than conventional AO systems. Extreme AO systems have relied on hardware developments in three key areas to operate high-performance AO correction: detectors, deformable mirrors, and computing hardware. Most extreme AO platforms utilize fast ultra-low noise detectors like Electron-Multiplying Charge-Coupled Devices (EMCCDs) to record stellar photons used for wavefront sensing with reduced measurement errors. High-actuator count DMs are now available. For example, the micro-electromechnical-type DMs (MEMS) offer unprecedented actuator density around 10 actuators per square millimeter, allowing for fairly compact extreme AO instruments. Other systems employ adaptive secondary mirrors (usually voice-coil DMs) whose low emissivity makes them especially well suited for exoplanet imaging in thermal IR. High performance computing hardware with low latency can handle the demanding computation requirements of fast, high order extreme AO systems. Extreme AO systems also differ from conventional AO systems in their optical design and system architecture. Wavefront sensors (WFS) optimized for speed, sensitivity, accuracy and precision such as the Pyramid wavefront sensor have been adopted by many leading extreme AO systems (top panel of Figure four, while the more established Shack-Hartmann WFS approach has been upgraded with spatial filtering to improve performance. Wavefront correction is often performed in two steps: a conventional coarse “woofer” correction is followed by a faster and more accurate “tweeter” correction. Extreme AO systems also include dedicated sensing and control of pointing for precise co-alignment of the star and coronagraph mask, which is essential to maintain high contrast, especially at the smallest angular separations. Advances in wavefront control algorithms are also enabling significant improvements in high-contrast imaging performance. Recent advances include predictive control and sensor fusion. These and other new software, hardware advances are described in Section 7.1. 2.2.2. Optical Starlight Suppression. Starlight suppression is primarily performed by dedicated coronagraph optics: phase and amplitude masks deployed in the beam to remove starlight while preserving planet light. These optical elements include occulters in the image plane to directly block out on-axis starlight, as well as optical elements in the pupil plane to manage the telescope diffraction pattern. Many coronagraph designs and approaches are available providing a wide range of performance characteristics. For example, the well-established conventional Lyot coronagraph combines an occulter in the image plane with a Lyot stop in the pupil plane to deliver robust performance at moderate contrast and large angular separations. Phase-mask coronagraphs or interferometric designs, for example, the vector vortex coronagraph, can be highly optimized for deep contrast at small inner working angles, Figure four, bottom panel. The highest-performing coronagraphs are also the most demanding in terms of wavefront quality and stability. Consequently, extreme AO systems first adopted Lyot coronagraphs, while more recent systems or upgrades deploy higher performance solutions as the corresponding wavefront quality improves. Wavefront control is also becoming an important component of starlight suppression in the form of speckle control. In speckle control, a feedback loop from the post-coronagraph image to an upstream deformable mirror allows for residual speckles to be measured and canceled. This approach has been used in laboratory testbed to reach deep contrast levels, ten to the minus seven to ten to the minus ten, in support of future space-based missions. Deployment on ground-based systems remains challenging due to the dynamic nature of atmospheric turbulence, but has been successful in removing a fraction of static and slow speckles. Sensing and compensation of non-common path aberrations can also be performed using a dedicated sensor, for example a Zernike phase-mask sensor. 2.3. Observing, Post-Processing, and Spectral Extraction Methods. At sub-arcsecond separations, the raw, instrument delivered, planet-to-star contrasts obtained by the first generation of facility and now extreme AO systems, ten to the minus three to minus four point five, are still too shallow to yield decisive detections of many young Jovian exoplanets. Residual quasi-static speckle noise, due to nanometer-scale imperfections in telescope optics, thermal flexure, etc., limits instrumental contrasts and fails to simply average out over time like photon, white, noise. Speckle noise statistics follow a modified Rician distribution whose long positive tail can lead to many false positives. Therefore, novel observing techniques coupled with advanced post-processing methods are critical to optimizing our ability to image exoplanets by further suppressing stellar halo light by orders of magnitude and making residual noise more Gaussian “whitening”. Various forms of differential imaging techniques are utilized to remove speckle noise for direct imaging. Commonly used techniques include: Angular Differential Imaging, ADI. ADI exploits the quasi-static nature of residual speckle noise in AO corrected high-contrast images from the ground and diffraction-limited imaging from space. For ground-based AO imaging, quasistatic speckles evolve on a characteristic timescale of 10 seconds to minutes to an hour or more. For space-based imaging, PSF breathing due to thermal variations and other small mechanical changes cause the quasi-static halo to evolve, but the halo for a given target can remain well correlated for months or years. By turning off the instrument rotator that keeps north fixed on the detector (on the ground) or obtaining observations at different roll angles (space), astrophysical objects off-axis from a star change position angle on the detector while the speckle halo remains fixed. Subtracting images obtained in an ADI sequence can then suppress speckle noise without fully suppressing planet signals. ADI efficiently suppresses speckles on the ground and in space. It is the most widely used observing method in direct imaging. ADI’s advantages are most limited at small angular separations where the displacement of the planet’s PSF is smallest for a given parallactic angle change or roll angle change. Typically, ADI induces signal loss, which must be corrected to achieve absolute spectrophotometric calibration for planets (see below). Spectral Differential Imaging, SDI. At raw contrasts shallower than the ten to the minus seven levels needed to image self-luminous Jovian exoplanets, achromatic phase errors dominate the wavefront errors responsible for the speckle halo. For data obtained simultaneously at multiple wavelengths for example dual-channel imaging or an integral field spectrograph (IFS), the speckle halo as a function of wavelength, scaled in radius by the central passband wavelength, is extremely well correlated. Since the images at different wavelengths are obtained simultaneously with the target, they do not suffer from are temporal decorrelation as with ADI. SDI then suppresses the speckle halo in a given passband by constructing a reference PSF drawn from (rescaled) images at other wavelengths. Like ADI, SDI induces signal loss and is least effective at small angular separations, where bandpass rescaling yields small displacements of a (de-)magnified planet PSF. SDI’s efficacy is also limited by non-common path errors, differential sampling of the point-source PSFs across wavelength channels, and the spectrum of the planet to be detected. Extracting throughput-corrected spectra from data processed with SDI is also more challenging than with other differential imaging techniques. Reference Star Differential Imaging, RDI. RDI subtracts the PSF of a companion-less reference star from the target. It is widely used in the ultra-stable environment of space. While ADI and SDI may yield superior speckle suppression at moderate to wide separations, RDI may be advantageous at very small angular separations where other techniques suffer from self subtraction. RDI requires a very stable PSF to efficiently operate. Using it on ground-based telescopes may require fast switching between the target and reference star(s) for maximum contrast gain, in broad bandpasses, it also requires a reference star that is extremely well color matched. These differential imaging techniques are used in combination with PSF subtraction algorithms. Many widely-used, advanced PSF subtraction methods are different forms of least-squares algorithms, which construct a reference PSF that minimizes the variance when subtracted from a given target image. The locally-optimized combination of images (LOCI) algorithm constructs a reference PSF from a linear combination of reference images weighted by coefficients c determined from the solution to the matrix equation C equals A inverse times b, where A is the (square) covariance matrix for the reference library and b is the column matrix populated by elements multiplying each reference image by the target image. Another approach exploits principal component analysis (PCA), computing the Karhunen-Lo`eve transform of the reference image set and projects this set onto the target image to construct a combination of weighted eigen-images to subtract from the target (Karhunen-Loeve Image Plane algorithm, henceforth KLIP. Multiple successor algorithms such as TLOCI and A-LOCI draw from the lineage of LOCI and, or KLIP, employing advances such as correlation-based frame selection, pixel masking, various rank-truncations of the covariance matrix, free parameter optimization or exploiting high-performance computing to solve for the variance-minimizing coefficients directly. Separate approaches involve maximum-likelihood methods to model and remove the stellar PSF or statistically modeling non-stationary covariances in small regions of images to improve PSF subtraction. The relative performances of these algorithms vary in the literature and may depend on their exact implementation and suitability for a particular data set, although some work suggests that the newer algorithms descended from LOCI and KLIP can offer significant improvements. In general, all of these algorithms enable significant contrast gains over simple, classical methods, especially at small angular separations. ADI, SDI, and RDI combined with PSF subtraction methods can attenuate and distort planet signals. The first methods to measure these biases and recover true planet flux measurements and astrometry injected synthetic planets into data at other locations or iteratively subtracted negative copies of planets at their apparent positions. Recently, several authors have developed efficient forward-modeling methods to estimate photometric and astrometric biases at the planet’s location. Planet forward models in turn can be used as matched filters to improve planet detection capabilities themselves. Analyses of images whose speckle noise is partially suppressed by advanced PSF subtraction techniques have revised notions of how to quantify detection significances and spectroscopic uncertainties. For instance, finite sample sizes impact our definitions for contrast limits drawn from images with noise whitened by KLIP, LOCI, and other algorithms, especially at small, one to three lambda over D, separations. In IFS data, residual speckle noise may be spatially and spectrally correlated. Considering the full spectral covariance has a substantial impact on deriving planet atmospheric parameters from model comparisons. Figure five shows the combined effect of high-contrast imaging hardware and software on imaging exoplanets, stepping through the successive improvements found from using extreme AO, coronagraphy, and PSF subtraction. 3. Direct Imaging Detections. 3.1. Taxonomy of Directly-Imaged Companions. Figure one shows the demographics of planetary mass companions to stars detected via various methods. Community consensus on the planethood of many imaged objects, such as HR 8799 bcde, Beta Pictoris bc, 51 Eridani b, and so on, is clear. However, identifying the exact criteria needed to distinguish between planets and brown dwarfs is challenging. Planets have often been identified as objects with masses below the deuterium-burning limit, nominally 13 times the mass of Jupiter, JUPITER MASS, the IAU Working Group Definition. However, this simple criterion is poorly motivated. In addition to being time and metallicity dependent, deuterium burning arguably does not identify a meaningful boundary for the evolution of low-mass objects, as some objects below the deuterium burning limit have been found in configurations which imply formation by cloud formation and other objects above the deuterium burning limit have been found in configurations which imply formation like a planet in a disk. For instance, recent imaging surveys have found objects that are members of quadruple systems, clearly formed by molecular cloud fragmentation, with inferred masses down to 5 Jupiter masses and free-floating objects with sub-deuterium burning masses as well. In contrast, RV surveys have identified some systems, for example the 2.7 solar mass star nu Oph, with companions at around 150 Giga-meters, one astronomical unit, with masses of 22 and 24 Jupiter masses that are nevertheless locked in a mean-motion resonance indicating formation in a disk, meaning like a planet. The physics of planet formation does not require that gas accretion shuts off once 13 Jupiter masses of material is accreted. An alternate definition leverages formation processes: a planet is an object formed in a circumstellar disk around a young star. Demographic analyses of substellar objects can provide empirically-motivated criteria for separating planets from brown dwarfs. Studies of the substellar mass function from previous RV surveys and recent ones, meaning the California Legacy Survey, show a local minimum at m sine (i) around 16 to 30 Jupiter masses. The minimum in the companion mass function may be proportional to the primary mass, indicating that companion mass ratio (q) could be a key discriminator. Theory also suggests that the semi major axes (ap) and mass ratios (q) of companions also help distinguish between bona fide planets and brown dwarfs. Binary companions to more massive stars with q greater than about 0.025 are exceptionally rare. Distributions of protoplanetary disk radii peak at around 200 au and fall to low frequencies by 30 Tera meters, 300 Astronomical Units. Companions at wider separations are far less likely to have formed from a disk unless scattered to their current locations by unseen companions. Thus, we set the following limits for a planet versus a brown dwarf: Mass less than 25 Jupiter Masses, q less than 0.025, and ap less than about 45 Tera-meters (300 Astronomical Units. Our linked spreadsheet lists the current inventory of directly imaged exoplanets, protoplanets, higher mass ratio, wider separation planet-mass companions, and controversial cases, hyperlink in text. As of 19 October 2022, we identify 22 directly imaged exoplanets that fit our criteria, three of which are imaged protoplanets. We also list another 34 dozen companions that may instead be better considered as brown dwarfs instead of planets, including companions orbiting brown dwarfs, and 6 “controversial” cases. Some of these individual classifications will undoubtedly change pending new analysis as may the exact values used to separate planets from brown dwarfs. Objects in each category will certainly be added over the next few years. Below, we describe general properties of fully-formed directly imaged planets within 45 Terameters, 300 astronomical units and discuss three well-studied, emblematic cases, HR 8799, Beta Pictoris, and 51 Eridani. We also summarize our current knowledge of protoplanets and challenges with identifying imaged exoplanets. 3.2. Fully-formed Exoplanets. 3.2.1. General Properties. Most directly imaged planets known thus far have near-IR contrasts ranging between ten to the minus four, kappa Andromedae b, and ten to the minus six, 51 Eridani b. All of them are self-luminous, made visible by thermal radiation released as the planets cool and contract. On the sky, the planets generally lie at angular separations of point two to two seconds of arc, or about one to ten micro radians in grown up units, around 5 to 40 lambda over D for an 8m telescope. Stars hosting imaged exoplanets typically have ages of 10 to 100 Mega years, many are found in young nearby stellar associations, often less than 100 parsec, 3,000 Peta-meters, with ages less than one hundred Mega years, that share similar kinematics (proper motions, space motions) and formed in the same star-forming region, such as Scorpius Centaurusor the beta Pictoris Moving group. See Section 6.1 for more details on such associations as sites for exoplanet searches. Most stars with imaged planets to date are B, A, or F stars, at least 50 percent more massive than the Sun. Most systems with imaged planets, including systems with the first detections, HR 8799, beta pictoris, also have Kuiper belt-like debris disks. Planets detected to date share similar spectral types and temperatures with more massive field brown dwarfs, but often show evidence of low surface gravity in their spectra. Brown dwarfs and exoplanets alike cool monotonically with age, beginning life as hot M type objects, cooling to the L spectral type (with very red near-IR colors and silicate condensate clouds, the cooler T spectral type (with blue near-IR colors and strong methane absorption at 1.6 micron and 2.2 micron, and eventually to the very cool Y spectral type. Thus, there is an age, mass, temperature degeneracy for these objects, rendering mass estimates based on models very sensitive to the age assumed for the system. Most mass estimates for imaged planets depend on luminosity evolution models, challenges for which are described in Section 3.4. Some planets have dynamical mass measurements or limits. Masses inferred from luminosity evolution or derived from dynamics are typically around 5 to 10 Jupiter masses. Some planets have masses near the deuterium-burning limit, kappa Andromedae b, HD 206893 b. 51 Eridani b may have a far lower mass, as little as about 2 Jupiter Masses. Mass ratios for most imaged planets are q around 0.005 to 0.01. 51 Eridani b may have the lowest mass ratio, with q about 0.001. Even leading extreme AO systems are typically not sufficiently sensitive to detect young Jupiter-mass planets, let alone Saturn-mass planets. However, future capabilities will close these gaps (See Section 7). 3.2.2. Emblematic Systems. While planetary mass companions to brown dwarfs had previously been detected as early as 2004 (most notably 2M1207b, the near simultaneous announcements of HR 8799bcd and Beta Pictrois b are widely regarded as the first bonafide directly-imaged exoplanet detections. Planets in both systems were detected from ground-based facility AO systems in the near-to-mid IR in thermal emission, not scattered light. Just over 7 years later, Macintosh et al announced the first exoplanet discovered with extreme AO, 51 Eridani b. HR 8799 is a nearby, around 40 parsec, 1200 Peta-meters, mid-A field star with an estimated age of about 40 Mega years. The star hosts a massive, resolved Kuiper belt-like debris disk and a warm debris population interior to 1.5 Tera meters, ten Astronomical units consistent with an asteroid belt, possible signposts of massive, perturbing planets. In 2008, Marois et al. announced the direct imaging discovery of HR 8799 bcd followed by a fourth planet discovery in 2010, HR 8799 e, located at projected separations of 2.25 to 10.5 Tera meters, 15 to 70 astronomical Units, Figure 6, left panel. Soon after HR 8799 bcde’s announcements, other studies identified one or more planets in archival or separately obtained data. A decade of Keck Observatory monitoring showed that HR 8799 bcde orbit close to the 1 to 2 to 4 to 8 resonance. Current analyses suggest that the planet orbits are nearly coplanar with the disk, with a small inclination of about 27 degrees. However, HR 8799 e may not orbit on a plane strictly coplanar with HR 8799 bcd Originally, masses inferred from the planets’ luminosities spanned a wide range of values, 5 to 13 Jupiter masses. However, dynamical stability modeling strongly favors masses below 10 Jupiter Masses, 7 Jupiter Masses for HR 8799 cde, HR 8799 b. Using Hipparcos and Gaia, measured a dynamical mass for HR 8799 e of about ten Jupiter masses, consistent with these limits, see Section 3.5. Additional planets may explain the shape of the inner edge of HR 8799’s cold belt, especially if they are near or below Jupiter’s mass. Any planets interior to HR 8799 e must be below three or four Jupiter masses at one to one point five Tera meters, 7 to 10 Astronomical Units, and five to six Jupiter Masses at six hundred to a thousand Giga-meters, or 4 to 7 Astronomical Units. The HR 8799 planetary system resembles a scaled-up version of our own outer solar system. HR 8799 bcde have been benchmark objects for understanding the atmospheres of young Jovian planets. Their photometry and low-resolution spectra differ from those of older, field substellar objects thought to have similar temperatures, identifying features diagnostic of clouds, chemistry, and gravity. Higher resolution spectra probe molecular abundances connected to formation mechanisms. Section 4 discusses HR 8799 bcde’s spectra and atmospheres in more detail. Beta Pictoris. The A6V star Beta Pictoris is the eponymous member of a collection of kinematically-associated stars known as the Beta Pictoris Moving Group, with an age of around 20 million years. The star hosts an edge-on debris disk, imaged since the mid-eighties from the ground and space. The disk consists of a planetesimal ring at ten to 15 Tera-meters responsible for the bulk of the dust through collisions and an outer extension comprised of small dust grains blown out by radiation pressure. Lagrange et al. first identified Beta Pictoris b from data obtained in 2003 with VLT, NaCo; follow-up data obtained one year later confirmed the planet. Thanks to Beta Pictoris’ b’s small semi-major axis 1 point 35 Tera-meters, or around 9 Astronomical Units, over 75 percent of its orbit has been monitored. More recently, a second planet Beta Pictoris c was identified from RV data and then recovered with high contrast inteferometric imaging with VLTI, GRAVITY. Beta Pictoris b may partially explain the observed warp in the edge-on debris disk and the many evaporating exocomets identified over the past 30 years. Planets besides Beta Pictoris b c could explain why the inner 70 Astronomical Units, 10 Tera-meters is relatively devoid of debris dust; localized features in the disk also suggest additional planets could be present. From RV and astrometric data estimate a mass of 9.3 and 8.3 Jupiter Masses for Beta Pictoris b and c respectively. Although these values heavily depend on the assumed uncertainties on the RV data. RV and direct imaging data combined exclude additional planets more massive than 2.5 JUPITER MASS from 15 Giga meters to Tera meters, 0.1 to hundreds of Astronomical Units. Beta Pictois b is more luminous and hotter than the HR 8799 planets, effective temperature 1700 to 1800 Kelvin, just slightly redder than field early L-type dwarfs, and is likely cloudy, dusty with a low gravity. Beta Pictois c probably has a temperature intermediate between its sibling and the HR 8799 planets. 51 Eridani. 51 Eridani is a 29 parsec,900 Peta meter, distant early F star, a member of the about 20 million years old beta pictoris Moving Group, and a member of a wide hierarchical triple system that includes an M dwarf binary GJ 3305. 51 Eridani has a detected infrared excess, modeled with a cold dust belt located approximately between 750 to twelve thousand Giga-meters, 5 to 80 Astronomical Units. The GPI campaign (GPIES) team discovered 51 Eridani b, a faint planet at projected around 2 Tera-meters, 13 Astronomical Unit, Figure 6, right panel. The planet is the first discovered using extreme AO and the first incontrovertible T dwarf planet, showing strong methane absorption in H band. The planet is likely more eccentric than either beta pictoris b or HR 8799 bcde. If confirmed, an eccentric orbit could indicate the presence of an additional massive body or could be due to gravitational perturbations from GJ 3305AB. Assuming a hot-start luminosity evolution, current data rule out other planets more massive than 4 JUPITER MASS beyond 5 Astronomical Units and more massive than 2 JUPITER MASS beyond 9 Astronomical Units. The mass of 51 Eridani b is not well constrained: values derived from comparing 51 Eridani b’s luminosity and age to evolutionary models favor about 2 JUPITER MASS, while atmospheric modeling may favor larger values, up to about 9 JUPITER MASS . Different characterization studies also find slightly diverging atmosphere properties, illustrating the challenge associated with characterizing very faint exoplanets with direct imaging Analysis of Gaia and Hipparcos astrometry set an upper limit of 11 JUPITER MASS for 51 Eridani b The planet likely has a temperature of around 700 Kelvin and either lacks clouds or is only partially covered by clouds. 3.3. Protoplanets. Direct images of planets in active assembly, protoplanets, around stars that still retain gas and dust-rich protoplanetary disks clarify how and where planets form. The large distances to the nearest star-forming regions, around 150 parsecc, mean that protoplanets orbiting their host stars at solar system scales are located at very small angular separations. However, protoplanets can be bright, Luminosities around a hundredth to a thousandth of the solar luminosity, especially if they are surrounded by their own circumplanetary disks. The 5 megayear old 0.87 solar mass star PDS 70 hosts the first incontrovertible detections of Jovian protoplanets: PDS 70 b and PDS 70 c. Both protoplanets are located within the PDS 70 disk cavity, at angular separations Point eighteen and point twenty four arc seconds and estimated semimajor axes 20 and 34 Astronomical Units, 3 and 5.1 Tera-meters. Dynamical arguments strongly favor masses less than 10 JUPITER MASS for PDS 70 b, while PDS 70 c’s mass is more poorly constrained. Masses inferred from SED modeling range between 1 and a few Jovian masses. PDS 70 b is slightly eccentric, eccentricity 0.17, while PDS 70 c’s orbit is consistent with being circular. The protoplanets’ IR data are best fit by model atmospheres with substantial dust, extinction. PDS 70 bc show H alpha emission consistent with accretion at rates of one to two solar masses per hundred million years, slightly less than the stellar accretion rate, but lack evidence for Br gamma accretion. PDS 70 c shows direct evidence for a circumplanetary disk with an estimated mass of 0.007 to 0.03 Earth Mass. Thermal IR data may suggest that PDS 70 b is surrounded by a circumplanetary disk. Recently, data from Subaru, SCExAO and the Hubble Space Telescope over 13 years reveal evidence for a wide separation, 93 astronomical unit embedded protoplanet around the one to three million year old, 2.4 solar mass star AB Aurigae. AB Aurigae b is consistent with a protoplanet responsible for the millimeter dust cavity and CO gas spirals both seen by ALMA. It appears spatially extended, plausibly due to light from the central source reprocessed by the star’s protoplanetary disk. The best-fit composite model explaining AB Aurigae b’s optical to near-IR emission includes a 2.75 Jupiter Mass, 9 Jupiter mass source with a surface gravity of log(g) equals to 3.5, emitting at a much hotter temperature than PDS 70 bc, around 2000 to 2500K, and accreting at a rate of one point one Jupiter masses per million years. The source is detected in H alpha although it is unclear whether this emission is due to accretion. Embedded in a massive disk with numerous spiral arms at over three times Neptune’s disk instead of in a fully cleared cavity like PDS 70 bc, the properties of AB Aurigae b may point to a planet formation mechanism by disk instability (see Section 6). Prior to the discovery of PDS 70 bc, other studies claimed detections of protoplanets located within the gaps of or embedded in disks around young stars. The 2-solar mass, protoplanetary disk-hosting star HD 100546 has a protoplanet candidate at around 7.5 Tera meters, 50 astronomical units and another at around 2 tera meters, 13 astronomical uunits, just interior to the gap in the protoplanetary disk (HD 100546 bc. HD 100546 b has been detected in multiple data sets but evidence for orbital motion is not yet clear. HD 100546 c has been imaged in a single data set and inferred through spectroastrometry but not yet imaged in subsequent data. Interpreting both candidates, whether a planet or disk feature, is challenging. Both candidates require further study and confirmation. Two studies presented detections of protoplanets around the young, Sun-like star LkCa 15 through a combination ofsparse aperture masking interferometry (SAM) and H alpha differential imaging, LkCa 15 bcd. However, later direct imaging observationsshowed that the SAM detections correspond to disk features LkCa 15 b technically remains a candidate for now due to its single epoch H alpha. Other claimed protoplanet detections have been revealed to likely be misidentified disk signals instead of planets. 3.4. Challenges with Interpreting Detections. 3.4.1. Confirming Companionship. Direct imaging observations reveal many point sources that are unrelated background stars instead of bound companions, especially for systems in the Galactic plane. Confirming candidate planets as bound, meaning sharing common proper motion with and orbiting their stars, often requires multi-year observations, depending on the star’s proper motion. In some pathologicalcases, background stars can have a non-zero proper motion and thus are more easily confused with bona fide planets. In the absence of full confirmation, an object’s near-IR spectrum can provide strong evidence that it is a directly-imaged exoplanet, for example for 51 Eridani b. 3.4.2. Estimating Accurate Masses. Masses for imaged planets and planet candidates are typically not directly measured but are instead estimated using luminosity evolution models that map between an object’s brightness and mass as a function of age. Accurate mass estimates therefore are affected by uncertainties in these models and require precise system ages. Hot start models for planet luminosity evolution assume a high initial entropy, resulting in bright planets for the first one to a hundred Mega years. Cold start models assume a low initial entropy, resulting in planets that are substantially fainter for the first 100 Mega years. Originally, hot start models were used to describe planets formed by disk instability, while cold start models described planets formed by core accretion. However, recent models show that core accretion formed planets can be compatible with hot start-like luminosity evolutions, and Jovian planets may form with a range of initial entropies. Aside from 51 Eridani b, all planets imaged thus far are only consistent with a hot-start luminosity evolution. Precise ages can be exceptionally challenging to derive for isolated stars. Members of young moving groups or other nearby associations can be age-dated using a variety of methods: for example fitting group Hertzsprung-Russell (HR) diagram positions with stellar evolutionary models, Lithium abundances. Thus, moving group or association membership can yield a star’s age. However, even stars with a motion similar to bona fide moving group members could instead be interlopers, especially if they have dissimilar space positions. For isolated Sunlike stars, stellar rotation and activity can give age estimates, albeit with significant scatter. For early-type stars, HR diagram positions have provided approximate ages: optical interferometry can now provide more precise estimates by resolving the stars themselves. Revisions in the stellar age often affects the interpretation of an imaged companion. For example, based on the primary’s claimed membership in the Columba association, Kappa Andromedae b was originally thought to be 12 to 13 JUPITER MASS. In contrast, the primary’s HR diagram position resembles an older system, 220 Mega-years, which would imply the companion is around 50 JUPITER MASS. However, through optical interferometry, it has been shown that the star is likely (nearly) coeval with Columba even if it is not a member around47 Mega years, implying the object is a planet mass companion. Later, near-IR spectra of Kappa Andromedae b were found to be consistent with a planet interpretation. In contrast, the imaged companion to GJ 504 was announced as a 3 to 8.5 JUPITER MASS planet with an age of 100 to 510 Mega years. Based on interferometric, RV, and high contrast imaging data, derived a mass range of 1 to 23 JUPITER MASS, while other analysis of the star suggested an age of about 2.5 Gyr and a mass well into the brown dwarf regime. 3.4.3. Planet or disk feature? Distinguishing between highly-structured disk signals and bona fide protoplanets is a steep, chronic challenge. Advanced algorithms needed to detect protoplanets can attenuate both disk and planet signals: forward-modeling is required to ensure that a claimed planet detection is not a partially subtracted piece of the disk. For systems with hot dust, T around 1000 to 2000 K, near the star that intercepts and reprocesses emission, the disk’s scattered light spectrum may strongly resemble spectra of bona fide protoplanets. Orbital motion over multi-year timescales can better establish that a signal is an orbiting planet and not a static disk feature H alphaission can also pinpoint actively accreting protoplanets however, accretion onto protoplanets embedded in disks may be unidentifiable, since optical extinction likely renders Hα undetectable. Fomalhaut b may represent the first of yet another class of objects with a challenging interpretation The object was initially identified as a directly imaged planet, made visible by both reflected light from a circumplanetary disk and thermal emission, and responsible for sculpting the star’s Kuiper belt-like debris disk. However, later analysis showed that Fomalhaut b’s spectrum is completely explained by scattered starlight and its orbit likely crosses the ring the object is lower in mass and likely made visible purely by circumplanetary dust. Recently, from analyzing archival and unpublished data, Gaspar and Rieke (2020) proposed that Fomalhaut b may be fading and dispersing, consistent with a massive planetesimal collision. Other analyses find no clear evidence for these two trends, although they were conducted only over a subset of available data. Future observations of Fomalhaut b and, or reanalyses of recent data may clarify its true nature. 3.5. Synergies with Other Techniques. The limitations of direct imaging affect our ability to interpretdata for individual objects and to draw conclusions from large-scale population studies. For individual objects, mass estimates depend on both the age of the planet and the chosen luminosity evolutionary model (see Section 3.4). At a population level, the angular resolution of the telescope and the achievable contrast of the instrument limit the range of detectable planetary masses and semi-major axes with direct imaging. Combining direct imaging with other planet detection techniques partially mitigates these limits. Optical interferometry provides a means to directly detect bright, modest planets at small angular separations. As a prime example, GRAVITY interferometer coherently combines the light of all four VLT telescopes, yielding the equivalent angular resolution of a 130-m telescope, GRAVITY. While the GRAVITY field-of-view is small (around 50 mas), indirect detection techniques, such as RV, cna help predict the position of an exoplanet candidate. GRAVITY has yielded exceptionally high-SNR spectra and ultra-precise astrometry of known planets, the combination of GRAVITY and indirect techniques have now resulted in new planet discoveries Relative astrometric measurements of a planet around mass of the orbiting planet; the semi-major axis of the relative orbit encodes the system’s total mass, not the individual components’ masses. The system’s mass ratio, and thus the mass of the planet, can be measured if the semi-major axis of the orbit of the star around the system barycenter is known. This orbit can be measured either using absolute astrometric measurements from catalogues such as Hipparcos and Gaia, or spectroscopic observations to measure the Doppler shift of the star’s spectral lines over the course of the orbit. In some cases, high-resolution spectroscopy can measure the Doppler shift of the planet’s spectral lines, also yielding the mass ratio. Combining the star’s orbit around the barycenter with the relative astrometry between star and planet yields the mass ratio, and a dynamical mass for the planet. In multi-planet systems with high-precision astrometry, the planets’ mutual gravitation can be detected by fitting for deviations from Keplerian motion, yielding their mass estimates. The Beta Pic system is the current benchmark example of combining planet detection techniques to measure model independent masses. Relative astrometry of Beta Pic b and c combined with the star’s astrometric and RV measurements provides dynamical masses for the planets Measurements of non-Keplerian motion due to the mutual gravity of Beta Pic b and c are also now possible given the precision of recent interferometric monitoring campaigns. Combining astrometry and, or RV and direct imaging data can yield dynamical masses for other directly-imaged planets, as well as for numerous brown dwarf companions. The upcoming Gaia data releases for the full and extended mission will be used extensively in future detection and characterization studies of directly-imaged planets, yielding further dynamical masses for directly imaged companions. The extended mission will provide precision astrometric measurements of the host star over around 10 years. Combining direct imaging with other detection techniques can improve planet occurrence rate measurements and can better determine the planet frequency distribution over a range of masses, orbital periods, and host star properties. Long-term Doppler surveys are largely complete to giant planets within about 750 Giga-meters, 5 astronomical units, and partially complete to giant planets out to around10 astronomical units, the current inner limit of sensitivity of direct imaging surveys. Thus, combining results from both techniques reveals giant planet demographics out to around100 astronomical units (Section 6.3). RV studies of young stars allow occurrence rates to be compared for stars of similar ages, tracing the extent of giant planet migration over system lifetimes and its effect on planet frequency. The final Gaia planet catalog will also help place demographic measurements of young, wide-separation giant planets into context, as astrometry will be better able to probe intermediate separation giant planets. 