Preterm birth is a major cause for neonatal morbidity and mortality, and is frequently associated with adverse neurological outcomes. The transition from intrauterine to extrauterine life at birth is particularly challenging for preterm infants. The main physiological driver for extrauterine transition is the establishment of spontaneous breathing. However, preterm infants have difficulty clearing lung liquid, have insufficient surfactant levels, and underdeveloped lungs. Further, preterm infants have an underdeveloped brainstem, resulting in reduced respiratory drive. These factors facilitate the increased requirement for respiratory support. A principal cause of preterm birth is intrauterine infection/inflammation (chorioamnionitis), and infants with chorioamnionitis have an increased risk and severity of neurological damage, but also demonstrate impaired autoresuscitation capacity and prevalent apnoeic episodes. The brainstem contains vital respiratory centers which provide the neural drive for breathing, but the impact of preterm birth and/or chorioamnionitis on this brain region is not well understood. The aim of this review is to provide an overview of the role and function of the brainstem respiratory centers, and to highlight the proposed mechanisms of how preterm birth and chorioamnionitis may affect central respiratory functions. Preterm birth, defined as childbirth <37 weeks gestation, is a leading cause of neonatal morbidity and mortality worldwide  nucleus and the parabrachial (PB) complex , BÖTC, retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG), as well as the rostral and caudal ventral respiratory groups , paracrine influences (ATP) by pH-sensitive astrocytes, as well as inputs from peripheral chemoreceptors  is largely comprised of excitatory inspiratory premotor neurons which provide neural input to spinal phrenic and intercostal motorneurons that innervate the diaphragm .in utero, FBMs are detectable at 10 weeks gestational age  profoundly stimulate FBMs , coincides not only with an immature lung, but also an immature respiratory control center.Whilst autonomous neonatal breathing is critical for sustaining life, the brainstem respiratory centers are not completely mature at birth, and instead, continue to develop and refine postnatally  and glycine are inhibitory neurotransmitters which modulate inspiratory activity to allow for the proper transition to the expiratory phase. The developmental expression of GABA in rats has been shown to steadily decrease in the NTS, but increase in the pBÖTC , which stimulate downstream signaling pathways leading to pro-inflammatory cytokine production Beutler, . The actin vitro hippocampal neurons demonstrate that IL-1β alters neuronal function in a concentration-dependent manner; at low concentrations IL-1β inhibits voltage-gated calcium currents, and lowers intracellular calcium concentrations (thereby decreasing neurotransmitter release), whereas high concentrations of IL-1β increase ionotropic glutamate receptor expression (NMDA), and decreases transmission at GABAergic and glycinergic receptors in rat hippocampal and cerebellar cultures , prostaglandin D2, prostaglandin F2α, and prostacyclin  stimulated  signaling can enhance COX isozyme expression leading to elevated prostaglandin synthesis . Binding to these receptors can result in distinct signaling pathways  activation which is a major transcription factor that can induce synaptic plasticity, and neuroprotection , and an increase in intracellular calcium which can affect neuronal excitability and firing rate (Bos et al., 2 production and subsequent binding to EP3R has been shown to reduce excitatory vagal neurotransmission to the NTS in rats (Marty et al., EP3R has the greatest affinity for PGE2 signaling through the EP4R is similar to the EP2R pathway as it offers neuroprotection in models of excitotoxic or hypoxic-ischemic injury, however, it may can also modulate pro-inflammatory and anti-inflammatory responses, as observed in the rat, and in mouse EP4R knockout studies (Zhang and Rivest, EP4R is highly expressed within the hypothalamus and brainstem, and functions similarly to EP2R (Andreasson, 2 specifically has been shown to cause functional changes of respiratory-related neurons within the brainstem. Dysregulation and damage to these brainstem centers may be implicated in the multifaceted pathophysiology of respiratory depression in preterm neonates. Understanding how chorioamnionitis may affect these central respiratory centers could lead to effective therapeutic interventions within the delivery room, with the goal of reducing brain injury and preserving the neural circuitry controlling rhythmic and coordinated respiratory functions.Preterm birth is associated with suboptimal development of the brainstem, and subsequently reduced respiratory control. Further, chorioamnionitis is strongly associated with preterm birth, and leads to an increased risk and severity of respiratory complications. There is extensive research on chorioamnionitis-induced white matter brain injury, however, the effects of inflammation on the brainstem, which contains central respiratory centers, remains unclear. Inflammation is associated with elevated prostaglandin synthesis, and PGEVS: wrote the manuscript and made figures. SLM, SBH and GRP: revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


High-fructose intake in healthy men is associated with characteristics of metabolic syndrome. Extensive knowledge exists about the differences between hepatic fructose and glucose metabolism and fructose-specific mechanisms favoring the development of metabolic disturbances. Nevertheless, the causal relationship between fructose consumption and metabolic alterations is still debated. Multiple effects of fructose on hepatic metabolism are attributed to the fact that the liver represents the major sink of fructose. Fructose, as a lipogenic substrate and potent inducer of lipogenic enzyme expression, enhances fatty acid synthesis. Consequently, increased hepatic diacylglycerols (DAG) are thought to directly interfere with insulin signaling. However, independently of this effect, fructose may also counteract insulin-mediated effects on liver metabolism by a range of mechanisms. It may drive gluconeogenesis not only as a gluconeogenic substrate, but also as a potent inducer of carbohydrate responsive element binding protein (ChREBP), which induces the expression of lipogenic enzymes as well as gluconeogenic enzymes. It remains a challenge to determine the relative contributions of the impact of fructose on hepatic transcriptome, proteome and allosterome changes and consequently on the regulation of plasma glucose metabolism/homeostasis. Mathematical models exist modeling hepatic glucose metabolism. Future models should not only consider the hepatic adjustments of enzyme abundances and activities in response to changing plasma glucose and insulin/glucagon concentrations, but also to varying fructose concentrations for defining the role of fructose in the hepatic control of plasma glucose homeostasis.  Eating habits and lifestyle are subject to change and are determined by socio-economic factors. In modern societies, the surplus offer of food leads to an excess caloric intake that may go along with reduced physical activity. The consequences are obesity with associated health risks such as nonalcoholic fatty liver disease (NAFLD) or type 2 diabetes. The continuously increased fructose consumption as sucrose and high fructose corn syrup over the past decades coincides with the raised prevalence of obesity, NAFLD, and type 2 diabetes ,2,3. TheStudies about different forms of nutrition point out the importance of a food mix adjusted to our metabolic/biochemical disposition. During evolution, the human metabolism became adapted to gain energy from different sources for survival in an environment with a seasonally changing food offer. It is prepared to gain energy from a variety of simple and complex carbohydrates (consisting of a range of sugar types) as well as from fatty acids. The organism provides cellular pathways to obtain flexibility in the use of various energy carriers. However, may an unbalanced diet stretch cellular regulatory mechanisms to their limits and eventually impair health? What is the optimal mix of energy carriers for the human organism in terms of type of carbohydrates and fat for preserving health? There is no widely accepted paradigm in this respect.The liver, as a central organ in carbohydrate metabolism, serves as an energy hub. It represents a major sink for carbohydrates. Hepatic glucose uptake contributes to the restoration of blood glucose homeostasis after a meal. Carbohydrates are stored as glycogen or converted into fatty acids which are then released into the plasma and deposited in adipose tissue. During fasting, glucose from the breakdown of glycogen, and gluconeogenesis is released by the liver maintaining blood glucose homeostasis. Impairment of such essential hepatic functions manifests as disturbed insulin sensitivity, increased hepatic glucose production and de novo lipogenesis. This article aims at highlighting the role of different types of sugars and their potential specific adverse effects on the liver. We refer to data from animal studies that examined diet induced gene expression and underlying molecular mechanisms, but we also included data from nutritional human studies investigating metabolic outcome based on tracer methods. We focus on the interconnections of fructose and glucose metabolism. Prior to going into the details of liver lipid metabolism and its adaptation to carbohydrate rich diets, some general differences between glucose and fructose and their potential roles as competitors need to be pointed out. It cannot be stressed enough that the cellular absorption and distribution of glucose and fructose differ markedly. Only two transporters, namely GLUT2 and GLUT5, of the 14 members of the glucose transporter (GLUT) family are permeable for fructose ,8. They GLUT5 expression is induced during the postnatal development. Its expression is dramatically stimulated by the introduction of dietary fructose and it seems to be modulated by paracrine and endocrine signals leading to a diurnal variation of expression in adults ,16,17,18Hepatic uptake of both glucose and fructose is mediated by the GLUT2 transporter, which is constitutively expressed on hepatocytes and facilitates diffusion . The affAfter hepatic uptake, fructose and glucose are phosphorylated by different kinases. In the liver, fructose is phosphorylated by the high-activity keto-hexokinase isoform C (KHK-C), which is more effective than the ubiquitous low-activity KHK-A, and subsequently rapidly converted into trioses . The rapIn summary, hepatic influx rates of fructose and glucose are determined by their plasma concentrations and GLUT2 affinities, but also by the KHK activity for fructose and the GK and GS activity for glucose. The hepatic response to rising intracellular hexose concentrations is orchestrated by hormones, key regulatory transcription factors, and metabolic intermediates that adjust both enzyme abundances and activities. The relative importance of the regulation of enzyme abundances and activities, respectively, in the control of the metabolic hexose flux and finally the restoration and maintenance of glucose homeostasis is debated. However, the results from mathematical modeling suggest that the regulation of enzyme abundances and of enzyme activities is equally important for the regulation of hepatic hexose metabolism .Certainly, a comparison of hepatic metabolism of glucose and fructose requires the consideration of the diverse actions of insulin in order to identify possible sites of interaction of glucose and fructose metabolism. We cover this issue by starting from the widely accepted actions of insulin on liver metabolism namely blocking glycogenolysis and gluconeogenesis, stimulating glycogen synthesis and thus regulating fasting glucose levels , and pyruvate kinase (PK) and inhibits the expression of gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK), F-1,6-bisphosphatase , and glucose-6-phosphatase (G6Pase). The regulatory mechanisms of gene expression by insulin involve sterol regulatory element binding protein 1c (SREBP1c) and forkhead box O1 (FOXO1). On the other hand, the immediate action of insulin is the regulation of enzyme activities namely of GS and citrate lyase by changing their phosphorylation state. Hepatic glucose metabolism is not only subject to insulin-dependent regulation, as a variety of glucose intermediates as well as adenosine mono-phosphate (AMP), adenosine tri-phosphate (ATP), and citrate are known to be potent direct or indirect modulators of glycolytic, gluconeogenic, and glycogenic enzyme activities. Fructose specific intermediates also play an important role in the regulatory system of glycolysis/gluconeogenesis and glycogenesis . To be mSmall amounts of dietary fructose have been shown to dramatically increase the hepatic glucose uptake through GK activation ,53. It iHowever, the excess consumption of fructose (corresponding to the 90th percentile of fructose intake of the US adults and above) has been shown to impair glucose uptake, reducing GK activity and liver glycogen content . While iIt has been demonstrated that the consumption of dietary fructose compared to glucose induces increased expression of the fructose metabolizing enzymes KHK, aldolase B and the glycolytic enzymes PFK1 and PK  Figure . Among tSimultaneous enhancement of glycolytic enzyme expression by insulin and fructose, while opposing effects on the expression of gluconeogenic enzymes appears paradoxical and raises the question about the metabolic outcome when fructose and glucose are co-ingested. Does dietary fructose promote futile cycling at the site of conversion of F6P into F-1,6-bisphosphate  and vice versa  66]? Wh? Wh66]? n = 6, healthy volunteers). However, if co-ingested with glucose, fructose contributes significantly to glycogen synthesis suggesting a critical role of insulin [Newly generated glucose from fructose is released into the plasma, but is there also deposition in the glycogen storage? Glycogen deposition depends on the activity of GS that catalyzes the rate limiting step in glycogen synthesis and the activity of glycogen phosphorylase. It is known that insulin and G6P activate the GS and F1P inhibits glycogen