150 to 1,500 Giga meters, around one to ten Astronomical units, Masses greater than around one Jupiter masses, around younger, higher-mass stars compared to RV. These target stars more closely match the hosts of imaged planets. A Gaia-selected survey of accelerating stars has now led to the first joint direct imaging and astrometry discovery of an exoplanet. Combining Gaia observations of younger, higher-mass stars with direct imaging will illuminate trends in occurrence rate as a function of stellar mass and age. Direct imaging also shares an important overlap with planet detection by microlensing. Both techniques are sensitive to planets beyond the snow line, with microlensing probing lower planet-star mass ratios, while the host mass of imaged planets can be directly determined, and the planet mass inferred from models. In addition, singly lensed short-period microlensing events can represent either a free-floating planet or a wide separation bound planet. Constraints on the wide-separation giant planet population can be determined from imaging, thus more definitively constraining the free-floating planet occurrence rate. 4. Atmospheric Characterization of Directly Imaged Exoplanets. Direct imaging enables characterization of young, age less than 200 Mega years Jovian exoplanets at wider separations with negligible irradiation compared to most transiting planets. In this section, we first summarize from an empirical standpoint what we have learned about the atmospheres of the current cohort of young directly imaged giant planets from their photometry and spectroscopy. We then consider the theoretical side of the picture, in particular, the state-of-the art in how we model these complex, cool atmospheres. 4.1. Empirical Constraints from Time-Averaged Observations. 4.1.1. Photometry. Photometry in the major near-to-mid IR passbands, J, H, K, Lp, Ms, covers the bulk of emission for young super Jovian planets and provided the first empirical diagnostic of young, directly-imaged planet atmospheric properties. Figure 8 displays a typical near-IR color magnitude diagram for selected directly-imaged planets and other planet mass companions compared to older, more massive field brown dwarfs and younger brown dwarfs whose ages and masses partially overlap with those of most imaged planets. Imaged planets and planet-mass companions span the full luminosity range characteristic of mature late-M, L, and T field brown dwarfs (vertical axis). However, young brown dwarfs, planets, and planet-mass companions typically have redder colors than field objects. The most pronounced differences between planet and methane-poor L-type dwarfs to methane-absorbing T-type dwarfs. The first imaged planet-mass companion, 2M 1207 B, and some of the first imaged exoplanets, for example, HR 8799 b) are particularly discrepant, appearing to populate a previously empty part of these diagrams consistent with a reddened extension of the L dwarf sequence to lower luminosities. Over the full 1 to 5 micron spectral range, photometry for these objects appears redder and more blackbody-like, Figure 10, top-left. More recent studies show that a number of other young imaged planets, planet-mass companions, for example HD 95086 b, TYC 8998-760-1 c, HD 206893 b, 2M 2236 plus 4751 B, also populate this region. Differences between field and young objects with T spectral types are less clear. Few young, T-type planet-mass companions have been identified in the last decade, for example 51 Eridani b. While GU Piscium B follows the sequence of field brown dwarfs in near-IR color-magnitude diagrams, the exoplanet 51 Eridani b is redder than field brown dwarfs. Four additional companions orbiting primary stars with intermediate ages, less than around one Giga-years. HN Pegasi B, ROSS458C, BD204-39B, and others, show similar but less pronounced deviations with respect to field dwarfs in the same luminosity range. Some directly imaged L, T transition planets and planet-mass companions have color-magnitude diagram positions discrepant from field objects in thermal IR passbands probing methane absorption (3.3 microns, Ms) but have positions similar to field objects in other passbands. 4.1.2. Spectroscopy. Over the past decade, IFS instruments, especially those used in combination with extreme AO systems (P1640, SPHERE, GPI, SCExAO-CHARIS) have provided critical low-resolution (R around 20 to 80) near-IR, 1 to 2.5 micron spectra of most directly-imaged exoplanets, a representative sample of which is displayed in Figure 9. Near-IR spectra provide key diagnostics of brown dwarf and planet atmospheres. Spectral indices derived from low-resolution data like H20 yield coarse estimates of spectral types. The H-band continuum index and (possibly) the H2-K index is a diagnostic of surface gravity. Following trends from photometry, the spectra of young directly-imaged planets and other planet-mass companions show significant differences with the spectra of field brown dwarfs. Among the most notable are the following: Chemistry. The L, T transition traces the onset of methane absorption. However, spectra of some L, T transition exoplanets and planet-mass companions, for example the HR 8799 planets, 2M 1207 B, show a lack of methane absorption compared to field dwarfs with similar temperatures and CMD positions. Thermal IR spectra confirm that the HR 8799 planets also have weaker absorption in the 3.3 microns methane filter. Gravity. The H-band spectra for L dwarf and L, T transition directly imaged exoplanets, planetarymass objects such as HR 8799 b, Kappa Andromedae b, ROXs 42Bb show highly peaked H-band spectra and, or red K-band spectra compared to field dwarfs. The H and K-band shapes probe collisionally-induced absorption (CIA) of hydrogen; lower gravities result in weaker CIA and thus peaked H-band peaks and redder K-band slopes. Dust. At least some of the HR 8799 planets have spectral properties and molecular absorptions that are well matched by those of young free-floating objects at the L, T transition (see also Section 4.3). However, no object yet reproduces the available 1 to 2.5 micron spectra of HR8799b, which may be due to the lack of identified young free-floating T-type objects. A handful of early-T-type objects such as the AB Doradus member 2MASS, number in text, can reproduce the spectral bands of HR 8799 b provided that an extra layer of extinction by sub-micron dust particles is applied to these empirical template spectra to match the spectral slope of the planet. HD 206893 b presents an even more extreme example, with an even flatter, more blackbody-like spectrum yielding an extremely red spectrum from 1 to 2.5 micron, likely due to substantial atmospheric dust. Spectra of the coolest and lowest mass imaged exoplanet known to date, 51 Eridani b, display a clear and so-far unique detection of a methane absorption at 1.6 micron in the spectrum of an imaged exoplanet, coincident with an enhancement in the K-band flux. Similar K-band flux enhancements have already been noted in young mid to late-T dwarfs but the enhancement is particularly extreme in the case of 51 Eridani b and is due to the reduced collision induced absorption of H2 in 51 Eridani b’s lower-pressure atmosphere. Protoplanets display far more featureless, black bodylike spectra. PDS 70 bc’s spectra reveal an extremely red spectral continuum devoid of the strong water-band feature, 1.3 to 1.4 micron expected given the observed luminosity of the planets. Higher resolution K-band spectra show a lack of molecular absorption in PDS 70 b’s spectrum. The circumplanetary disks and, or cocoon surrounding each planets, see Fig 7, produce significant foreground extinction and may produce spectroscopic properties similar to what is seen in the near-infrared spectra of enshrouded class one protostars. The near-IR spectrum of AB Aurigae b is reproduced by a 2000 to 2500 Kelvin blackbody but likewise lacks clear evidence for molecular absorption common in fully-formed substellar objects with similar temperatures. Aside from a few isolated cases, for example HR 8799 bcde, the spectroscopic properties of imaged exoplanets are more poorly constrained at wavelengths longward of 2.5 micron. The 3-5 micron range is particularly interesting since it contains both methane and 12 Carbon Monoxide absorption features diagnostic of carbon chemistry, Carbon Monoxide, Methane and cloud structures. The Lp-band spectra of hotter young M7 to L3 companions, show no significant difference with those of field dwarf counterparts. The Lp spectrum of kappa Andromedae b is well matched with that of yo

Post-Vaccination Syndrome: A Descriptive Analysis of Reported Symptoms and Patient Experiences After Covid-19 Immunization. https://rumble.com/v3t4yzj-index-of-science.-music-by-dan-vasc.html Harlan M. Krumholz, MD, SM, and others. Center for Outcomes Research and Evaluation, Yale New Haven Hospital, New Haven, Connecticut. Corresponding author: Dr. Harlan M. Krumholz 195 Church Street, Fifth Floor New Haven, CT 06510 203-497-1246 Email: harlan.krumholz@yale.edu medRxiv preprint doi: https://doi.org/10.1101/2023.11.09.23298266 https://www.medrxiv.org/content/10.1101/2023.11.09.23298266v1 ABSTRACT. Introduction: A chronic post-vaccination syndrome (PVS) after covid-19 vaccination has been reported but has yet to be well characterized. Methods: We included 241 individuals aged 18 and older who self-reported PVS after covid-19 vaccination and who joined the online Yale Listen to Immune, Symptom and Treatment Experiences Now (LISTEN) Study from May 2022 to July 2023. We summarized their demographics, health status, symptoms, treatments tried, and overall experience. Results: The median age of participants was 46 years, interquartile range, IQR, 38 to 56, with 192 (80%) identifying as female, 209 (87%) as non-Hispanic White, and 211 (88%) from the United States. Among these participants with PVS, 127 (55%) had received the BNT162b2, Pfizer-BioNTech, vaccine, and 86 (37%) received the mRNA-1273, Moderna vaccine. The median time from the day of index vaccination to symptom onset was three days, IQR 1 day to 8 days. The time from vaccination to symptom survey completion was 595 days (IQR: 417 to 661 days). The median Euro-QoL visual analogue scale score was 50, IQR 39 to 70. The five most common symptoms were exercise intolerance (71%), excessive fatigue (69%), numbness (63%), brain fog (63%), and neuropathy (63%). In the week before survey completion, participants reported feeling unease (93%), fearfulness (82%), and overwhelmed by worries (81%), as well as feelings of helplessness (80%), anxiety (76%), depression (76%), hopelessness (72%), and worthlessness (49%) at least once. Participants reported a median of 20, with the IQR from 13 to 30, interventions to treat their condition. Conclusions: In this study, individuals who reported PVS after covid-19 vaccination had low health status, high symptom burden, and high psychosocial stress despite trying many treatments. There is a need for continued investigation to understand and treat this condition. INTRODUCTION. The vaccines against SARS-CoV-2 have saved many lives, but adverse events have been reported. The Centers for Disease Control and Prevention notes the possibility of rare complications, including anaphylaxis, thrombosis with thrombocytopenia syndrome, specifically after viral vector vaccines, Guillain-Barre Syndrome, specifically after viral vector vaccines, and myocarditis and pericarditis. These complications were not reported in the vaccine clinical trials, emphasizing the limitations of these studies in capturing rare adverse events and highlighting the critical role of post-market surveillance. A less well-characterized adverse event is a chronic syndrome with symptoms that begin soon after vaccination. A recent preprint from the US National Institutes of Health described 23 people who reported neuropathic symptoms starting within 21 days after vaccination. The cause of this syndrome is undefined, diagnostic tests and evidence-based interventions are lacking, and its connection with vaccination remains controversial. A first step in understanding what these patients experience is a description of their symptoms, treatments, and health status. This information can lay the groundwork for enabling prevention, mitigation, and treatment. Accordingly, we sought to describe the characteristics, symptoms, health status, treatment, and experience of individuals who report post-vaccination syndrome (PVS) using data from the Yale Listen to Immune, Symptom and Treatment Experiences Now (LISTEN) study, an online observational study. METHODS. Study Design. LISTEN is a cross-sectional study that collects participant-reported, participant-generated, and clinical data. Deep immune phenotyping is carried out for a subset of individuals, which involves comprehensive profiling of immune cell subtypes, their related products, and their functional states. LISTEN participants were recruited from Hugo Health Kindred, an online patient community. This platform, for which recruitment occurs mainly through social media and word of mouth, provides opportunities for information exchange, and allows members to share survey responses and health data with research studies. The LISTEN study began recruiting participants who reported PVS in May 2022. PVS was defined by self-report in response to whether the individual thought the vaccine had injured them. We summarized these individuals' symptoms, diagnoses, treatments, and experiences. Patient Involvement Patients (DH, BD) were research partners in the study and participated in identifying and prioritizing the research question. Patient partners, including DH, were involved in designing the survey, assessing the burden and time required to participate in the study, recruiting participants, and interpreting results, and will be involved in disseminating the research findings. Study Sample The study sample includes participants aged 18 years and older who reported PVS from May 2022 through July 2023. We did not include people who concurrently reported long covid. See Supplemental Figure 1. Data Collection. The Kindred platform provided a series of surveys, see Supplemental Appendix that collected demographic, infection, vaccination, clinical, and social information. The surveys were developed using an iterative process, including feedback from potential participants reporting PVS, to ensure they were relevant and understandable to those participating. The surveys were provided only in English due to funding limitations. Surveys could be completed on computers or mobile devices, and reminders were sent to encourage completion. Surveys were completed between November 2022 and July 2023, with half completed by December 2022. See Supplemental Figure 2. Data were extracted on July 7, 2023. Variables. The surveys included questions about the participants' demographic information, health status, and prior SARS-CoV-2 infection and vaccinations. The duration from the day of index vaccination to the day of the survey completion was a median of 595 days, Interquartile Range, IQR 417 to 661 days; range: 40 to 1058 days. Prior medical conditions were assessed using the question, "Have you ever been told by a doctor before January 2020 that you have any of the following?" followed by a list of 30 medical diagnostic categories, eight psychiatric diagnostic categories, "other," and "none of the above." Current medical conditions were assessed similarly, with 31 medical diagnostic categories, 8 psychiatric diagnostic categories, "other," and "none of the above." The question that assessed vaccine-associated symptoms was, "Please select all the following health conditions that you have had as a result of vaccine injury," followed by a list of 96 specific symptoms, "other," and "none of the above." Self-reported health status was assessed on a 5-point scale, excellent, very good, good, fair, or poor, based on individuals' self-perceived general health. The Euro-QoL visual analogue scale (EQ-VAS) was used to quantify the quality of life, with 100 representing the best. Symptom severity was collected by asking, "On your worst days, how bad are your symptoms, 0 being trivial illness and 100 being unbearable?" Statistical Analysis. We characterized participants by their demographics, age, gender, race, country of residence, marital status, pre-pandemic household income, employment status, and insurance status, vaccine type received, pre-pandemic comorbidities, PVS symptoms, symptom severity, duration of symptoms since vaccination, treatments tried, new-onset medical conditions since vaccination, and psychological and socioeconomic status. These characteristics are described using percentages for categorical variables and median and IQR for continuous variables. We then described the differences in characteristics by age, younger than 60 years versus 60 years or older, gender, and type of index vaccine. We also summarized the EQ-VAS score on people without prior substantial comorbidities. Such comorbidities did not include common allergies generally perceived as non-life threatening, dyslipidemia, and hypertension. All statistical analyses were performed in R version 4.3.1, June sixteenth, 2023. The Yale University Institutional Review Board approved the LISTEN study. We followed STROBE reporting guidelines. Harlan Krumholz, a co-founder of Hugo Health, developed the Hugo Kindred platform, and the Yale Conflict of Interest Committee oversees his involvement. RESULTS. Study Sample. The study population comprised 241 individuals with self-reported PVS, Supplemental Figure 1. Among the participants reporting PVS, 127 (55%) were those who had taken the Pfizer-BioNTech vaccine, followed by 86 (37%) who had taken the Moderna vaccine, shown in Table 1. The participants' demographic and psychosocial characteristics are shown in Table 2. The median age was 46 years, IQR from 38 to 56; 192 (80%) identified as female; 7 (3%) reported that they were non-Hispanic Black and 209 (87%) as non-Hispanic White. There were 127 (55%) participants who received the BNT162b2, Pfizer-BioNTech vaccine, 86 (37%) the mRNA-1273, Moderna vaccine, 15 (6%) the Janssen Ad26.COV2.S Johnson and Johnson vaccine, 1 (0.4%) the NVX-CoV2373, Novavax, vaccine, and 4 (2%) the ChAdOx1 nCoV-19, AstraZeneca vaccine. Additionally, 82 (34%) participants reported being infected by the SARS-CoV-2 virus at least once. The participants were mainly from the United States (88%), and in this group, there were 5 uninsured individuals, 176 with private commercial insurance, and 8 Medicaid beneficiaries. Before the pandemic, 194 (82%) were employed. Regarding income, 166 (69%) reported an annual income greater than or equal to 75,000 dollars. Approximately three quarters had one or more pre-pandemic comorbidities including gastrointestinal issues reported by 67 (28%), anxiety by 61 (25%), asthma by 49 (20%), depression by 49 (20%), and migraines by 46 (19%). There were 59 (25%) participants who did not have any prior substantial comorbidities. The frequencies of pre-pandemic comorbidities are shown in Supplemental Table 1. The duration from the day of index vaccination to the day of the survey completion was a median of 595 days, IQR from 417 to 661 days; range: 40 to 1058 days. Participants reported numerous challenges in their daily lives, Supplemental Table 2. In the week before survey completion, 221 (93%) reported feeling unease at least once, 194 (82%) felt fearful, 192 (81%) felt overwhelmed by worries, and 180 (76%) struggled with anxiety. Furthermore, 190 (80%) felt helpless, 182 (76%) depressed, 171 (72%) hopeless, and 116 (49%) worthless at least once in the week before survey completion. Additionally, 233 (98%) felt rundown and 216 (91%) reported sleep problems. Pain interfered with the daily activities of 204 (86%) participants. Regarding social support, 98 (41%) had two or fewer supportive individuals to rely on for help. Getting help from neighbors was described as challenging or very challenging by 86 (36%), while 24 (10%) rarely or never had assistance for tasks like shopping or visiting the doctor. Loneliness was prevalent, with 47 (20%) often feeling a lack of companionship, 55 (23%) feeling left out, and 77 (32%) feeling isolated. Furthermore, 28 (12%) often or always felt lonely. Concerns related to living situations and food security were also prominent. Among all participants, 21 (9%) feared running out of food before they could afford to buy more, and 16 (7%) had stable housing but expressed worry about future loss. Transportation issues impeded 13 (5%) participants from carrying out essential non-medical tasks and 10 (4%) from attending medical appointments. Timing of Symptom Onset Following Vaccination. The median time from vaccination to the onset of any symptoms was 3 days, with an IQR of 1 to 8 days, Table 2, Supplemental Figure 3. Symptoms began after the first, second, third, and fourth (or more) vaccinations for 106 (44%), 80 (33%), 33 (14%), and 22 (9%) participants, respectively. Health Status. The median EQ-VAS score among participants was 50, IQR of 39 to 70, Figure 1. We evaluated the distributions of EQ-VAS scores in men. The median was 50, the IQR was 37 to 70, and women, median 51; IQR 39 to 69, age groups, less than 60 years old median: 50; IQR: 39 to 65. For Greater than or equal to 60 years old the median was 61, the IQR: 39 to 76. And Pfizer-BioNTech, the median was 51, the IQR was 35 to 70, and Moderna the median was 50, the IQR was 40 to 62, index vaccination types, Figure 1. The median EQ-VAS score in the participants without previous comorbidities was 52, with an IQR of 36 to 70. The median EQ-VAS score in the participants with any prior comorbidities was 50, with IQR of 39 to 66. There were 106 (44%) participants who rated their current health as fair or poor, Supplemental Table 3; Supplemental Figure 4, and their median EQ-VAS score was 40, with an IQR of 30 to 52. Symptom Severity. When asked to quantify symptom severity on their worst days, 0 representing a trivial illness and 100 for an unbearable condition, participants reported a median severity of 80, IQR of 69 to 89, Supplemental Figure 5; Supplemental Table 3. On their worst days, participants who rated their current health as fair or poor reported a median symptom severity of 80 points, with the IQR from 70 to 90. Symptom Characteristics. The symptoms reported by the participants are shown in Figure 2, Supplemental Figure 6, and Supplemental Table 4. The median number of symptoms attributed to PVS was 22, the IQR was 13 to 35. The most common symptoms were exercise intolerance reported by 170 (71%) participants, excessive fatigue by 167 (69%), numbness by 153 (63%), brain fog by 151 (63%), neuropathy by 151 (63%), insomnia by 148 (61%), palpitations by 145 (60%), myalgia by 132 (55%), tinnitus or humming in ears by 131 (54%), headache by 128 (53%), burning sensations by 121 (50%), and dizziness by 121 (50%). New Diagnoses since the Pandemic. The most common new diagnoses in the study sample since the beginning of the pandemic were anxiety, 49 (36%) participants, neurological conditions, 79, 33%, gastrointestinal issues 73, 30%, and postural orthostatic tachycardia syndrome (POTS), 70, 29%. Supplemental Table 5. There were 53 (22%) participants who reported migraine and 49 (20%) who reported depression. Treatments. Participants reported having taken many treatments, Figure 3, Figure 4, and Supplemental Table 6. The total number of unique treatment types was 209, which we grouped into 40 categories. The median number of individual treatments tried was 20, IQR of 13 to 30; range: 0 to 65. The most common prescription therapies reported were oral steroids 116, 48% participants, gabapentin 61, 25%, low-dose naltrexone 48, 20%, ivermectin 44, 18%, propranolol 27, 11%, and bronchodilators 26, 11%. More than 500 additional treatments were reported by participants, Supplemental Table 6. The most common non-pharmacological treatment included limiting exercise or exertion by 124, 51% of participants, quitting alcohol or caffeine, 105, 44%, hydration and increasing salt intake 105, 44%, and intermittent fasting 95, 39%. DISCUSSION. This study is the largest to describe people who report a severe, debilitating chronic condition following covid-19 vaccination. This chronic condition began soon after covid-19 vaccination and persisted in many people for a year or more. The symptoms reported are diverse and severe. Despite having tried many treatments, the median EQ-VAS score was low. Although the cause of PVS has yet to be established, this report is a step toward acknowledging the suffering it creates. It signals the need for scientific investigation to help us understand its mechanism and develop prevention, mitigation, and cure strategies. This observational study of self-referred individuals cannot determine causality or provide estimates of the incidence and prevalence of PVS. Although there is a background rate for conditions unrelated to vaccination that can produce many symptoms reported by participants, these individuals do not have other diagnoses to explain their symptoms. Many participants did not have chronic conditions before the pandemic. Also, we excluded LISTEN participants who reported long covid. PVS could be caused by several potential mechanisms, including a mechanism related to the vaccination or manufacturing process. It may represent a rare response to vaccines in susceptible individuals. Some investigators have concluded, based on their self-referred case series, that vaccines may cause immune system dysfunction. They focused on people with neuropathy after vaccination and could not identify other causes. In 2 of 5 participants, the cerebrospinal fluid had oligoclonal bands, and they found immune complexes in some skin biopsies. Nevertheless, the syndrome could be unrelated to the vaccination, occurring by chance during the vaccination period. However, the temporal relationship with clustering of symptom onset within the first 1 to 18 days from the index vaccine suggests a potential relationship. The possibility that the syndrome may be related to the vaccination has implications for future vaccine development and safety surveillance. Research in this area has the risk of being embroiled in debates about vaccinations. The net benefit of the covid-19 vaccination program is clear, and there are concerns about vaccine hesitancy. But fears of inciting vaccine hesitancy should not impede efforts to research this condition, and make progress for people who are suffering. This study has several limitations. It is an uncontrolled observational cross-sectional study of self-referred participants who reported that they had the onset of symptoms soon after vaccination, which, in many cases, persisted for more than a year. The participants are not representative, so it is not possible to estimate the incidence or who might be most susceptible to this condition. Participating in LISTEN required people to join the community, consent to the study, and complete the surveys. This approach may have skewed our sample away from people who were too ill to participate or had substantial cognitive dysfunction. Also, the study required online access, some digital literacy, and English fluency, further limiting participation and representativeness. Since participants were strongly skewed toward those who reside in the United States and self-identify as White, efforts are needed to study a more inclusive group. Future investigations need more outreach and partnerships with diverse groups, low-income communities, communities of color, and other countries to assemble a more representative participant group. CONCLUSION. In conclusion, people reporting PVS after covid-19 vaccination in this study are highly symptomatic, have poor health status, and have tried many treatment strategies without success. As PVS is associated with considerable suffering, there is an urgent need to understand its mechanism to provide prevention, diagnosis, and treatment strategies.

Welcome to the Dalek chanel, where we value your mind, and not just your eighteen year old daughters body. Movies are wonderful things. The Marvels Cinematic Universe is a marvelous thing. Superhero Marvel movies have apparently made a lot of money. But, we ask, exactly how crappy are they? How crappy is the Marvel Crapatronic Universe? All data taken from the website The Numbers, and only domestic US sales are included. Here we utilize the Iso-Crap model of movie revenue. Equally crappy movies have the same proportionally crappy evolution. Whether they interest the same people, or because of the same marketing. Or perhaps the Hurricanes in California, they have a few similar features. These financially underperforming fantasy genre movies of the Marvel Cinematic Universe, or M C U are examined here. All of the domestic box office revenue is normalized, meaning divided by the first three days of revenue, or the day three as reported from the numbers. As an example the MCU phase five movie: Blue Beetle had an initial three day opening of: 25 point zero million. And a total domestic box office of: 72 point 49 million. Therefore, the iso-crap of blue beetle is a value of 1 after three days, and 72.48 divided by 25.0 or 2.896 at day 77. To put it another way, for every dollar Blue beetle bought in in the first three days, it needed another two and a half months to get the next two dollars. But what was it like back in the old days? Well let’s have a look at MCU phase one. The MCU Phase one consisted of: Iron Man, Hulk, Iron Man two, Thor, Captain America and the Avengers. The evolution of these films is shown in figure one. Normalizing their three day revenue to one, the average over the MCU phase one is 2.75. What does this curve illustrate? Well, after the three day opening, at some value determined by buzz, advertising, fan interest, or a piece of dirt on the radar screen at Norad, the movie revenue evolves. Reviews are written. Reviews are read. And the toxic fandom word of mouth spreads, and people are drawn into the theatre. The iso-crap here is the revenue divided by whatever the movie made in its first three days, so all the films are comparable to their initial interest. The best long term performer, the one with the highest iso crap, was Iron Man, with an Iso Crap of three point one two. In figure one point one, we add an average movie. And it’s a very average movie, as we will reveal later. As we can see, even in the glory days of MCU phase one, none of the films bought in more movie goers compared to the three day opening than an average movie. The plot of this average movie is shown on the numbers “Average-ness graph”, to display how thoroughly average it was. Time went on, and along came MCU Phase two. It bought us such memorable gems as Iron Man one hundred and eleven, or perhaps that’s a three, Thor, the Dark World, Captain America, The Winter Soldier, Guardians of the Galaxy, the Avengers, AOU, and I can’t remember what AOU stands for, and last but not least, Ant-man. All their revenue is shown in Figure 2. The best Isocrap was Guardians of the Galaxy, which reached the level of averageness with an Iso-crap of three point five four. Overall, the iso-crappiness of the MUC phase two was: two point seven four. So Guardians of the Galaxy bought in as many people after its three day opening, as any average film. It achieved the peak transitional breakthrough of being average. Figure three: Along came MCU phase three, from twenty sixteen to twenty nineteen. Phase three had such wonderful little gems as Captain America and the semi colons, and the Civil war. Doctor Strange, and a semi colon and something else in the title. Guardians of the Galaxy vengeance Waffe two, Spiderman Homecoming. Thor and the Semi Colon of Ragnerok. Black Panther, Avengers Infinity War, Ant man and the Wasp, Captain Marvel and Avengers Endgame, which also had the memorable semi colon. Here again, the average iso-crap of the MCU was two point seven six. Almost indistinguishable from the earlier phases. The peak performer was Black Panther with an Iso-Crap of three point four six. Figure Four. And the came MCU phase four or five, along with the inevitable semi colons. We had movies like Blue Beetle, Ant-man and some more semi colon quantum things, The Flash, The Marvels, That blonde chick and her aunt, which was released under the title of “Black Widow”, Shang Chi, Doctor Strange, in some other movie, Thor and the love of Chunder, Black Panther in some other sequel movie, the Eternals, and the aforementioned Guardians Volume three. Guardians of the Galaxy Volume three peaked three point zero one, and the whole Phase four to five Had an isocrap of two point four five. Over all, the Entire MCU to date, Ignoring the Marvels, which has not crapped to completion in theatres, the MCU averages two point six five. The best performers? With iso-craps near three, we have Blue beetle, Shang Chi, and Guardians of the Galaxy. Indiana Jones and the Rotary phone time travel thing as also iso-crapic with these three. The worst? The Flash and Ant-man, both iso-craps of each other at around two point zero. So what? Well, if an MCU movie is released, and makes a hundred million in three days, the average final domestic box office will be two point six five million. Or in other words, for every dollar bought in during the first three days, the rest of the domestic box office run will bring in one dollar sixty five cents. If we ignore overseas income, which is usually a smaller fraction of box office revenues, a movie studio will receive as income about half of the box office revenue. For an Average MCU movie, this is about half of the average iso-crap, or one point three two times its three day opening. So, if an average MCU movie is released, and makes one hundred million over three days, the Movie studio is likely to receive, on average, one thirty two million back. Which means, that average MCU movie is going to have to cost less than one thirty two million to make if the Studio wants to see any profit. We might then ask, what did the MCU movies make as a three day opening, over all phases? Here we include the Marvels, since it has emphatically had its three day opening. And the number is: MCU phase one, average three day 103 million. MUC Phase two, three day opening 116 million. MCU Phase three, average of 165 million. MCU Phase 4 to 5, a value of 95.9 million. Or about 120 million, over all phases, and close enough to one hundred million if we consider phase one or phase four to five by themselves. For optimistic accounting, if we took the MCU phase three average of 165 million three day opening, and the Average MCU phase three iso-crap of two point seven six, then for a fifty percent return of the domestic gross, and ignoring foreign sales, the average movie would have to cost less than two hundred and eighteen million to break even. Well, the best of the best performers of the MCU, the original Guardians of the Galaxy, and Black Panther, bought in movies like an average move, with iso-craps of about three point five. Whatever happened with reviews, twitter-bots, word of mouth, those two movies grew the initial pool of movie goers like the average flick does. What was that average movie with an iso-crap of about 3.5 that beat almost everything, and tied with the best the MCU could produce? Indiana Jones and the Crystal Skull. 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Movies are wonderful things. Movie revenue starts on an opening weekend, and then the income decreases, and the movie is pulled from theatres. Here we introduce the Iso-Crap model of movie revenue. Equally crappy movies have the same proportionally crappy opening, and so might be scaled. That’s the theory, and for five minutes or less it might be sorta accurate. We take as examples, Indiana Jones and the Dial of Destiny, Blue Beetle, Ant man Quantum-mania, and Blue Beetle. These are financially underperforming recent movies of the same fantasy genre. All of the domestic box office revenue is normalized, meaning divided by the first three days of revenue. Indiana Jones and the Dial of Destiny has an initial three day opening of: 60 point 4 million. And a total domestic box office of: 174 million. Therefore, the iso-crap of Indiana Jones has a value of 1 at three days, and 174 divided by 60.4 or 2.89 at day 77. Blue Beetle had an initial three day opening of: 25 point zero million. And a total domestic box office of: 72 point 49 million. Therefore, the iso-crap of blue beetle is a value of 1 after three days, and 72.48 divided by 25.0 or 2.896 at day 77. Blue Beetle and Indiana Jones and the Dustbin of density have very similar iso-craps. Ant Man Quantum-mania had an initial three day opening of: 106 point one million. And a total domestic box office of: 214 point five million. Therefore, the iso-crap of Ant Man Quantum-mania has a value of 1 at three days, and 214.5 divided by 106 or 2.02 at day 119. The Flash had an initial three day opening of: 55 point zero million. And a total domestic box office of: 108 point one million. Therefore, the iso-crap of The Flash has a value of 1 at three days, and 108.1 divided by 55.0 or 1.96 at day 63. Ant Man Quantum-Mania and The flash have similar iso-craps, Figure One. The iso-craps for these four movies are shown in the figure. So, Where does captain marvel and the Mids come in? The Marvels had an opening three day of 46 point one million. Assuming it is iso-crappy with Antman and the Flash, it will have a domestic iso-crap of around 2 when it finally is pulled, and hence a domestic box office of 92 million. If the Marvels has an Isocrap of Indian Jones and the thing we’ve already forgotten about, it will top off at three times the initial three day opening, or three times forty six, or a domestic revenue of about 138 million dollars. The performance of The Marvels is shown in Figure two. At this point, after ten days, it does not appear that the Marvels is noticeably different from the four other randomly chosen movies. As always, we love all of our subscribers.