phosphorylase. A further tracer study showed that fructose contributes little to glycogen synthesis when ingested alone  and fatty acid synthesis pathways. Fructose was primarily described as “lipogenic substrate” in the context of hepatic metabolism, however, studies demonstrate that gluconeogenesis and glycogen deposition also play an important role in fructose metabolism. The metabolic effects of fructose consumption are dose-dependent. Small amounts of fructose may substantially enhance the uptake of glucose into the liver and glycogen deposition, while large amounts of fructose may reduce the hepatic glucose uptake. The presence of insulin determines the fate of fructose. The fact that co-ingestion with glucose directs fructose to the glycogen storage probably through the action of insulin would suggest that glucose takes temporarily the load off the other fructose disposal pathways.De novo lipogenesis (DNL) is the conversion of carbohydrates or proteins into fatty acids, enabling the organism to store energy in an unlimited way as fat. DNL takes place in the cytosol of hepatocytes and adipocytes if there is an excess of the substrate acetyl co-enzyme A. Critical enzymes of the synthesis pathway of fatty acids are the Acetyl CoA Carboxylase (ACC), the fatty acid synthase (FAS), elongation of long-chain fatty acids family member 6 (ELOVL6), as well as stearoyl-CoA desaturase 1 (SCD1) ,76,77,78There are several master transcription factors regulating the expression of partly the same set of lipogenic enzymes. ChREBP induces the transcription of FAS, SCD1, ELOVL6, ACC, and PK ,80. SREBHepatic ChREBP expression is induced by a high-carbohydrate diet ,84. FeedOverall, any hexose pathway that ends in the gluconeogenetic pathway at the level of trioses  potentially enhances ChREBP dependent expression of lipogenic enzymes. Fructose may directly activate ChREBP through fructose derived intermediates or indirectly through activation of GK enhancing the glycolytic flux. The importance of ChREBP for the regulation of hexose metabolism is reflected in ChREBP deficient mice which show glycogen accumulation and decreased triglyceride (TAG) synthesis in the liver . There are three SREBP isoforms, SREBP-1a, -1c and 2. SREBP1c is known to induce lipogenic gene expression and its expression itself is induced by carbohydrate feeding. Ad libitum chow-feeding of mice  increased SREBP1c expression in wildtype mice and to a lesser extent also in liver insulin receptor knock-out mice (LIRKO) . SREBP1cThe potential of fructose to induce SREBP1c expression has also been examined in mice. The impact of a 1-week high-fructose diet (60% fructose) was compared to a standard chow diet on SREBP1c expression. Fructose diet increased SREBP1c mRNA levels and consequently the mRNA levels of lipogenic enzymes more potently than standard chow diet in wildtype, as well as in LIRKO mice. Levels of nuclear SREBP1c protein in LIRKO mice are lower than in wildtype mice after both fructose and standard chow diet indicating the important role of insulin signaling.Insulin binding to the insulin receptor activates the phosphoinositid-3-kinase/protein kinase B (PI3K/PKB) pathway and results in the proteolytic activation of SREBP1c through the interaction of SCAP (SREBP cleavage-activating protein) and INSIG1 (insulin-induced gene-1), and its release from the Golgi apparatus . Mice wiXBP-1 is an active transcription factor which increases the ER folding capacity in response to ER stress (unfolded protein response (UPR)) . Apart fAlthough mechanisms are described that involve insulin in the regulation of the expression of both ChREBP and SREBP1c, feeding studies with insulin receptor knock-out mice show that insulin is required neither for the induction of ChREBP nor for SREBP1c. However, in the case of SREBP1c the presence of insulin is required for a full induction of SREBP1c expression upon carbohydrate consumption. ChREBP, SREBP1c and XBP-1 are more potently induced by a high-fructose compared to high-glucose diet ,65,83,85Rapid phosphorylation of fructose by KHK decreases ATP levels, paralleled by an increase in AMP levels ,98. It hn = 5). A hypercaloric carbohydrate diet (around 2.5 times energy expenditure) during four days generated moderate hyperinsulinemia and increased VLDL-TAG concentrations. It caused an increase in the secretion rate of VLDLs, newly synthesized palmitate and preformed fatty acids, and a decreased VLDL-TAG catabolism [n = 8). After six days of this diet, there was no significant difference in respect to the fasting fractional DNL [It is an open question, to which extent de novo lipogenesis (DNL) from carbohydrates is contributing to TAG synthesis/secretion or eventually to the TAG accumulation in hepatocytes. In the past, studies have addressed this issue by using isotope-based methods for the measurement of de novo lipogenesis. After the consumption of different kinds and amounts of sugars, rates of secretion of newly synthesized palmitate, or the fractional de novo lipogenesis  have been calculated in different experimental settings. Here, two studies should be mentioned that applied the mass isotopomer distribution analysis (MIDA) approach for which 13C-acetate is infused and 13C-palmitate isotopomers from isolated VLDLs are measured. In one study, the short-term effects of a hypercaloric carbohydrate (hydrolyzed corn starch) feeding on fatty acid synthesis were examined in healthy men  is a master regulator of hepatic lipid metabolism and its expression is induced in the fasted state . It is iPPAR α controls the fatty acid import into and processing within the mitochondria by carnitine palmitoyltransferase 1 (CPT-1) and important enzymes of the beta oxidation pathway, such as acyl-CoA dehydrogenases (Acad) ,106. FruThe rate limiting step of beta oxidation is the translocation of LCFA into the mitochondrium mediated by CPT-1. Malonyl-CoA, produced by carboxylation of acetyl CoA by ACC2, is inhibiting CPT-1. Malonyl-CoA is the primary substrate for lipogenesis and by inhibiting CPT-1 it avoids a futile cycle ,111,112.n = 5) by using stable-isotope methodology and indirect calorimetry. Two weeks of a high-carbohydrate diet  significantly reduced the total and plasma fatty acid oxidation when compared to a 2-week low-carbohydrate diet  [The effect of low- and high-carbohydrate diets on substrate oxidation has been investigated in healthy men (protein) . It was protein) . OxidatiThe primary determinant for TAG synthesis and secretion is the availability of free fatty acids in the liver ,116. FatThe rate of the hepatic uptake of NEFAs is given by the plasma NEFA concentration and the amount and activity of transporter protein . NEFAs fThe effects of fructose consumption on TAG concentrations has not been comprehensively clarified. On the one hand, there is evidence that carbohydrate and high-fructose intake significantly increase the blood TAG concentrations probably due to stimulation of DNL and VLDL secretion in weight-maintaining diets ,124,125.A fructose-induced increase of de novo fatty acid synthesis and a decrease of beta oxidation is supposed to increase hepatic FA content and to enhance TAG synthesis and VLDL secretion . While aPotential