Research in Astronomy and Astrophysics. Type One-A Supernova Explosions in Binary Systems: A Review. Zheng-Wei Liu and others. Yunnan Observatories, Chinese Academy of Sciences, Kunming, China; Abstract Type One-A supernovae, SNe One-A, play a key role in the fields of astrophysics and cosmology. It is widely accepted that SNe One-a arise from thermonuclear explosions of white dwarfs, WD’s in binary systems. We can distinguish two kinds of supernovae, corresponding to two kinds of star death: Type one-a, thought to be the thermonuclear explosions of accreting white dwarf stars, and all the rest, Type two, one-b, one-c and so on), which happen when the iron core of a massive star collapses to a neutron star or black hole. Observationally, Type one is defined by a lack of hydrogen lines in its spectrum, lines that Type two has. However, there is no consensus on the fundamental aspects of the nature of SN One-a progenitors and their actual explosion mechanism. This a fundamental limit in our understanding of these important astrophysical objects. In this review, we outline the diversity of SNe One-a and the proposed progenitor models and explosion mechanisms. We discuss the recent theoretical and observational progress in addressing the SN One-a progenitor and explosion mechanism in terms of the observables at various stages of the explosion, including rates and delay times, pre-explosion companion stars, ejecta, companion interaction, early excess emission, early radio, X-ray emission from circumstellar material (CSM) interaction, surviving companion stars, late-time spectra and photometry, polarization signals, and supernova remnant properties, etc. Despite the efforts from both the theoretical and observational side, the questions of how the WD’s reach an explosive state and what progenitor systems are more likely to produce SNe One-a remain open. No single published model is able to consistently explain all observational features and the full diversity of SNe One-a. This may indicate that either a new progenitor paradigm or the improvement of current models is needed if all SNe one-a arise from the same origin. An alternative scenario is that different progenitor channels and explosion mechanisms contribute to SNe One-a. In the next decade, the ongoing campaigns with the James Webb Space Telescope, Gaia and the Zwicky Transient Facility, and upcoming extensive projects with the Vera C Rubin Observatory Legacy Survey of Space and Time and the Square Kilometre Array will allow us to conduct not only studies of individual SNe One-a in unprecedented detail but also systematic investigations for different subclasses of SNe One-a. This will advance theory and observations of SNe One-a sufficiently far to gain a deeper understanding of their origin and explosion mechanism. One. INTRODUCTION. Supernovae (SNe) are highly energetic explosions of some stars, that are so bright that they can outshine an entire galaxy. Their typical bolometric luminosities reach the order of ten to the forty three ergs per second, which is about ten billion times the solar luminosity. SNe play an important role in the fields of astrophysics and cosmology because they have been used as cosmic distance indicators, and they are heavy-element factories, especially for intermediate mass and iron-group elements, kinetic-energy sources, and cosmic-ray accelerators in galaxy evolution. SNe are also key players in the formation of new-generation stars by triggering the collapse of molecular clouds. SNe are generally classified into two main categories according to their spectroscopic features, Type One and Type Two SNe. Type One SNe have no hydrogen, H, lines in their spectra whereas Type two SNe contain obvious H lines. Type One-a SNe, SNe Ione-a are a subclass of Type One which exhibit strong singly ionized silicon, Si, absorption, Si two at 6150, 5800 and 4000 Angstroms feature in their spectra. SNe One-a are widely thought to be thermonuclear explosions of white dwarfs (WDs) in binary systems, Hoyle and Fowler 1960. They have been found to occur in all galaxy types. Their typical peak luminosity in the B-band is about MB equals minus 19.5 magnitudes, and the typical kinetic energy is around ten to the fifty one ergs, or equivalently, ten to the forty four Joules. The light curves of SNe One-a are powered by the Compton scattering of gamma rays produced by the radioactive decay of Nickel 56 to Cobalt 56 to Iron 56, with respective half-life times of 6.1 and 77 days SNe One-a have been successfully used as cosmic distance indicators to constrain cosmological parameters, which has led to the discovery of the accelerating expansion of the Universe, a breakthrough rewarded with the 2011 Nobel Prize in physics. Despite their importance and far-reaching implications, the specific progenitor systems as well as the explosion mechanism of SNe One-a remains enigmatic. This affects the reliability of necessary assumptions such as those of universality of their calibration as distance indicators. Recently, it was found that the local measurements of the Hubble constant H sub zero, based on SNe one-a is inconsistent with the value inferred from the cosmic microwave background radiation observed by the Planck satellite assuming a Capital Lambda CDM cosmological world model. To determine whether this so-called “H sub zero tension” hints to new physics, it is critical to improve our understanding of SNe One-a and, more specifically, their progenitors and explosion mechanisms. Two. THE DIVERSITY OF SNe One-a. A large fraction of observed SNe one-a, around seventy percent, is found to show remarkable homogeneity and quantifiable heterogeneity, and they exhibit a clear empirical relationship between light curve width and peak luminosity, meaning the so-called “Phillips relation”, sometimes known as the width–luminosity relation, WLR. These SNe one-a are usually referred to as “normal SNe one-a”, and they have long been used as standard candles for measuring cosmological distances. However, an increasing number of SNe one-a has been observed that does not follow the Phillips relation, see Figure one, and they are diverse in their observational characteristics such as light curveshape, peak luminosity and spectral features. For these reasons, SNe one-a have been classified into different sub-classes diverging from normal events, which include 1991T-likes 1991bg-likes, SNe One-ax, meaning, SN 2002cx-likes, 2002es-likes, Carich Objects, meaning SN 2005E-like, super-Chandrasekhar objects, meaning SN 2003fg-likes; SNe One-a-CSM and fast decliners. The diversity of SNe One-a has recently been reviewed by Taubenberger in 2017, and so we only skim the surface here. 1991T-like objects form a luminous, slow-declining subclass of SNe One-a, named after the well-observed SN 1991T. Their optical spectra at pre-maximum phases show extremely weak Calcium Two H and K and Silicon two wavelength 6355 and strong Iron three absorption features. 91T-like SNe are expected to be on average zero point two to zero point five magnitudes more luminous than normal SNe One-a with similar decline rate. 1991T-like SNe are found preferentially in late-type galaxies, suggesting that they are likely associated with young stellar populations. It has been suggested that 1991T-like SNe could contribute two to nine percent to all SNe One-a in the local Universe. 1991bg-like objects are a cool, subluminous, and fast-declining subclass of SNe One-a with low ejecta velocity. Typically, they are fainter than normal SNe One-a in optical band up to 2.5 magnitudes. Their spectra at maximum light show strong Titanium two absorption, indicating a relatively cool photosphere. 1991bg-like SNe are found preferentially in early-type, meaning, passive galaxies. Only few 1991bg-like SNe have been found in spiral galaxies. This suggests old stellar populations for the progenitors of 1991bg-like SNe. There is no agreement about the rates of 1991bglike SNe in the literature, estimates range from six to fifteen percent of all SNe One-a. SNe One-a-x are proposed as a hot, sublumious, subclass of SNe One-a. SNe One-ax are fainter than normal SNe One-a and highly skewed to late-type galaxies. Their explosion ejecta are characterized by low expansion velocities and show strong mixing features. Their maximum-light spectra show similar features to those of 1991T-like SNe, which are characterized by weak Silicon two wavelength 6355 Angstrom features and dominated by Iron three lines. In addition, strong Helium lines are identified in spectra of two events, meaning, SN 2004cs and SN 2007J. The late-time spectra of SNe One-a-x are dominated by narrow permitted Iron two. It has been suggested that they contribute about one third of total SNe One-a. 2002es-like objects are another cool, rapidly fading, subluminous subclass of SNe One-a which have a peak luminosity and ejecta velocity around 6000 kilometers per second, similar to SN 2002cx. Their spectra at near maximum light phases share some characteristics in common with the subluminous 1991bg-like SNe, which are clearly characterized by strong Titanium two, Silicon two, and Oxygen two absorption features. However, 2002es-like SNe do not have the fast-declining light curves characteristic of 1991bg-like events. White suggested in 2015, that 2002es-like events tend to explode preferentially, but not exclusively, in massive, early-type galaxies Ganeshalingam in 2012 suggested that SN 2002es-like objects should account for around two point five percent of all SNe One-a. Calcium-rich objects, Ca-rich, constitute a peculiar subclass of SNe One-a with SN 2005E as a prototype. Ca-rich SNe are primarily characterized by peak magnitudes of minus 14 to minus 16.5 magnitudes, rapid photometric evolution with typical rise times of 12 to 15 days, and strong Calcium features in nebular phase spectra. They exhibit low ejecta and Nickel 56 masses of less than a half Solar mass, and less than a tenth of a solar mass, respectively. The majority of Calcium-rich SNe has been observed in early type galaxies and the inferred rates of such SNe are likely in the range of five to twenty percent of the normal SN One-a rates. Super-Chandrasekhar objects are sometimes known as SN 2003fg-like SNe. They are referred to as “super-Chandrasekhar SNe” because a differentially rotating WD with a super-Chandrasekhar mass of around two solar masses was used to interpret the observations of SN 2003fg. The main features of this subtype are summarized by Ashall in a 2021 article: They are generally characterized by high luminosities, B-band peak absolute magnitudes of minus 19 to minus 21 magnitudes, broad light curves, delta m 15 B less than 1.3 magnitudes, defined as the decline in the B-band magnitude light curve from peak to 15 days later, and relatively low ejecta velocities. To repeat, the quantity delta M 15 B is the decline in the B-band magnitude light curve from peak to 15 days later. This is puzzling for a theoretical explanation: the first two properties point to a powerful explosion which seems to be at odds with the low ejecta velocities. They have only one i-band maximum which peaks after the epoch of the B-band maximum, but with weak (or without) i-band secondary maximum. Their maximum-light spectra do not show a Titanium two feature; in addition, their nebular-phase spectra are characterized by a low ionization state. Super-Chandrasekhar SNe seem to be preferentially found in low-mass galaxies, indicating that they prefer a low-metallicity environment. They seem to make up a small fraction of SNe One-a, but their exact rates are still unknown SNe One-a-CSM are a subclass named after the discovery of SN 2002ic, although there is still a debate on whether these objects are SNe One-a or in fact core-collapse SNe. A list of several common features of SNe One-a-CSM has been compiled by Silverman in a 2013 article. They have a range of R-band peak absolute magnitudes of MR minus 19 to minus 21.3 magnitudes, and they exhibit narrow hydrogen emission features in their spectra. The presence of narrow H lines is thought to arise from circumstellar material, CSM, which is strongly indicative of mass loss, or outflows, of the progenitor system prior to the SN explosion. An initial systematic study of this subclass has been presented by Silverman in a 2013 article, and it has been recently updated by Sharma in 2023. SNe One-a-CSM are preferentially found in late-type spirals and irregular galaxies, indicating the origin from a relatively young stellar population. The rate of SNe One-a CSM is estimated to be no more than a few per cent of the SN one-a rates. Fast decliners are rare and the extremely rapidly declining SNe. So far, this class includes SN 1885A, SN 1939B, SN 2002bj, SN 2005ek, SN 2010X. Whether these peculiar objects arise from thermonuclear explosions of WD’s or core-collapse explosions of massive stars remains open. There is no conclusion on whether or not all of these objects actually belong to the same class of events Three. PROGENITORS AND EXPLOSION MECHANISMS. It is widely accepted that SNe One-a arise from thermonuclear explosions of white dwarfs, WD’s in binary systems, Hoyle and Fowler 1960. However, there is no consensus on the fundamental aspects of the nature of SN One-a progenitors and their explosion mechanism from both, the theoretical and observational side. In this section, potential progenitor models and explosion mechanisms of SNe One-a are briefly summarized. Three point one. Progenitor scenarios. Three point one, point one. Single-degenerate scenario. In the single-degenerate (SD) scenario, a WD accretes hydrogen-rich or helium-rich material from a nondegenerate companion star through Roche-lobe overflow, RLOF, or stellar wind until its mass approaches the Chandrasekhar-mass, around one point four solar masses, at which point a thermonuclear explosion ensues. The companion star could be either a main-sequence, MS, star, a subgiant, SG, a red giant, RG, an asymptotic giant-star, AGB, or a Helium star. It has been suggested that a Chandrasekhar-mass WD can undergo a deflagration, or a detonation or a delayed detonation to lead to a SN one-a explosion In the SD scenario, SNe One-a are thought to arise from Chandrasekhar-mass WD’s, double detonation explosions of sub-Chandrasekhar mass WD’s could happen when accreting from a Helium-star companion, the homogeneity of the majority of SNe One-a therefore can be well explained by this scenario. A schematic illustration of main binary evolutionary paths for producing SNe One-a in the SD scenario is given in Figure two. One of the key questions in the SD scenario is how the WD retains the accreted companion material and grows in mass to approach the Chandrasekhar limit, meaning the mass-retention efficiency of onto the WD. The SD scenario requires that the WD accretes material at a relatively narrow range of accretion rates of a few times ten to the minus eight or seven times the mass of the sun per year, to allow steady burning of accreted material, which causes difficulties for explaining the observed nearby SN one-a rate, see Section four. Moreover, some recent observations seem to pose a challenge to the SD scenario, see Section five, such as the missing of surviving companion stars in supernova remnants, SNR’s. The absence of swept-up Hydrogen, Helium in their late spectra and low X-ray flux from nearby elliptical galaxies. In addition, although the SD scenario makes the explosion rather homogeneous, it turns out to be difficult to cover the observed ranges in brightness and decline rates in this scenario. However, to conclude whether the SD scenario is promising for producing the majority of SNe One-a requires comparing a full range of predicted observational consequences from this scenario with the observations of SNe One-a. A number of candidate progenitors have been suggested for the SD scenario, including cataclysmic variable stars like classic novae, recurrent novae and dwarf novae, supersoft X-ray sources, symbiotic systems and WD plus hot-subdwarf binaries. In the SD scenario, a WD accretes and retains companion matter that carries angular momentum. As a consequence the WD spins with a short period which leads to an increase of the critical explosion mass. If the critical mass is higher than the actual mass of the WD, the SN explosion could only occur after the WD increases the spin period with a specific spin down timescale. This scenario is known as the “spin-up, spin-down model”. In this model, if the spin down timescale is longer than about one million years, the CSM around the progenitor system could become diffuse and reach a density similar to that of the ISM. This could explain the lack of radio and X-ray emission from SNe One-a in agreement with the current radio and X-ray observations. Also, the H rich or Helium-rich companion star, meaning, MS, subgiant, RG and Helium stars, may shrink rapidly before the SN one-a explosion occurs by exhausting most of its Hydrogen-rich or Helium-rich envelope during a long spin-down, greater than one hundred million year phase to become a WD or a hot subdwarf star. This would explain the non-detection of a pre-explosion companion star in SNe One-a and the absence of swept-up Hydrogen, Helium in their late spectra. However, no, or weak, interaction signature of shocked gas is predicted in this scenario, which makes it difficult to explain the early excess luminosity seen in some SNe One-a such as iPTF14atg and SN 2012cg. The exact spin down timescale of the WD in this model is uncertain, but it is a key to the success of the model. Three point one point two. Double-degenerate scenario. In the original double-degenerate, DD, scenario, two carbon-oxygen, CO, WD’s in a binary system are brought into contact by the emission of gravitational wave radiation and merge via tidal interaction into one single object, triggering a SN One-a explosion if the combined mass exceeds the Chandrasekhar-mass limit. There are a number of evolutionary paths that can lead to SN one-a explosions in the DD scenario, see Figure two. The key question of the original DD scenario is whether the merger of two WD’s could successfully lead to an SN one-a explosion. Different calculations have predicted that the merger of two White Dwarves would likely cause the formation of neutron stars through accretion-induces collapse, AIC, rather than SN one-a explosions. The accretion from the secondary WD onto the primary WD during the merger process may lead to burning in the outer layers of the WD rather than central burning, which would turn the original carbon-oxygen WD into an oxygen-neon-magnesium, O-Ne-Mg WD. A Chandrasekhar-mass One WD is thought to be prone to collapse into a neutron star via AIC. However, there are possibilities to avoid AIC after the merger of two CO WD’s. For instance, Yoon, in 2007 concluded that the merger of two Carbon-Oxygen White Dwarves could avoid off-center C-burning and explode as an SN one-a in the thermal evolution phase if the rotation of the WD’s is taken into account. In the past decades, a number of numerical simulations have investigated the merger of two WD’s More importantly, some recent theoretical studies have shown that the merger of two WD’s can eventually trigger an SN one-a explosion in ways that are different from the original DD scenario. For instance, a carbon detonation can be directly triggered by the interaction of the debris of the secondary WD with the primary WD during the violent merger phase of two CO WD’s to eventually trigger an SN one-a explosion, meaning the “violent merger model”. If the secondary WD in a DD binary system is a pure Helium White Dwarf, an initial Helium detonation could be triggered by accumulating a Helium shell on top of the primary Carbon-Oxygen White Dwarf through stable mass transfer, eventually triggering the C-core detonation near the center to successfully cause an SN one-a. This corresponds to the sub-Chandrasekhar-mass double detonation scenario. In addition, unstable mass transfer could also lead to the presence of Helium in the surface layers of the primary CO WD if the secondary WD is either a Helium WD or a hybrid Helium Carbon Oxygen White Dwarf, which could successfully give rise to an SN one-a during the coalescence itself through the double detonation mechanism, meaning the Helium-ignited violent merger model described in Section three point two point five. There are some evidences in favor of the DD scenario, see Sections 4 and 5 for a detailed discussion. Binary population synthesis, BPS, calculations have shown that the predicted SN one-a rates and delay times from the DD scenario could well reproduce those inferred from the observations In addition, the non-detection of pre-explosion companion stars in normal SNe One-a the lack of radio and X-ray emission around peak brightness the absence of a surviving companion star in SN one-a remnants, and the fact that no signatures of the swept-up Hydrogen, Helium have been detected in the nebular spectra of SNe One-a and the lack of X-ray flux, meaning supersoft X-ray sources, expected for accreting WD’s seem to favour the DD scenario. Also, it has been suggested that some super luminous SNe One-a that have ejecta masses of greater then two solar masses may arise from the merger of two WD’s However, the DD scenario predicts a relatively wide range of explosion masses and thus makes it difficult to explain the observed homogeneity of the majority of SNe One-a. Double WD’s, DWD’s are the primary targets of some upcoming space gravitational-wave missions and observatories such as the Laser Interferometer Space Antenna, LISA, Tianqin and Taiji. Searches for DWDs have been carried out by different surveys like the dedicated ESO Supernovae type One-a Progenitor survey, SPY. The Sloan Digital Sky Survey, SDSS, the SWARMS survey, the Extremely Low Mass, ELM, survey, the Kepler-K2 survey, and the large all-sky survey Gaia, Gaia Collaboration. However, to date, only about 150 DWD systems have been detected with detailed orbital parameters. A comprehensive list of close DWD systems, periods below 35 days, containing two low-mass WD’s are given by Schreiber in a 2022 article. Only a few DWD’s have been reported to be possible SN one-a progenitors that would merge in a Hubble time, including two systems with sub-Chandrasekhar total masses obtained by SPY (WD2020 dash 425 and HE2209 dash 1444, two super-Chandrasekhar progenitor candidates composed of a WD and a hot sub-dwarf, KPD 1930 plus 2752 and HD 265435; CD minus 30, 11223, Henize 2 dash 428 system, 458 Vulpeculae, SBS 1150 plus 599A and GD 687. Besides, Kawka in 2017 suggested that NLTT 12758 is a super-Chandrasekhar DWD system, but it would merge in a timescale longer than the Hubble time. Three point one point three. Other proposed progenitor scenarios. Some subtypes of the SD model and other possible progenitor scenarios have been proposed for SNe One-a, including: One. The CE wind model, in which the SD models are assumed to drive CE winds rather than optical thick winds when the mass transfer rate exceeds the critical accretion rate Two. The hybrid C-O-Ne WD model, in which a hybrid carbon-oxygen-neon, C-O-Ne, WD with a mass of greater than around one point three solar masses, accretes material from its companion star to approach the Chandrasekhar-mass limit and explodes as faint SNe One-a. Three. The M dwarf donor model, in which the WD accretes material from an M-dwarf star so that it approaches the Chandrasekhar-mass limit and triggers an SN one-a explosion. Four. The core-degenerate model, in which an SN One-a is produced from the merger of a COWD with the core of an AGB companion star during a common envelope, CE, evolution. Five. The triple channel, in which thermonuclear explosions in triple-star systems are triggered through both the SD and DD channels. Six. The single-star model, in which AGB stars or Helium stars with a highly degenerate CO core near the Chandrasekhar mass ignite carbon at the center to subsequently cause an SN one-a explosion if they have lost their H-rich or Helium-rich envelopes. Note that this list may not be complete and that new channels may still be proposed. Ultimately, the question of SN one-a progenitor systems has to be settled by observations. For a coarse and sketchy overview of the different progenitor scenarios of SNe One-a, we compile the different characteristics in Table one. We would like to caution here, that usually the arguments to be made in favor or against specific scenarios are more complex than what can be listed in a table. Therefore we emphasize that they are only intended for a quick overview. The main benefit of our table is to highlight open research questions that are marked with “unclear”. Three point two. Explosion models. The explosion mechanism depends mainly on the question of whether the WD explodes near the Chandrasekhar mass, or at a mass below this limit the “sub-Chandrasekhar mass” explosion scenario. To provide clues on the yet poorly understood origin and explosion mechanism of SNe One-a, one needs to compare the observational features predicted by different explosion mechanism in the context of the progenitor models discussed in Section three point one with the observations. A number of explosion models have been proposed to cover various progenitor scenarios of explosion. SNe One-a, including near Chandrasekhar-mass deflagrations, near Chandrasekhar-mass delayed detonations, gravitationally-confined detonations, sub-Chandrasekhar-mass double detonations, and violent mergers. A schematic overview of various SN One-a explosion models proposed in the framework of either Chandrasekhar-mass or sub-Chandrasekhar-mass explosion is shown in Figure three. Section three point two point one. Chandrasekhar-mass pure deflagrations. Near Chandrasekhar-mass explosions in the SD scenario have long been proposed as a potential model for SNe One-a because they could reproduce some observational features such as the light curves and spectra. Moreover, Yamaguchi and others in 2015 suggested that the detection of strong K-shell emission from stable Iron peak elements in SN one-a remnant 3C 397 requires electron captures at high density that can only be achieved by a near-Chandrasekhar mass explosion. In such a configuration, a supersonic prompt detonation would turn essentially the entire star into iron-group elements which is inconsistent with the observed features of SNe One-a: To produce the intermediate-mass elements, IME, such as Silicon and Sulphur, observed in their spectra, burning must start out as a subsonic deflagration. The WD then expands prior to being incinerated. Compared with a prompt detonation, this reduces the production of Nickel 56 and can in principle increase the IME yields. The outward propagation of the subsonic deflagration flame leads to Rayleigh-Taylor instabilities that generate turbulence at the contact between hot ashes and cold fuel. This enlarges the surface area of the burning front and accelerates it. One of commonly used near Chandrasekhar-mass explosion models is the so-called “W7 model” of Nomoto from 1984. The W7 model is a one-dimensional, 1D, pure deflagration explosion of a Chandrasekhar mass WD, in which a parametrized description was used for the turbulent burning process. To avoid free parameters in the model, multidimensional simulations, for an example, see top panels of Figure four, have been carried out. The result of these simulations is that pure deflagrations are not able to reproduce the majority of normal SNe One-a. In the framework of the the Chandrasekhar-mass deflagration model, it is difficult to produce the canonical half solar mass nickel 56 for normal SNe One-a, because the flame ultimately cannot catch up with the expansion of the WD and much of its material remains unburned. Enhancing the burning efficiency with multi-spot ignitions had only limited success. Moreover, the ignition process itself is rather uncertain and multi-spot ignition does not seem very likely according to the simulations of Nonaka from a 2012 article. However, off-center ignited weak deflagration models have been suggested to explain the particular sub-class of SNe One-ax Figure four presents an example of a 3D explosion simulation for a Chandrasekhar-mass pure deflagration model from Lach in 2022. In the weak pure deflagration model of Chandrasekhar mass WD’s, sometimes known as the “failed detonation model”, an off-center ignited pure deflagration of a Chandrasekhar-mass CO WD, or hybrid CONe WD, fails to completely unbind the entire WD, leaving behind a bound WD remnant. It has been shown that pure deflagrations in near-Chandrasekhar-mass CO WD’s and hybrid CONe WD’s can respectively reproduce the observational light curves and spectra of brighter SNe One-ax such as SN 2005hk, and, less confidently, the faint Iax event SN 2008ha have shown that the maximum light polarization signal observed in SN 2005hk can be explained in the context of a weak deflagration explosion of a Chandrasekhar-mass WD if asymmetries caused by both the SN explosion itself and the ejecta-companion interaction are considered. Therefore, the weak deflagration explosion of a Chandrasekhar-mass WD seems to be a potential model for SNe One-ax, at least the brighter members of this sub-class. Interestingly, the weak pure deflagration model of Chandrasekhar-mass WD’s predicts the existence of a surviving bound WD remnant which is significantly heated by the explosion and highly enriched by heavy elements from SN ejecta. Searches for such surviving WD remnants would be very helpful for assessing the validity of this explosion model. Section three point two point two. Chandrasekhar-mass delayed detonations. Besides pure deflagration models, pure detonations of near-Chandrasekhar-mass WD’s have also been proposed for SNe One-a. As already mentioned, the first numerically studied pure detonation model of a near-Chandrasekhar mass WD in hydrostatic equilibrium showed that this model produces too much Nickel 56 and too little IME’s to explain the observations of normal SNe One-a. This conflict indicates that an expansion of the WD is needed prior to the detonation in order to reduce the production of Nickel 56 and to increase that of IME’s. To achieve this, the “delayed detonation model” of a near Chandrasekhar-mass WD was proposed by Khokhlov in 1989: The WD expands first due to an initial deflagration and causes the subsequent detonation to burn at relatively low fuel densities, reducing the production of Nickel 56 and enhancing the yields of IME’s compared with the earlier pure detonation models. This therefore makes the delayed detonation model more favorable for explaining normal SNe One-a. Figure four shows an example of 3D explosion simulations for a Chandrasekhar-mass delayed detonation model, meaning a gravitationally confined detonation model. Several scenarios for the transition from the initial deflagration to a subsequent detonation have been proposed for SNe One-a such as the deflagration to detonation transition model, or DDT; the pulsating delayed detonation model, PDD; gravitationally confined detonation model, GCD, and the pulsational reverse detonation model. Despite substantial effort, none of the simulations could demonstrate from first principles that the transition of the deflagration to a detonation really occurs. Section three point two point three. sub-Chandrasekhar-mass double-detonations. Sub-Chandrasekhar mass WD’s can be ignited through a double detonation mechanism to give rise to thermonuclear explosions in the context of either the SD or DD progenitor scenario. The initial detonation in this model is triggered by accumulating a Helium shell on top of the primary WD through either stable mass transfer, meaning the sub-Chandrasekhar mass double-detonation model; or unstable mass-transfer, meaning the so-called D6 model; from a secondary in a binary system. In the sub-Chandrasekhar-mass double-detonation scenario, shown in figure three, the WD accretes material from a Helium-burning star or a Helium WD companion via stable mass-transfer to accumulate a Helium-layer on its surface. If the Helium shell reaches a critical mass of around zero point zero two, to zero point two Solar masses, which is, however, quite uncertain, an initial detonation of the Helium shell is triggered and eventually ignites a second detonation in the core. This leads to a thermonuclear explosion of the entire sub-Chandrasekhar mass WD. On the one hand, several binary systems composed of a WD and a Helium-rich companion star have been detected observationally, for example KPD 1930 plus 2752, V445 Pup, HD 49798, and others; which seems to support this scenario. For example, CD minus thirty, 11223 is a binary system containing a WD and a sub-dwarf-B, sdB, star, in which the WD mass is MWD equals zero point seven six solar masses, the companion mass is MsdB equal to zero point five one solar masses, and the orbital period is only Porb around one point two hours. Venneset and others suggested that CD minus thirty, 11223 will likely explode as a SN One-a via the sub-Chandrasekhar double-detonation mechanism during its future evolution. Very recently, Kupfer in 2022 predicted that PTF1 J2238 plus 7430 would lead to a thermonuclear explosion in the context of the sub-Chandrasekhar double-detonation scenario with a thick Helium shell of around zero point seventeen solar masses. On the other hand, different studies in the literature have shown that the sub-Chandrasekhar-mass double detonation models with a thick Helium shell zero point one to zero point two solar masses, produce an outer layer of SN ejecta enriched with titanium, Ti, chromium, Cr, and nickel, Ni, leading to predicted spectra and light curves that are inconsistent with the observations of SNe One-a. However, numerous complications remain to be solved in such a model, and both the production of IGEs in the outer layers and the predicted observables, such as spectra and color, are rather sensitive to the total mass, the thermal and the chemical conditions of the Helium shell, and to details of the treatment of radiative transfer modeling. For instance, Kromer showed that pollution of the Helium shell with carbon 12 helps to bring the predicted observables into better agreement with observations of normal SNe One-a. More recently, some updated simulations have shown that double detonations of sub-Chandrasekhar mass WD’s with a thin and C-polluted Helium shell holds promise for explaining SNe One-a, including normal SNe One-a and peculiar objects. Figure five shows an example of the sub-Chandrasekhar-mass double-detonation simulation of a one solar mass CO WD with a thin Helium shell of zero point zero sixteen solar masses from have suggested that the sub-Chandrasekhar-mass double-detonation scenario might be viable for producing spectroscopically normal SNe One-a if the Helium layer is sufficiently thin, around one hundredth of a solar mass, and modestly enriched with core material. This indicates that double detonations of sub-Chandrasekhar-mass WD’s may contribute the bulk of observed SNe One-a. However, the exact critical Helium shell mass required for successfully initiating double detonations of the entire sub-Chandrasekhar mass WD remains uncertain. In addition, the exact Helium retention efficiency of the accreting WD in the progenitor system is still poorly constrained. Section three point one point four. Carbon-ignited violent mergers. The “C-ignited violent merger model” of figure three is one of the modern versions of the DD scenario. In this model, unstable dynamical accretion of material from the secondary, less massive, WD on to the primary WD causes compressional heating sufficient to directly trigger a detonation of a CO core in primary WD, producing an SN one-a. While the original DD scenario assumes an explosion of a merged object exceeding the Chandrasekhar mass limit, in the violent merger model the explosion triggers already during the merger process before the two stars are completely disrupted. Therefore it proceeds in sub-Chandrasekhar mass WD’s. This scenario avoids the problem of a potential collapse to a neutron star in an AIC. It has been shown that the violent mergers of two CO WD’s that involve a single carbon detonation in the primary star can generally explain the observational properties of a sub luminous SNe One-a, such as 1991bg-like events SN 2010lp and the SN 2002es-like event iPTF14atg. However, the triggering of the detonation during the violent merger phase is still poorly constrained. Section three point two point five. Helium-ignited violent mergers. The Helium-ignited violent merger model, or “dynamically driven double-degenerate double-detonation” (D6) model, is another modern version of sub-Chandrasekhar explosions in the DD scenario, in which SNe One-a are produced through the double detonation mechanism during the merger of two WD’s, see Figure three. In the D6 model, an initial Helium detonation triggers on the surface of a heavier CO WD primary due to unstable dynamical Helium accretion from the less massive secondary which could be either another CO WD with thin surface Helium layers, a Helium WD, or a hybrid Helium-CO WD. Via a double-detonation mechanism, the initial Helium detonation initiates into a detonation of CO core, producing an SN one-a, shown in figure six. Because the Helium detonation in this model proceeds in a dynamic stage and not in a massive Helium layer at hydrostatic equilibrium conditions, the impact of the Helium detonation products on the observables is reduced compared to the classical sub-Chandrasekhar mass double detonation scenario. For instance, it has been shown that the double detonation explosion in the violent merger of two COWD’s with masses ofzero point nine and one point one solar masses can closely resemble normal SNe One-a, indicating that the D6 model has the potential to explain the bulk of normal SNe One-a. Interestingly, the secondary WD may survive from the explosion in the D6 model and become a hypervelocity WD with a velocity of greater than around 1000 kilometers per second, suggested that three hypervelocity runaway stars with a velocity of greater than around 1000 kilometers per second detected in the Gaia survey are likely to be WD companions that survived the D6 SNe One-a scenario. However, the fate of the secondary WD in this model is rather unclear. Recent investigations of the fate of secondary WD’s with self-consistent 3D hydrodynamical simulations, have confirmed that the primary WD can explode as an SN one-a. But there is a large uncertainty on the question of whether the secondary WD detonates or not. In contrast, others claim that an initial Helium detonation does not ignite a carbon detonation in the underlying WD. Section three point two point six. Other proposed explosion models. In the framework of either Chandrasekhar-mass or sub-Chandrasekhar-mass explosion, some other possible explosion models have been proposed for SNe One-a, including: One. The core-degenerate model, in which the WD merges with the core of an AGB star during the CE phase, triggering a thermonuclear explosion inside the envelope. Two. Tidal disruptions, in which the tidal interaction of a WD with a black hole triggers a thermonuclear explosion. Three. Head-on collisions of two WD’s, in which two WD’s collide in a binary or triple-star system, leading to a thermonuclear explosion due to the resultant shock compression. Four. The spiral instability model, in which a spiral mode instability in the accretion disk forms during the merger of two WD’s and leads to a detonation on a dynamical timescale resulting a SN one-a. In Table 2, we present an overview of the main characteristics of different explosion mechanisms of SNe One-a. Again, the same cautionary remark as for Table 1 applies. Four. RATES AND DELAY TIMES. The observationally-inferred SN one-a rate in our Galaxy is about 2.84 plus or minus zero point six times ten per thousand years. The observed delay-time distribution of SNe One-a, DTD’s, meaning the distribution of durations between star formation and SN one-a explosion, covers a wide range from around ten mega years to ten giga-years. By comparing the expected rates and DTD’s of SNe One-a from BPS calculations for different proposed progenitor models with those inferred from the observation, several studies attempted to place constraints on the nature of SN one-a progenitor systems In summary, no single proposed progenitor model is able to consistently reproduce both the observed SN one-a rates and the DTD’s, see Figure seven. The DD progenitor model generally predicts a broad range of delay times that follow an inverse t power-law, which is similar to the overall behavior of the observed DTD. But a sharp decrease of SN one-a rates for delay times shorter than 200Myr is seen in the DD model. This is inconsistent with a significant detection of prompt SNe One-a with delay times of t bounded by 35 to 200 mega years. BPS calculations have predicted that SD models with a MS or a RG donor mainly contribute to intermediate delay times of a hundred million to a billion years, and long delay times of greater than 3 Giga-years, respectively. SD models with a Helium star donor are expected to contribute to delay times shorter than a hundred mega years. The SD scenario generally tends to predict much lower SN one-a rates than those of the DD scenario However, a large variation of the results among different BPS studies is seen. One should always keep in mind that there are significant uncertainties in the theoretical predictions of SN one-a rates and delayed times from BPS calculations. On the one hand, constraints on the mass-retention efficiencies in the SD scenario are still rather weak yet studies show that there is a significant impact of the mass-retention efficiencies on BPS results such as rates and DTD’s. On the other hand, the predictions of BPS calculations sensitively rely on the assumed parameters in specific BPS codes such as the CE evolution, star-formation rate and initial mass function. However, to date, strong constraints on these parameters, for example the CE efficiency, are still lacking. This limits the predictive power of the BPS results. Section five. OBSERVABLES OF THERMONUCLEAR SUPERNOVAE. The approach to compare the observational features predicted by different progenitor models with observations has long been used to provide important clues to the yet poorly understood origin and explosion mechanism of SNe One-a. Over the past decades, substantial effort in modeling SNe One-a aimed at the prediction of optical observables light curves and spectra; The main goal was to distinguish between explosions of Chandrasekhar mass and sub-Chandrasekhar mass WD explosions as well as different mechanisms of thermonuclear combustion in these events. Despite all efforts, degeneracies make it difficult to draw firm conclusions. Besides optical light curves and spectra predicted by radiative transfer calculations in the context of different explosion mechanisms, certain other observational signatures are also expected to be indicative for different progenitor scenarios, including the detection of pre-explosion companions, H, Helium lines in SN one-a late-time spectra caused by material stripped from the companion during its interaction with the SN ejecta, early excess emission due to the ejecta–companion interaction, narrow absorption signatures of circumstellar material, CSM, radio and X-ray emission from CSM interactions, surviving companion Stars, and WD remnants, polarization signals, SN remnant, SNR, morphology, etc. In this section, we will give a detailed overview to the observables predicted for different phases, from the pre-explosion phase to the SNR phase, of SNe One-a from currently proposed progenitor scenarios and their comparisons with the observations. In particular, we focus on the question of how a binary companion star in the SD scenario shapes the observables of SNe One-a. Section five point one. Pre-explosion companion stars. The companion stars in potential progenitor models of SNe One-a fall into two categories One. Nondegenerate companion stars (MS, SG, RG, AGB or Heburning stars) in the SD scenario; Two. WD companions in the DD scenario. Becuase a non-degenerate companion star is much brighter than a WD, a luminous source is expected to be detected in pre-explosion images at position of the SNe One-a if they are generated from the SD progenitor scenario. Therefore, analyzing pre-explosion images from the SN position provides a direct way to test the SD progenitor scenario. On the theoretical side, Han in 2008 has comprehensively addressed the pre-explosion observable properties, luminosities, effective temperatures, masses, surface gravity, orbital and spin velocities, of MS companion stars at the moment of SN one-a explosion by performing BPS calculations for the WD + MS progenitor model. Following this work, Liu in 2015, extended the calculations to present pre-explosion properties of different non-degenerate companion stars, including the MS, SG, RG companions in the SD scenario, and the Helium-burning companion stars from both the SD and sub-Chandrasekhar mass double-detonation scenarios. Wong in 2021 also made predictions for the properties of the Helium-star donors at the time of explosion for a set of progenitor systems involving a CO WD and a Helium star. On the observational side, different studies have attempted to search for the expected non-degenerate companion stars by analyzing pre-explosion images at the SN position, for example, those taken by the Hubble Space Telescope, HST. To date, however, no progenitor companion star has been firmly detected in the analysis of pre explosion images of normal SNe One-a But there are some possible pre-explosion detections recently reported in several SNe One-ax. For instance, in 2014 McCully detected a blue luminous source in pre-explosion image of an SN one-ax event, SN 2012Z. As shown in Figure eight, the properties of this pre-explosion luminous source, SN 2012Z minus S1, have been found to be consistent with those of a Helium-star companion to the exploding WD. Interestingly, latetime observations taken about 1400 days after the explosion by the HST have shown that SN 2012Z is brighter than the normal SN 2011fe by a factor of two at this epoch. Comparing with theoretical models, this suggests the excess flux to be a composite of several sources: the shock-heated companion, a bound WD remnant that could drive a wind, and light from the SN ejecta due to radioactive decay. Analyzed pre-explosion HST images of another SN one-ax, SN 2014dt, but no source could be detected in this case. Section five point two. Ejecta–companion interaction. After the explosion in the SD scenario, the ejecta expand freely for a few minutes to hours before hitting the non-degenerate companion star, engaging into ejecta– companion interaction. The effect of a SN explosion on a nearby companion star has been studied since the 1970’s There are several ways in which the SN blast wave can modify the properties of companion stars during the ejecta-companion interaction, giving rise to observables that can be used to constrain SN one-a progenitors. First, the SN ejecta significantly interact with the companion star after the explosion, stripping some H-rich and Helium-rich material from its surface. This effect is caused either by the direct transfer of momentum or by the conversion of the blast kinetic energy into internal heat, meaning, by evaporation, ablation. As a consequence, some H, Helium lines caused by the stripped material may be present in late-time spectra of SNe One-a. Second, the shock heating injects thermal energy into the companion star during the interaction, leading to a dramatic expansion of the surviving companion star so that it displays signatures that are different from a star without experiencing the ejecta–companion interaction. For example, it could become more luminous and have a lower surface gravity. Third, radiative diffusion from shock-heated ejecta during the interaction is expected to produce an early excess in optical, UV or X-ray emission, see Kasen 2010. Fourth, the surface of a companion star may be enriched with heavy elements, for example, Nickel, Iron or Calcium, deposited by the SN One-a ejecta, which might be detectable in the spectra of a surviving companion star. Finally, the companion star survives from the explosion and retains its pre-explosion orbital velocity after the SN explosion, which leads a high peculiar velocity compared with other stars in the vicinity. The typical pre-explosion orbital velocities of the Hydrogen rich and Helium-rich companions in the SD Chandrasekhar mass scenario are eighty to two eighty kilometers per second, and around two fifty to five hundred kilometers per second, respectively. The Helium star companions in the sub-Chandrasekhar mass double detonation scenario and the Helium WD, or the CO WD which transfers its outer Helium layers, companions in the D6 model are respectively expected to have pre-explosion orbital velocities of around four hundred to one thousand kilometers per second and greater than a thousand kilometers per second. Section five point two point one. Searches for stripped hydrogen and helium. The earliest study of the effect of a SN explosion on a companion star was done by Colgate in 1970. Helium suggested that the companion star receives a kick that is mainly caused by the evaporation from the stellar surface, meaning the ablation, although there is also a small kick from the direct collision with the SN ejecta. Cheng in 1974 further investigated the impact of a SN shell onto a two point eight two and twenty solar mass MS companion star for various binary separations, SN shell masses, and velocities. Helium concluded that the MS companion star could survive from the interaction with SN shell. Upon these two works, several analytical models were developed to estimate the amount of stripped H mass and the kick velocity received by the companion star during the ejecta–companion interaction for MS companion stars with an n equals three and and n equals two thirds polytrope, which is appropriate for a low-mass MS, and for RG companion stars. To test the analytic prescription of Wheeler from 1975, several numerical simulations were performed for low-mass MS companions and RG stars. In particular, Livne suggested in 1992 that almost the entire envelope of a RG star could be stripped off by the SN blast, imparting a velocity to the stripped material around a thousand kilometers per second, much smaller than that of SN ejecta, of around ten thousand kilometers per second. Following the 1975 work of Wheeler, Meng in 2007 semi-analytically estimated the amount of stripped Hydrogen mass due to SN One-a explosions by adopting the binary and companion properties constructed with detailed binary evolution calculations. However, they underestimated the total stripped companion masses because of neglecting the effect of the ablation on the companion surface. More recently, updated two-dimensional 2D and 3D simulations with grid-based or smoothed particle hydrodynamics, SPH, methods have been presented that investigate the details of the interaction between SN one-a ejecta and the companion star. For instance, Marietta performed high-resolution 2D simulations in 2000 to comprehensively study the interaction of SN one-a ejecta in a variety of plausible progenitor systems with MS, SG and RG companions. However, they assumed the structure of single MS, SG, RG stars for the companion in their simulations. The 2000 study of Marietta for MS companion stars was updated to 3D simulations with the smoothed particle hydrodynamics (SPH) method by Pakmor in 2008, in which they considered the effect of pre-explosion mass transfer on the structures of a companion star at the moment of SN explosion. However, they computed their companion star models by constantly removing mass while evolving a single MS star to mimick the detailed mass transfer processes in a binary system. This makes their MS star model much more compact than one constructed from a full binary evolution calculation. Therefore, they predicted a small amount of stripped H masses of one to six percent of a solar mass for MS donor model. Liu further developed the work of Pakmor by adopting more realistic companion star models constructed from detailed, state-of-the-art binary evolution calculations. They also extended simulations to cover different companion stars, MS, SG and Helium-star, and a range of binary separations and explosion energies. Pan in 2012 employed adaptive mesh refinement, AMR, simulations to study the ejecta, companion interaction for MS, RG and Helium-star companions with different binary separations and explosion energies. In their simulations, however, they did not follow the full binary evolution but used initial conditions with a constant mass-loss rate when constructing their companion stars. The main results of ejecta–companion interaction of SNe One-a in the literature can be summarized as follows. One. 2D or 3D hydrodynamical simulations have predicted that about 5 per cent to 30 per cent of the companion mass, meaning greater than around ten percent of a solar mass can be stripped off from the outer layers of a MS or SG companion star, see top panel of figure ten. For RG companions, almost the entire envelope is removed by SN one-a blast wave. In the case of a Helium companion star, about one to three per cent of the mass is lost in the interaction. Two. The SN impact affects not only the companion star, but also the SN ejecta themselves. The presence of a companion star strongly breaks the symmetry of the SN one-a ejecta after the interaction. The stripped companion material is largely confined to the downstream region behind the companion star, creating a hole in the SN debris with an opening angle of about thirty to a hundred and fifteen degrees. Three. Depending on the different stellar types, the companion stars receive kick velocities of a few ten kilometers per second to one hundred kilometers per second , which are lower that their pre-explosion orbital velocities. This indicates that the surviving companion star should move with a velocity which is largely determined by its pre-explosion orbital velocity. Four. The characteristic velocities of stripped companion material for the MS, SG, RG and Helium star companions are five hundred to eight hundred kilometers per second , less than around nine hundred kilomeers per seonc, four to seven hundred kilometers per second, and eight hundred to a thousand kilometers per second respectively, which are slower than the maximum velocity of SN one-a ejecta, ten thousand kilometers per second, by about one order of magnitude. This implies that Hydrogen, Helium lines caused by stripped companion material become visible only at late-times when the photosphere recedes and moves to low velocity regions, revealing the inner SN one-a ejecta. Five. For a given companion model, the amount of stripped companion mass and kick velocity received by the companion star during the interaction decrease as the binary separation increases, which can be fitted by power-law relations. Six. The dependence of the amount of stripped mass and kick velocity on the explosion energy is in agreement with linear relations. Both quantities increase as the explosion energy increases. Seven. The companion star is generally expected to survive the explosion and becomes a runaway or hypervelocity star. However, whether a Helium WD companion in the double-detonation model would survive the explosion is still unclear. Eight. The companion surface could be enriched with heavy elements, contamination, from the low-expansion velocity tail of SN one-a ejecta, which provides a way to observationally identify the surviving companion stars in SNRs. However, the exact level of contamination is still rather uncertain in current models because of uncertainties of mixing of the contaminants in the envelope. One of the key questions of the SD scenario is whether the signatures of swept-up H or Helium due to the interaction can be detected in late-time spectra of SNe One-a. On the theoretical side, by performing the 1D parameterized spherically symmetric radiative transfer calculations, Mattila concluded in 2005 that Balmer lines should be detectable in SN one-a nebular spectra if the stripped H masses are greater than around three percent of a solar mass. In their models, they artificially added some uniform density solar-abundance material with a low expansion velocity of a thousand kilometers per second at the center of SN one-a ejecta of the W7 model from Nomoto 1984. Recently for the first time were performed, 3D Monte Carlo simulations with a non-local thermodynamic equilibrium, NLTE, radiative transport code to determine the signatures of stripped companion material in nebular spectra of SNe One-a as a function of viewing angle. In this study, more realistic distributions of stripped companion material and post-explosion SN One-a ejecta structures were adopted based on 2D hydrodynamical simulations of the ejecta–companion interaction. However, the Sobolev approximation as well as the simplified treatment on line overlap and multiple scattering causing some uncertainties in the results. Extension of previous calculations with a set of 1D NLTE steady-state radiative transfer simulations by covering a broader parameter space, a large range of masses for the ejecta, Nickel 56, and stripped material, and computing line overlap and line blanketing explicitly. These models adopted a 1D parameterized spherically symmetric SN ejecta structure. In summary, all radiative transfer calculations in the literature for SNe One-a with stripped companion material have concluded that the ejecta-companion interaction in the SD scenario produces significant and detectable signatures of stripped Hydrogen, Helium in late-time spectra. They further provided the dependence of line luminosities from stripped H-Helium-rich material on the amount of stripped H-Helium mass. This indicates that searching for Hydrogen-Helium emission due to stripped companion material in late-time spectra of SNe One-a is promising for identifying the SD or DD nature of the progenitor system. On the observational side, a series of observations have attempted to search for narrow, low-velocity H, Helium emission lines expected to be caused by swept-up H, Helium in late-time spectra of SNe One-a. But to date, no strong evidence for such H, Helium emission has been found in late-time spectra of most SNe One-a, even for the nearby SNe One-a with very high quality observations, meaning, SN 2011fe and SN 2014J. A detection was reported only for two fast-declining, sub-luminous events, SN 2018cqj and ASASSN 18tb. However, the H alpha emission lines detected in SN 2018cqj and ASASSN-18tb have been suggested to be caused by either CSM interaction or by H material stripped from a companion star. Furthermore, by analyzing late-time spectra of SNe One-a, one can convert the line luminosity limits to limits on the mass of H, Helium in SN one-a progenitors based on the current radiative transfer calculations for stripped companion material. Statistical limits on stripped H, Helium mass by analyzing a number of SN one-a late-time spectra have been given by Tucker in 2020, and they are summarized in Figure eleven. Comparing these statistical limits from the observation with the stripped H, Helium masses derived from numerical simulations, we can examine the validity of SD scenario for SNe One-a. As shown in Figure eleven, the observational constraints on the swept-up H, Helium masses are generally much lower than those from theoretical predictions, which poses a serious challenge for the SD scenario. Current radiative transfer simulations for SNe One-a with stripped material are still afflicted with uncertainties, because they either simply assume parameterized spherically-symmetric SN ejecta, or the treat line overlap and multiple scattering in an approximate way. For stricter predictions of the strength of H and Helium lines in late-epoch spectra, multi-dimensional NLTE radiative transfer calculations are needed that use the output ejecta model from 3D impact simulations and treat line overlap and multiple scatterings in detail. Moreover, there are some other possibilities that may explain to the lack of H-He emission in late-time spectra. For instance, the “spin-up, spin-down” model may lead to a compact companion star whose H, He-rich envelope has been stripped before the explosion, causing the absence of stripped H, Helium material during the interaction. Section five point two point two. Early excess emission. Different progenitor models and explosion mechanisms of SNe One-a may produce distinct early light curves. Therefore, early light curves of SNe One-a have been thought to play an important role in constraining their progenitor systems and explosion mechanism. For example, Nugent used the early light curves of the nearby SN 2011fe to constrain the radius of its exploding star, confirming that it must have been a WD. In the literature, different mechanisms have been proposed to cause an excess emission, meaning a “bump”, in early light curves of SNe One-a within the days following explosion, which will be described in detail below. Companion interaction: Kasen (2010) predicted that the shock caused by the ejecta–companion interaction significantly heats SN one-a ejecta to high temperatures, which causes a strong excess emission during the first few days after the explosion that is observable in the light curves within certain viewing angles in the SD scenario. This early-time excess emission is expected to be brightest in the ultraviolet (UV) wavelengths and becomes subordinate at longer optical wavelengths. However, it can still cause a blue color evolution in the optical light curve. By applying BPS results to the analytical models presents the distributions of expected early UV emission for different SD progenitor systems. Because the DD scenario does not predict such early UV emission, detecting early strong UV emission within the days following explosion has long been considered a smoking gun for the SD scenario of SNe One-a. For a given explosion model, early UV emission caused by the ejecta– companion interaction is strongly dependent on the ratio of binary separation to companion radius, assuming RLOF, at the moment of SN explosion. Therefore, the properties of this early UV emission are expected to provide a clue to the types of non-degenerate companions. Sub-Chandrasekhar-mass double-detonations: The burning of the initial Helium shell in sub-Chandrasekhar-mass double-detonation explosions can leave heavy, radioactive material in the outermost ejecta. A more massive Helium shell is expected to produce more radioactive material. The decay of this heavy, radioactive material could create an excess luminosity in the early light curves of SNe One-a. This may produce the early gamma emissions detected in SN 2014J Nickel-shell models: Piro and Nakar in 2013 suggested that the location of Nickel 56 in SN one-a ejecta could have noticeable impact on early-time light curves of SNe One-a. Further investigation showed how the distribution of Nickel 56 in the outer layers of the ejecta shapes early light curves of SNe One-a. More recently, comprehensive predictions of early-time curves of SNe One-a from a series of models containing Nickel 56 shells with different masses and widths in outer layers of SN one-a ejecta. They have shown that a Nickel 56 shell in outer SN one-a ejecta will lead to an early excess luminosity at a few days after the explosion. CSM interaction: The presence of CSM is expected in different progenitor scenarios, which can also significantly affect early light curves of SNe One-a. The presence of CSM can lead to a significant shock cooling emission during the first few days after the explosion, which can affect the early-time rise of the light curves of SNe One-a. Depending on the degree of mixing of Nickel 56 in the exploding WD and the detailed configurations of the CSM, this shock cooling emission can lead to early-time signatures, such as the early colour evolution, similar to those caused by the eject

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A simple proof of Bell’s inequality. Lorenzo Maccone. Dip. Fisica, University of Pavia, via Bassi 6, I-27100 Pavia, Italy Bell’s theorem is a fundamental result in quantum mechanics: it discriminates between quantum mechanics and all theories where probabilities in measurement results arise from the ignorance of pre-existing local properties. We give an extremely simple proof of Bell’s inequality: a single figure suffices. This simplicity may be useful in the unending debate of what exactly the Bell inequality means, since the hypothesis at the basis of the proof become extremely transparent. It is also a useful didactic tool, as the Bell inequality can be explained in a single intuitive lecture. Introduction: Einstein had a dream. He believed quantum mechanics was an incomplete description of reality and that its completion might explain the troublesome fundamental probabilities of quantum mechanics as emerging from some hidden degrees of freedom: probabilities would arise because of our ignorance of these “hidden variables”. His dream was that probabilities in quantum mechanics might turn out to have the same meaning as probabilities in classical thermodynamics, where they refer to our ignorance of the microscopic degrees of freedom (e.g. the position and velocity of each gas molecule): he wrote, “the statistical quantum theory would, within the framework of future physics, take an approximately analogous position to the statistical mechanics within the framework of classical mechanics”. A decade after Einstein’s death, John Bell shattered this dream: any completion of quantum mechanics with hidden variables would be incompatible with relativistic causality! The essence of Bell’s theorem is that quantum mechanical probabilities cannot arise from the ignorance of local pre-existing variables. In other words, if we want to assign pre-existing (but hidden) properties to explain probabilities in quantum measurements, these properties must be non-local: an agent with access to the non-local variables could transmit information instantly to a distant location, thus violating relativistic causality and awakening the nastiest temporal paradoxes. It is important to emphasize that we use “local” here in Einstein’s connotation: locality implies superluminal communication is impossible. In contrast, often quantum mechanics is deemed “non-local” in the sense that correlations among properties can propagate instantly, thanks to entanglement. This ‘quantum non-locality’ cannot be used to transfer information instantly as correlations cannot be used to that aim. In the remainder of the paper we will only use the former meaning of locality, Einstein non-locality and we warn the reader not to confuse it with the latter, quantum non-locality. Modern formulations of quantum mechanics must incorporate Bell’s result at their core: either they refuse the idea that measurements uncover pre-existing properties, or they must make use of non-local properties. In the latter case, they must also introduce some censorship mechanism to prevent the use of hidden variables to transmit information. An example of the first formulation is the conventional Copenhagen interpretation of quantum mechanics, which, thanks to complementarity, states that the properties arise from the interaction between the quantum system and the measurement apparatus, they are not pre-existing: “unperformed experiments have no results”. An example of the second formulation is the “de Broglie-Bohm interpretation” of quantum mechanics that assumes that particle trajectories are hidden variables, they exist independently of position measurements. Bell’s result is at the core of modern quantum mechanics, as it elucidates the theory’s precarious co-existence with relativistic causality. It has spawned an impressive amount of research. However, it is often ignored in basic quantum mechanics courses since traditional proofs of Bell’s theorem are rather cumbersome and often over-burdened by philosophical considerations. Here we give an extremely simple graphical proof of Mermin’s version of Bell’s theorem. The simplicity of the proof is key to clarifying all the theorem’s assumptions, the identification of which generated a large debate in the literature. Here we focus on simplifying of the proof. Bell’s theorem: Let us define “local” a theory where the outcomes of an experiment on a system are independent of the actions performed on a different system which has no causal connection with the first. As stated previously, this refers to locality in Einstein’s connotation of the word: the outcomes of the experiment cannot be used to receive information from whoever acts on the second system, if it has no causal connection to the first. For example, the temperature of my room is independent on whether you choose to wear a purple tie today. Einstein’s relativity provides a stringent condition for causal connections: if two events are outside their respective light cones, there cannot be any causal connection among them. Page Two: Let us define “counterfactual-definite” a theory whose experiments uncover properties that are pre-existing. In other words, in a counterfactual-definite theory it is meaningful to assign a property to a system, e.g. the position of an electron, independently of whether the measurement of such property is carried out. Sometime this counterfactual definiteness property is also called “realism”, but it is best to avoid such philosophically laden term to avoid misconceptions. Bell’s theorem can be phrased as “quantum mechanics cannot be both local and counterfactual-definite”. A logically equivalent way of stating it is “quantum mechanics is either non-local or non counterfactual-definite”. To prove this theorem, Bell provided an inequality (referring to correlations of measurement results) that is satisfied by all theories that are both local and counterfactual-definite. He then showed that quantum mechanics violates this inequality, and hence cannot be local and counterfactual-definite. It is important to note that the Bell inequality can be derived also using weaker hypotheses than “Einstein locality” and “counterfactual definiteness”: such a proof is presented in Appendix A, where Einstein locality is relaxed to “Bell locality” and counterfactual definiteness is relaxed to “hidden variable models”. However, from a physical point of view, the big impact of Bell’s theorem is to prove the incompatibility of quantum mechanics with local counterfactual-definite properties, and we will stick to these hypotheses in the main text, see also Appendix B for a schematic formalization of all these results. A couple of additional hypothesis at the basis of Bell’s theorem are often left implicit: (1) our choice of which experiment to perform must be independent of the properties of the object to be measured, technically, “freedom of choice” or “no super-determinism”, meaning for example, if we decided to measure the color of red objects only, we would falsely conclude that all objects are red. (2) future outcomes of the experiment must not influence which apparatus settings were previously chosen, whereas clearly the apparatus settings will influence the outcomes. A trivial causality requirement, technically, “measurement independence”. These two hypothesis are usually left implicit because science would be impossible without them. All experiments performed to date have shown that Bell inequalities are violated, suggesting that our world cannot be both local and counterfactual definite. However, it should be noted that no experiment up to now has been able to test Bell inequalities rigorously, because additional assumptions are required to take care of experimental imperfections. These assumptions are all quite reasonable, so that only conspiratorial alternatives to quantum mechanics have yet to be ruled out, where experimental imperfections are fine-tuned to the properties of the objects, namely they violate the “freedom of choice”). In the next couple of years the definitive Bell inequality experiment will be performed: many research groups worldwide are actively pursuing it. Figure one. Proof of Bell inequality using areas to represent probabilities. In Panel “A”, the dashed area represents the probability that property Alpha of the first object and Beta of the second are equal, either they are both one or both zero. P same (Alpha, Beta). The white area represents the probability that they are different: P different (Alpha, Beta). The whole circle has area one equals P same (Alpha, Beta) plus P different (Alpha, Beta). In panel “B” the gray area represents the probability that Alpha and Gamma are equal, and the non-gray area represents the probability that Alpha and Gamma are different. If Alpha of the first object is different from both Beta and Gamma of the second, dotted area, then Beta and Gamma of the second object must be the same. Hence, the probability that Beta and Gama are the same must be larger than, or equal to, the dotted area: since Beta is the same for the two objects, P same (Beta, Gamma) must be larger than, or equal to, the dotted area. Panel “C”, the quantity P same (Alpha, Beta) plus P same (Alpha, Gamma) plus P same (Beta, Gamma) is hence larger than, or equal to, the sum of the dashed plus gray plus dotted areas, which is in turn larger than, or equal to, the full circle of area 1. This proves the Bell inequality. Note One. The reasoning fails if we do not employ counterfactual definite properties, for example if complementarity prevents us from assigning values to both properties Beta and Gamma of the second object. It also fails if we employ non-local properties, for example if a measurement of Beta on an object to find its value changes the value of Alpha of the other object. Proof of Bell’s theorem: We use the Bell inequality proposed by Preskill, following Mermin’s suggestion. Suppose we have two identical objects, namely they have the same properties. Suppose also that these properties are predetermined, counterfactual definiteness, and not generated by their measurement, and that the determination of the properties of one object will not influence any property of the other object, locality. We will only need three properties Alpha, Beta, and Gamma that can each take two values: “0” and “1”. For example, if the objects are coins, then Alpha equals zero might mean that the coin is gold and Alpha equals one that the coin is copper. Property Alpha, material. Beta equals zero means the coin is shiny and Beta equals one that it is dull. Property Beta, texture. And Gamma equals zero means the coin is large and Gamma equals one it is small. Property Gamma, size. Suppose I do not know the properties because the two coins are a gift in two wrapped boxes: I only know the gift is two identical coins, but I do not know whether they are two gold, shiny, small coins. Page Three: Alpha equals zero, Beta equals zero, Gamma equals one. Or two copper, shiny, large coins (1, 0, 0) or two gold, dull, large coins (1, 1, 0), and so on. I do know that the properties “exist”, namely, they are counterfactual-definite and pre-determined even if I cannot see them directly, and they are local, namely, acting on one box will not change any property of the coin in the other box: the properties refer separately to each coin. These are quite reasonable assumptions for two coins! My ignorance of the properties is expressed through probabilities that represent either my expectation of finding a property, Bayesian view, or the result of performing many repeated experiments with boxes and coins and averaging over some possibly hidden variable, typically indicated with the letter lambda, that determines the property, the frequentist view. For example, I might say the gift bearer will give me two gold coins with a 20 percent probability. He is stingy, but not always. Bell’s inequality refers to the correlation among measurement outcomes of the properties: call P same (Alpha, Beta) the probability that the properties Alpha of the first object and Beta of the second are the same: Alpha and Beta are both 0, the first coin is gold and the second is shiny, or they are both 1, the first is copper and the second is dull. For example, P same (Alpha, Beta) equals a half tells me that with 50 percent% chance Alpha equals Beta, namely they are both either zero 0 or both one. Since the two coins have equal counterfactual-definite properties, this also implies that with 50 percent chance I get two gold shiny coins or two copper dull coins. Note that the fact that the two coins have the same properties means that P same (Alpha, Alpha) equals P same (Beta, Beta) equals P same (Gamma, Gamma) equals one. If one is made of gold, also the other one will be, or if one is made of copper, also the other one will be, and so on. Bell’s inequality. Under the conditions that three arbitrary two-valued properties Alpha, Beta or Gamma satisfy counter-factual definiteness and locality, and that P same (X, X) equals 1 for X equals Alpha, Beta or Gamma. This means that the two objects have same properties, the following inequality among correlations holds, P same (Alpha, Beta) plus P same (Alpha, Gama) plus P same (Beta, Gamma) is greater than, or equal to one, equation one. Namely, a Bell inequality. The proof of such inequality is given graphically in Figure one. The inequality basically says that the sum of the probabilities that the two properties are the same if I consider respectively Alpha and Beta, Alpha and Gamma, and Beta and Gamma must be larger than one. This is intuitively clear: since the two coins have the same properties, the sum of the probabilities that the coins are gold and shiny, copper and dull, gold and large, copper and small, shiny and small, dull and large is greater than one: all the combinations have been counted, possibly more than once. In Figure two the events to which the probabilities represented by the Venn diagrams of Figure one refer are made explicit. This is true, of course, only if the two objects have same counterfactual-definite properties and the measurement of one does not affect the outcome of the other. Figure Two: Explicit depiction of the properties whose probabilities are represented by the areas of the Venn diagrams in figure one. The properties are represented by a triplet of numbers, Alpha, Beta, Gamma, that indicate the counterfactual-definite, local values of the properties Alpha, Beta, and Gamma for both objects. Note that in the dotted area Alpha must be different from both Beta and Gamma, so that Beta and Gamma must be equal there. Beta and Gamma are equal also in the intersection between the two smaller sets, but that is irrelevant to the proof. If we lack counterfactual-definite properties, we cannot infer that the first coin is shiny only because we measured the second to be shiny, even if we know that the two coins have the same properties: without counterfactual definiteness, we cannot even speak of the first coin’s texture unless we measure it. Moreover, if a measurement of the second coin’s texture can change the one of the first coin, non-locality, again we cannot infer the first coin’s texture from a measurement of the second: even if we know that the initial texture of the coins was the same, the measurement on the second may change such property of the first. Both the “counterfactual definiteness” and the “Einstein locality” hypotheses we used here can be relaxed somewhat, as shown in Appendix “A” suggested only to more advanced readers. To prove Bell’s theorem, we now provide a quantum system that violates the above inequality. Consider two two-level systems, qubits, in the joint entangled state: Phi plus equals zero, zero plus one, one over square root of two, and consider the two-valued properties Alpha, Beta, and Gamma obtained by projecting the qubit on the states, equation two: Alpha is composed of two states alpha zero and alpha one, is alpha zero which is the zero state, alpha one which is the one state. Beta is beta zero which is one half the zero state, plus square root three over two times the one state. And beta one which is square root three over two times the zero state, plus one half the one state. And Gamma is Gamma zero, and gamma one, similar combinations of normalized states Where gamma zero equals a half times the zero state minus square root of three over two times the one state. And gamma one equals square root of three over two times the zero state plus a half times the one state. Where it is easy to check that beta one is orthogonal to beta zero and gamma one is orthogonal to gamma one. Since the state zero and one are orthonormal this is, the inner product of state zero and state zero is one, and the inner product of state one and state one is one. And the product of the zero state and the one state is zero. The inner product of the states b zero and b one is: A half times square root of three over two times the magnitude of the state zero. Plus square root f thee over two times minus a half, times the magnitude of state one. Which is the square root of thee over four minus the square root of three over four, which is zero. Unfortunately it is common to talk of states being composed of states in quantum mechanics, and this can often lead to confusion. It is seldom made explicit that a compound state is composed of sub states, and it is merely implied. In figure two point one, consider the system of a switch being in the configuration of open, meaning zero, or closed, meaning one. The system is in the state of zero, or one. A compound system of two switches can be formed, or new state, where the individual components are open or closed, which is equivalent to zero or one. Alternatively, the system could be composed of spinning magnets with a North or South Pole, or units of electrical charge, or standing vibrational waves in a crystal, or anything two which a label of zero or one can be given. And, as usual, the entire ensemble configuration is typically referred to, with profound indifference to communicability, as some state, which is in some state, written with a Greek letter psi. If the reader is familiar with the animated series the “Smurfs” this will all be completely clear. Page Four: It is also easy to check that the state Phi plus can be written in terms of the states Alpha, Beta or Gamma, equation three. So that the two qubits have the same properties, namely P same (Alpha, Alpha) equals P same (Beta, Beta) equals P same (Gamma, Gamma) equals one. The measurement of the same property on both qubits always yields the same outcome, either both 0 or both 1. We are now ready to calculate the quantity on the left of Bell’s inequality, equation one. Just write the state Phi plus in terms of the eigenstates of the properties Alpha, Beta and Gamma. For example, writing Phi plus as alpha zero, alpha zero plus alpha one, alpha one, all over square root of two, and calculating its inner product Phi plus transpose phi plus, we obtain: Alpha zero, alpha zero transpose on alpha zero, alpha zero, plus beta zero, beta zero transposed on beta zero, beta zero, all over two, since the square root of two time the square root of two is two. Which is one plus one over two, equals one, or P same (Alpha, Alpha) equals one. Note that the state zero is equivalent to one half of the sum of b zero plus square root thee b one. And the state one is equal to a half of the difference of square root of three b zero minus b one. Substituting into the definition of Phi plus, it is easy to find the value of P same (Alpha, Beta) if we write: Phi plus in terms of alpha zero, alpha one, and beta zero, beta one. In fact, the probability of obtaining zero for both properties is the square modulus of the coefficient of alpha zero, beta zero, namely one eighth while the probability of obtaining one for both is the square modulus of the coefficient of alpha one, beta one, again one eighth. Hence, P same (Alpha, Beta) equals one eighth plus one eighth equals one quarter. Analogously, we find that P same (Alpha, gamma) equals a quarter and that P same (Beta, Gamma) equals a quarter by expressing the state as Phi plus in terms of alpha, and gamma and finding its inner product with Phi plus expressed in terms of beta and gamma. Summarizing, we have found: P same (Alpha, Beta) plus P same (Alpha, Gamma) plus P same (Beta, Gamma) equals three quarters, which is less than one, equation four. And this violates Bell’s inequality of equation one. This proves Bell’s theorem: all theories that are both local and counterfactual-definite must satisfy inequality one which is violated by quantum mechanics. Then, quantum mechanics cannot be a local counterfactual- definite theory: it must either be non-counterfactual-definite, as in the Copenhagen interpretation, or non-local, as in the de Broglie-Bohm interpretation. APPENDIX “A.” Hidden variable models. This appendix is addressed only to more advanced readers. In the spirit of the original proof of Bell’s theorem, one can relax both the “counterfactual definiteness” and the “Einstein locality” hypotheses somewhat. In fact, instead of supposing that there are some pre-existing properties of the objects (counterfactual definiteness), we can suppose that the properties are not completely pre- determined, but that a hidden variable lambda exists and the properties have a probability distribution that is a function of lambda. The “hidden variable model” hypothesis is weaker than counterfactual definiteness: if the properties are pre-existing, then their probability distribution in lambda is trivial: there is a value of lambda that determines uniquely the property, for example a value lambda zero such that the probability P, i (“A” equals zero, given Alpha, lambda zero) equals one and hence P, i (“A” equals one, given Alpha, lambda zero) equals zero, namely it is certain that property Alpha for object i has value “A” equals zero for lambda equals lambda zero. We can also relax the “Einstein locality” hypothesis, by simply requiring that the probability distributions of measurement outcomes factorize, referred to as “Bell locality”. Call P (of x, x prime given capital X, capital X prime, lambda the probability distribution, due to the hidden variable model, that the measurement of the property capital X on the first object gives result x and the measurement of capital prime on the second gives x prime, where Capital X, X prime equals Alpha, Beta, Gamma denote the three two-valued properties Alpha, Beta, and Gamma. By definition, “Bell locality” is the property that the probability distributions of the properties of the two objects factorize, namely equation five: Probability of x, x prime, given Capital X, capital X prime, lambda equals P one x, given capital X, lambda, p two, x prime, capital x prime, lambda. The factorization of the probability means that the probability of seeing some value x of the property capital X for object one is independent of which property capital X prime one chooses to measure and what result x prime obtained on object two, and vice versa. The “Bell locality” condition, equation five, is implied by and, hence, it is weaker than, Einstein locality. In fact, Einstein locality implies that the measurement outcomes at one system cannot be influenced by the choice of which property is measured on a second, distant, system. So, the probability of the outcomes of the first system P one must be independent of the choice of the measured property of the second system capital X prime, namely: P one (x, give Capital X, X prime, lambda) equals P one of x, five capital X, lambda). The same reasoning applies to the second system, which leads to condition (5). We now show that a Bell-local, hidden variable model together with the request that the two systems can have identical properties, implies counterfactual definiteness. This means that we can replace “counter-factual definiteness” with “hidden variable model” in the above proof of Bell theorem, which, with these relaxed hypothesis states that “no local hidden variable model can represent quantum mechanics”. If two objects have the same property, then P same (capital X, capital X) equals one, namely the probability that a measurement of the same property X on the two objects gives opposite results, say, x equals one and x prime equals zero, is null. In formulas, equation six, the sum over lambda of Probability of x equals one, x prime equals zero, given Capital X, capital X, lambda times p (Of lambda) equals zero. Where the sum over lambda emphasizes that we are averaging over the hidden variables, since they are hidden, and p of lambda is the probability distribution of the hidden variable lambda in the initial joint state of the two systems. Note that in Equation six we are measuring the same property capital X on both objects but we are looking for the probability of obtaining opposite results x prime not equals to x. Using the Bell locality condition of equation five the probability factorizes, namely Equation six becomes equation seven: The sum over lambda of P one of x equals one, given Capital X, lambda times P two of x prime equals zero, given Capital X, lambda times p (Of lambda) equals zero. Since P one, P two, and p, are probabilities, they must be positive. Consider the values of lambda for which p of lambda) is greater than zero. The above sum can be null only if either P one or P two is null. Page Five: Namely if P one (x equals one, given| Capital X, lambda) equals zero, which implies that X has the predetermined value x equals zero, or if P two (x prime equals zero, Capital X, lambda equals zero, which means that capital X has predetermined value x prime equals zero. We remind the reader that counterfactual definiteness means that P I (of x, give capital X, lambda) is either zero or one. It is equal to 0 if the property capital X of object i does not have the value x, and it is equal to 1 if it does have the value x. We have, hence, shown that Equation seven implies counterfactual definiteness for property X: its value is predetermined for one of the two objects. Summarizing, if we assume that a Bell-local hidden variable model admits two objects that have the same values of their properties, then we can prove counter- factual definiteness. This means that we can relax the “counterfactual definiteness” and “Einstein locality” hypotheses in the proof of the Bell theorem, replacing it with the “existence of a hidden variable model” and with “Bell locality” respectively, so that the Bell theorem takes the meaning that “no Bell-local hidden variable model can describe quantum mechanics”, the hypothesis that two objects can have the same values for the properties is implicit in the fact that such objects exist in quantum mechanics, see Equation three. Namely, if we want to use a hidden variable model to describe quantum mechanics, as in the de Broglie-Bohm interpretation, such model must violate Bell locality. Otherwise, if we want to maintain Bell locality, we cannot use a hidden variable model, as in the Copenhagen interpretation. APPENDIX B: Summary of the hypotheses used and logic formalization of Bell’s theorem: We have given two different proofs of the Bell inequality based on different hypotheses. In this appendix we summarize the logic behind the Bell inequality proofs. Hypotheses we used, rigorously defined above. (A) “Counterfactual Definiteness”. (B) “Einstein locality”. (C) “No super-determinism” (D) “Measurement independence” (A’) “Hidden variable model”, implied by (A) and by the fact that systems with same properties exist (see Appendix A). (B’) “Bell locality”, implied by (B) (see Appendix A). In the main text we have proven (Fig. 1) the following theorem: (A) AND (B) AND (C) AND (D) therefore Bell inequality, therefore NOT QM, where with “NOT QM” we mean that quantum mechanics (QM) violates the Bell inequality and is, hence, incompatible with it. Using the fact that “X AND Y implies NOT Z” is equivalent to “Z implies NOT X OR NOT Y” (modus tollens), we can state the above theorem equivalently as QM implies NOT (A) OR NOT (B) OR NOT (C) OR NOT (D). Since one typically assumes that both (C) and (D) are true, they can be dropped and the theorem can be written more compactly as QM implies NOT (A) OR NOT (B). Namely, assuming “no super-determinism” and “measurement independence”, quantum mechanics implies that either ‘‘counterfactual definiteness’’ or ‘‘Einstein locality’’ must be dropped. This is the most important legacy of Bell. We have also seen that the hypotheses (A) and (B) can be weakened somewhat, so that the Bell inequality can also be derived using only (A prime) and (B prime). Namely, we can prove (see Appendix A): (A prime) AND (B prime) AND (C) AND (D) implies Bell inequality implies NOT QM. Namely, assuming “no super-determinism” and “measurement independence”, quantum mechanics is incompatible with Bell-local hidden variable models. References: [1] A. Einstein, B. Podolsky, N. Rosen, Can quantum- mechanical description of physical reality be considered complete?, Phys. Rev. 47, 777 (1935). [2] A. Einstein, in A. Einstein, Philosopher-Scientist, ed. By P.A. Schilpp, Library of Living Philosophers, Evanston (1949), pg. 671. [3] J. S. Bell, On the Einstein Podolsky Rosen Paradox, Physics 1, 195 (1964); Bell J. S., On the problem of hidden variables in quantum mechanics, Rev. Mod. Phys. 38, 447 (1966). [4] J. S. Bell, Speakable and Unspeakable in Quantum Mechanics (Cambridge Univ. Press, Cambridge, 1987). [5] N.D. Mermin, Hidden variables and the two theorems of John Bell, Rev. Mod. Phys. 65, 803 (1993). [6] “Bell’s telephone” in R. Werner, Quantum Information Theory—an Invitation, Springer Tracts in Modern Physics 173, 14, (2001), available from http://arxiv.org/pdf/quant-ph/0101061. [7] A. Peres, Unperformed experiments have no results, Am. J. Phys. 46, 745 (1978). [8] N.D. Mermin, Bringing home the atomic world: Quantum, mysteries for anybody, Am. J. Phys. 49, 940 (1981). [9] J. Preskill, lecture notes at http://www.theory.caltech.edu/people/preskill/ph229. [10] G. Auletta, Foundations and Interpretation of Quantum Mechanics (World Scientific, Singapore, 2000). [11] P.G. Kwiat, L. Hardy, The mystery of the quantum cakes, Am. J. Phys. 68, 33 (2000). [12] Kurt Jacobs and Howard M. Wiseman, An entangled web of crime: Bell’s theorem as a short story, Am. J. Phys. 73, 932 (2005). [13] A.L. Macdonald, Comment on ”Resolution of the Einstein-Podolsky-Rosen and Bell Paradoxes, Phys. Rev. Lett. 49, 1215 (1982). [14] A. Peres, Existence of “Free will” as a problem of Physics, Found. Phys. 16, 573 (1986). [15] H.P. Stapp, S-Matrix Interpretation of Quantum Theory, Phys. Rev. D 3, 1303 (1971); Bell’s theorem and world process, Nuovo Cimento 29B, 270 (1975); Are superluminal connections necessary?, Nuovo Cimento 40B, 191 Page Six. More References. (1977); Locality and reality, Found. Phys. 10, 767 (1980); W. De Baere, On Some Consequences of the Breakdown of Counterfactual Definiteness in the Quantum World, Fortschr. Phys. 46, 843 (1998). [16] Y. Aharonov, A. Botero, M. Scully, Locality or non-locality in quantum mechanics: Hidden variables without “spooky action-at-a-distance”, Z. Natureforsh. A 56, 5 (2001). [17] A. Aspect, P. Grangier, G. Roger, Experimental Realization of Einstein-Podolsky-Rosen-Bohm Gedankenexperiment: A New Violation of Bell’s Inequalities, Phys. Rev. Lett. 49, 91 (1982). [18] P.G. Kwiat, E. Waks, A.G. White, I. Appelbaum, P.H. Eberhard, Ultrabright source of polarization-entangled photons, Phys. Rev. A 60, R773 (1999). [19] M. Giustina, A. Mech, S. Ramelow, B. Wittmann, J. Kofler, J. Beyer, A. Lita, B. Calkins, T. Gerrits, S. Woo Nam, R. Ursin, A. Zeilinger, Bell violation with entangled photons, free of the fair-sampling assumption, Nature 497, 227-230 (2013). [20] P.M. Pearle, Hidden-Variable Example Based upon Data Rejection, Phys. Rev. D 2, 1418 (1970). [21] J.S. Bell, Atomic-cascade Photons and Quantum mechanical nonlocality, Comments Atom. Mol. Phys. 9, 121 (1980); reprinted as Chap. 13 of [4]. [22] G. Ghirardi, On a recent proof of nonlocality with- out inequalities, Found. Phys. 41, 1309 (2011), also at arXiv:1101.5252. [23] T. Norsen, Bell Locality and the Nonlocal Character of Nature, Found. Phys. Lett. 19, 633 (2006).