implications of high-fructose consumption on insulin sensitivity and blood lipids have been examined extensively. Fructose has generally been recognized as sugar that decreases insulin sensitivity and promotes dyslipidemia ,132.n = 9). The study assessed the effects of moderate amounts of fructose and sucrose as compared with glucose on insulin sensitivity and lipid metabolism. Moderate amounts of fructose consumption impair insulin sensitivity in healthy young men, without affecting plasma glucose and TAG levels [Focusing on the effects of fructose consumption in healthy men, a study by Aeberli et al. examined the implications of fructose ingestion in amounts close to usual daily consumption when soft drinks replace unsweetened beverages  confirmed a pivotal role of KHK in the regulation of hepatic fructose uptake. The deletion of KHK abolished hepatic fructose uptake and led to a dramatic hyperfructosemia, but prevented fructose-induced hyperglycemia . In partThe precise mechanisms underlying fructose induced hepatic insulin insensitivity are still unknown. Lines of research currently focus on mechanisms driving gluconeogenesis by increasing expression of gluconeogenic enzymes namely PEPCK, F1,6BPase and G6Pase. Yet, there are also studies examining the impact of high-fructose diets on hepatic glucose uptake as well as glycogen formation  Figure .In rats, a long-term high-fructose, but not -glucose  diet caused hepatic deposition of TAG and impaired insulin signaling and glucose tolerance, but did not change the expression of PEPCK (fasted state) . Also inFurther studies suggest that fructose feeding increases the expression of the gluconeogenic enzymes F1,6BPase and also G6Pase driving hepatic gluconeogenesis and glucose release ,65. It iFurthermore, as mentioned earlier, excessive fructose consumption may lead to decreased GK levels, hepatic glucose uptake, and further decreased GS activity resulting in decreased glycogen deposition . w/v fructose or glucose solutions for 2 months, ad libitum fed rat) increased hepatic TAG content and decreased hepatic insulin receptor 2 (IR-2) expression and Akt activation, the impact on hepatic metabolism has to be further clarified [Overall, even though long-term fructose consumption (supplementation of eucaloric 10% larified . Namely,larified . The implarified ,138. A sResearch about fructose induced insulin insensitivity has been strongly focused on the gene expression of metabolic enzymes. The impact of fructose on the hepatic allosterome and thereby on the regulation of enzymes and glucose flux should also be considered for further research.After 50 years of research, there is sound evidence that fructose can be qualified as glucogenic and lipogenic sugar. A central role in the hepatic hexose metabolism play the transcription factors ChREBP and SREBP1c, both more potently induced by fructose than by glucose. This seems to reflect the importance of the liver for fructose metabolism. Consequently, fructose consumption may impair glucose homeostasis by increased expression of the gluconeogenic enzymes counteracting the effect of insulin. On the other hand, fructose may lead to an increased expression of lipogenic enzymes with implications on blood TAG and intrahepatic TAG concentrations. The impact of fructose on metabolic parameters, as assessed by feeding-trials, is dose-dependent, with probably little effect when low dosages of fructose are used as isocaloric substitution for glucose ,142,143.Overall, fructose consumption has been shown to promote hepatic insulin resistance in non-diabetic men ,133, as However, the debate about the limit between harmless and harmful quantities of fructose is still ongoing, and larger and longer feeding trials are needed in order to complete existing evidence and to increase our understanding of the topic. In the future, mathematical models may enhance our understanding of the role of fructose in glucose flux control and will be useful for the design of new experiments .


The founding member of the Sirtuin family, S. cerevisiae Sir2p, and its orthologs have important roles in senescence and aging, with manipulation of their activities resulting in modest lifespan extension, as verified in several model organisms. Sirtuins have a wide range of nuclear and cytoplasmic substrates, and are implicated in the transcriptional and epigenetic regulations of cellular and systemic processes of energy metabolism, stress response, death and survival, as well as pathological conditions such as malignancy  . These mice were previously shown to have stunted postnatal growth, but ultimately become obese with time  from low potassium treatment, it was found that in contrast to SIRT1, SIRT6 over-expression reduced the viability of CGNs (Pfister et al., In yet another report, SIRT6 over-expression in rat hippocampal neurons was found to attenuate AKT-GSK-3β phosphorylation (Mao et al., From the discussion above it appears that the neuroprotective effect of SIRT6 may be somewhat context-dependent rather than universal. The basis of this context dependency is not yet clear. However, it is notable that context-dependence of neuroprotection was also observed to some degree with SIRT1, and it would thus be of interest to see if these occur via similar mechanisms. SIRT1's role in AD is well known (Wong and Tang, +, which may become limiting in stressed conditions (Liu et al., It has been previously argued that while sustained SIRT1 activity may benefit chronic stresses associated with neurodegenerative disorders, elevation of SIRT1 activity or levels may instead adversely affect acutely injured neurons (Ng and Tang, Other than intrinsic enzymatic activity and differential substrate preference, there are also other important differences between CNS SIRT6 and SIRT1, for example the spatial and temporal difference in their expression, as well as substrate specificity. As mentioned earlier, SIRT6 levels decline in the aging brain as oppose to SIRT1's increase. SIRT6 levels are in fact positively regulated by SIRT1 and FOXO3a in metabolically active peripheral tissues (Kim et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sudden death is a major health problem all over the world. The most common causes of sudden death are cardiac but there are also other causes such as neurological conditions , drugs, catecholamine toxicity, etc. A common feature of all these diverse pathologies underlying sudden death is the imbalance of the autonomic nervous system control of the cardiovascular system. This paper reviews different pathologies underlying sudden death with emphasis on the autonomic nervous system contribution, possibilities of early diagnosis and prognosis of sudden death using various clinical markers including autonomic markers (heart rate variability and baroreflex sensitivity), present possibilities of management and promising prevention by electrical neuromodulation. Yet, a plethora of data suggests that the imbalance of the autonomic nervous system control of the cardiorespiratory system is a common phenomenon linking various pathologies underlying SUD syndrome.Sudden death (SUD) is a major health problem all over the world. Estimations are that SUD is responsible for one fifth of all deaths. The most common causes of SUD are cardiac, but there are also non-cardiac reasons, such as neurogenic conditions, catecholamine toxicity, asthma attacks etc.