Local gravitational physics of the Hubble expansion. Einstein’s equivalence principle in cosmology. By Sergei M. Kopeikin, Of the Department of Physics and Astronomy, University of Missouri, Columbia, Missouri, and Siberian State Geodetic Academy, Novosibirsk, Russia. Abstract. We study physical consequences of the Hubble expansion of Friedmann-Lemaıtre-Robertson- Walker (FLRW) manifold on measurement of space, time and light propagation in the local inertial frame. We use results of this study to analyse the solar system radar ranging and Doppler tracking experiments and time synchronization. The FLRW manifold is covered by the coordinates (t, and y), where t is the cosmic time coinciding with the proper time of the Hubble observers and identified with the barycentric coordinate time (TCB) used in ephemeris astronomy. We introduce local inertial coordinates x alpha equals (x zero, x i) in the vicinity of a world line of a Hubble observer with the help of a special conformal transformation that respects the local equivalence between the tangent and FLRW manifold. The local inertial metric is Minkowski flat and is materialized by the congruence of time-like geodesics of static observers being at rest with respect to the local spatial coordinates x i. The static observers are equipped with the ideal clocks measuring their own proper time which is synchronized with the cosmic time t measured by the Hubble observer. We consider the geodesic motion of test particles and notice that the local coordinate time x zero, equals x zero (Of t) taken as a parameter along the world line of particle, is a function of the Hubble’s observer time t. This function changes smoothly from x zero equals t for a particle at rest (observer’s clock), to x zero equals t plus a half H t squared for photons, where H is the Hubble constant. Thus, motion of a test particle is non-uniform when its world line is parametrized by the cosmic time t. NASA JPL Orbit Determination Program operates under assumption that spacetime is asymptotically flat which presumes that motion of light (after the Shapiro delay is excluded) is uniform with respect to the time t but it does not comply with the non-uniform motion of light on cosmological manifold. For this reason, the motion of light in the solar system analysed with the Orbit Determination Program appears as having a systematic blue shift of frequency, of radio waves circulating in the Earth-spacecraft radio link. The magnitude of the anomalous blue shift of frequency is proportional to the Hubble constant H that may open an access to the measurement of this fundamental cosmological parameter in the solar system radiowave experiments. In other words, the assumption of a local, asymptotically flat Minkowski space-time in which the solar system physics evolves does not take into account the effect of the expansion of the universe, and measureable deviations can be obtained. Introduction. Modern physics is intensively looking for the unified field theory that might explain the origin of the universe and the underlying fundamental nature of space-time and elementary particles. This work requires deeper understanding of the theoretical and experimental principles of general relativity. It is challenging to find a new type of experiments that broaden the current knowledge. An appealing problem is to examine a presumable link between the local gravitational phenomena and the global cosmological expansion of the universe that is to test the foundational basis of the Einstein equivalence principle (EEP) in application to a conformal cosmological metric with a time-dependent scale factor. The EEP in general relativity is universally valid because the gravitational field in general relativity has a purely geometric nature. It is always mathematically possible to find a local diffeomorphism which reduces any global metric to a Minkowski metric in a sufficiently small neighborhood of a time-like world line of an observer if tidal forces are neglected. This mathematical fact was a clue that led Einstein to the formulation of his general principle of relativity, also known as the principle of covariance, and, later on, to the discovery of general relativity as a physical theory of gravitational fields. The apparent mathematical nature of EEP caused some physicists to deny its physical significance. The present paper neither shares this extremal point of view nor confronts the solid mathematical foundation of EEP. We focus on physical aspects of EEP, namely: One. The comparison of the inertial motion of test particles on cosmological manifold considered from the local point of view of a Hubble observer, Two. The derivation of experimental consequences that can be used for testing the Hubble law in local solar system experiments. So far, all gravitational experiments in the solar system have been interpreted under a rather natural assumption that the background spacetime geometry is asymptotically flat covered by coordinates (t, and y) with the background Minkowski metric: D s squared equals minus d t squared plus delta I, J d y I, d y j, equation one. With Delta I, J the unit matrix, with a diagonal of one, one, one, and we have used a convention for the speed of light, c equals one. The time t entering the metric is identified with the barycentric coordinate time (TCB) of the solar system according to the IAU 2000 resolutions. On the other hand, theoretical and observational cosmology postulates that the background space-time is described by the Friedmann-Lemaıtre-Robertson-Walker (FLRW) metric: D s squared equals minus d t squared plus R squared (Of t) over the square of one plus one quarter k r squared, times delta I, J, d y I, d y J, equation two. Which is to say that the Minkowski metric is effectively multiplied by a function of t, r, and k, Where: t is the universal cosmic time, y i are the global isotropic coordinates, k is either minus one, zero or one and defines the curvature of space, and the scale factor R( Of t) is a function of time found by solving Einstein’s equations. The cosmic time t is the proper time measured by observers having fixed spatial coordinates y i. Therefore, it is exactly the same as time t in the flat metric (equation one) and identifies with TCB of fundamental astronomy in the solar system. In what follows, we admit k equals zero in accordance with observations and limit ourselves with the linearized Hubble approximation. In other words, we consider only terms being linear with respect to the Hubble constant H and neglect all terms that are quadratic with respect to H or proportional to its time derivative H dot, as H dot is around H squared. We shall also neglect post-Newtonian gravitational effects of the solar system which must be included in the realistic data analysis of observations in the solar system. These effects are described in other references, and can be easily accounted for by superposition, if necessary. The FLRW metric (equation two) is not asymptotically-flat and has a non-vanishing space-time curvature tensor R alpha, beta, gamma, delta where the Greek indices alpha, beta, gamma, delta take values zero, one, two, three. Nonetheless, the Weyl tensor of FLRW metric, C alpha, beta, gamma, delta is by definition zero. Hence the metric of equation two can be reduced to a conformally-flat metric for any value of space curvature k. When k equals zero, it is achieved by transforming the cosmological time t to a conformal time, eta equals eta (Of t), defined by an ordinary differential equation: D t equals “A.” (Of eta) d eta, Where the scale factor “A.” (Of eta) is R Parameterized by T (Of eta). This time transformation allows the rewriting of the LMRW metric into the conformally Minkowski form of equation four: D s squared equals “A.” squared (Of eta) f alpha, beta d y alpha, d y beta. Here y alpha is y zero, y I, equal to eta, y I, the global conformal coordinates, F alpha, beta has the diagonal of (minus one, one, one, one), and is the Minkowski metric. In other words, although a flat space Minkowski metric is used by the IAU, theoretical and observational cosmology postulates the LMRW metric, and by transforming by the use of a scale factor “A.” (of eta), the LMRW can be approximated by a Conformally Minkowskian metric, with the Minkowski metric merely multiplied by a function of eta. According to Einstein’s general relativity and the definition of FLRW metric, the cosmological time t is a physical proper time of the Hubble observer and can be measured with the help of the observer’s atomic clock while the conformal time eta is a convenient coordinate parameter which is calculated from the clock’s reading but cannot be measured directly. Typically, the cosmological metric (equation four) is applied to describe the properties of spacetime on the scale of galaxy clusters and larger. On small scales of the size of the Milky Way, the solar system and terrestrial lab, the background spacetime is believed to be flat with any cosmological effect being strongly suppressed. Nevertheless, the question remains open: if we admit FLRW metric to be valid on any scale, can the cosmological expansion be detected in local gravitational experiments? Following others, we postulate that FLRW metric (equation four) is a physical metric not only in cosmology but for the description of the local physics as well. It describes the background spacetime geometry in the global coordinates y alpha on all scales spreading up from the cosmological horizon to the solar system and down to a local observer. The small parameter in the approximation scheme used in the present paper, is the product of the Hubble constant, H, with the interval of time used for physical measurements. All non-linear terms of the quadratic order with respect to the small parameter (formally, the terms being quadratic with respect to H) will be systematically neglected because of their smallness. We introduce the reader to the concepts associated with the Einstein equivalence principle in section 2 and discuss construction of the local inertial coordinates in section 3. The inertial frame is built in sections 4. Light geodesics in local coordinates are derived in section 5. We solve these equations in section 6 and employ them for investigation of observability of cosmological effects in the solar system. Discussion is provided in section 7. Two: Einstein’s principle of equivalence. A thorough treatment of the local astronomical measurements on cosmological manifold inquires a scrutiny reexamination of Einstein’s equivalence principle (EEP) which states: “In a given gravitational field, the outcome of any local, non-gravitational experiment is independent of the freely-falling experimental apparatus’ velocity, of where and when in the gravitational field the experiment is performed and of experimental technique applied”. Mathematical interpretation of EEP suggests universality of local geometry in the sense that at each point on a spacetime manifold with an arbitrary gravitational field, it is possible to choose the local inertial coordinates such that, within a sufficiently small region of the point in question, all laws of nature take the same form as in non-accelerated Cartesian coordinates. EEP is applicable in general relativity to any kind of spacetime manifold, in particular, to the manifold of the FLRW universe, which is described by the metric of (equation four). We noticed in previous work, that due to the expanding nature of space in an FLRW manifold, the parametric description of the propagation of light given in local inertial coordinates in terms of the proper time of observer, differs from that in the Cartesian coordinates of flat spacetime. Let us consider a Hubble observer who is at the origin of a local inertial coordinate system (LIC), x alpha equals (x zero, x i). The Physical metric g alpha, beta equals “A.” squared (Of eta) f alpha, beta, given by (equation four) in the global coordinates, is reduced to the Minkowski metric, f alpha, beta, at the origin of LIC with the affine connection being nil, Gamma alpha, mu, nu (Of x) equals zero, on the observer’s world line. EEP asserts that the worldlines of freely falling (electrically-neutral) test particles and photons are geodesics of the physical metric g alpha, beta with an affine parametrization. Because the affine connection is nil in LIC, it presumes that the geodesic equations of motion of all test particles – massive and massless, can be written down as follows: D two x alpha, d sigma squared equals zero, Where sigma is the affine parameter along the geodesic. Equation (five) neglects the tidal (caused by the Riemann curvature of FLRW spacetime) effects, which produce terms of the order of H squared which we discard. Solving (equation five) for the time component shows that the parameter sigma can be chosen equal to the coordinate time x zero of LIC, that is: Sigma equals x zero. The local coordinate time, x zero, must be further operationally connected to the proper time t measured in LIC by the central Hubble observer. The time x zero is often identified with the proper time of observer t but one must keep in mind that this identification is true only for static observers being at rest with respect to the central Hubble observer. In general, the local coordinate time x zero is a nonlinear function of t on world-lines of moving test particles. Therefore, changing the affine parameter sigma to the non-affine (but directly measurable) parameter t brings (five) to the following form, of equation six: D two x alpha, d t squared equals d x alpha, d x zero, times d two x zero, d t squared. The cosmic time t coincides with the proper time of the central Hubble observer in the absence of any gravitational perturbations caused by massive bodies of the solar system like Sun and planets. Real experiments demand to include the effect of gravitational field of the solar system on time transformations but they are well-known and can be easily taken into account. In ephemeris astronomy the time t is identified with the barycentric coordinate time (TCB) which is considered as a uniform global time scale. Equations for transformation of the proper time tau of any observer within the solar system to TCB are given by the corresponding IAU resolutions. This transformation shows that tau differs from t by small relativistic terms which are not essential for further discussion. The NASA JPL Orbit Determination Program that is used for spacecraft navigation and calculating planetary and lunar ephemerides, assumes that for any particle including photons, x zero equals t. It means that the right side of (six) is postulated to be nil, or, equation seven: D two x alpha, d t squared equals zero. This equation yields the photon’s world line as: X zero equals t, X I equals x zero I plus U I t, where U I is a unit vector in the direction of the photon’s propagation and we assumed that light passes through the point x I zero of the local inertial coordinates at instant t equals zero which fixes the integration constants. It establishes a linear relationship between the spatial coordinates x i of the photon and the proper time t of the observer at the origin of LIC, which is a directly measurable quantity, after accounting for the IAU time transformations. Equation (seven) does not show the presence of the Hubble constant, H. It led scientists to believe that EEP cancels out all cosmological effects of the linear order of the Hubble constant H, and that prevents astronomers from observing them in solar system experiments. However, equation (seven) and its solution are incomplete as the relation between x zero and t is not a linear function of time so that light does not propagate uniformly with constant velocity. This non-uniform propagation of light in local coordinates may appear as an anomaly and a violation of EEP for photons but this is just a mathematical consequence of the geometric expansion of space in FLRW universe. This effect makes possible measurement of the Hubble expansion in the solar system in the local experiments like the Doppler tracking of spacecraft in deep space. The local inertial coordinates. In order to interpret local astronomical measurements, like radar ranging, spacecraft Doppler tracking, and so on, we have to construct the local inertial coordinates (LIC) in the neighborhood of a time-like world line of an observer. We focus on building LIC in the vicinity of a Hubble observer, which by definition has constant spatial coordinates: Y I equal a constant value, of the FLRW metric and moves along a time-like geodesic world-line. Real observers move with respect to the Hubble flow and experience gravitational forces from the massive bodies of the solar system. Therefore, construction of LIC for a real observer requires determining additional coordinate transformations which are known and can be found, so that they are not a matter of concern of the present paper. Let us put the Hubble observer at the origin of the LIC, with X I equals zero, so that the world-line coincides, then, with the timelike geodesic of the observer. The Hubble observer carries an ideal clock that measures the parameter of the observer’s worldline, which is the observer’s proper time t. The proper time t of the Hubble observer coincides with the cosmological coordinate time t in the definition of the Lemaıtre-Robertson-Walker (FLRW) metric of equation two. The Einstein equivalence principle suggests that in a small neighborhood of the worldline of the observer, called a tangent spacetime, there exists a local diffeomorphism from the global, y alpha, to local, x alpha, coordinates such that the physical metric: G alpha, beta (Of y) equals “A.” squared (Of eta), times f alpha, beta, Is transformed to the Minkowski metric, f alpha, beta, as follows in equation eight: “A.” squared (Of eta) f mu, nu d y mu, d x alpha, d y nu, d beta equals f alpha, beta. This is an essential point, wherein the local flat space the metric must transform to the global FLRW metric, in which the transformation is using terms proportional to the Hubble constant H, and ignoring higher order terms. The suggestion is of a local transformation, which therefore will change on position, from global to local inertial coordinates. Where all tidal terms of the order of Hubble squared have been omitted as negligibly small. In the tangent space-time where (eight) is valid, the physical space-time interval of the conformally-Minkowskian form of equation four can be written down in local inertial coordinates. It reads: D S squared equals f alpha, beta d x alpha, d x beta. Where f alpha, beta is understood as the physical, global metric g alpha, beta (Of x) expressed in the local coordinates. Equation (eight) looks similar to the special conformal transformation establishing a conformal isometry of the Minkowski metric, which is that of equation ten: Omega squared (Of x) f mu, nu d y mu, d x alpha, d y nu, d x beta equals f alpha beta. Since the required diffeomorphism is local, it is a function of the local x co-ordinates, and hence the transformation can be identified with the conformal isometry of the Minkowski metric. “A.” squared (Of eta) is here replaced with Omega squared (Of x). The transformation Omega (Of X) can be written as: Omega (Of x) equals one minus two times the contraction of B alpha with x alpha, plus b squared times x squared. In this transformation: B alpha is a constant four-vector yet to be specified, X squared is defined as f alpha, beta X alpha, X beta, and: B squared is defined as f alpha, beta B alpha, B beta. The special conformal transformation includes inversions and translations, and is equivalent to equation thirteen: Y alpha equals x alpha minus b alpha x squared, all divided by Omega (Of x). Let us assume for simplicity that the origin of LIC, x I equals zero, coincides with the point having the global spatial coordinates, y I equals zero. As the background manifold is assumed to be analytic, equation (eight) should match (ten) in a small neighbourhood of the origin of the LIC. The matching can be achieved by demanding the scale factor of the FLRW metric, “A.” (Of eta) equals Omega. This equality is valid in any arbitrary cosmological model if we discard the curvature terms being proportional to around H squared and, or the time derivative of H. Indeed, for small values of the conformal time eta we have the expansion: “A.” (Of eta) equals “A.” (Zero) plus d “A.”, d eta (At zero) times eta, plus higher terms in the derivatives. Where we assume that the present epoch corresponds to eta equals zero in the conformal time. We normalize the scale factor at the present epoch to “A.” (At zero) equals one. Then, at the present epoch the Hubble constant H equals d “A.”, d eta (At zero). The second time derivative of the scale factor two “A.”, d eta squared equals d H, d eta plus two H squared, and we drop it off as being negligibly small. Assuming that the constant vector, b alpha is of the order of the Hubble constant H, we approximate the conformal factor, Omega (Of x) equals one minus two b alpha, x alpha, by neglecting terms of the order of b squared. In keeping to first order in terms of the Hubble constant H, we have the equivalence that: “A.” (Of eta) equals “A.” (Zero) plus d “A.”, d eta (At zero) times eta equals: Omega (Of x) equals one minus two b alpha, x alpha. Since “A.” (zero) has been set equal to one, and d “A.”, d eta (at zero) equals H, this yields: “A.” (Of eta) equals: One plus H eta, equals Omega (Of x), which equals: One minus two b alpha, x alpha. Or eta equals x zero plus linear terms of Order (B). Equating terms, b alpha equals H over two times: U alpha, equals a four vector of components: H, over two, zero, zero, zero. It is directed along the four-velocity u alpha equals (one, zero, zero, zero) of the Hubble observer, and is time-like. The reader may notice that the special conformal transformation has a singular point, x alpha equals minus b alpha over b squared, that goes over to t equals two over H. It means that the special conformal diffeomorphism (thirteen) is approximately limited in time domain by the Hubble time, T H equals one over H, calculated for the present value of the Hubble parameter: H is approximately two point three times ten to the minus eighteen per second. However, because LIC have been derived under assumption that the series in “A.” (Of eta) is convergent, H t is far less than one, the period of time for which the local inertial frame is really valid is much smaller than the Hubble time and is given by, t far less than T H. Because of this limitation imposed on the time of applicability of the local frame, the local coordinates are also bounded in space by the radius, r far less than Hubble radius, where R H equals c times T H is the Hubble radius of the universe. The conclusion of this paragraph is that the LIC can be employed only for sufficiently close objects in the universe with the redshift factor z far less than one, which excludes the most distant quasars and galaxies. Therefore, the formalism of the present paper is not applicable to the discussion of global cosmological properties and, or effects like the red shift of quasars. More stringent results on the domain of applicability of the local inertial coordinates in cosmology can be found in the literature. In review: Because the local coordinates X I, utilizing a Minkowski metric, are embedded in a global Friedmann-Lemaıtre-Robertson-Walker (FLRW) that utilizes a coordinate system Y I, and limiting the transformation between them to first order in the Hubble constant, or dimensions small compared to the Hubble radius, there is a linear transformation between the local time t, and the Cosmological time eta. The conformal factor thus obtained, Omega (Of x) is one minus H over two U alpha, x alpha, or: one minus H, times U four dot X four. The association ensures that LIC can be constructed in the linearized Hubble approximation from the global coordinates, y alpha, by means of the special conformal transformation (thirteen) that respects EEP as the procedure demonstrates. In what follows, we accept the equalities, Omega (Of x (Of eta)) equals “A.” (Of eta), that are valid in the linearized Hubble approximation. Moreover, we work in the vicinity to the present epoch where “A.” (Of t) equals one plus H t plus terms of Order (H squared, t squared), and in this approximation we are allowed to use t equals eta in terms which are proportional to H. It means we can equate “A.” (Of eta) equals “A.” (Of t). Four. The local inertial frame. The local inertial coordinates, x alpha, are mathematical functions on FLRW manifold which have no immediate physical meaning unlike the Cartesian coordinates in Euclidean space. To make the local coordinates physically meaningful they should be further specified and operationally connected with measuring devices (clocks, rulers) of a set of some reference observers. This materialization yields access to the local inertial frame. The corresponding relations between the measuring tools and the local coordinates are known in differential geometry as inertial (or projective) structure. The Minkowski form of the physical local metric: D s squared equals f alpha, beta d x alpha, d x beta, suggests that LIC can be associated with the Gaussian normal coordinates based on the congruence of time-like geodesics of (electrically-neutral) test particles being at rest with respect to LIC. The first step, is to find a relation between the coordinate time x zero and the proper time t of the Hubble observer at the origin of LIC. Because the space-time interval: D s squared equals minus d x zero squared for x I equals zero, and D s squared equals minus d t squared, By the definition of the proper time, we come to the conclusion that x zero equals t on the world-line of the origin of LIC. The grid of the Gaussian coordinates start from the initial hypersurface, t equals zero, that is orthogonal to the world line of the Hubble observer. We identify the spatial Gaussian coordinates with the orthogonal (in the Euclidean sense) spatial coordinates X I of LIC on the initial hypersurface. Extension of the spatial coordinates from the initial hypersurface to arbitrary values of the time coordinate x Zero equals t is performed by means of time-like geodesics. The Christoffel symbols of the local metric are nil in a neighborhood of the origin of LIC in accordance with diffeomorphism (thirteen) by which LIC were introduced. Because all Christoffel symbols are nil, the time-like worldlines of particles having constant spatial coordinates, X I equals constants, are geodesics given by (five). Or, if X I equals a constant, d two X I, d sigma squared equals zero. The proper time of the particle with the constant spatial coordinate X I coincides with the time coordinate X zero which was identified with the proper time of the Hubble observer. Hence, the parameter sigma in (five) can be identified with the proper time t as well. After that equation (five) describing worldlines of the static observers takes on the following simple form or equation nineteen: D two x alpha, d t squared equals zero. The meaning of the time-like geodesic equation (nineteen) is as follows. The world lines X alpha equals X zero equals t, x I equals constant. These world lines are identified with the network of static reference observers which play a fundamental role in local physical measurements. We admit that each static observer is equipped with an ideal (atomic) clock measuring their proper time, which coincides with a time-like parameter, x zero, along the observer’s world line. Solving (nineteen) reveals that X zero equals t is the proper time of the Hubble observer located at the origin of LIC. We assume that the ideal clocks of the static observers are synchronized. It can be done with Einstein’s procedure of exchanging light signals as we will confirm in section six point two. The Gaussian normal coordinates form a local inertial frame that is used for doing local physical measurements of time and space along time-like world lines of static observers and on space-like hypersurfaces of constant time. The frame is defined operationally in terms of the proper time of the ideal clocks and rigid rulers. The rulers are made of an ordinary matter whose rigidity is determined primarily by the chemical bonds having an electromagnetic origin. We have proved in other references that in the linearized Hubble approximation the electromagnetic (Coulomb) forces in an expanding universe remain the same as in a flat spacetime. For this reason, the rigid rulers and rods are not subject to the cosmological expansion and can serve for physical materialization of LIC. Another physical realization of the local Gaussian coordinates is achieved by the celestial ephemerides of the solar system bodies since their orbits are not affected by the Hubble expansion either. Five. The light geodesics. The most precise measurements of spacetime events are made with electromagnetic waves and light. Therefore, we have to solve the equations of light geodesics of equation six: D two x alpha, d t squared equals: d x alpha, d x zero, times d two x zero, d t squared. Where equation six is parameterized with the proper time t of the central observer, which is a directly measurable quantity. First of all, we need to evaluate the right side of (six). The function x zero taken on the light cone, is given by x zero equals “A.” (Of eta) times eta. Since the conformal time eta and the cosmic time t are related by: T equals the integral of “A.” (of eta) d eta, equals eta plus H, over two eta squared plus Order Hubble squared, and the cosmic time coincides with the proper time t of the central Hubble observer, we get on the light geodesic, equation twenty one: X zero equals t plus H over two t squared. Taking the second derivative from x Zero in (twenty one) yields d two x zero, d t squared equals H. Hence, equation of light geodesics (six) takes on in the local coordinates the following form, equation twenty two: D two x alpha, d t squared equals H d x alpha, d t squared. Here we have made use of a legitimate approximation, that d x alpha, d x zero equals d x alpha, d t, in the right side of (twenty two). Equation (twenty two) predicts the existence of a cosmological force in the tangent space of FLRW universe, exerted on a freely-falling photon. It should not be misinterpreted as a violation of general relativity or Newtonian gravity like the “fifth force” or whatever else. Equation (twenty two) is a direct consequence of general relativity applied along with the cosmological principle stating that the global cosmological time t is identical with the proper time measured by the Hubble observer. It explains how and why the Hubble expansion of the universe may appear locally. We discuss the observational aspects of this local cosmological effect in the next section in more detail. Solution of (twenty two) is given by a quadratic function of time: X alpha equals X alpha, zero plus k alpha times t plus H over two t squared. Where x alpha zero is the position of photon at time t equals zero, and k alpha equals (one, K i), is a constant null vector with the unit vector K I pointing out in the direction of propagation of light. The reader may notice that the coordinate speed of light, v alpha equals k alpha times one plus H t, exceeds the fundamental value of c equals one for t greater than zero. There is no violation of special relativity here because this effect is non-local, the speed is given with respect to the origin of the local coordinates. The local value of the speed of light measured at time t at the current position of the photon, is always equal to c equals one. This is because the group of the conformal isometry includes the Poincare group as a sub-group which allows us to change the initial epoch and the initial position on the background manifold without changing the differential equation (twenty two). Non-uniform propagation of light in the local frame may look counterintuitive as compared with our experience with special relativity. Nonetheless, this is how light propagates in the expanding universe. Equation (twenty three) is just a direct consequence of a standard light propagation formula in cosmology which reads in the global conformal coordinates, y alpha equals k alpha times eta, where k alpha equals (one, k i) is the null vector. The non-uniform propagation of light in the local frame can be observed in the solar system, thus, making it possible to measure the Hubble expansion rate locally as contrasted to the cosmological observations of distant quasars. Six. Cosmological effects in the local frame. Six point one. Radar and laser ranging. Precise dynamical modelling of the orbital and rotational motion of astronomical bodies in the solar system, major and minor planets, asteroids, spacecraft, and so on, is inconceivable without radar and laser ranging. The ranging is an integral part of the experimental testing of general relativity and alternative theories of gravity in the solar system. We are to check if the Hubble expansion can be measured in the ranging experiments. The equation of light propagation in the local Gaussian coordinates x alpha is given by equation twenty two. Let us consider the radial propagation of light. The radial, and always positive, spatial coordinate of a photon is: R equals square root of delta I, J times x I, X J. Let a light pulse be emitted at time t zero at point r zero, reach the target at the radial coordinate r greater than r zero at time t, and is immediately retransmitted to the point of observation being at radial distance to which it arrives at time t one. Propagation of the outgoing and incoming light rays are obtained from twenty three, where we demand that at the time of emission, t zero, the coordinate speed of light r dot (Of t zero) equals one for both outgoing and incoming light rays. The equation of propagation for outgoing light ray is: R equals r zero plus (t minus t zero) plus H over two times the square of t minus t zero. And propagation of the incoming light ray is described by R One equals r minus (t one minus t) minus H over two times the square of t minus t zero. Let us assume for simplicity that the radar ranging is conducted by the Hubble observer at the origin of the local coordinates so that both the points of emission and observation of the light signal are at the origin and have the radial coordinate, r zero equals r equals zero. We define the radar distance L by a standard equation: L is one half of (T One minus T Zero). L is a relativistic invariant due to the covariant nature of the proper time t and the constancy of the fundamental speed c equals one in the geometrized system of units adopted in the present paper. After solving for incoming and outgoing r (Of t) we obtain: T equals a half T zero plus T One, plus a half H r squared, and: L equals R minus H R squared. Where the residual terms of the order of Hubble squared have been neglected, r is the radial distance of the point of reflection of a radar signal at time t. In other words, the approximation of a locally flat Minkowski space is corrected, to first order by linear terms, in which the propagation of light signals is modified by the cosmological expansion. This calculation reveals that the difference between the coordinate distance r and the invariant radar distance L is of the order of H r squared. Planetary ranging is done for the inner planets of the solar system so we can approximate r around one astronomical unit, A U and H around two point three times ten to the minus eighteen inverse seconds. Hence the difference H r squared of around zero point one seven millimeters which is a factor of around ten thousand smaller than the current ranging accuracy two meters to interplanetary spacecraft. In case of lunar laser ranging to the Moon, the coordinate radius of the lunar orbit, r of 384 thousand km, and the estimate of the residual term H R squared is about one point one times ten to the minus six mm which is one million times less than the current accuracy (around one millimeter) of LLR. We conclude that in radar, laser ranging experiments: (One) Within the measuring uncertainty the coordinate radial distance r equals L, (Two) The radial distance r in the local frame of reference has an invariant geometric meaning in agreement with the definition of the proper distance accepted in cosmology, (Three) the radar, laser ranging metrology is insensitive to the Hubble expansion in the local coordinates. Hence, the celestial ephemerides of the solar system bodies built on the basis of radar, laser ranging data are not crippled by the Hubble expansion. They represent a dynamical reference frame with a fixed value of the astronomical unit, A U, which is not changing in time and can be treated as a rigid ruler for measuring distances between celestial bodies within the solar system in accordance with a recent resolution of IAU General Assembly (Beijing 2012) on the meaning and value of the astronomical unit. Six point two. Einstein’s synchronization of clocks. Let us now consider the Einstein procedure of the synchronization of two clocks based on the exchange of light signals between the clocks. We want to synchronize the clock of the central Hubble observer with the clock of a static observer located at a point with the Gaussian radial coordinate r. We apply exactly the same procedure as in the case of radar ranging described above. By Einstein’s definition, when the photon reaches the reflection point with the radial coordinate r at the instant of time t, the clock of the Hubble observer at the point, r equals zero, reads the time: T star equals a half T Zero plus t one, Because the time rate of the ideal clock of the Hubble observer is uniform. The instant of time t∗ is defined as being simultaneous with the time reading, t, of a second clock located at the position with a radial coordinate, r, at the instant when the light signal is reflected. The time t star as a function of t, can be found immediately from equation twenty seven: t star equals t minus a half H r squared. This relation reveals that in order to synchronize two clocks separated by a radial distance r, we have to subtract the time difference H r squared from the reading t of the clock of the static observer at the point with radial coordinate r in order to make the time readings of the two clocks identical. Because the radial distance r coincides with the invariant radar distance L, which is a measurable quantity, the Einstein synchronization of clocks in such experiment is operationally possible. The two clocks will remain synchronized as time goes on, if and only if, the radial distance between the clocks does not change. For example, a clock at a geocenter will remain synchronized with clocks on-board of a geostationary satellite moving around Earth on a circular orbit. On the other hand, an ultra-stable clock on board of spacecraft which moves with respect to the primary time standard on Earth may detect the de-synchronization effect due to the Hubble expansion of the universe if the radial distance between Earth and the spacecraft changes periodically. If the change in the radial distance amounts to delta r, the overall periodic time difference caused by the clock’s de-synchronization amounts to: Delta t equals delta of t star minus t, which equals: Two H times R squared, over c squared times delta r, over r. Expressing r in astronomical units we can find a numerical estimate of the de-synchronization between the readings of the two clocks, equation thirty one: Delta t equals one point seven times ten to the minus twelve times R in units of A U squared, delta R, over r seconds. This local cosmological effect may be detectable by NIST and, or other world-leading timekeepers. Six point three. The Doppler Effect in the local frame. Next step is to consider the Doppler Effect that is a change in frequency of propagating electromagnetic wave (light) emitted at one spacetime event and received at another one, as caused by various physical reasons, relative motion of observer and the source of light, gravity field, expansion of the Universe, etc. A monochromatic electromagnetic wave propagates on a light cone hypersurface of a constant phase Phi, that is a function of spacetime coordinates, Phi equals Phi (Of x alpha). The wave one form is L alpha equals d alpha of Phi, and frequency of the wave measured by an observer moving with four velocity, u alpha is: Omega equals minus L alpha u alpha. The frequency of electromagnetic wave can be calculated directly as soon as we know L alpha and u alpha, equals: d x alpha, d tau, where tau is the proper time along the world-line of emitter (or receiver) of light. Indeed, Omega equals minus the contraction of L alpha, and u alpha, which equals, equation thirty three: D phi, d x alpha, d x alpha, d tau equals d phi, d tau. Which is just the rate of change of the phase of the electromagnetic wave along the world line of emitter (or receiver). Let us denote the point of emission of the wave by P one, and the point of its observation as P two, and the emitted and observed wave frequencies as omega one and omega two, respectively. The proper time of the emitter is denoted as tau one, the proper time of receiver is tau two, and the time measured by the central Hubble observer is the cosmic time t. The ratio of received to emitted frequency, equation thirty four: Omega one over omega two, equals: L alpha, u alpha P two, over l alpha, u alpha P one. Which quantifies the Doppler Effect. Because the phase of the electromagnetic wave remains constant along the light rays we can use equation (thirty three) to reformulate (thirty four) in terms of the time derivatives. This is another way of saying, along a geodesic the phase in the proper time of a light pulse is constant. Omega two over omega two equals d tau one, d t one, times: d t one, d t two, times: d two, d tau two. We can introduce the unit vector N two one, which is X two minus X one, over the magnitude of X two minus X one, which points out from the point of emission, P one, to the point of reception, P two, of the light signal. After some algebra, expanded upon in the paper, we obtain the Doppler shift of frequency of electromagnetic wave in the expanding universe for the emitter and receiver being moving with respect to the LIC, equation forty two: Omega two, over omega one equals: One minus N two one dot V two, Over one minus N two one dot V one times: The square root of one minus v one squared, over one minus v two squared, times: One plus H t two minus t one. Where we have dropped off all residual terms of the order of H V One and H V Two as negligibly small. Notice that (forty two) does not depend on the choice of the initial epoch t zero. Equation (forty two) consists of two groups of terms. The first group depends on velocities of emitter and receiver, and represents a special relativistic Doppler effect. The second group (in square brackets) depends on the Hubble constant H and represents an additional shift of frequency caused by the cosmological expansion of space. The gravitational field of the solar system bodies should be also taken into account in realistic experiments. We have excluded the gravitational shift of frequency as it brings about many more terms to (forty two) and makes it unnecessarily complicated. These terms are well-known and can be found, for example, in the literature. For a static emitter and receiver we have V One equals V two equals zero, and the Doppler shift equation (forty two) drastically simplifies to: Omega two, over omega one equals one plus H times T two minus t one. It tells us that the cosmological Doppler shift measured by the local static observers is blue because T two is greater than t one and, consequently, Omega two is greater than Omega one. It works opposite to the cosmological red shift for distant quasars, but there is no contradiction here. Cosmological red shift is measured with respect to the reference objects (quasars) which have fixed values of the global coordinates, y I, while the local Doppler shift is measured with respect to static observers having fixed Gaussian coordinates x i. Thus, the Doppler shift measurements in the global cosmological spacetime and in the local tangent spacetime refer to two different sets of reference observers moving one with respect to another with the velocity of the Hubble flow. Therefore, it is natural to expect a different signature of the Doppler effect, red shift for light coming from distant quasars and blue shift for light emitted by the astronomical objects, for example spacecraft, within the solar system. Our theory provides an exact answer for the signature and magnitude of the cosmological blue shift effect measured in the local inertial frame. The Doppler effect in the tangent spacetime of FLRW universe has been considered by a number of other authors, most notably by Carerra and Giulini. They claimed that the cosmological expansion does not produce any Doppler effect in the local radio-wave frequency measurements. Their conclusion is invalid as they implicitly identified the local Minkowskian time coordinates x zero with the proper time t of the Hubble observer on a worldline of any freely-moving particle including photons. However, this identification is not applied to photons (or any other moving particle) but solely to the static clocks of the Hubble observer. Six point four. Measuring the Hubble constant with spacecraft Doppler-tracking. Results of previous section suggest that precise and longterm Doppler tracking of space probes in the solar system may offer a new, fascinating opportunity to measure the local value of the Hubble constant H in the solar system. It is highly plausible that the “Pioneer anomaly” detected by John Anderson with the JPL deep-space Doppler tracking technique in the hyperbolic orbital motion of Pioneer spacecraft has a natural explanation given in terms of the Hubble expansion which changes the frequency of radio waves in spacecraft radio communications in an amazing agreement (both in sign and in magnitude) with our equation (forty three). We have analyzed the cosmological origin of the “Pioneer anomaly” effect in another paper, making use of the local equations of motion for charged and neutral test particles as well as for photons in the FLRW universe. We have proved that in the local frame of reference the equations of motion for interacting massive neutral and, or charged particles do not include the linear terms of the first order in the Hubble constant, only tidal terms of the order of H squared remain. On the other hand, equations of motion of photons parameterized with the TCB time t do contain such linear terms of the order of H which have dimension of acceleration. The present paper confirms results of the paper from the point of view of a set of local observers doing measurements in tangent space of the FLRW manifold. Transformation to the local coordinates x alpha, equals (x zero, x I) allows us to transform the FLRW metric to the Minkowski metric: D s squared equals minus d x zero squared plus Delta I, J, d x I, d x J, but the coordinate time x zero can be identified with the proper time t of the central Hubble observer only for static observers while for moving particles X zero equals x zero (Of tau) is a non-linear function of time t which is given for photons by (equation twenty one). Equation (forty two) explains the “Pioneer anomaly” effect as a consequence of the expansion of space bringing about the blue frequency shift of radio waves on their round trip from Earth to spacecraft and back. Indeed, let us denote omega zero the reference frequency emitted from Earth to spacecraft, omega two the frequency received at spacecraft and transmitted back to Earth, and omega two the frequency received on Earth. Then, according to (forty two), the shift between omega zero and omega two can be determined. Let us simplify further consideration by assuming that the measurement is done by the central Hubble observer located at the origin of LIC. Then, V zero equals V two equals zero, and the unit vector is N one zero, equals minus N two one points out in the positive radial direction toward spacecraft moving with velocity v equals V One. After noticing that T Two minus T zero is approximately two times (T one minus T zero), and neglecting quadratic with respect to velocity terms, formula for omega two over omega one takes on the following form of equation forty five: Omega two, over omega zero equals one minus two V over C, minus H times (T One, minus T zero). Where V equals V dot N is the radial velocity of spacecraft, and we prefer to retain the speed of light c explicitly. The Doppler shift is defined as: Z equals a half omega two over omega one minus one. We get from equation forty five: Z equals minus v over C plus H Two minus t zero. That shows that the cosmological shift of frequency appears as a tiny blue shift on top of a much larger red shift of frequency caused by the outward motion of the spacecraft. It was observed by J Anderson, and confirmed in a number of papers. The time rate of change of the Doppler shift is Z dot equals d X, d t one, which yields: Z dot equals minus one over c times “A.” minus H C. Where “A.” equals d v, d t one is the magnitude of the radial acceleration of the Pioneer spacecraft due to the attraction of the solar gravity field. The Hubble frequency-shift term, H c, is subtracted from the spacecraft acceleration and can be interpreted as a constant, inwardly directed acceleration, “A.” P equals H c, in the motion of the spacecraft. In fact, the true cause of the “anomalous” acceleration is associated with the motion of photons but not the spacecraft. This is the reason why the vigorous attempts to find out the explanation for the “anomalous gravity force” exerted on the Pioneer spacecraft were unsuccessful. The observed value of acceleration observed equals eight point five times ten to the minus ten meters per second per second, and is in a good agreement, both in sign and in magnitude, with the theoretical value of: H C is around seven times ten to the minus ten meters per second per second. Therefore, we believe that our result provides a strong evidence in favour of general-relativistic explanation of the “Pioneer anomaly” as opposed to numerous attempts to explain it by thermal recoil force. The thermal recoil definitely makes contribution to the acceleration of the Pioneer spacecraft because the observed value of observed acceleration P exceeds theoretical value by 20 percent. Recent studies indicate that the numerical value of the Pioneer anomalous acceleration may be slightly decreasing over time which may be associated with the radioactive decay of the power generators of the Pioneer spacecraft. The question about how much the thermal recoil force contributes to the overall effect remains open. The literature states that the Pioneer effect is 100 percent thermal but they have not taken into account the geometric effect of the expanding space on the propagation of light in the local frames in cosmology which suggests that the numerical value of the Pioneer effect cannot be smaller than the theoretical value of seven times ten to the minus ten. The thermal emission always adds to the general-relativistic prediction. Observations indeed show an observed acceleration larger than theoretical values by 20 percent. The theory of the present paper explains 80 percent of the overall effect by the effect of the expanding geometry leaving for the thermal recoil contribution no more than 20 percent. Seven. Discussion. One. We have built the LIC by applying the special conformal transformation (thirteen). Comparison with other approaches to build the LIC in cosmology reveals that all of them bring about the same coordinate transformation in the linearized Hubble approximation. Therefore, there is no difference between various approaches to build the local inertial coordinates in cosmology so far as the quadratic terms in the expansion with respect to the Hubble parameter are not considered. Our approach to build LIC helps to realize that the transformation to the local coordinates on the expanding cosmological manifold is, in fact, an infinitesimal special conformal transformation which establishes one to one local mapping between the local and conformal coordinates. Two. Introducing a local physical distance X I equals R (Of t) Y I allows recasting equation two into the following form: D s squared equals minus one minus H squared X squared d t squared minus: Two H X I, X I d t plus delta I, J, d X I, d X J. This can be rearranged as: D S squared equals minus d t squared plus Delta I, J times (d X I minus Chi I d T), times (d X j minus Chi J d t). Where vector field Chi I equals the Hubble constant times X I. The former metric is exactly the warp-drive metric that was suggested by Alcubierre to circumvent the light-speed limit in general relativity. All mathematical properties of the warp-drive metric that have been analyzed, and they are valid in the local coordinates (t, x i) where t is the proper time of the local static observers (X I equals constant) coinciding with the cosmic time. The metric is non-inertial but it can be converted to the flat Minkowski metric in a neighborhood of the coordinate origin with the help of an additional transformation of the proper time t to a local time coordinate x zero as shown in (equation thirteen). The local time coordinate x zero coincides with the proper time t of the static observers but deviates quadratically from t on the light cone as demonstrated in equation twenty one. Three. The analysis of EEP given in the present paper, was focused on the solar system experiments as contrasted with pure cosmological tests. There are other possible tests which can be potentially conducted for testing the formalism worked out in the present paper, for example, with binary pulsars. Timing measurements establish a very precise local frame for the binary pulsar system which is not affected by the Hubble expansion as explained in the literature. On the other hand, we expect that the cosmological expansion influences the time of propagation of radio pulses from the pulsar to observer on Earth, and this effect should be seen in the secular change of the orbital period P b of binary pulsars of the order of: The time derivative of P B, over P B equals H around two point three times ten to the minus eighteen seconds. This effect is superimposed on the effect of the orbital decay due to the emission of gravitational waves by the binary system and introduces a bias to the observed value of P B dot in addition to the Shklovskii effect. However, the orbital decay of binary pulsars with wide orbits is negligibly small, hence, we may expect to observe the Hubble expansion effect in the secular change of the orbital period. Four. It is worth mentioning that the Cassini spacecraft was also equipped with a coherent Doppler tracking system and it might be tempting to use the Cassini telemetry to measure the universal “anomalous Cassini acceleration” of around seven times ten to the minus ten meters per second per second. Unfortunately, there are large thermal and outgassing effects on Cassini that would make it difficult or impossible to say anything about the “Cassini anomaly” from Cassini data, during its cruise phase between Earth and Saturn. Due to the presence of the Cassini-on-board-generated systematics, the recent study of radio science simulations in general relativity and in alternative theories of gravity is consistent with a non-detection of the “Cassini anomalous acceleration” effect. 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The ten nearest stars. Sol. The Sun. Sol and its system will be used to introduce the units. Often properties of nearby stars are listed in fractions of solar units, such as luminosity, or solar mass. The quoted effective stellar temperature refers to the temperature of the outer photosphere. By definition the photosphere is the outer surface of the star through which half of the light escapes un-scattered by collisions with matter. The photosphere of Sol is approximately 100 kilometers thick, and has a density of zero point three milligrams per cubic meter. The temperature in the photosphere is between 4500K and 6000 K, with the effective temperature stated as 5772 K. In the photosphere, bubbles of plasma, or granules, rise, cool and fall back into deeper depths. Each granule is around one thousand kilometers in size. The spectral classification of a star is often reported in the Morgan-Keenan, MK system, with O being the hottest, brightest and having the shortest lifespan, and M being the coolest, smallest, and longest persisting. Sol, of spectral type G, is a star with an effective temperature between 5200 and 6000 degrees Kelvin. The sub-categorization of “Two”, or G-Two means that the temperature is around 5770K, with a G-One having a temperature of 5860 K, and a G-three a temperature of 5720K. The luminosity class of “V”, often referred to as dwarf means that the star is on the Main-Sequence, and is consuming hydrogen to create Helium. As the Sun ages, the amount of Helium will increase. Currently the ratio of Hydrogen to Helium in the sun is around three to one. Hence, by the definitions, Sol is a spectral class G two dwarf star, with a photosphere temperature of 5772 Kelvin. Sol has a mass of 1.99 Rona tons, RT, or 1.99 times ten to the thirty Kilograms, and a radius of 0.969 Giga meters. Sol is 4.6 Giga years old, and has an equatorial rotational period of 24 point 47 days. As stars are not solid, the rotational period decreases towards the poles, which can result in differential shearing of the atmosphere. The average orbital period around the Galactic core is two hundred and forty million years. In its lifetime, the Sun has orbited the Galactic center just over nineteen times. The composition of a star is often reported as its metallicity, which is the ratio of number of Iron atoms to the number of Hydrogen atoms. For the Sun, the ratio of Iron atoms to Hydrogen atoms is about one to twenty thousand. Expressed as a logarithm, the solar metallicity Log ten of Iron to Hydrogen is minus 4.33. The metallicity ratio of Iron to Hydrogen for a star, is the log to base ten of the Iron to Hydrogen ratio, minus the log to base ten of the Iron to hydrogen ratio of the Sun. Expressed as a ratio, the logarithmic metallicity Iron to Hydrogen of the sun is Zero. A metallicity of plus one represents ten times the Iron to Hydrogen of the Sun. A value of minus one corresponds to one tenth of the Iron to hydrogen ratio of the sun. And a value of zero indicates that the star has the same ratio of Iron to Hydrogen as the sun. An additional specification of stellar composition is the fraction of Hydrogen, Helium and other items. Fractional hydrogen is denoted as X, fractional Helium is labelled as a Y, and all other elements are denoted as Z, with the sum X plus Y plus Z equal to one. For the Sun, X, the fractional Hydrogen is zero point 70. Y, the fractional Helium is zero point 28, and Z, all other elements are zero point zero two. The 2015 Resolution B2 of the International Astronomical Union defined a standard luminosity, the zero point luminosity L zero of 3.0128 times ten to the 28 Watts, or approximately 78.7 times the luminosity of Sol. Minus two point five times the logarithm, of a stars luminosity over this standard zero point value is now known as the bolometric magnitude of the star. For the sun, this bolometric luminosity is 4.74, and is a dimensionless number. The absolute magnitude of a star is the apparent luminosity of the star if it was viewed at exactly ten parsecs, 30.857 Peta-meters, without scattering and absorption of light by any interstellar material. The visual absolute magnitude, M V, considers only the light emitted in the visible spectrum. For Sol M V is the dimensionless number 4.83. An alternative unit introduced here is the “LILO”, or logarithm to base ten of the total solar isotropic luminosity L, which is the bolometric or integrated emitted energy over all frequencies in Joules, divided by four pi. LILO is just the log to base ten of the Radiant Intensity, or logarithm of the Watts per steradian. For Sol the LILO is: Log L divided by four pi equals 25.484. As an example of the use of LILO, at a distance of ten Giga meters, the logarithmic fluence is 25.484 minus twenty, or 5.484, or about three hundred kJ per square meter. The standard gravitational parameter is the Cavendish gravitational constant G, times the mass, usually denoted by a Greek letter mu. The Log to base ten of mu, which is log to base ten of G, M, for the sun is 20 point 12. In units of Giga-meters, the semi-major axis of Sol’s inner planets are located at 58, 108, 150 and 228 giga-meters. The major outer planets are located at 778, 1,433, 2,871 and 4,500 Giga meters. The Habitable zone of a star is the range of distances where the surface temperature of planets will allow liquid water. The inner habitable zone of the solar system is zero point nine five “A-U”, or 142 Giga-meters. The outer habitable zone is one point four “A-U”, or 208 Giga meters. These distances will increase or decrease with the square root of the luminosity of the star relative to the sun. Alternatively, using the LILO of the star, the log to base ten of the distances in meters can be determined. As the star ages, the luminosity will increase. Since the inner radius of the habitable zone is zero point nine five “A-U”, a ten percent increase in luminosity of the sun will place the Earth at the boundary of the habitable zone. At the suns current rate of hydrogen burning, this will occur in approximately one billion years. Conventionally star distances are given in one of two units. Light years, the distance light can travel in one Julian year of 365 point 25 days. The other unit is the parallelax-seconds, or “Parsec”, derived from one Astronomical Unit, the distance from the Earth to the sun, divided by one second of arc, or a thirty six hundredth of a degree. A Parsec is approximately 3 point 26 light years. One light year is 9.45 times ten to the fifteen meters, or slightly less than ten Peta-meters. One Peta meter is ten to the fifteen meters, or a million Giga-meters. Ten Peta-meters equals one point zero five light years. Distances are here quoted in Peta meters. Many of the extra solar planets discovered, were detected using the radial velocity method. Since the star and the unseen planets revolve about their common barycenter, there is a Doppler shift imparted upon the spectral lines of the parent star. One form of the equation for the radial velocity K, measured in meters per second, is given by Wright, in his 2017 paper, included in the references. The constants are numerical values, and the Gravitational constant G. The system specific values include the eccentricity of the orbit, usually zero to a half, the Period of the rotation P, and the mass of the parent star. And the variable, “Em-sine-eye”, or the mass of the planet times the sine of the angle of inclination to the observer. Hence if a planet was inclined with an inclination angle of zero, the radial velocity measurement would not be able to detect the planet. It must also be noted that the radial velocity shift can be quite small. The 2023 limit is a motion of a system of around one meter per second. The motion of the Earth on the sun is 9 centimeters per second, which would not be detectable with 2023 technology. By comparison, the Jovian scale planet of zero point four Jovian masses around 51 Pegasi b, with a 4.2 day orbit imparts a radial velocity of around 60 meters per second, and a correspondingly large Doppler shift. With a slight rearrangement of terms, the equation for the Doppler velocity K can be written as K is: Cube root of two pi G over M star squared, which are the star specific parameters, times: M planet over cube root of period P, times the square root of one minus eccentricity, Which are the planet specific terms, and sine inclination I, which is the observer specific term. In practice the combination of the planet mass and inclination, “Em-sine-Eye” is usually the determining factor for the detection of an extrasolar planet. Typical results, from Hurt 2022 for GJ 411, also known as Lalande 21185, show the radial velocity perturbations of planets after processing. Alternative methods of extra-solar planet detection include transits of a planet across a star. And the Kepler space telescope was designed explicitly for this purpose. In this case, the difference on measured light intensity as the planet occludes part of the star is required, so non-transiting planets cannot be detected. Direct imaging of a planet can be obtained by image processing of stellar images. By subtracting the optical light, the scattered infra-red can display the position of orbiting planets. Processed images of GJ 504, also known as 59 Virginis, a G zero star about 570 Peta-meters from Sol, from 2011 to 2012 are shown, to display GJ 504 b. Alpha Centauri. The Alpha Centauri system consists of three stars. Alpha Centauri “A”, and Alpha Centauri B, and Proxima Centauri, gravitationally bound with the binary system. Alpha Centauri “A” is a G Two of 4.34 absolute magnitude at a distance of forty one point six Peta meters from Sol. Alpha Centauri B is a K Zero, 5.7 absolute magnitude star, revolving at 3,570 Giga-meters from Alpha “A”. Proxima Centauri is an M Five point 5 of absolute magnitude 15.45. It orbits Alpha Centauri at a distance of 1.5 Peta-meters. Alpha “A” has of Mass of 2.15 Ronna tons, a Radius of 0.85 Giga-meters, and a Temperature of 5790 Kelvin. The Fe over H Metallicity of 0.2. Its LILO is 25.66, and its Log mu is 20.16. It is very similar to Sol. The age of Alpha Centauri “A” is 5.17 Giga years, and its rotation period is 22 days. Alpha “B” has a Mass of 1.81 Ronna tons, a Radius of 0.60 Giga-meters, and a Temperature of 5260 Kelvin. Its Iron to Hydrogen Metallicity is 0.21. Alpha Centauri “B” has a LILO of 25.181, and a Log GM of 20.08. Alpha Centauri B is 2.53 Giga-years old, with a rotation period of 41 days. Proxima’s details are that it has a mass of 0.24 Ronna tons, a Radius of 0.11 Giga-meters, a temperature 2879 K, and its Metallicity Iron to Hydrogen is 0.1. Proxima is also a flare star of type BY Draconis. Proxima Centauri has an age of 4.85 Giga-years and a rotation period of 92.1 days. The orbits of Alpha and Beta Centauri and Proxima are at an inclination to an observer from Earth, so the Orbit can be readily distinguished through a telescope. Proxima’s orbit around the alpha and beta Centauri barycenter has an eccentricity of 0.5, with a semi major axis of 1,300 Terra-meters, with an orbital period of 547 thousand years. Proxima Centauri has three confirmed planets, Proxima B, C and D. Proxima D has a mass of 0.26 of Earth, and its orbital parameters are: semi-major axis. 4.32 Giga-meters, eccentricity 0.04, period 5.122 days. Proxima B has a mass of 1.07 of Earth, and its orbital parameters are: semi-major axis 7.27 Giga-meters, eccentricity 0.109, period 11.184 days. Proxima C is disputed, but would have a mass of seven Earths, and its orbital parameters would be: semi-major axis 222 Giga-meters, eccentricity 0.04, period 1928 days. “B-Y” Draconis flare stars have flares driven by sunspots and stellar rotation. The prototype is “B-Y” Draconis “A” and “B”, two stars fifty three light years away in the constellation of Draco. Flare periods are quasiperiodic with frequencies close to the stellar rotation period, with typical amplitudes of approximately half a visual magnitude. The flare oscillations are likely the interplay of the stellar magnetic field and the turbulent surface convection. Flares which increase the local temperature up to 8000K are known, which can increase the luminosity by up to a half a visual magnitude. Energy fluxes can reach ten to the twenty two Joules of UV or X ray energy. Bernard’s Star. Bernard’s Star is an M five, of absolute magnitude 13.24, located at a distance of 56.2 Peta-meters. The Mass of Bernard’s Star is 0.32 Ronna-tons, its Radius is 0.13 Giga-meters, and it has a Temperature of 3,278 K. Bernard’s star is a “B-Y” Draconis type flare star with the designation of V2500 Ophiuchi. Bernard’s stars age is 10 Giga-years, and its rotational period is 130.4 days. Bernards Star is smaller, cooler and older than the sun, with an Iron to Hydrogen metallicity of minus 0.12 Dex. LILO 23.015, Log GM, 19.33. As yet there are no positively confirmed planets orbiting Bernard’s Star, and the sensitivity of the studies would require any undetected candidates to be approximately earth mass or less. For example, see the paper by Jack Lubin, 2021: “Stellar Activity Manifesting at a One Year Alias Explains Barnard b as a False Positive”. Also Artigau, 2022, in “Line-by-line velocity measurements, an outlier-resistant method for precision velocimetry” stated: We confirm with measurements spanning 2.7 years that the candidate super-Earth on a 233-day orbit around Barnard’s star is an artifact due to a combination of time-sampling and activity. Wolf 3-5-9. Wolf 3-5-9 is an M six of absolute magnitude 16.56, located at 73.8 Peta-meters. Wolf 3-5-9 has a mass 0.22 Ronna-tons, a Radius 0.10 Giga-meters, and an effective Temperature of 2,749 K. Because of its small size, Wolf 3-5-9 is fully convective, so that the internal material physically cycles through the star, maintaining the spectral type for a considerable time period. Wolf 3-5-9 is likely to remain a main sequence for two trillion years. Wolf 3-5-9 is a young star, between one hundred to three fifty million years old. It is a UV Ceti type flare star with the fares driven by magnetic activity in the atmosphere. Such flares can be quite short, in terms of minutes. The magnetic field of Wolf 3-5-9 is around 0.2 Tesla’s, and can vary over times scales of hours. Flares can be around ten to twenty percent of the size of the star, compared to the few thousand for solar flares. X ray luminosities can be up to one percent of the bolometric luminosities. Flares of two magnitudes of luminosity have been observed. Wolf 3-5-9 rotates once every 2.70 days, and has a metallicity of 0.18, a LILO of 22.509, and a Log GM of 19.16. The candidate planets of Wolf 3-5-9 consist of Wolf 3-5-9 B and C. Wolf 3-5-9 B has a predicted mass of around forty four Earths, with an orbit radius of 275 Giga-meters. The existence of Wolf 3-5-9 C is dubious, but it would have a mass of around four Earths, with a period of two point seven days, and an orbit radius of just over seven Giga-meters. Lalande 21185. Lalande 21185 is an M two of absolute magnitude 10.46, at a distance of 78.6 Peta-meters. Mass 0.77 Ronna-tons, Radius 0.27 Giga-meters, and a Temperature of 3547 Kelvin. Lalande 21185 is a “B-Y” Draconis type variable star, with the variable star designation NSV 18593. Lalande 21185 is 5 to 10 Giga-years old, and rotates once every 56 days. Its metallicity, Iron to H, is -0.09. LILO 23.774, log ten GM 19.71. Lalande has three candidate planets, located at 11.8, 76.6 and 424 Giga-meters. Only one of the planets of Lalande 21185 is confirmed, Lalande 21185 b, with an orbital period of 12.93 days. Lalande 21185 b has the orbital elements of eccentricity 0.12, Semi major axis 11.8 Giga-meters, and an orbital period of 12.9 days. Estimated equilibrium temperature is 370 Kelvin. Sirius. Sirius is an A one star of absolute magnitude 1.43 at a distance of 81.4 Peta-Meters, and is the brightest star in the night sky. Sirius B is a “D” “A” two dwarf, of absolute magnitude 11.33, gravitationally bound to Sirius “A”, at a distance of 3000 Giga-meters. Sirius “A” has a Mass of 4.1 Ronna-Tons, a Radius of 1.19 Giga-Meters and a Temperature of 9,940K. The metallicity ratio is minus zero point 13 Dex. Sirius “A” is very young, with an age of 240 Mega years. Due to its high luminosity, the inner edge of the habitable zone for Sirius “A” is around the same distance as from the Sun to Jupiter. Sirius B has a Mass of 2.02 Ronna-Tons, a Radius 6 Mega-meters, and a Temperature of twenty five thousand kelvin. Whereas Sirius “A” is slightly larger than the Sun, the size of Sirius B is approximately equal to Earth. Sirius “B” is a young star, with an age of two hundred and thirty mega years. Sirius “A” and “B” orbit around a common Barycenter, and the respective orbits can be seen quite readily from Earth. At present the maximum mass of any planets in the Sirius system would have to be less than around fifteen times the mass of Jupiter. Luyten “7 2 6” dash 8, also known as GJ 69, or Gliese 65. Luyten “7 2 6” dash 8 “A” is an M five point 5 of absolute magnitude 15 point 27, located at 82.6 Peta meters from Earth. Luyten “7 2 6” dash 8 “B” is an M five point 5 of absolute magnitude 16 point 11, at a separation of 2340 Giga-meters from Luyten “A”. Luyten “7 2 6” dash 8 “B” was originally named UV Ceti, and is a UV Ceti flare star. Luyten “7 2 6” dash 8 “A” is a BL Ceti type flare star, with a rotation period of 0.2 days. Lutyen “7 2 6” dash 8 “A” has a LILO of 22.262, and a log ten GM of 19.13, Luyten “7 2 6” dash 8 B has a LILO of 21.086, Log ten GM 19.12. Since they are both small and dim, their habitable zones extend from one to one point five Giga-meters. At present there are no known planets for this system. Ross 154. Ross 154 is an M four point 5 of absolute magnitude 13, at 91.7 Peta meters. Ross 154 is an active UV-Ceti type flare star, with the designation V1216 Sagitarri. Light curve variations of up to 1.5 percent. Flares of up to 2.3 times 10 to the 26 Joules have been detected. There are significant x-ray components. It does not display any excess infrared absorption, which would indicate planetary companions. Ross 154 has a Mass of 0.352 Ronna-tons, Radius of 0.139 Giga-meters, and a Temperature of 3,248 Kelvin. Its Metallicity is minus 0.25, and it is around zero point seven Giga-years old. Ross 154 has a rotation rate of 2.85 days. Ross 248. Ross 248 is an M four point 6 of absolute magnitude 14 point 79, located at a distance 97 point 7 Peta meters. Due to its high- relative velocity Ross 248 will be the closest star to earth in 36,000 years time. Ross 248 is a B, Y Draconis type variable star, with the designation H, H Andromedae. B, Y Draconis variables have quasi-periodic light curves due to rotation and active chromospheres. Ross 248 has a Mass of 0.288 Ronna-tons, Radius 0.132 Giga-meters, and a Temperature 2930 Kelvisn. Its Metallicity is 0.23, and its age is 2.6 Giga-years. Ross 248 has a rotation period of 99.58 days. Epsilon Eridani. Epsilon Eridani is a K two of absolute magnitude 6 point 18, and is located at a distance of 99.3 Peta meters. Epsilon Eridani has a rotation period of 11.4 days and is a B Y Draconis variable star. It is around six hundred million years old. The X-ray luminosity of Epsilon Eridani is 2 times ten to the 21 Watts. The metallicity of Epsilon Eridani is minus zero point one three. Epsilon Eridani is 4 to 8 hundred million years old, and has a rotational period of 11.4 days Epsilon Eridani is a very dusty star, with significant infrared absorption indicating an asteroid belt at 200 to 600 Giga-meters, another asteroid belt at 1,000 to 3 thousand Giga-meters, and a dust disk extending out to 15 Tera-meters. It has one confirmed planet Epsilon Eridani b, with a mass of 0.78 of Jupiter. Epsilon Eridani b’s parameters are eccentricity 0.07, period 7.37 years, with a semi major axis of 521 Giga-meters. Lacaille 9352, Also known as GJ887. Lacaille 9352 is an M zero point five of absolute magnitude 9 point 76, at 101 point 5 Peta meters from Earth. Lacaille 9352 was the first red-dwarf to have its diameter measured. Lacaille 9352 is 4.57 Giga-years old, with an Iron to Hydrogen Metallicity of minus 0.06, and a temperature of 3688 K. The mass of Lacaille 9352 is 0.952 Ronna-tons, and its diameter is 0.330 Giga-meters. At least two planets are reported for this system, at 10.1 Giga-meters mass greater than four times earth, and 17.9 Giga-meters, mass greater than seven times Earth. Lacaille 9352 is a suspected variable star, although with no observation evidence of variability. GJ887b has a mass of 4 earths, a period of 9.262 days, an eccentricity of 0.09, and a semi major axis of 10.3 Giga-meters. Its equilibrium temperature would be 468 Kelvin. GJ887c has a mass of 7.6 earths, a period of 21.789 days, an eccentricity of 0.22, and a semi major axis of 17.9 Giga-meters. Its equilibrium temperature would be 352Kelvin. Ross 128. Ross 128 is an M four dwarf, F 1 Virginis type flare star, with an absolute visual magnitude of 13.53. The rotational period is 121 days. The flare activity is low compared to BY Draconis type flares, with an amplitude of a thousandth of a magnitude, mainly in the X-ray spectrum with very little UV or visible variation. Ross 128 is 103 Peta-meters from Sol, Is 5 Giga-years old and has a temperature of 3192 K. Ross 128 has a mass of 0.334 Ronna-tons, a diameter of 0.137 Giga-meters, and an Iron over Hydrogen metallicity of minus 0.02. A confirmed rocky planet, Ross 128 b, is located at 7.4 Giga-meters, with an orbital period of about 9.866 Earth days. As the metallicity of Ross 128 is close to that of the sun, the dimensions of the planet are expected to be similar to Earth, with a similar surface temperature. Orbital parameters for Ross 128 b are eccentricity 0.116, period 9.866 days, Semi major axis 7.42 Giga-meters. Ross 128 b has a mass of 1.8 of Earth, and an equilibrium temperature of 294 Kelvin. Chahal 2022. Statistics of BY Draconis Chromospheric Variable Stars (https://arxiv.org/pdf/2206.05505.pdf) Recalculating the Orbit of α-Centauri AB https://arxiv.org/ftp/arxiv/papers/1007/1007.2293.pdf Proxima's orbit around Alpha Centauri https://arxiv.org/abs/1611.03495 Zhao 2017 PLANET DETECTABILITY IN THE ALPHA CENTAURI SYSTEM https://arxiv.org/abs/1711.06320 Precision Millimeter Astrometry of the α Centauri AB System https://arxiv.org/pdf/2104.10086.pdf Breezing through the space environment of Barnard's Star b https://arxiv.org/abs/1901.00219 Stellar Activity Manifesting at a One Year Alias Explains Barnard b as a False Positive https://arxiv.org/abs/2105.07005 Searching for Planets Orbiting Alpha Centauri A with the James Webb Space Telescope https://arxiv.org/abs/1910.09709 Toldeo 2018 Stellar activity analysis of Barnard's Star: Very slow rotation and evidence for long-term activity cycle https://arxiv.org/abs/1812.06712v3 EDEN: Flare Activity of the Nearby Exoplanet-hosting M Dwarf Wolf 359 Based on K2 and EDEN Light Curves. https://iopscience.iop.org/article/10.3847/1538-3881/abf933/pdf The Astronomical Journal, 162:11 (14pp), 2021 July Pineda 2021 The M-dwarf Ultraviolet Spectroscopic Sample I. Determining Stellar Parameters for Field Stars https://arxiv.org/abs/2106.07656 Ribas 2023 The CARMENES search for exoplanets around M dwarfs. Guaranteed time observations Data Release 1 (2016-2020) https://arxiv.org/abs/2302.10528 Stock 2020 The CARMENES search for exoplanets around M dwarfs. Three temperate to warm super-Earths https://arxiv.org/abs/2010.00474 The Sirius System and Its Astrophysical Puzzles: Hubble Space Telescope and Ground-based Astrometry The Astrophysical Journal, 840:70 (17pp), 2017 May 10 https://arxiv.org/abs/1703.10625 Wargelin 2007 X-Ray Flaring on the dMe Star, Ross 154 https://arxiv.org/abs/0712.2791 Brown 2023 Coronal X-Ray Emission from Nearby, Low-mass, Exoplanet Host Stars Observed by the MUSCLES and Mega-MUSCLES HST Treasury Survey Project The Astronomical Journal, 165:195 (19pp), 2023 May https://iopscience.iop.org/article/10.3847/1538-3881/acc38a/pdf Santos 2003 Spectroscopic [Fe/H] for 98 extra-solar planet-host stars: Exploring the probability of planet formation https://arxiv.org/abs/astro-ph/0311541 Mawet 2019 Deep Exploration of Epsilon Eridani with Keck Ms-band Vortex Coronagraphy and Radial Velocities: Mass and Orbital Parameters of the Giant Exoplanet. The Astronomical Journal, 157:33 (20pp), 2019 January https://arxiv.org/abs/1810.03794 Jeffers 2020 A multiple planet system of super-Earths orbiting the brightest red dwarf star GJ887 https://arxiv.org/abs/2006.16372 Bonfils 2017 A temperate exo-Earth around a quiet M dwarf at 3.4 parsecs https://arxiv.org/abs/1711.06177 Wright 2017 Radial Velocities as an Exoplanet Discovery Method. https://arxiv.org/pdf/1707.07983.pdf Koch 2010 Kepler Mission Design, Realized Photometric Performance, and Early Science. https://arxiv.org/ftp/arxiv/papers/1001/1001.0268.pdf Bonnefoy 2018 The GJ 504 system revisited Combining interferometric, radial velocity, and high contrast imaging data https://arxiv.org/pdf/1807.00657.pdf

A Fully Validated, Multi-Kilogram “c-G-M-P” Synthesis of “M-D-M-A”. Jay B Nair, Linda Hakes, Berra Yazar-Klosinski, and Kathryn Paisner. ACS Omega. 2022, number 7, pages 900 to 907. ABSTRACT: “M-D-M-A” is increasingly used in clinical research, but no “c-G-M-P”, Current Good Manufacturing Practice, process has yet been reported. We describe here the first fully validated “c-G-M-P” synthesis of up to 5 kilograms, about thirty thousand patient doses, of “M-D-M-A” in a four-step process beginning with a non-controlled starting material. The overall yield was acceptable, 41 to 53 percent, over four steps, and the chemical purity of the final product was excellent, exceeding 99.9 percent of the peak area by “H-P-L-C” in each of the four validation trials. The availability of “c-G-M-P” compliant “M-D-M-A” will facilitate ongoing clinical trials and provide for future therapeutic use, if encouraging results lead to “F-D-A” approval. INTRODUCTION. Interest in the clinical utility of psychedelic compounds has increased dramatically in recent years. Although medical usage of these substances, in tandem with psychotherapy, was briefly and controversially explored, in the 1950s and 1960s, increased regulatory oversight and social disapprobation effectively eliminated such research until the late 1990s, when tentative efforts to revive it commenced. Promising early results very slowly stimulated additional engagement, both experimentally and culturally, provoking recent regulatory shifts that have further stimulated engagement by making research chemicals more accessible and expanding the permissible scope of clinical studies. This second wave of so-called psychedelic studies more expansively includes compounds like entactogen 3, 4-methylenedioxymethamphetamine (“M-D-M-A”). Like traditional psychedelics, “M-D-M-A” had previously enjoyed a brief period of encouraging early-stage exploration, in the 1970s and 1980s, which was similarly curtailed by social and regulatory backlash. In contrast to psychedelics like LSD and psilocybin, however, the addition of “M-D-M-A” to the U.S. Drug Enforcement Administration’s (DEA) Schedule One appeared to be largely related to “M-D-M-A”’s popularity as an illicit “party drug,” rather than to significant concerns regarding either contemporary research efforts or its therapeutic utility. Indeed, in clinical trials conducted since the U S Food and Drug Administration (“F-D-A”) and DEA first granted research approval, in 2004, “M-D-M-A” has shown promise as a psychotherapeutic aid for patients suffering from “P-T-S-D”, autism-related social anxiety, and alcoholism. As the research environment grows steadily more supportive of clinical exploration, and as successful clinical trials open the door for fully approved treatments, the need for pharmaceutically acceptable “M-D-M-A” continues to expand. To ensure that patients receive safe, effective drugs, the manufacture of pharmaceutical substances is closely regulated by the “F-D-A”, under a structure called Current Good Manufacturing Practice (“c-G-M-P”). These rules delineate standards for every aspect of the manufacturing process, including facility design, establishment and documentation of operating procedures, process monitoring, and chemical analysis. Because only small samples of each pharmaceutical batch are submitted for destructive quality control testing, a well-controlled manufacturing process is the best-known way to ensure that all drugs distributed to consumers are of predictably high quality, consistency, and efficacy. “c-G-M-P” compliant synthetic processes are typically developed for drug candidates in tandem with progressing clinical trials. Unlike many drug candidates, “M-D-M-A” enjoyed a robust synthetic history prior to receiving any serious consideration as a pharmaceutical substance. “M-D-M-A” was first synthesized by Merck, in 1912, as an intermediate to the styptic compound methyl-hydrastitine. Scientists periodically explored its pharmacological effects over the intervening half-century, both at Merck and in the United States Army, but “M-D-M-A” does not appear in either the patent or the chemical literature again until 1960, when Biniecki and Krajewski published a synthesis identical to Merck’s in Poloniae Pharmaceutica, it is unlikely that they were aware of the Merck patent. This synthetic route proceeded via hydrobromination of the natural product safrole (Scheme 1, TWO), yielding the Markovnikov adduct (Scheme 1, THREE), which was then converted to “M-D-M-A” using methylamine in methanol. A variety of synthetic approaches from methyl piperonyl ketone (Scheme 1, FOUR), which was commercially available at the time, and which can be easily prepared either from safrole typically via Wacker oxidation, or piperonal (Scheme 1, FIVE) typically by reducing its nitroalkene derivative with iron in hydrochloric acid, were summarized by Shulgin, in 1986. A novel approach from piperonal, via Curtius rearrangement, was reported by Schulze, in 2010, and a handful of asymmetric syntheses of (S)-”M-D-M-A”, some relying on alternate starting materials, have also appeared in the literature (Scheme 1). Clandestine chemists preparing “M-D-M-A” for the black market have additionally developed a number of synthetic routes from readily available starting materials like catechol (Scheme 1, 7), eugenol (Scheme 1, 8), isosafrole (Scheme 1, 9), and piperine (Scheme 1, 10), though most still approach “M-D-M-A” through a safrole or (less frequently) piperonal intermediate. These synthetic methods often rely on chemicals readily available to ordinary consumers, in an effort to circumvent controlled substance precursor regulations. Most of these clandestine syntheses are well documented, both by anonymous chemists, in online forums, and by forensic scientists, who often identify clandestine production methods by their distinct impurity profiles. To date, none of the synthetic explorations into “M-D-M-A” appear to have considered “c-G-M-P”s. While intended for pharmaceutical production, Merck’s early investigations occurred less than a decade after the “F-D-A” was founded, and well before its “c-G-M-P” rules were developed. Some clandestine labs reliably produce large quantities of high-quality “M-D-M-A”; however, these facilities necessarily operate outside of regulatory frameworks and certainly do not report or document “c-G-M-P”-compliant procedures. Most other synthetic explorations of “M-D-M-A” have been geared toward the production of “M-D-M-A” as a research chemical, usually for small-scale studies in animals or for forensic analysis (Figure one). Indeed, prior to a recently completed Phase 3 trial for “P-T-S-D”, it is likely that few even contemplated a need for “c-G-M-P”-compliant “M-D-M-A”. As a Schedule One substance, it officially had no recognized medical utility up until now. As a well-known compound with a lengthy history in the public domain and a short treatment regimen, it also had little apparent commercial value. RESULTS. We report here the first “c-G-M-P” synthesis of “M-D-M-A” and its hydrochloride salt (“M-D-M-A”, ”Hydrochloride”), which is used in pharmaceutical formulations. In this fully validated, four-stage process, up to 5 kilogram of “M-D-M-A”, ”Hydrochloride” was reproducibly synthesized, with an overall yield of 41.8 to 54.6 percent and a minimum purity of 99.4 percent (weight by weight) by “H-P-L-C” assay. Over a minimum of four consecutive trials, for each stage, the established