+, L type Ca++, ultra rapid and slow delayed rectifier K+) and key proteins involved in intracellular calcium handling  endorsing arrhythmogenesis under unfavourable, pathological conditions. On the other hand, vagal nerves that innervate the heart most densely in atria release acetylcholine that binds to muscarinic, M-2 receptors coupled to inhibitory G proteins, activates K+ channels and inhibits cyclic adenosine 3′, 5′-monophosphate-dependent stimulation of L type Ca++ channels. Cholinergic transmission is further supported by nitric oxide release from cardiac nerves further preventing the occurrence of life-threatening arrhythmias.2Two components of the autonomic nervous system, the sympathetic and parasympathetic , act in concert to modulate electrical and mechanical properties of the heart. The balance between the two is fine-tuned by the brain, which processes the inputs from the periphery  and higher brain structures . Postganglionic sympathetic nerves that reach the heart release noradrenalin and activate β adrenergic receptors (β1<<β2<β3) to produce the following effects: synthesis of adenylate cyclase, formation of cyclic adenosine monophosphate and activation of protein kinase A. Protein kinase A then phosphorylates structural proteins of ion channels  and heart failure, and that enhanced sympathetic activity and impaired cardiac parasympathetic responsiveness are negative prognostic indicators for both morbidity and mortality associated with SUD.5The vast majority of the cases of SUD occur in the elderly population suffering from cardiac diseases, of which coronary artery disease is the most prevalent. Frequent cardiac diseases associated with SUD also include cardiomyopathies (both hypertrophic and dilated forms) and congestive heart failure.6 In the past few decades the mechanisms have been elucidated. Cardiac ischaemia has been shown to injure both nerves and myocardial tissue and to elicit denervation and re-innervation of the myocardium, as advocated by sympathetic scintigraphy.7 Acute ischaemic necrosis induces sympathetic denervation and the upregulation of β adrenergic receptors at cardiomyocytes inducing receptor hypersensitivity8 and hyperresponsiveness to catecholamines. Moreover, in the infarcted area, nerve growth factor (NGF) is released locally in abundance and this triggers increased expression of NGF and of growth associated protein 43 (GAP43). These are transported retrogradely to the left stellate ganglion where they induce nerve sprouting in non-infarcted sites.10 Uneven sympathetic innervations of the myocardium, and release of neurotransmitters  in hyper-innervated sites in excess,11 induce ionic imbalance and action potential prolongation that increases the risk of arrhythmias and the incidence of SUD.12During cardiac ischaemia, sympathetic hyperactivity is crucial for the generation of spontaneous ventricular ectopy and life-threatening arrhythmias.13 Areas of interstitial fibrosis slow or block electrical conduction and its peripheral zone is thought to act as a node for re-entry wave fronts.15Ischaemic and other causes of cardiomyopathy, such as infection , drug toxicity (mostly antarcycline cytotoxicity), inflammation, autoimmune diseases, and transmissible gene mutations  are associated with the formation of scars and fibrosis. Histological studies identified two forms of fibrosis: replacement fibrosis due to cell death and interstitial fibrosis due to collagen accumulation, both of which are governed by the activation of the renin–angiotensin system and β adrenergic system.16 Increased sympathetic activity directed to the failing heart increases heart rate (HR) and causes diastolic calcium leak through ryanodine type 2 (RyR2) receptors. Focally released calcium initiates more calcium release and propagates as a calcium wave that can cause delayed afterdepolarisation and engender premature beats evolving to sustained ventricular tachycardia.17 The neuroendocrine arousal in congestive heart failure incites adaptive but harmful mechanisms, NGF downregulation, reduction of sympathetic innervations18 and downregulation of β adrenergic receptors.19 Loss of adrenergic support is detrimental to the heart inotropism. An alternative scenario was observed in an experimental model of the right ventricular hypertrophy induced by monocrotaline-induced pulmonary hypertension in rats by Kimura and colleagues.20 In that model, in spite of the NGF upregulation and sympathetic hyper-innervation, there was a decrease of noradrenalin synthesis and re-uptake suggesting functional denervation occurs due to nerve rejuvenation.21Regardless of the cause of heart failure , the reduction of cardiac output triggers a reflex increase of sympathetic outflow, as well as an increase of renin–angiotensin–aldosterone synthesis and the release of antidiuretic hormone from the hypothalamus, in order to maintain the circulation.22The experimental findings on the role of the sympathetic nervous system in the occurrence of life-threatening arrhythmias is clearly supported by the efficacy of β-blockers in the prevention of SUD in patients suffering from MI, as well as the effectiveness of cardiac sympathetic denervation in patients suffering from ventricular tachycardia and fibrillation storms refractory to drug treatment.23 At autopsy, usually no morphological abnormalities are identified and the underlying cause of SUD remains uncovered. Molecular forensic pathology revealed that the population of young people that succumbed to fatal arrhythmias is affected by hereditary, genetic, disorders caused by mutations in genes encoding structural elements of cardiac ion channels responsible for abnormal electrical activity of the heart.24 There is no doubt that the autonomic activity associated with exercise and stress can trigger SUD in individuals suffering from congenital channelopathies, usually presenting normal ECG under baseline physiological conditions. The most common hereditary disorders are congenital long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT) and Brugada syndrome (BrS).A very small percentage (less than 1%) of the total population affected by SUD are young people, below the age of 35 years.25 Among these, the chromosome 7 associated hERG  mutation was the first reported and is the most prevalent.27 This gene encodes the constitutive α subunit of voltage-gated K+ channels ; defective gating; or defective ion permeation.29Congenital LQTS is characterised by delayed repolarisation of the myocardium and is genetically a heterogeneous disorder. It is often inherited in an autosomal dominant mode. Hundreds of mutations have been identified in 13 genes including KCNQ1 (LQT1), hERG (LQT2), SCN5A (LQT3), ANK2(LQT4), KCNE1 (LQT5), KCNE2 (LQT6), KCNJ2 (LQT7), CACNA1C (LQT 8), CAV3 (LQT9), SCN4B (LQT10), AKAP9 (LQT11), SNTA1 (LQT12) and GIRK (LQT13).channels . The α s+ channel is responsible for ‘rapid’ delayed rectifier K+ current, which plays an important role in ventricular repolarisation. Its malfunction is associated with prolongation of the QRST interval. Long