targets for yields and impurity profiles were achieved, and, in most cases, exceeded. Chemical impurities in the final product (“M-D-M-A”, ”Hydrochloride”) averaged 0.04 percent of the total peak area, by “H-P-L-C”, and no single impurity ever exceeded 0.03 percent of the total peak area. Of all of the organic solvents used in the production process, only isopropanol (Class 3, 409 to 509 parts per million), tetrahydrofuran (Class 2, less than 7 parts per million), methanol (Class 2, less than 6 parts per million), and n-heptane (Class 3, less than 67 parts per million) were detected in the final product, all in concentrations well below the permitted daily exposure (PDE) per “F-D-A” guidance. The scale and reliability of this “c-G-M-P” process will improve access to “M-D-M-A” for ongoing and future clinical trials, and potentially for licensed therapeutic use, pending “F-D-A” approval. DISCUSSION. Increased demand for pharmaceutical-grade “M-D-M-A” encouraged us to develop a “c-G-M-P”-compliant production process, both to supply our own Phase three clinical trials, for “P-T-S-D”, and to ameliorate existing supply constraints for the broader research community. While large-scale clandestine production is common, to the best of our knowledge, no multi-kilogram synthesis of pharmaceutical-grade “M-D-M-A” has yet been reported in the literature. We therefore needed to develop a practicable synthetic route while simultaneously addressing “c-G-M-P” requirements. “M-D-M-A” is not a particularly complex molecule, and many synthetic pathways have been reported. Most begin from either safrole or piperonal, which are highly regulated and consequently difficult to obtain; for the sake of convenience and efficiency, we elected to avoid these. We identified 5-bromo-1, 3-benzodioxole (Compound 11), which does not appear on any geopolitical entity’s list of controlled substance precursors, as a useful starting material for our synthesis. The 1, 3-benzodioxole moiety appears in a variety of natural products, including oils, spices, and traditional plant-based medicines. Many compounds containing this structural feature are known to interact with cytochrome P450 enzymes in mammals, producing a range of clinically notable effects, both pharmacologically useful and neurotoxic. Compound 11 is synthesized via the bromination of benzodioxole with NBS; analysis of multiple batches, from a range of suppliers, indicated that the only significant impurities present in the batch are 5,6-dibromo-1,3-benzodioxole and succinimide, which is insoluble in Compound 11 and consequently present in only very trace amounts. We additionally screen for the presence of 4-bromo-1, 3-benzodioxole, which would likely present separation challenges during production, but we have never observed this isomer in the starting material. At the levels observed, neither of the two significant impurities interfered with the downstream chemistry. Compound 11 has been previously used in at least two (reported) approaches to “M-D-M-A”: as a starting material in the asymmetric synthesis of (S) ”M-D-M-A”, through a protected aziridine intermediate, and as a precursor to safrole, via Grignard reaction with allyl bromide (Scheme 2). Instead of approaching “M-D-M-A” conventionally, via safrole, we elected to generate a 2-propanol substituent via a ring opening addition between the same aryl Grignard reagent used to synthesize safrole, above, and 1, 2-propylene oxide (Compound 12), which is both inexpensive and readily available. Reactions of Grignard reagents and epoxides are well-known, but, to the best of our knowledge, this particular synthetic pathway has not previously been used to produce “M-D-M-A”. Our familiarity with this type of reaction made us optimistic that scale-up would proceed smoothly, and it did. Although Grignard formation is slow, the bulk reaction can be expedited via initiation with a small amount of previously prepared Grignard reagent. The 5, 6-dibromo-1, 3-benzodioxole impurity present in the starting material does not undergo Grignard formation and is removed during workup as part of the organic layer. The workup at this stage was quite efficient, and distillation via a wiped-film evaporator, two to three passes, yielded 1-(3,4-methylenedioxy-phenyl)-2-propanol (Chemical 13) in excess of 96 percent chemical purity by “H-P-L-C”. The adjusted yield, based on “H-P-L-C” assay, was 79.22 to 87.39 percent (weight by weight) over five trials. The next three steps relied on well-known synthetic transformations. Compound 13 was oxidized to methyl piperonyl ketone (Scheme1, 4) with a biphasic (DCM, H2O) TEMPO, Potassium Bromide, bleach reagent system, which was followed by aqueous workup and filtration to remove remaining solids. The solvent was removed via a rotatory evaporator, and the crude product was of sufficient purity to proceed to the next process stage, without an additional purification step (100.2 to 108.2 percent yield over four trials; 84.9 to 90.01 percent weight by weight by “H-P-L-C” assay). Stage 3, reductive amination of Compound 4, was accomplished with aqueous methylamine and Sodium Hydroxide, Sodium Borohydride. Workup was somewhat complex, using an acid and base treatment to remove the vast majority of impurities, followed by acidification with “Hydrochloride” in isopropanol which yielded 71.6 to 75.8 percent “M-D-M-A”, ”Hydrochloride” (Compound 14), over eight trials, with chemical purity exceeding 99.26 percent of peak area, by “H-P-L-C”. Recrystallization in isopropanol (Stage 4) yielded 85 to 86.2 percent of a white, crystalline solid, with a minimum purity of 99.95 percent by “H-P-L-C” and a minimum assay of 99.40 percent (weight by weight), also by “H-P-L-C” (Table 1). “M-D-M-A”, ”Hydrochloride” was previously known to form one major crystal form (Form One) and at least four hydrates that incorporate 0.25 to 1 waters of hydration. Our polymorphic screening process identified two new anhydrous crystal forms (Forms Two and Three) and established Form One as the most stable of the three. Form Two can be reproducibly produced from a variety of alcoholic solvents, as well as in the presence of ethyl acetate and an ethereal antisolvent. Unlike Form Three, which spontaneously converted to Form One after 2.5 weeks at ambient conditions, and could not be reproduced, Form Two is shelf stable, though it will convert to Form One under competitive equilibration conditions. Interestingly, both Form One and Form Two reversibly convert into the known monohydrate; upon dehydration, the monohydrate formed from Form One will revert back to Form One, and the monohydrate formed from Form Two will revert back to Form Two. If crystallized from a concentrated aqueous solution with no form memory, the monohydrate will thermally dehydrate exclusively into Form One. X-ray powder diffraction spectra for all three forms are shown in Figure 2. To maintain compliance with “c-G-M-P” regulations, all reagents were visually inspected and tested, prior to use. Conformance to established specifications was documented, reagents were labeled with identifying raw material numbers, and these identifying numbers were recorded whenever a reagent was used, in-process. Organic reagents were typically confirmed by FT-IR, as well as by other methods specific to their chemical identity and various process needs, for example Karl Fischer titration to establish water content, and so on, in accordance with established procedures. Inorganic reagents were confirmed by appropriate chemical identification tests. Reagents that failed to meet all established specifications were not used at any stage of the process. Another concern for “c-G-M-P” manufacturing is the presence of residual solvents, which must be below solvent-specific concentration thresholds defined in USP 467. The limits set for residual solvent concentrations are based on anticipated daily exposure to a pharmaceutical product. In clinical use, “M-D-M-A” is never recommended for daily, or even regular, consumption; instead, it is ingested during a small number of therapy sessions, spread over weeks or months. Nevertheless, our monograph utilizes the USP 467 PDE limits as acceptance criteria, and our process yielded residual solvent concentrations significantly below these limits, over four consecutive validation trials (Table 2). The limit of detection for all tested solvents was 1 part per million; solvents detected in concentrations below the quantitation limit were reported as such. In addition to meeting residual solvent concentration limits, “c-G-M-P” pharmaceuticals must have acceptable impurity profiles. Any single impurity exceeding 0.1 percent must be both characterized and quantified. Over four trials, our process yielded “M-D-M-A”, ”Hydrochloride” with chemical purity in excess of 99.9 percent of peak area by “H-P-L-C”; no single impurity ever exceeded 0.05 percent of the total peak area. While impurity characterization was consequently not required, we routinely screened for two known impurities (Figure 3), both of which were generated via low level electrophilic addition during the Stage 2 oxidation of Chemical 13. Chlorination was only significant when the bleach was overcharged, and the reaction conditions used in Stage 2 prevent this. Bromination, which also increased with excess bleach, was a more significant side reaction, but it was successfully minimized using Potassium Bromide in catalytic, rather than stoichiometric, quantities. Neither impurity was ever found in excess of 0.03 percent of the total peak area, by “H-P-L-C”, in any of the four Stage 4 validation trials. Heavy metal impurities in finished pharmaceutical products are also an area of potential concern. As with residual solvents, “c-G-M-P”-compliant limits are established with the assumption that a medication will be consumed on a daily basis, a usage pattern that we do not anticipate will ever be in effect for clinically administered “M-D-M-A”. Nevertheless, we used the oral daily dose PDEs from USP 232 when determining acceptability parameters. As shown in Table 3, the greatest quantifiable amount of any heavy metal impurity was 97 percent less than the permissible daily intake limit, and most were well below that level. To validate this “c-G-M-P” process, each stage was successfully completed at the 8 kilogram scale (based on the starting charge of benzodioxole) at least four consecutive times, in accordance with the documented procedures. All reagents, products, intermediates, common impurities, and, as required, reaction end points were validated using “c-G-M-P”-compliant analytical methods, some of which were specifically developed for this synthetic process. Any deviations from the documented procedures or parameters were noted, and the anticipated impact on the final product, if any was characterized. No documented deviation appeared to affect either the final product or the outcome of the Stage 4 recrystallization step, which yielded remarkably consistent results throughout the validation process (Table 1). We are confident that our “c-G-M-P” protocols are sufficient to reliably produce enough pharmaceutically acceptable “M-D-M-A” to meet expanding research and therapeutic needs. EXPERIMENTAL SECTION. General. Reactions were performed using commercially available raw materials and solvents. Unless otherwise stated, all commercially obtained reagents were qualified prior to use and then used as received. Reactions were conducted in a 50 Liter reaction vessel. The small-scale production of the Grignard reagent, used to initiate the bulk reaction, was conducted in a 2 Liter reactor fitted with a reflux condenser. A Huber Unistat was used for temperature control and logging. In-process analysis was conducted by “H-P-L-C”, with supplemental H one NMR analysis used to quantify residual solvent content during evaporation steps. A wiped-film evaporator was used for distillation. All processes were conducted under nitrogen (target: less than 5 percent O2). Residual solvent testing was performed on an Agilent J and W DB-624 HRGC column (60 meters times 0.32 millimeters, 1.80 micron film thickness). Stage 1, the Synthesis of 1-(3, 4-Methylenedioxyphenyl)-2-propanol. Grignard Formation. Into a 2 Liter vessel was charged 16.6 grams of magnesium turnings (0.68 mole, 1.1 equivalents), followed by 500 milliliters of THF (181 parts per million H2O by KF titration) at 20 degrees C. Stirring was initiated after the introduction of THF, and the vessel was heated to a gentle reflux. To the vessel was then charged 125 grams of 5-bromo-1, 3-benzodioxole, 0.62 moles, 1 equivalent, chemical purity greater than 98.70 percent by “H-P-L-C”) in two unequal portions. The first portion weighed 6.3 grams and was stirred for 12 hours at a gentle reflux until an exotherm was observed. Following this initiation step, the remaining 118.4 grams was added, dropwise, to the reaction vessel, and the resultant Grignard solution was stirred at reflux for 40 minutes and then cooled to 25 degrees C. Into a separate 50 Liter reaction vessel was charged 1.06 kilograms of magnesium turnings (44 moles, 1.1 equivalent) and 32 Liters of THF. The suspension was stirred and then heated to a gentle reflux. To the reaction vessel was then added 400 grams of 5-bromo-1, 3-benxodioxole (2.0 moles), followed by 400 milliliters of the small batch Grignard solution described above. Reflux was maintained. After 5 minutes, a significant increase in the reflux rate was observed in the glass condenser, indicating initiation. While maintaining reflux, 7.6 kilograms of 5-bromo-1, 3-benxodioxole (38 moles) was then added to the reaction vessel, using a dropping funnel, and the batch was stirred at a gentle reflux for 40 minutes. Addition of Propylene Oxide. The bulk Grignard solution was cooled to 10 degrees Celsius, and 128.8 grams of copper iodide (1.5 moles, 0.4 equivalent) was added to the 50 Liter vessel. A solution of 2.5 Liter (plus, minus) propylene oxide (37 moles, 0.93 equivalents) in 2.5 Liters of THF was then added to the reaction vessel while maintaining the temperature at 0 to 10 Celsius. The container and dropping funnel were rinsed with an additional 800 milliliters of THF, which was then added to the 50 Liter reaction vessel. The batch was stirred for 40 minutes at 5 to 20 degrees C, forming a dark brown solution and a crystalline suspension. Completion analysis performed by “H-P-L-C” confirmed the reaction end point (0.30 percent 5-bromo-1,3- benzodioxole; target limit was less than or equal to 1 percent). The batch was then divided into two 20.4 Liter portions for workup. For each portion, 5.45 Liters of a 10 percent (weight per weight) sodium chloride solution, followed by 1.37 Liters of acetic acid, was added to the 50 Liter reaction vessel while maintaining the temperature at 10 to 25 degrees. The half-batch portion was then transferred from carboy into the reaction vessel while ensuring that the temperature remained below 40 degrees. Following this addition, the half-batch portion was stirred at 30 to 40 degrees for 45 minutes; then, the pH was adjusted to less than 5 by sequential addition of three 200 milliliters of aliquots of acetic acid. The batch was allowed to settle, and the aqueous layer was removed. 8.2 Liters of n-heptane were then charged into the 50 Liter reaction vessel, followed by an additional 8 Liters of the sodium chloride solution. The batch was stirred and allowed to settle, and the aqueous layer was again removed. The dark brown organic layer was filtered over a vacuum, using a plate filter with a 11 micron filter mesh. Following workup, the two half-batches were combined, and the solvent was removed in a 20 Liter rotatory evaporator. The crude yield was 7442.7 grams and analysis by H one NMR revealed 2.5 percent residual THF, n-heptane not detected; target limit is less than or equal to 10 percent total amount of both solvents. The crude product was charged with 1488.5 grams of PEG400, 0.2 equivalent weight per weight and mixed to ensure homogeneity. This mixture was then distilled at 150 to 185 degrees and 0.1 to 1.5 milli bars, using a wiped-film evaporator. Two passes yielded 6293.2 grams of a pale yellow oil (94.2 percent yield; 96.44 percent area, 89.78 percent weight per weight by “H-P-L-C”). Stage 2. Oxidation to 1-(3, 4-Methylenedioxyphenyl)-propan-2-one. A 50 Liter reaction vessel was charged with 2760.1 grams of crude 1-(3, 4-methylenedioxyphenyl)-2-propanol from Stage 1 (88.56 percent weight per weight by “H-P-L-C” assay; active charge is 2444.3 grams, 13.6 moles, 1 equivalent) and 9780 milliliters of dichloromethane at 10 to 25 degrees. Stirring was initiated, and 178.5 grams of potassium bromide was added, followed by 233.2 grams of TEMPO (0.11 equivalents). The batch was cooled to 0 degrees, and 7280 milliliters (60 percent) of a solution of sodium hydrogen carbonate (0.25 equivalent) in 12120 milliliter of bleach, 1.6 equivalent, diluted to 12.5 percent weight per volume) was added, dropwise, while stirring efficiently and maintaining the temperature at minus 10 to 10 degrees. A 1 milliliter sample was then removed, for analysis by “H-P-L-C”, and four additional 610 milliliter (5 percent) of aliquots of the Sodium Bicarbonate bleach solution were then added, dropwise, to the reaction vessel. A sample was collected after each addition, and “H-P-L-C” analysis was used to monitor the reaction progress. After the fourth aliquot was added, 1.61 percent of Stage 1 starting material remained (target limit is less than 5 percent). Stirring was halted, and the layers were allowed to settle. The layers were separated, and the organic layer was returned to the 50 Liter vessel. For workup, the organic layer was cooled to 0 Celsius, and 4890 milliliters of a 12 percent (weight per weight) solution of aqueous sodium hydrosulfite was added while maintaining the temperature at 0 to 10 degrees. The reaction mixture was then warmed to 19.5 degrees and stirred for 15 minutes. The layers were separated, and the organic layer was returned to the 50 Liter reaction vessel. Then, 4900 milliliters of freshly prepared 0.5 Molar aqueous Sodium Hydroxide was added, and the reaction mixture was stirred for 15 minutes. The layers were separated, and the brown organic layer was returned to the 50 Liter reaction vessel. To this was added 4900 milliliters of 11 percent (weight per weight) aqueous Sodium Chloride, followed by 98 milliliters of concentrated Hydrochloric acid 36 percent weight per weight aqueous solution. After stirring for 15 minutes at 18.5 degrees, the layers were separated, and the organic layer was returned to the reaction vessel. Two more washes, the first with another 4900 milliliters of the 11 percent Sodium Chloride solution, the second with 4900 milliliters of a saturated Sodium Chloride solution were completed, following the same procedure. The organic layer was filtered over a Buchner funnel fitted with a filter cloth rinsing with 500 milliliters of DCM and then transferred to a 20 Liter rotatory evaporator. The solvent was removed under vacuum, yielding 2442.1 grams of a yellow-to brown oil (101.0 percent crude yield; 94.52 percent peak area, 89.80 weight per weight by “H-P-L-C”). Stage 4. Recrystallization of “M-D-M-A”·”Hydrochloride”. To a 50 Liter reaction vessel was added 4107.3 grams of crude “M-D-M-A”, ”Hydrochloride” and 41 Liters of 2-propanol. The batch temperature was increased to 67.2 degrees, while stirring, and the mixture was then stirred for 30 minutes at 67.2 degrees until all of the solids dissolved. Stress tests had demonstrated stability for 72 hours at 70 to 80 degrees proving the thermal stability of “M-D-M-A”, ”Hydrochloride”. The batch was then transferred through a 1.2 micron in-line filter capsule, using positive pressure, to a clean, 50 Liter reaction vessel, fitted with a jacket that had been preheated to 66.1 Celsius. In this new reaction vessel, the batch was cooled to 55.3 degrees, over the course of 90 minutes. Then, 41.1 grams of “M-D-M-A”, ”Hydrochloride” Form 1 seed crystal (0.18 moles, 0.008 equivalents) was added, and the batch was stirred at the same temperature for 30 minutes. The batch was cooled to 15.2 degrees at a rate of 3 degrees per hour and then stirred at this temperature for an additional 10 hours. The white suspension was removed from the mother liquor via vacuum filtration over a filter plate fitted with a filter cloth and then washed with 8220 milliliters of 2-propanol. The filter cake was transferred to a drying oven and dried under vacuum (140 millibars) for 19 hours at 56.6 degrees. The collected “M-D-M-A”, ”Hydrochloride” was a white solid weighing 3548.3 grams (85.5 percent yield; 99.95 percent peak area, 99.64 percent weight by weight by “H-P-L-C”). No single impurity exceeded 0.02 percent of the peak area by “H-P-L-C”, and residual solvents, methanol, less than six parts per million; 2-propanol, 490 parts per million, were found to be within the target range.

A review of the newly identified impurity profiles in methamphetamine seizures. Forensic Science International: Synergy two (2020) pages 194 to 205. Isaac Onoka and pals. Abstract. Forensic intelligence of synthetic illicit drugs suffers a problem of continuous introduction of new synthetic methods, modification of the existing routes of manufacture, and adulterations practiced by criminal networks. Impurity profiling has been indispensable in methamphetamine intelligence based on precursors, synthetic routes, and chemical modifications during trafficking. Law enforcement authorities maintain the credibility and integrity of intelligence information through constant monitoring of the chemical signatures in the illicit drug market. Changes in the synthetic pattern result in new impurity profiles that are important in keeping valuable intelligence information on clandestine laboratories, new synthetic routes, trafficking patterns, and geographical sources of illicit Methamphetamine. This review presents a critical analysis of the methamphetamine impurity profiles and more specifically, profiling based on impurity profiles from Leuckart, Reductive amination, Moscow, Emde, Nagai, Birch, Moscow route; a recent nitrostyrene route and stable isotope signatures. It also highlights the discrimination of ephedrine from pseudoephedrine sources and the emerging methamphetamine profiling based on stable isotopes. One. Introduction. Methamphetamine is a schedule two controlled substance according to the Single Convention on Narcotic drugs and the United Nations Convention against Illicit Traffic in Narcotic Drugs and Psychotropic Substances, 1988. It is a highly addictive drug with a potent central nervous system (CNS) stimulant properties. The United Nations Office on Drugs and Crime, UNODC, report MA as the most abused drugs worldwide. For the last two decades, the use of MA has been increasing in many countries worldwide. In Japan, about 15,000 drug arrests were from cases related to MA accounting for 90 percent of all reported violations. Previous studies have documented the prevalence of methamphetamine over other synthetic drugs elsewhere. The impurity profiling of MA provides the linkage of illicit drug seizures based on the chemical signatures contained in the seized illicit drugs. The method uses organic and inorganic impurities which are by-products of reactions in the final formulation of MA. It has successfully been used to establish intelligence information in France, Australia, Thailand, China, Philippines, Japan, USA, Spain, Korea and in many other places worldwide. Recently, the emerging complementary profiling method based on stable isotopes has drawn the interest of many researchers; its details will be included in this review. The chemical analysis of illegal drugs provides valuable information about the conspiracy links and trafficking routes, categorizing the seizures based on the signatures, thereby identifying their origins. As a complementary law enforcement investigative work, it provides a background intelligence information concerning the number of sources of drugs, whether those sources are within a country or are internationally based and also unveiling the points of distribution and distribution networks. Similarly, the impurity profiles identify the emergence of new clandestine laboratories and their associated synthetic methods, which, in turn provides background intelligence information. Chemical profiling marks the principal purpose of the intelligence of illicit drugs by establishing a link between the clandestine laboratories, suppliers, and users. The chemical information obtained from a drug can indicate its synthetic method, adulterations during trafficking, and the operations of the criminal networks. Generally, the fundamental role of a forensic chemist in drug profiling is to extract the chemical signatures that can be used to establish the degree of commonality of seizures with their origins or a specific group of other samples, as well as linking the signatures with the possible synthetic methods, conditions, and post-production modifications. A comprehensive examination and comparison of the chemical signatures has found growing applications strategically at the macro level and tactically at the micro-level. These advances have helped the police and criminal investigators at both national and global scales to establish the dynamics of illicit drug markets, locate the drug traffickers, establish conspiracy links between dealers and users. In this decade, extensive number of research studies on the chemical profiling of MA and its derivatives are focusing on the determination of organic and inorganic impurities, determination of synthetic routes, and synthesis of the impurities, identification of the impurities concentration in body fluids, characterization and the extraction and separations of the impurities. In this paper, we critically analyze the impurity profiles of MA synthesized from ephedrine, pseudoephedrine, and 1-phenyl-2-propanone “P2P” precursors and subsequently analyze their potential use for intelligence perspective. One point one. The synthetic schemes of methamphetamine and the specific route impurities. The Forensic intelligence of illicit drugs is an exciting subject and very challenging. In totality, it embraces the determination, identification, and characterization of the individual components in the final formulation of illicit drugs regarded in this review as the impurities, intermediates, and the contaminants. Depending on the level of operation of a clandestine laboratory, an illegal drug is an assemblage of constituents carrying the information about the synthetic route, condition-specifics, reagents, adulterations during trafficking, synthetic batches and sometimes the chemical process level of the cooks (purity). Like in any other reaction, each synthetic scheme has byproducts emanating from the conversion of the precursors to MA. For instance, some constituents are by-products of the reaction conditions; others are formed from the conversion of precursors to intermediates and from intermediates to MA while others are intentionally added as cutting agents for potency or weight and as artifacts described by Broseus. Therefore, the final formulation of MA is characterized by a variation of the relative abundance of the major by-products, intermediates, and impurities that define a chemical signature. Although different MA seizures produced from the same precursor, using similar route and the same reagents have related impurity profiles; some intra and inter batch variations may still occur due to varying reaction conditions. This variation is essential to distinguish chemists (cooks) by attaching a specific profile to reaction conditions practiced by a particular clandestine laboratory. Based on a certain probability of a link corresponding to the calculation of a threshold, two or more exhibits will have the same chemical profiles supporting the fact that they originate from the same batch, with the strength of support increasing as profiles become more complex. Otherwise, a range not meeting the threshold can only distinguish the samples rather than discriminating against their cooks. For quite a long time, the clandestine synthesis of MA employed three major precursors, namely ephedrine, pseudoephedrine, and the 1-phenyl-2-propanone “P2P”. With “P2P”, Figure one, the Leuckart route (Six) and reductive amination are the most commonly used routes for the synthesis of MA. In contrast, ephedrine, pseudoephedrine precursors, Figure two, convert to MA through the Nagai, one, Emde, two, Hypo, three, Moscow, Six, Rosenmund, Five, and Birch, Nazi, seven pathways. As one of the main MA precursors, the “P2P” reaction scheme involves the reductive amination reactions Figure one, one to four, and the Leuckart (HCL, H2O). The reductive amination reaction of “P2P” to MA is achieved through Palladium Hydrogen, and methylamine, NaBH4, and methylamine, NaBH3CN, and methylamine, HCl, H2O, Platinum, H2, and methylamine, and Mercury, Aluminum, and methylamine, however, the aluminum, mercury amalgam in a slightly acidic media method is reported to be the most commonly used method in Europe and USA. Although the method has long history, 1-phenyl-2-propanol formed from the direct reduction of the precursor, “P2P”, remains the potential intelligence impurity profile. Described by Verweij in 1989, the Leuckart route, figure one, six, is achieved by the addition of N-methylformamide, methylamine or formic acid followed by H2SO4 or HCl to form MA. By means of N-methyl-formamide, the reaction results in a Leuckart route determinant; the N-formyl-methamphetamine disputed by Qi and others, and Barron; and non-synthetic route determinants namely: Di-benzyl-ketone, R-benzyl-N-methylphenethylamine, and N-methyl-di-phenethylamine. The synthetic-route character of N-formyl-methamphetamine argued by Barron and Qi was resolved by the identification of alpha, alpha prime, dimethyl-di-phenethylamine and N, alpha, alpha prime tri-methyl-di-phenethylamine by Barron. The two impurities were later confirmed by Vanitha having identified them in Leuckart based MAs only. The Nagai route figure two, one is associated with the formation of several chemicals. The impurities are formed from the nucleophilic substitution reaction of the OH group of ephedrine pseudoephedrine to form ido-ephedrine or iodo-pseudoephedrine. The intermediary iodine is liable to internal nucleophilic attack from the adjacent nitrogen to form cis- and trans phenylaziridines which is reduced to MA or hydrolysed to “P2P”. In prolonged acidic conditions, the latter undergo condensation to form methylnaphthalenes, reported to be the specific synthetic route signatures. The conversion of ephedrine, pseudoephedrine to MA via Emde route is the dominant synthetic route in South East Asia. In contrast to the Nagai route, the Emde reaction scheme is augmented by SN1 substitution, intramolecular nucleophilic displacement, or SN2 substitution, intermolecular displacement, of the OH in ephedrine, pseudoephedrine with chloride to form a racemic mixture ephedrines of variable impurity concentrations. The plus-chloro-pseudoephedrine and minus-chloro-ephedrine can then undergo a cyclic ring closure to form cis and trans phenylaziridines, respectively. Accordingly, plus-nor-pseudo-ephedrine and minus-norephedrine alternative precursors undergo similar reaction to form plus-chloro-methyl-pseudoephedrine and minus-chloro-methyl-ephedrine. These intermediates may eliminate the HCl to form 1-propenyl-benzene and 2-propenylbenzene or can undergo a rearrangement to form 1-dimethylamino-1-phenyl-2-chloropropane. The route specific potential of the intermediary aziridines was contradicted by Ko and Salouros having identified 1-methylamino-1-phenyl-2-chloropropane as a vapour-phase nucleophilic product of the aziridine and another amine. The latter was recogonized in “Moscow” and Nagai related methods and could not qualify as a route specific impurity for Emde route. Ko instead identified and proposed chloro-propane, chloro-ephedrine, chloro-pseudoephedrine as route specific impurity for the Emde method. Other non-route specific impurities are included in the text. The “Moscow” method Figure two, four, is achieved by a reaction between ephedrine, pseudoephedrine with red phosphorus and iodine in water. Its mechanism is treasured in the regenerative role of red phosphorus. Skinner as supported by NicDaeid proposed a scheme based on the oxidation of P by I2 to di-phosphorus tetra-iodide, P2I4, followed by the decomposition of P2I4 in water to form phosphoric acid and phosphonium iodide. The mixture them converts to hydroiodic acid (HI) and phosphine (PH3) upon heating. The former protonates the OH of ephedrine, pseudoephedrine to form aziridine intermediates which potentially reduce to MA as in the case of the Nagai route. Birch, Nazi route, figure two six, is a reduction reaction of ephedrine, pseudo-ephedrine using excess alkali metal for example lithium, sodium in liquid ammonia to form (CMP) , notated as a propanamine. The impurity is the most commonly encountered MA impurity prepared by the Birch route. Its reaction scheme is based on the role of alkali metals preferably lithium as a proton source for the OH of ephedrine, pseudoephedrine. As lithium protonates the precursor, NH3 facilitates the reduction of the aromatic rings to form a methyl-amino-propane. This primary impurity associated with the lithium-ammonia method normally results in high CMP to MA ratio limiting the isolation of the impurity. Martinez proposed potassium permanganate and aqueous base for effective CMP isolation. One point two. A paradigm shift in methamphetamine precursor production. As a result of the crackdown measures taken against the production, trafficking and the availability of the “P2P” and ephedrine, pseudo-ephedrine, access to the precursors has shifted to the illicit manufacture of the precursors through readily available starting materials with new routes leading to the emergence of new impurity profiles. For quite a long time, the synthesis of phenyl-2-propanone was through a vast number of starting materials such as alpha-phenyl-aceto-acetonitrile, alpha-phenyl-beta-methyl-eneglycol, aphenylisopropyl alcohol phenyl-acylmalonic ester, phenylacetyl chloride, alpha-methylstyrene with thallium nitrate, and benzene via o,o-diprotonated nitro olefin, beta-methyl-beta-Nitrostyrene, and phenylacetic acid “PAA”. Although several “P2P” synthetic schemes were available in the 1980s, the illicit production of “P2P” was mainly through Phenylacetic acid (PAA) via acetic anhydride and lead two acetate; and beta-methyl-beta-Nitrostyrene via iron, H plus. A recent twist in the production of “P2P” has recently involved the nitrostyrene method (NTS). This emerging synthetic scheme results in nitrostyrene recently identified in MA samples seized in Mexico and the USA. The NTS method uses benzaldehyde and nitroethane in Knoevenagel reaction to form a nitrostyrene, yellow solid, which converts to “P2P” in the presence of iron powder and hydrochloric acid. The evolution of the “P2P” clandestine chemistry is further confirmed by the re-emergence of a new impurity profile in place of alpha-benzyl-N-methylphenethylamine and trans-N-methyl-4-methyl-5-phenyl-4-penten-2-amine from the usual foul-smelting of a crystalline PAA. The synthesis of “P2P” from PAA, figure three “A”, utilizes the then easily available ethyl phenyl-acetate “ETPA”. However, a recent decline of “ETPA” and its associated esters and amides resulted in a shift in the “P2P” precursors, figure three b, resulting in the emergence of new characteristic impurities recently reported in Australia and observed in the USA. The “P2P” produced from the PAA method and nitrostyrene (NTS) convert to MA with route-specific markers intelligently used to trace the sources of “P2P”. The dynamics of the operations of criminal MA networks is one of the exciting profiling topics that appeals to close monitoring by intelligence agencies. A recent Drug Enforcement Administration (DEA) MA Profiling Program (MPP) done in the USA recorded trade-off impurity profiles assigned to pseudoephedrine route to those assigned to “P2P” precursors. According to the MA impurity profiles documented by this program, the impurity profiles derived from pseudoephedrine decreased significantly since 2007 with increasing impurity profiles derived from “P2P”. This shift was associated with a spike in unknown synthetic route assignments and a sharp decrease, 84 percent, in samples assigned to a “P2P”-based recipe in the first quarter of 2015. Section one point three. The emerging methamphetamine impurity profiles. In response to the crackdown measures imposed on the production and trafficking of MA and its precursor chemicals, clandestine laboratories circumvent the law enforcement authorities by deriving the precursors from uncontrolled substances such as phenyl acetic Acid (PAA), nitrostyren, and legal medicine resulting into the emergence of new impurity profiles. The emergence of impurity profiles such as dimethyl-amphetamine and p-methoxy-amphetamine was recently documented by Stojanovska and supported by a literature collection of impurity profiles and synthetic route of manufacture of methyl-amphetamine, 3,4-methylenedioxymethylamphetamine, amphetamine, di-methyl-amphetamine, and p-methoxy-amphetamine as well as the recently identified less potent l-methamphetamine in place of d-methamphetamine in the United States. Since their identification in seized MA, several MA impurity profiling reports reveal profiles that are potentially important for strategic, tactical and operational intelligence of MA in the USA, France, Australia, Korea, Iran, China, Philippines, Japan and Thailand. One point three point one. Impurities from metal catalytic hydrogenation. Metal catalytic hydrogenation of ephedrine, pseudoephedrine and “P2P” is one of the oldest MA synthetic methods. Using ephedrine, pseudoephedrine, the clandestine laboratories often use palladium via the Rosenmund route, lithium and NH3 via Birch route, and nickel via Emde route. The reaction involves the reduction of the C-X, X-halo, phosphate, and sulfate, figure four, rather than the benzylic OH group to form methamphetamine. Using “P2P”, an imine intermediate MA base is formed from a reaction of between “P2P” with methylamine. The MA freebase is then distilled and directly converted to hydrochloride salt. Figure five represents a reductive amination for the conversion of “P2P” to MA hydrochloride. Reaction (c) occurs through heterogeneous reactions with internal or external sources of hydrogen in the presence of Palladium, Palladium Carbon, Palladium BaSO4, Platinum, Platinum, Carbon, Copper Oxide, CaSO4, BaSO4, Raney Nickel. Tracking the traces of metals in the final formulation of MA has been used to determine the synthetic routes. MA manufactured from ephedrine, pseudoephedrine through the Emde and Nagai methods was found to contain two specific chemicals as route-specific impurities. Since their identification, they have been used as Emde route-specific signatures. Furthermore, a chloro-propane derivitive has recently been used as an additional route specific marker impurity synthesized from ephedrine via chloro-ephedrine by the Emde route, figure six. The metal catalysis reaction of (1R, 2S)-plus-ephedrine or (1S, 2S)-plus-pseudoephedrine results in the formation of chloro-ephedrine, chloro-pseudoephedrine which is hydrogenated to (S)-plus-Methamphetamine. Recent profiling of reductive amination of “P2P” made from PAA, lead two based MA, elucidated trans-N-methyl-amine and alpha-benzyl-N-methylphenethylamine. Trans-N-methyl-4-methyl-5-phenyl-4-penten-2-amine used as a route-specific marker was presumed as an acetone and P2P condensation product, however, this assumption could not explain why the little amount of the impurity and its associated intermediates produced even if the “P2P” was refluxed in acetone for a long time. Figure six. Metal catalytic reduction of ephedrine and pseudo-ephedrine. Furthermore, an attempt to produce “P2P” using a Dakin-West and lead two acetate conditions were futile and could not yield the expected: Trans-N-methyl-4-methyl-5-phenyl-4-penten-2-amine as an impurity. The best reasoning so far centers the argument on the role of a low-level 4-carbon acetate unit as an intermediate. Since acetic acid undergoes decarboxylation in aqueous solution over a range of temperatures, a route-specific marker impurity amine, results from an intramolecular reaction of lead acetate with P2P via chelation controlled transition states followed by decarboxylation. Figure seven shows the proposed mechanism for the formation of this route specific marker. The reductive amination of P2P is also associated with the formation of: Alpha-benzyl-N-methylphenethylamine, as a synthetic route characteristic impurity. The MPP identified the contaminant at the DEA’s Special Testing and Research Laboratory. The emergence of new impurity profiles in MA analysis suggests, possibly, a change in the synthetic route parameters or the synthesis of precursor chemicals. The foul-smelting of a crystalline PAA results in new impurity profiles monitored in seized MA samples. The impurities associated with the modified “P2P” synthetic pathway are: Alpha-benzyl-N-methylphenethylamine, and: Trans-N-methyl-4-methyl-5-phenyl-4-penten-2-amine. They have been used to track MA synthesized from PAA. The emergence of N-butyl-amphetamine and N-cyclo-hexyl-amphetamine in seized MA has recently triggered interest in the nitroalkene chemistry. The two impurities result from a Knoevenagel reaction of benzaldehyde and nitroethane to form a nitrostyrene. Toske referred to this method as a nitrostyrene method (NTS) or a nitropropene method. The catalytic activities of butylamine, cyclohexylamine influence the conversion of the “P2P” precursors. The catalysts react with benzaldehyde to form imine, which then reacts with the nitroalkane to form a nitrostyrene as an intermediate. The reaction mixture at this step contains nitrostyrene and extractable cyclohexylamine, butylamine with a significant reaction potential. Based on a reaction proposed by Hass, nitrostyrene converts to “P2P” in the presence of iron powder and hydrochloric acid. The extractable cyclohexylamine, butylamine can then react with “P2P” to form the stable N-butyl-amphetamine, and N-cyclo-hexyl-amphetamine elucidated in MA seizures. Figure eight “A” and “B” represents the formation of N-butyl-amphetamine and N-cyclo-hexyl-amphetamine. Since 2015, the two impurities were detected in MA seizures collected in the USA. The identification of the two impurities has been fundamental in tracking the “P2P” based MA synthesized by the nitrostyrene chemistry. Section one point three point two. Emerging impurity profiles from pharmaceutical compounds. In response to the crackdown measures taken against controlled substances, ephedrine, and pseudoephedrine, other adaptation strategies used by clandestine laboratories are co-ingredients of legal medicines, direct extraction from ephedra plants, as well as direct synthesis from easily available starting materials. Although Lee reports less common MA crystals containing pharmaceutical impurities, Barker and Antia had a different opinion on the most common sources of ephedrine and pseudoephedrine used to synthesize crystal MA. The latter as supported by Liu who also considered medicinal drugs as the most common sources of pseudoephedrine and ephedrine. Synthesis from legal medicines is the most common coping strategy practiced by clandestine laboratories to avoid strict measures from the law enforcement authorities. The legal medicines approach result in MA whose final formulation contains pharmaceutical signatures that used, to reflect the trends in precursor chemicals, manufacturing sources, and the trafficking patterns organized by the criminal networks. Unlike by-products, there is limited literature linking pharmaceutical impurities to the synthetic route of MA. More recently, the MA profiling based on synthetic pharmaceutical