QRST reflects the prolongation of the phase 3 action potential representing the process of cardiac cell repolarisation. As repolarisation becomes prolonged, the left ventricle becomes susceptible to a premature (i.e. early) afterdepolarisation, which triggers rapid polymorphic ventricular tachycardia ‘torsades de pointes’, the phenomenon first described by French physician François Dessertenne in 1960s. The name denotes the twisting of the points of QRS complexes around the isoelectric baseline of the ECG.The hERG KCPVT1 60%) cardiac ryanodine receptor,30 or a rare form with mutations of cardiac calsequestrin encoded by the CASQ2 gene (CPVT2). Calsequestrin is a protein in the endoplasmatic reticulum that binds calcium and releases it during depolarisation-associated calcium-induced calcium release. Mutations of the CASQ2 gene disrupts the linear polymerisation of the calsequestrin and reduces its ability to retain calcium inside the reticulum. It is usually diagnosed during exercise or stress by the appearance of ventricular ectopy typically clinically manifested with syncope or SUD.31CPVT is another heritable arrhythmia syndrome transmissible in autosomal recessive manner, as a result of mutations of genes encoding RyR2 (SCN5A gene that encodes cardiac sodium channel (BrS1).24 At rest, ECG records of the precordial V1–V3 leads show elevation of the ST segment and negative T wave. They have an increased risk of episodes of polymorphic ventricular tachyarrhythmia leading to SUD.BrS is in 30% of cases inherited as an autosomal dominant trait cause by loss-of-function mutation of the 3235 The mechanism of drug-induced arrhythmias seems to involve direct blockade of the hERG K+ channel or inhibition of hERG trafficking.36 Knowing this, an in-vitro IKr/hERG assay using the patch clamp technique has been adopted for early screening of drug candidates in a cardiac safety testing programme. 37 Also, in clinical settings a risk score for predicting QTc prolongation has been proposed and validated.39It is noteworthy mentioning that the LQTS and torsades de pointes can be acquired and induced by drugs, both cardiac (class I and class III anti-arrhythmics) and non-cardiac drugs (treatments for widely differing conditions)..40 Cannon postulated that, under special circumstances involving social pressure and susceptible psychological profile, death can occur from fright in an otherwise healthy person. He believed that fright can elicit intense activation of the sympatho-adrenal system.41 His student Curt Richter challenged his view with evidence that showed that increased vagal tone, without sympatho-adrenergic activation, provoked massive SUD in a colony of rodents whose whiskers were clipped.42 It is now established that the sympathetic and parasympathetic part of the autonomic nervous system complements each other in cardiovascular regulation and that life-threatening stressors induce sympathovagal imbalance.43In 1942, the famous professor of physiology at Harvard Medical School, Walter B. Cannon, published a paper entitled ‘Voodoo Death’ largely known as death from fright44 In these animals thrombotic coronary occlusion was not present, and the pattern of myocardial necrosis described by Selye was coagulative myocytolysis whereby myocytes die in a hypercontracted state with early myofibrillar damage and irregular cross-band formation . Lesions were multifocal, and the localisation was preponderantly subendocardial, corresponding to the distribution of sympathetic nerve endings. A confirmation for the deleterious effects of excessive adrenergic activation on the heart came from the rat model for stress-induced cardiomyopathy. Infusion of high doses of isoprenaline, a β adrenergic receptor agonist, has been shown to induce injury of the rat heart, pointing to a major role of β adrenergic receptors in catecholaminergic cardiotoxicity.45In the 1970s, clear experimental evidence that cardiac lesions can be produced as a result of nervous system disease was provided. Hans Selye was the first to produce heart injury in rats by exposing them to stressors and increased concentrations of hormones.46 towards the end of the 20th century in the Japanese population. It affected elderly women who experienced great grief. It is clinically indistinguishable from acute coronary syndrome. However, it is not associated with coronary thrombosis and the pathognomonic sign at echocardiography is apical ballooning (akinetic part) of the left ventricle resembling the shape of a ‘tako-tsubo’ ceramic pot used to trap octopus in Japan.47 Further clinical evidence for neurocardiogenic injuries came from patients suffering from pheochromocytoma, in which chronic exposure to increased concentrations of circulating catecholamines was evident.48 Multifocal subendocardial injuries were also observed in reperfusion injuries during heart transplantation, so-called ‘stoned heart syndrome’, as well as during percutaneous coronary intervention.49 In past decades advances have been made in understanding these mechanisms but not yet in providing effective cardioprotection.50Decades later, neurocardiogenic heart injuries were reported in humans, giving clinical confirmation to the experimental finding. Acute cardiomyopathy known as broken heart syndrome or Takotsubo cardiomyopathy was first described by Japanese cardiologists51 produced bradycardia and myocardial necrosis in cats by stimulation of the anterior hypothalamus. Later on, Kannan and collaborators52 in 1989 induced an increase in blood pressure (BP) and renal sympathetic outflow in conscious rats by bilateral electrical stimulation of the paraventricular nucleus (PVN) of the hypothalamus or by microinjecting L-glutamate, an excitatory amino acid neurotransmitter, into the PVN. Morphological studies complemented functional studies and provided evidence of direct PVN axonal connections, from the parvocellular part to the respiratory, pre-Bötzinger complex, vasomotor and cardiac neurons in the rostro-ventro-lateral medulla and preganglionic neurons in the inter-medio-lateral column of the spinal cord. The PVN also receives abundant inputs from other part of the hypothalamus, the limbic system and higher brain regions as well as from the periphery.53 Such neural network enables the PVN to be a key site in the coordination of behavioural and neuroendocrine response to stress. Furthermore, it has been assumed, both in animals and humans, that the increase of L-glutamate concentrations in the cerebrospinal fluid during cerebral ischaemia stimulates N-methyl-d-aspartic acid receptors in PVN and enhances sympathetic drive to the heart5457 suggesting a mechanism of SUD associated with stroke. The PVN is a major source of vasopressin and oxytocin and vasopressin receptors (V) and oxytocin receptors (OT) are found in abundance in the PVN.58 Their role is to prime magnocellular neurons and also to integrate the activity of neurons located in the magnocellular and the parvocellular parts of the PVN,59 involved respectively in humoral and autonomic neural control of the circulation.60 Lozić and colleagues62 have demonstrated that upregulation of vasopressin V1A and OT receptors in PVN modulates autonomic control of the cardiovascular system and changes the vulnerability of the rat phenotype to stress, thus pointing to possible targets for new drug development in cardioprotection from neurocardiogenic injuries.Cardiac lesions produced by central nervous system stimulation are morphologically indistinguishable from stress and catecholamine-induced cardiac damage. Back in 1963, Melville and colleagues63 and their occurrence correlates to a central increase of sympathetic activity.64 Other brain regions affected by stroke have also been associated with sudden autonomic death. Oppenheimer suggested a role for the insular cortex in the pathophysiology of SUD.65 It has been reported that right-sided ventromedial prefrontal cortex lesions bear a risk of an exaggerated cardiovascular response.66 Myocardial damage and an increase of troponins and Takotsubo syndrome have been reported in humans suffering from epileptic seizures.6769 McMillan and Teasdale70 reported a high incidence of sudden death in humans who had mild traumatic brain injury years ago. Clinical evidence of neurocardiogenic damage is usually reported in anterolateral and inferolateral leads of the ECG recording: repolarisation changes in ST segment, peaked T wave and U wave, known as hyperkalemic pattern.64In clinical practice arrhythmias are frequently seen in stroke patients without primary heart disease,71 Therefore, multifaceted evaluation using different risk markers increases the accuracy of detecting cardiac risk for life-threatening arrhythmias.72 Electrocardiographic measures such as standard 12-lead ECG QRS duration, QT dispersion, signal averaged ECG, microvolt-level T wave alternans (a beat-to-beat variability of T wave amplitude that reflects heterogeneity/dispersion of ventricular repolarisation), HR turbulence and HR deceleration capacity, have all been proposed.73 In electrophysiological testing, a programmed ventricular stimulation is used for risk stratification. Induction of sustained ventricular tachycardia during programmed ventricular stimulation is a marker of increased risk of recurrent sustained ventricular tachycardia and sudden cardiac death.74Patients at greatest risk for SUD are those with coronary artery disease and impaired left ventricular function, heart failure secondary to ischaemia or idiopathic dilated cardiomyopathy, hypertrophic cardiomyopathy, documented sustained ventricular tachycardia or fibrillation, and survivors of cardiac arrest. Assessing the cardiovascular function and the degree of impairment of left ventricular ejection fraction (LVEF) is currently used for prognosis in patients with structural heart disease. It is acknowledged that a LVEF of 40% or less is an independent risk factor. However, it should be noted that there is no linear correlation between the degree of left ventricular dysfunction and the prevalence of fatal arrhythmias, the occurrence of which is higher among patients with mid to moderate heart failure, rather than in those with severe failure.75 but it became recognised in the late 1980s when it was confirmed that HRV is a strong and independent predictor of mortality after MI.7678 Many methods in both the time and frequency domain are used to calculate HRV as detailed elsewhere.79 However, the true nature of HRV was discovered by the method of spectral analysis in animal models.80 In animals and humans, the lower frequencies of the power spectrum of HRV are created by the activity of the sympathetic nervous system directed to the heart, whereas higher frequencies are created by vagal activity modulated by respiration and depict respiratory sine arrhythmia.81 In elderly individuals, the lower frequency components of the HR spectrum are observed because respiratory sine arrhythmia is lost with ageing.82 In diseased individuals, in patients surviving an acute MI and in patients suffering from chronic heart failure or left ventricular dysfunction, reduced or abnormal HRV are indicators of an increased risk of mortality within a few years after an acute MI or after a diagnosis of congestive heart failure or left ventricular dysfunction.8385 HRV has proved to be useful in predicting SUD in patients suffering from non-cardiac causes.86 Picturesque methods for the assessment of HRV are colour spectrograms  and baroreflex sensitivity (BRS). The clinical relevance of HRV was first observed in 1963 by Hon and Lee,trograms  and Pointrograms . When th91 Baroreceptors located in the upper part of the body, aortic arch and carotid sinuses sense subtle changes in central BP and adjust circulation to all organs, but especially to the brain, on a beat-to-beat basis. The more sensitive the baroreflex, the less variable is the BP, and the lower is the risk of stroke.92 Under physiological conditions, BRS decreases during exercise and stress,94 in order to permit concomitant increase of arterial BP and HR to supply adequate circulation to the body. However, a decrease in BRS occurs in disease, in survivors of MI and in patients suffering from chronic heart failure, and represents an independent risk factor for SUD.91The baroreflex is the main negative feedback corrector of arterial BP, and evaluation of BRS is an established tool for the assessment of autonomic control of the cardiovascular system.95 thus circumvents the use of drugs and is without risk for patients, so may be applicable in wide populations. Several useful parameters have been proposed to evaluate the function of the spontaneous baroreflex. Experimental evidence indicates that an increase in spontaneous BRS over an increase in operating range indicates allostatic overload and may precede the exhaustion of the neurogenic mechanisms.94BRS can be assessed in an old fashioned manner using pharmacological tools (Oxford method) and using novel methods from spontaneously occurring BP and pulse interval variability in the low frequency and high frequency spectral bands. The Oxford method requires injections of short-acting vasoconstrictor or vasodilating drugs to trigger a reflex HR response, but this carries serious risks for the patient and is thus not widely used in clinical practice. However, the sequence method assesses the BRS from spontaneously and concomitantly occurring systolic BP and pulse interval increasing/decreasing values,d-sotalol increases mortality.96 A new drug, dronedarone, is promising.97 β-Blockers have been applied with success in women suffering from hereditary long QT syndrome, in the postpartum period when the risk from cardiac events is several fold increased.98 In the therapy of coronary artery disease and congestive heart failure the so-called non-anti-arrhythmic drugs are useful in the prevention of SUD, although their effect is indirect and delayed. These include angiotensin-converting enzyme inhibitors and angiotensin II AT1 receptor blockers, statins, and nutrients such as fish oil.98The current management of SUD involves drugs and electrical devices for correction of arrhythmias. In patients with ischaemic heart disease and congestive heart failure β-blockers were found to be most effective and reduced total mortality. Other anti-arrhytmic drugs are not well tolerated because they have pro-arrhythmogenic action. Class I anti-arrhythmic drugs in patients with ischaemic