signatures has been done in Korea, Iran, China, Japan and Thailand. Several studies have been done in this field, more research is required to unveil the potential of pharmaceutical impurities beyond their existence as sole impurities into the final MA formulations. Tracking the by-products down to their origin and their point of entry may provide the potential of establishing synthetic routes using pharmaceutical contaminants. In a profiling program conducted in Korea between 2006 and 2011, Acetaminophen, Caffeine, Phenacetin, Ambroxol, Chlorpheniramine, Desloratadine, Barbital, Ketamine, Procaine, and Dimethylsulfone were elucidated as characteristic pharmaceutical impurities. The profiling program linked these impurities with cold medicines, cold relievers, ingredients of analgesic drugs, expectorant, and dietary supplements extracted together with ephedrine, pseudo-ephedrine. In contrast, others added as adulterants during trafficking. While the pharmaceutical medicines demonstrate growing intelligence phenomena, tracking the specific legal drugs used in the MA production and the post-production modifications is an area of utmost interest. Interestingly, in 2010, chlorpheniramine was identified in both Korea and Iran, indicating a cross-border operation of the criminal networks. The emergence of these impurities was a direct indication of the use of legal medicines and their associated analgesic and co-ingredients containing ephedrine or pseudoephedrine. Furthermore, Lee identified a pharmaceutical recipe based on dimethyl sulfone from seized MA in Korea. The impurity was associated with the recipe used in medicinal drugs containing ephedrine, pseudoephedrine as well as an adulterant used in cutting MA. The impurity was previously identified in Korea, 1996 to 2003, and USA, 1996 to 2003, emerged in Australia, 1998 to 2002, re-surfaced in USA 2007, Korea 2006 to 2011 and Japan 2006 to 2007. The observed trend in the occurrence and re-emergence of dimethyl sulfone in the seized MA is potentially important in linking the operation of criminals in these countries. Although the determination of homogeneity might be very challenging, linking the dimethyl sulfone to its common source is essential for integrated intelligence. Unlike other countries, a new profiling program based on pharmaceutical impurities conducted in China recorded a new trend of impurity profiles of MA synthesized from ephedrine, pseudoephedrine. Liu reported tablets with Theophylline-Ephedrine, Ephedrine-Diphenhydramine, Pseudoephedrine, Dextromethorphan, and Chlorpheniramine as a new set of legal medicines commonly used as a source of ephedrine, pseudoephedrine. These drugs contain alkaline substances such as chlortrimeton, diphenhydramine, dextromethorphan, and tri-prolidine with the potential to form characteristic impurities. Their profiles information is not only used for monitoring the routine trends in precursor chemicals but also for the identification of the seized materials, smuggling patterns, and the determination of the synthetic routes. Liu systematically determined a specific diamine, which was assigned a characteristic impurity derived from pharmaceutical products containing ephedrine, pseudoephedrine and diphenhydramine. Unlike other pharmaceutical contaminants, the impurity is a product of a reaction between MA and traces of diphenhydramine derivative, which is co-extracted with ephedrine, pseudoephedrine. As a pharmaceutical co-extract of ephedrine, ephedrine derived diphenhydramine, the traces of the “P2P” precursor undergo band dissociation with HI in an I-P route to form 2-iodo-N, N-di-methyl-ethan-amine as an intermediate and traces of diphenyl methanol. The 2-iodo-N, N-di-methyl-ethanamine then reacts with MA to form a specific diamine as shown in Figure nine. In this reaction, the diphenhydramine is present as co-ingredients of legal medicine used for the synthesis of ephedrine, pseudoephedrine. The control of such drugs is essential for monitoring and identification of illicit production of ephedrine, pseudoephedrine from legal medicines. Section one point three point three. Impurities discriminating ephedrine and pseudoephedrine synthetic routes. Ephedrine and pseudoephedrine are the basic precursors commonly used to synthesize MA beside the Phenyl-2-propanone. A synthetic method using each of the precursor chemicals is associated with specific impurities that can intelligently discriminate against the MA synthetic method. From a forensic chemist’s viewpoint, tracking the impurities down to the level of discrimination ephedrine and pseudoephedrine is an ultimate goal. Many MA profiling methods based on ephedrine, pseudoephedrine end up with non-discriminatory results, deriving their conclusions from an unresolved analytical process. Precursor discrimination based on identified impurities is another interesting intelligence work. In a recent study by Dujourdy, 43 target impurities in MA were successfully characterized and discriminated using chemometric methods. Through clustering, the impurities identified from ephedrine, pseudoephedrine, and Benzyl-methylketone. In their work, 1-benzyl-3-methyl-naphthalene, and 1, 3-dimethyl-2-phenyl-naphthalene were used to signify a route associated with ephedrine precursor. Previously, N-formyl-methamphetamine was considered a route-specific impurity for Leuckart route based MA; however, the impurity has recently been identified in a reductive amination based route for MA. A realization of this challenge was reported by Khajeamiri in their work involving the reduction of ephedrine, pseudoephedrine with HI-red P. In their viewpoint, both ephedrine and pseudoephedrine react with HI, red P to form iodoephedrine, which undergoes a ring-opening to form commonly used route-specific impurities; the cis and trans-1, 2-dimethyl-3-phenylaziridine. Khajeamiri articulated that 1, 2-dimethyl-3-phenylaziridine is derivatized into several other chemicals mentioned in the text. Reporting N-benzyl-2-methylaziridine as an emerging impurity, Khajeamiri associated its formation with the conversion of 1, 2-dimethyl-3-phenylaziridine into N-benzyl-2-methylaziridine during the formation of MA from ephedrine and pseudoephedrine. Additionally, Khajeamiri reported for the first time in 2012 the presence of Chlorpheniramine as a pharmaceutical-based impurity in MA. The impurity was later reported in Korea, 2013, and Iran. The reports associated the impurity with pharmaceutical tablets used to synthesize pseudoephedrine precursors. Because chlorpheniramine is a co-ingredient of pseudoephedrine tables only, it discriminates ephedrine and pseudoephedrine based MA. Section one point three point four. Emerging signatures from stable isotopes. Stable isotope composition in a MA sample has recently been used to profile MA seized in the USA and Japan. The technique employs natural abundance stable isotope compositions in samples to establish chemical signatures for evaluating the links between MA seizures and their production batches. The isotopes have specific natural ratios; however, compartmental isotope ratios vary as per the geographical origin of the source which is the basis for MA profiling based on stable isotopes. The stable isotope ratio is presented in delta values, delta in per mill, where a “mil” equals a thousand. The calculation of delta value as proposed by Barrie is shown in the equation: Delta equals on thousand times R sample, minus R standard, all over R standard. Where R Sample represents the ratio of the heavy to the light isotope measured for the sample while R Standard is the equivalent ratio for the standard. Although the conventional analytical techniques through the existing GC, HPLC, GC-MS, and LC-MS-MS, ICP-MS, NMR have been very effective in determining the type of precursor chemicals synthetic route, and adulteration of illicit MA; it has not been able to discriminate the precursors produced by different methods in the sense of identifying their origins. Furthermore, reductive amination routes usually have few impurity profiles that may not grant a successful impurity profiling. The conventional techniques are also ineffective in traceability beyond sources of the starting materials. In such circumstances, stable isotope analysis is a complementary technique that can individualize illicit MA samples based on the sources of their starting materials. Elsewhere, the method has been successfully used in tracing studies in food, identification of illegal migrants, past human activities, and reconstruction of human diet. In forensic intelligence of illicit drugs, stable isotopes technique unveil the hidden intrinsic precursor signatures that break the limits of the conventional impurity profiling by linking the seized MA and their batches to their synthetic origin. Stable isotopes add value to the intelligence of illicit drugs based on impurity profiling. MA produced by the same hidden laboratories, following the same method with the same kind of a precursor but different sources, can be distinguished by examining the Carbon-13 and Nitrogen-15 values of their precursors. In this respect, the variation of stable isotopes in seized MA can also trace the diversion of medicinal ephedrine for the illicit manufacture of MA. Illicit ephedrine based MA was initially produced through a biosynthetic approach from the ephedra plant Figure ten “A”; however, a growing trend of total chemical synthesis, Figure ten “B” and semi-synthesis, figure ten “C” have dominated the market. The ephedrine produced through methods “A”, “B” and “C”, above will have different stable isotopic compositions used to track MA seizures. In principle, isotopic variation is due to different enrichment factors during the biochemical synthesis of raw materials for the precursor chemicals as well as the isotopic fractionation during the synthetic processes. Although chemical synthesis is a reliable source of ephedrine, the extraction from natural sources is another potential source for clandestine laboratories. Therefore, an integrated approach with intelligence information collected beyond the starting materials is key to linking the operation of the criminal networks. A complementary study on the use of stable isotope techniques in evaluating the links between different MA seizures was reported by Iwata, Benson, and Billault. In a study by Billault, a variation of Carbon-13 and Nitrogen-15 was used to cluster seized MA and successfully established a link between different MA cases. The study successfully discriminated against semi-synthetic ephedrine from bio-synthetic or synthetic ephedrine by using Carbon-13. However, based on the fact the Carbon-13 values of acetaldehyde from sugar are more optimistic compared to C3- photosynthetic plants or products derived from petroleum, it could not differentiate biosynthetic ephedrine from synthetic ephedrine. Recently, a stable isotope technique was used to investigate the unique profiles of stable nitrogen isotopic composition in seized MA samples. In this work, the stable isotopes variation can be due to the isotopic variations in the starting materials, isotopic fractionation during the synthetic processes, and due to analytical errors. It is therefore essential to draw a conclusion based on the magnitude of the variation to eliminate the influence of isotopic fractionation and analytical errors. A variation of zero point nine mils Nitrogen-15 evidenced a difference in batches of production and subsequently, different ephedrine sources used as starting materials for the production of MA. Iwata further used the stable isotope technique to classify the MA seizures based on their synthetic batches. Based on the criteria proposed by Iwata, a change of zero point four mills represents a significant variation in batches. Interestingly, stable isotope technology is making an in-road towards the discrimination of illicit synthetic, semi-synthetic illegal drugs based on their synthetic routes and their associated reactions conditions beyond its conventional use in discriminating the sources for MA. As a growing profiling method, it complements the impurity profiling by linking the synthetic routes to isotopes ratios. Billault reported startling scientific research that seems to confirm the use of stable isotopes in linking MA batches to their respective synthetic routes. These results were the first to be reported in linking the Carbon-13 values of the precursors in distinguishing synthetic routes of seized MA and their derivatives. Having investigated the relationship between Carbon-13 and Nitrogen-15 of the precursors and those of 45 samples of MDMA, the authors demonstrated how the number of synthesis steps influenced the value of Carbon-13 in the seized MA samples and consequently discriminated the synthetic routes possessing more than one step. Similarly, the discrimination of synthetic routes using stable isotopes is established by comparing the Nitrogen-15 values of the origin precursors and the seized MA. The values of Nitrogen-15 in MA are dependent on the source of nitrogen used, the route by which the MDMA is synthesized, and the experimental conditions employed. Previous work by Billault discriminated MA based on their synthetic origins, synthetic routes, as well a close variation of the stable isotopes based reaction conditions of MA tested. Conclusion. In this review, we have discussed the impurities and stable isotopes signatures found in illicit MA. The signatures are critical in the intelligence of illegal drugs, linking the illegal drugs with their sources, synthetic methods, synthetic batches, and their geographical origin. Although stable isotopes have been influential in discriminating seizures based on their origin, it is evident from this review that its potential in profiling MA has not been fully explored. The review highlights further how the integration of impurity profiling with stable isotopes signatures coupled with chemometric techniques complements the existing intelligence gaps. The review illustrates how an assortment of legally available chemicals and medicines used to mask the controlled substances. It has also been shown in this review how precursor chemicals come with their corresponding stable isotopes and tracking them down to their stable isotopes has been essential in discriminating the seizures based on their original starting materials resulting in comprehensive profiling. Studies linking stable isotope technique in profiling MA are undoubtedly limited, perhaps because of the advanced instrumentations. In furtherance of monitoring the dynamics of the MA, drug markets, and the advances of the criminal networks, future research is indispensable. Future research should focus on the diversity of both licit and illicit starting materials, complementing impurity profiling with stable isotopes, building knowledge with regards to the newly identified MA profiles, and finally; coupling the methods with chemometric techniques.

(Re-upload to correct the mathematics) Movies are wonderful things. People pay money for movies. Looking at the movie gross receipts, they look like a logarithmic curve. Why would this be? Often in Physics if the individual bits of a system decide for themselves exponentials make a reasonable model. A radioactive nucleus will decay, independent of whether there are other atoms around it. The attenuation of light through a medium often follows Beer’s exponential law. And perhaps movie tickets. If we look at a typical box office return we might be tempted to say that: The change in money, in a unit of time decreases, for example with some coefficient K, and is inversely proportional to time. Or a differential equation d M, d t equals k over t. Integrating: we can write M, the money equals K, some coefficient, multiplied by time Log (T) A little jiggery-pokery is slipped in here, where we rescale the time, by adding one to the time to prevent the equation blowing up. Anyone familiar with quantum field theory or string theory will hardly care about such a sin. If we believe this equation of M equals K log t, we ought to be able to plot some results, and if physics means anything, make predictions. Firstly, for our crackpot theory, M, money, ought to be a straight line when plotted against log t. We present three movies. First Indiana Jones and the dial of destiny. For the first twenty days of this film, the coefficient K is approximately 52 million dollars, And the graph does appear reasonably linear, with an r squared factor around zero point 98. If the formula was still valid after 100 days, we would predict a domestic gross of 240 million dollars. Next, the little Mermaid. Using 55 days of data, the coefficient K is 77 million dollars, and the model has an r squared value of zero point 99. Applying the model, if the little mermaid stayed in theatres, then after 100 days the total domestic would grow to 355 million. Finally, taking something from out of the vault, Black Hawk down, from 20 years ago. We have data for days 22 to days 69 for Black hawk down, and the model again has a correlation r squared value of 0.97. The coefficient K for black Hawk down had a value of 27 million dollars. This is an interesting case, as its results for the first 21 days are not reported on the website “The Numbers”, and it was released around December 2001, on the heels of the September eleventh attacks. In its later stages, the model has a 97 percent r squared value. In terms of earnings, it might as well have been released on the seventeenth of January. Now, discussing these results, we might ask, we note several things. One, a correlation of 99 percent or so is obtained for our silly model, 97 percent for BHD. Meaning, about 99 percent of the data is explained by it. The model assumes the increase in earnings is inversely proportional to the time in the box office, or daily earnings equals K over t, where K is a coefficient, and t is the time since release. Two, for this model, the coefficient K, determines how far the movie goes. If we multiply the time by natural log of t, or two point seven one, we gain K units of money. For example, for Indiana Jones and the dial of destiny, for the first natural log of t days, or 2.71, days, the movie gains K dollars, or 52 million. The natural log equals two when we multiply that time by two point seven one again, or in 7.4 days, the income has grown by another K dollars, or to 104 million. After 20 days, which is the natural log three days, the gross income has grown to three K, or around 156 million. Using this model, to obtain another K units of income, we would have to wait two point seven one times as long, or to day 55. For five units of K income, we hang around to day 148. Naturally, any model eventually passes from usefulness. In analogy of the attenuation of light through a medium, once the light has travelled through it, there is no more attenuation. Similarly, once the movie is out of theatres, and the income per day no longer sustains the theatre, the movie is pulled, and the model lo longer applies. If Indiana Jones and the dial of destiny was to stay in theaters for 100 days, we would predict a domestic gross of 240 million dollars. And, According to "The Numbers": After 77 days, IJ5 topped: $174 Million Domestic. With the income on the final day being: $59 per theater in 100 reported theaters. Or about $66 million below our projection.

SYNTHETIC REDUCTIONS IN CLANDESTINE AMPHETAMINE AND METHAMPHETAMINE LABORATORIES: A REVIEW. ANDREW ALLEN and THOMAS S CANTRELL. Forensic Science International, Volume 42 (1989) Pages 183 to 199. Elsevier Scientific Publishers Ireland Limited. Summary. A review of synthetic reductions utilized in the clandestine manufacture of amphetamine and methamphetamine is presented. General discussions on the mechanism of heterogeneous catalysis, dissolving metals, hydrides and non-metal reductions used in the manufacture of amphetamine and methamphetamine with over 80 references are presented. Introduction. This review addresses reductions in clandestine methamphetamine and amphetamine synthesis. Central to the diverse routes published for the synthesis of methamphetamine and amphetamine is a reductive step at some point in the synthesis. Of 95 references surveyed concerning the synthesis of these controlled drugs, all but ten utilize a reductive approach. Since such diversity exists in these approaches, we felt that a composite literature review and discussion of the chemistry involved would help forensic chemists charged with investigating these clandestine laboratories. Secondly, we felt that a composite reference list would be of assistance in correlating notes or procedures found in clandestine laboratory sites to the open literature. Finally, only two open literature review articles in this forensic area have appeared and both were devoid of extensive references, reference one and two. An overview of synthetic approaches to methamphetamine and amphetamine utilizing reductive routes is outlined in Tables one and two. Table one is organized by the type of catalytic surface or reductive species; meaning Palladium, Platinum, Lithium aluminum Hydride, Formic acid and so on. Table two is organized by the synthetic route or intermediate; meaning Leuckart, Schiff base, oxime, nitrostyrene, and so on. Figures one to twelve illustrate the chemical formulas of the chemical reduction routes to amphetamine and meth-amphetamine. References three and seventy-two are annotated with the type of reductive catalyst, reagent and route utilized. Chemical Abstract citations, C, A Volume, page and year, are included for each reference for ease of cross reference with cryptic notes often found in clandestine laboratory sites. Finally, the recurrent use of the terminology "open literature" refers to legitimate, accredited journals as opposed to underground publications or notes passed between clandestine manufacturers. Heterogeneous catalysis. The role of heterogeneous catalytic hydrogenation and hydrogenolysis in organic synthesis is replete in the literature. However, the mechanism of the catalyst's role has remained elusive due mainly to the difficulty of studying such heterogeneous systems. Recent research in this area has shown that a system charged with H, and D, in the presence of a catalyst yields HD. This has been interpreted as the catalyst's coordination with molecular H, and weakening or disruption of the H-H bond, reference eighty-seven and eighty-eight. Studies by Maier et al, in a personal communication, in which the catalytic surface has been coated with Silicon dioxide, have revealed that the H-H (which penetrates the SiO2, layer to coordinate with the catalytic surface) is truly ruptured, yielding singlet hydrogen. Furthermore, hydrogenation of an organic species (incapable of penetrating the SiO2, layer) occurred. This suggests that coordination between the organic moiety and the catalytic surface may not be necessary. "Selectivity" for an organic substrate in some catalytic metal hydrogenation systems has recently been shown to be dependent upon the topology of the catalytic surface, reference eighty-nine. Further work in this area will be followed with interest. Heterogeneous catalytic reduction of ephedrine to methamphetamine in clandestine laboratories is most often achieved with palladium, references three to eight, fifteen, seventeen, thirty-nine. The use of platinum (Adams Catalysis) is second in frequency (Figure one). Similar correlations apply to the reduction of phenylpropanolamine to amphetamine utilizing palladium, platinum and Raney Nickel. Hydrogenolysis of ephedrine or phenylpropanolamine, here hydrogenolysis is defined as reduction of C-X) is not a result of reduction of the benzylic carbon-OH bond. The actual moiety reduced is C-X, where X refers to halogen, sulfate, phosphate or perchlorate esters, figure one. This moiety (C-X) may be produced in situ, or synthesized externally, isolated and then reduced. The stereochemistry and analytical methodology for methamphetamine prepared from ephedrine and pseudoephedrine has recently been addressed, reference ninety-two and three. Heterogeneous catalysis has been used to reduce the imine bond of Schiff Bases formed with phenyl-2-propanone and ammonia or methylamine in order to produce amphetamine, references twenty six to nine, or methamphetamine, references nine, ten, twenty to twenty two, twenty-five, figure two. When heterogeneous catalysis is utilized in this Schiff’s base reduction, a Competing reaction, that of P-2-P reduction to 1-phenyl-2-propanol limits the yield of amphetamine or methamphetamine. Additions of large excesses of the amine component in these reactions have been employed to suppress the ketone reduction. This has limited applicability, since the optimum pH for the Schiff’s base production is between pH 6 and 7. Other clandestine routes, although less popular, which have open literature references utilizing heterogenous catalysis for the synthesis of amphetamine are oxime reduction, figure three, nitro-styrene reduction, figure four, 2-keto-oxime reduction figure five and hydrazine reduction, figure six. Precursors to amphetamine (phenylpropanolamine) and methamphetamine (ephedrine) have been synthesized with the aid of heterogeneous catalysis references sixteen, thirty-eight, figure five. Dissolving metal reductions. Dissolving metal reductions, in particular aluminum, continue to be the most popular synthetic routes to methamphetamine and amphetamine in clandestine laboratories in the United States. Although molecular Hydrogen, is produced as the metal dissolves, this is generally considered a detriment to the reduction of the organic species. The actual reductive mechanism does not involve molecular Hydrogen, but is, in fact, a result of an "internal electrolytic process". Electron transfer from the metal to a heteroatom results in a radical carbon which abstracts hydrogen from solution to complete reduction. In metals where higher oxidation states are present, meaning Al, Mg, Zn, dimers may form as a result of intramolecular radical combination. Poisoning of catalysis is one approach used to minimize rapid dissolution of the metal and to abate evolution of Hydrogen gas. Amalgams made between sodium and mercury have the effect of diminishing the activity of the parent metal thus slowing dissolution of the reducing species. Amalgamation between aluminum and mercury has the added benefit of preventing oxide formation on the surface of aluminum in contact with air. Aluminum-mercury amalgam serves to poison the metal somewhere between the extremes of the over-active metal and the inactive metal oxide. In the clandestine manufacture of amphetamine and methamphetamine the most popular route is via aluminum-mercury amalgam reduction of the Schiff base adduct of phenyl-2-propanone, P-2-P, and the appropriate amine, see references forty to forty five and figure two. This popularity persists despite U.S. Government control (Schedule Two) of P-2-P in 1980. This controlled status has resulted in an upsurge in the clandestine manufacture of P-2-P. A variety of synthetic routes have surfaced in clandestine laboratories, primarily through phenyl-acetic acid, reference seventy-three to seventy seven figure seven. Alternatives to the phenyl-acetic acid, now on a reporting schedule in some states, synthesis of P-2-P have appeared. One approach to P-2-P utilizes a dissolving metal reduction of nitro-styrene with iron and hydrochloric acid, as shown in figure four. Clandestine laboratories which utilize other dissolving metal reduction routes have been infrequently encountered. However, reduction of a Schiff base to meth-amphetamine, figure two and of 5-phenyl-4-methyl-thiazole to amphetamine, figure nine using sodium in alcohol are cited in the open literature. Additionally, Sodium alcohol reduction of an oxime, figure three, Sodium Mercury amalgam reduction of a nitro-styrene, figure four or a 2-keto-oxime, figure five to amphetamine and zinc, HCl reduction of Chloro analogs of ephedrine to methamphetamine, figure one are also cited in the literature. Metal hydride reduction. Metal hydride reductions have not captured the imagination of clandestine laboratory chemists like the remainder of the scientific community. This fact is probably the result of their inability to utilize current Chemical Abstracts nomenclature, wherein most literature references to metal hydrides appear. Metal hydrides function by transfer of a hydride to the electron deficient center (typically carbon) of a double bond. Protonation is effected on the electron rich center via the solvent media in the case of Sodium Borohydride or product workup in case of Lithium Aluminum Hydride. The infrequent use of metal hydride reducing agents in clandestine laboratories cannot be attributed to the lack of open literature references in these agents, references fifty-five to sixty-two. Methamphetamine has been produced in clandestine laboratory sites via Sodium Borohydride reduction of the Schiff Base adduct of P-2-P and methylamine following a procedure outlined by Weichet et al, figure two. Unfortunately, the activity of Sodium Borohydride is sufficient to reduce the ketone of P-2-P and this is a competing reaction. This is not the case with the more selective reducing agent NaCNBH3 whose activity is dependent on the pH of the reaction media, reference fifty-seven. Lithium aluminum hydride, whose activity is greater and therefore less selective than Sodium Borohydride, has been used to produce methamphetamine or amphetamine through the reduction of a variety of functional groups; meaning formyl, figure eight, carbamate, figure ten, oxime, figure three, Nitro-styrenes figure four and halogen analogs, figure eleven. Sodium borohydride has also been used in a de-mercuration procedure route followed by acid hydrolysis to amphetamine (in a clandestine laboratory) as outlined in Figure twelve. Non-metal reductions. Non-metal reduction routes to methamphetamine and amphetamine have been what might be termed as "fads" in clandestine laboratory synthesis within the United States. In the early and mid-1970’s, the Leuckart Synthesis, which employs formic acid, was the most popular clandestine route to amphetamine and methamphetamine. For whatever reason, this route, which is still very common in Western Europe, lost popularity in the United States by the end of the 1970s. In the early 1980s, the hydriodic acid reduction of ephedrine to methamphetamine began increasing in frequency in the Southwestern and Western areas of the United States. Although several literature references link the Leuckart synthesis figure eight, to amphetamine references sixty seven to nine, and methamphetamine, reference sixty four to six, "no" open literature reference directly links hydriodic acid reduction of a benzylic alcohol to the production of methamphetamine figure one. Several general benzylic alcohols have been reduced to their aliphatic counterparts. However, this “Cross application” of chemical syntheses would require a level of chemical knowledge not common among clandestine chemists. The mechanism of the Leuckart reaction has been studied and shown to be a free radical process initiated by formic acid. Unfortunately, the mechanism of the hydriodic acid reduction has not been established. It seems clear that the benzylic alcohol of ephedrine undergoes a substitution reaction with iodine. However, the mechanism of the carbon-halogen reduction is in conjecture; i.e. hydride transfer, internal electrolysis via disproportionation of iodine, or elevated temperature decomposition of HI to H, and I, whereby H, reduces the C-I bond. Conclusion. In this review we have addressed reductive approaches to amphetamine and meth-amphetamine via heterogeneous catalysis, dissolving metals, metal hydrides and non-metal reductions. The chemistry of these varied approaches has been highlighted with emphasis on the role of the reducing species. It may be concluded that there are many options available to clandestine chemists, see Figures one to twelve. However, in actual practice, the three most frequently encountered routes in the United States are: One, the aluminum foil reduction of the Schiff Base adduct of P-2-P and methylamine, reference forty to forty four. Two, the palladium catalyzed reduction of the Chloro analog of ephedrine to methamphetamine, references four and five, and. Three, the hydriodic acid reduction of ephedrine to methamphetamine.

METHAMPHETAMINE SYNTHESIS VIA REDUCTIVE ALKYLATION. HYDROGENOLYSIS OF PHENYL-2 PROPANONE WITH N-BENZYLMETHYLAMINE. HARRY F SKINNER. Forensic Science International, Volume 60, 1993. Pages 155 to 162. Elsevier Scientific Publishers Ireland Limited. Drug Enforcement Administration, Southwest Laboratory, National City, California. Summary. Methamphetamine was synthesized by reductive alkylation hydrogenolysis of phenyl-2-propanone with N-benzyl-methylamine. The expected product N-benzyl-methamphetamine, once formed, undergoes hydrogenolysis to methamphetamine and toluene. The progress of the reaction, the intermediates formed during the reaction, and the products were analyzed by gas chromatography and mass spectrometry. Introduction. The synthesis of methamphetamine from phenyl-2-propanone, P2P, and methylamine is a common method utilized in clandestine laboratories. Reductive alkylation hydrogenolysis of P2P and N-benzyl-methylamine to form methamphetamine is not commonly encountered, reference one. Reductive alkylation involves the reaction of an aldehyde or ketone with a primary or secondary amine in the presence of hydrogen and a hydrogenation catalyst. The reductive alkylation of P2P and methylamine with hydrogen and palladium on carbon produces methamphetamine. The reductive alkylation of P2P and N-benzyl-methylamine produces N-benzyl-methamphetamine, also known as benzphetamine. Subsequent hydrogenolysis of benzphetamine produces methamphetamine. Experimental. During this study, the one step hydrogenation reactions were run on a Parr pressure reaction apparatus in hydrogenation flasks with shaking at a hydrogen pressure of 50 pounds. Hydrogen consumption was monitored by the pressure drop in the gas supply tank on the hydrogenator. Methanol was utilized as the solvent and 10 percent palladium on charcoal and, or palladium black was utilized as the catalyst. The reactions were monitored by removal of aliquots from the reactions with subsequent analysis by gas chromatography, GC, and gas chromatography, mass spectrometry, GC, MS. The progress of the reaction was followed by observing the decrease in the concentration of the reaction precursors, the increase in the concentration of the final product, detection of intermediate compounds formed during the reaction, and detection of side products formed during the reaction. The gas chromatograph used was a Hewlett-Packard 5890 equipped with a flame ionization detector. It was operated in the split mode, 100 to 1 ratio, using a 25 meters, times 0.20 millimeter fused silica capillary column with a 5 percent cross-linked phenyl-methyl-silicone liquid phase, 0.11 micron film thickness. The injector temperature was maintained at 280 degrees C. The oven temperature was programmed as follows: Initial temperature, 60 degrees C; initial hold 2.0 minutes; temperature program rate, 40 degrees C per minute; final temperature, 270 degrees C; final hold, 5.0 minutes. Helium was used as the carrier gas at a column flow rate of 1 milliliter per minute. The gas chromatography mass-spectrometry was performed on a Finnigan INCOS 50 Mass Spectrometer interfaced to a Hewlett-Packard 5890 gas chromatograph. The gas chromatograph was operated in the split mode, 50 to one ratio, using a 25 meter, times 0.20 millimeter fused silica capillary column with a 5 percent cross linked Phenyl-methyl-silicone liquid phase (0.11 micron film thickness). The injector temperature was maintained at 250°C. The oven temperature was programmed as follows: Initial temperature, 60 degrees C; initial hold 1 minute; or initial temperature 40 degrees C, initial hold two minutes. Temperature program rate, 30 degrees C per minute; Final temperature, 270 degrees C; final hold, 5.0 minutes. Helium was used as the carrier gas at a column flow rate of 1 milliliter per minute. The reaction mixtures were processed by filtering off the Palladium, Carbon, acidifying with hydrochloric acid and evaporating off the methanol and toluene to dryness. In the reactions in which the ratio of N-benzyl-methylamine was greater than that of P2P, the P2P was almost completely consumed and easily removed by washing with ether, acetone. The product was a mixture of d,l-methamphetamine hydrochloride and N-benzyl-methylamine hydrochloride. The stereochemistry was determined by polarimetry. Results and Discussion. Primary (1) and secondary amines can condense with aldehydes and ketones (2) to give different kinds of products. Primary amines give imines (4). The reaction as shown in Figure one is straightforward and proceeds through N-substituted hemi-aminals (3) in high yields, reference two. When secondary amines are added to an aldehyde or ketone, the initially formed N,N-disubstituted hemiaminals (5) cannot condense across the Carbon Nitrogen bond; however, if there is an alpha hydrogen, water is eliminated to give an enamine (6) Figure one. The water is usually removed azeotropically to drive the reaction to the enamine, reference three. If the resulting imines, or enamines, are subsequently reduced, then the overall reaction gives more complex amines. This general reaction is used in many methods found in clandestine laboratories such as methamphetamine, the 3,4-methylenedioxyamphetamines, as well as the fentanyls. The formation of more complex amines can be done in a one-step reaction by reductive alkylation. When an aldehyde or ketone is mixed with a primary or secondary amine in the presence of hydrogen and a hydrogenation catalyst, reductive alkylation takes place. For example, methamphetamine was synthesized by reductive alkylation of phenyl-2-propanone and methylamine in the presence of hydrogen and palladium on carbon. Mixing of an equal volume, mole ratio 1 to 1.7, of P2P (7) and 40 percent methylamine in methanol immediately produced the imine (8) shown in Figure two. After 24 hours, methamphetamine (9) is the main component (50 percent). The mass spectrum of the imine compound, 1-phenyl-2-methyl-2-methylimino propane, is shown in Figure three. The imine compound is fairly stable since it does not readily reduce under the reaction conditions, 50 pounds hydrogen, Palladium on Carbon. The reductive alkylation of phenyl-2-propanone and N-benzyl-methylamine surprisingly gives methamphetamine as the final product rather than N-benzyl-methamphetamine. Unlike the immediate formation of the imine compound in the reaction of P2P and methylamine, the analogous enamine is not seen by either GC or GC, MS during the reaction. Initially N-benzyl-meth-amphetamine is detected along with the precursors P2P and N-benzyl-methyl-amine. As the reaction progresses, toluene and methamphetamine are detected simultaneously. The concentrations of toluene and methamphetamine increase while the concentration of N-benzyl-meth-amphetamine remains relatively constant. As expected, increasing the ratio of N-benzyl-methylamine to P2P speeds up the reaction. Using twice the amount, mole ratio two to one, of N-benzyl-methylamine to P2P, the reaction is complete in 24 hours. Figure four shows the ion chromatogram of this reaction at 24 hours with all five components of the reaction detected. Small amounts of P2P (Scan 955) and N-benzyl-meth-amphetamine (scan 1569) remain as well as toluene (scan 390), N-benzyl-methylamine (scan 866) and methamphetamine (scan 1016). The mass spectrum of N-benzyl-methamphetamine is shown in Figure five. Production of methamphetamine is not unexpected if the reduction of benzyl-amines is considered. Reduction of benzyl moieties is known to result in hydrogenolysis, references four and five. In this case it is the cleavage of a benzylic Carbon Nitrogen bond with hydrogen addition to the resulting fragments. Benzyl-amines give toluene and the corresponding amine, reference six. Another illustration of this type of reaction is the synthesis of a primary amine-steroid from the corresponding keto-steroid. The reaction with ammonia, hydrogen, and Palladium on Carbon proceeds giving poor yields. However, the product can efficiently be synthesized by using benzyl-amine to produce a secondary amine intermediate which undergoes hydrogenolysis to yield toluene and the desired primary amine-steroid in a 71 percent yield, reference seven. The hydrogenolysis of benzyl-amine was also shown in this work by the reduction of N-benzyl-meth-amphetamine. Standard N-benzyl-meth-amphetamine was easily reduced completely to methamphetamine and toluene with hydrogen and Palladium on Carbon. The reaction route for methamphetamine synthesis via reductive alkylation hydrogenolysis of phenyl-2-propanone with N-benzyl-methylamine is shown in Figure six. P2P (7) and N-benzyl-methylamine (10) react to form the hemiaminal intermediate (11) which can be reduced directly to N-benzyl-methamphetamine (12). The hemi-aminal can also lose water and form the enamine (13) compound which would then reduce to N-benzyl-methamphetamine, according to a personal communication from B Klein. Clearly the formation of these intermediates are more difficult compared with the analogous P2P and methylamine reaction where the imine forms immediately and in large amounts. The intermediates must also reduce readily to N-benzyl-methamphetamine since they are not detected during the reaction. N-Benzyl-meth-amphetamine undergoes hydrogenolysis with cleavage of the C, N bond and adds a hydrogen on each fragment, thereby producing methamphetamine (9) and toluene (14) as the final products in the reaction. Since N-benzyl-methylamine can undergo hydrogenolysis and produce toluene, detected, and methylamine, unable to be detected directly, the route of reaction to produce methamphetamine could also proceed through the reaction of P2P and the methylamine produced in situ. This reaction readily proceeds through the imine intermediate which is easily detected. However, none of the imine (8) compound was detected during the course of the P2P and N-benzyl-methylamine reaction indicating the reaction route proceeds through N-benzyl-methamphetamine to methamphetamine. N-Benzyl-methamphetamine can be manufactured from methamphetamine and benzylchloride as well as from N-benzyl-methylamine and 2-chloro-1-phenylpropane. Obviously the use of methamphetamine would not be logical unless N-benzyl-methamphetamine, benz-phetamine, was the desired product. However, the second synthesis to produce N-benzyl-methamphetamine with subsequent reduction would be a non-typical synthetic route to methamphetamine. Conclusion. The synthesis of methamphetamine via reductive alkylation hydrogenolysis of phenyl-2-propanone and N-benzyl-methylamine has been shown to be a viable method. Both this method and the synthesis and reduction of N-benzyl-methamphetamine need to be considered as possible routes for the manufacture of methamphetamine in clandestine laboratories. References. One: H.F. Skinner, Methamphetamine synthesis via hydriodic acid/red phosphorus reduction of ephedrine. Forensic Sci. Int., 48 (1990) 123 to 134. Two: J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, McGraw-Hill, New York, 1968, page 667. Three: J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, McGraw-Hill, New York, 1968, page 668. Four: R. Baltzly and J. Buck, Catalytic debenzylation. The effect of substitution on the strength of the O-benzyl and N-benzyl linkages. J. Am. Chem. Sot., 65 (1943) 1984 to 92. Five: C. Bronislaw and R. Bartsch, Effects of amines on O-benzyl group hydrogenolysis. J. Org. Chem., 49 (1984) pages 4076 to 4078. Six: C. Buehler and D. Pearson, Survey of Organic Synthesis, Wiley and Sons, New York, 1970, page 430. Seven: L. Fieser and M. Fieser, Reagents for Organic Synthesis, Vol. One, Wiley and Sons, New York, 1967, page 51.