heart disease, especially class 1C, have been shown to increase mortality. Class III, dofetilide and amiodarone have no effect on mortality while 99In patients with very low LVEF, asymptomatic ventricular arrhythmias and inducible ventricular tachycardia/fibrillation, an implantable cardioverter-defibrillator (ICD) is superior to conventional drug therapy. In secondary prevention of SUD, in patients with sustained ventricular tachycardia/fibrillation or survivors of sudden cardiac death drug treatment alone is ineffective and can be used as an adjunct to ICD to decrease episodes of ventricular tachycardia/fibrillation and non-sustained ventricular tachycardia to reduce ICD discharge, to slow the rate of ventricular tachycardia and to increase haemodynamic stability.100 The first-into-man study, although performed on a small sample and with a short follow-up period confirmed the beneficial effects of BAT in advanced stages of chronic heart failure.101 In the first randomised and controlled trial patients with heart failure with reduced ejection fraction also benefited from BAT treatment.102 An alternative approach to restore the sympathovagal balance to the heart is to stimulate the vagus nerve directly. Experimental studies indicate that several mechanisms are likely to be involved in the beneficial effect of vagal stimulation.103 These include anti-adrenergic effects, anti-apoptotic effects, an increase of nitric oxide synthesis and anti-inflammatory effects.104 The first-in-man study with chronic heart failure confirmed that the method is safe and well tolerated, and significant improvement of the LVEF and life quality was observed.105 There are experimental indices in rabbit and rats that BRS may be increased by chronic exposure of baroreceptors to a medium intensity static magnetic field.106 Studies indicate that a medium intensity static magnetic field affects BRS and cardiovascular haemodynamics by modulating microcirculation, neurotransmission and the rate of biochemical reactions.107Patients with advanced heart failure and low LVEF in which symptoms progress despite optimal pharmacotherapy and ICD device implantation may benefit in future from devices engineered to restore sympathovagal balance to the heart by electrical neuromodulation. Several options have been investigated in animal models, involving baroreflex activation therapy (BAT) and vagal stimulation. In dogs with experimentally induced heart failure and very low LVEF, BAT reduced sympathetic tone and enhanced vagal tone, reduced susceptibility to life-threatening arrhythmias and prolonged survival.Finally, it can be concluded that in spite of the development of new anti-arrhythmic drugs and DCI devices, SUD remains a frequent cause of death worldwide. In the past few decades progress has been made in the multifaceted risk stratification approach by the introduction of autonomic markers into clinical practice. A new approach of treatment by electrical neuromodulation is under development for the prevention of SUD in advanced stages of congestive heart failure. The results of large scale clinical studies are still awaited to confirm the clinical efficacy and tolerability of electrical neuromodulation that may hopefully become a new cornerstone in the prevention of SUD in these patients.


Resveratrol is a polyphenol that has witnessed an unprecedented yearly growth in PubMed citations since the late 1990s. Based on the diversity of cellular processes and diseases resveratrol reportedly affects and benefits, it is likely that the interest in resveratrol will continue, although uncertainty regarding its mechanism in different biological systems remains.We hypothesize that insights on disease-modulatory activities of resveratrol might be gleaned by systematically dissecting the publicly available published data on chemicals and drugs. In this study, we tested our hypothesis by querying DTome (Drug-Target Interactome), a web-based tool containing data compiled from open-source databases including DrugBank, PharmGSK, and Protein Interaction Network Analysis (PINA). Four direct protein targets (DPT) and 219 DPT-associated genes were identified for resveratrol. The DPT-associated genes were scrutinized by WebGestalt . This enrichment analysis resulted in 10 identified KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. Refined analysis of KEGG pathways showed that 2 — one linked to p53 and a second to prostate cancer — have functional connectivity to resveratrol and its four direct protein targets. These results suggest that a functional activity network (FAN) approach may be considered as a new paradigm for guiding future studies of resveratrol. FAN analysis resembles a BioGPS, with capability for mapping a Web-based scientific track that can productively and cost effectively connect resveratrol to its primary and secondary target proteins and to its biological functions. Interest in resveratrol increased greatly following the report by Jang et al. in 1997 showing that it displays chemopreventive activities as evidenced by the inhibition of carcinogenesis using  studies –11. WhatIn recent years, the successful launch and implementation of numerous multi-center genomics studies, encompassing the use of microarrays, proteomics, and other high-throughput screening assays often produce hundreds to thousands of intriguing gene hits which can present formidable challenges for integration, phenotypically or thematically. A possible solution to this analytical obstacle in data mining may lie in the use of the network-based approach — a simple yet effective method that has found application in the analysis of massive data obtained from numerous human disease studies designed to explore and connect the relationship existing between a drug and its targets and interacting proteins, for association with disease –15. DTomIn this study, we introduce a FAN analysis capable of effectively analyzing and prioritizing candidate genes quickly for linkage to experimentally verifiable grouped pathways through the deployment of Web-accessible, open portal databases. As test of principle, we applied DTome to the widely studied polyphenol resveratrol. We found that 3 of its 4 identified direct protein targets are linked to the tumor suppressor gene p53. When the readout targets and their associated genes were further analyzed for functionality through pathway enrichment analysis, the p53 pathway was identified as one of top 10 enriched KEGG pathways containing 8 resveratrol-associated genes. We propose that the DTome-directed FAN analysis is a new paradigm for analyzing resveratrol:p53 interplay; the flagged genes involved in resveratrol-mediated p53 control can be experimentally verified and new insights on the biological context of activation/restoration of p53 function by resveratrol might be illuminated. The approach has implications for understanding the prevention of cancer by resveratrol and can aid in the treatment of malignant tumors harboring the dysfunctional p53.In biological systems, chemicals , proteins, and genes interact with one another to result in a physiologic function at the cellular and organ levels. This higher-order, complex interaction among molecules can be visualized as a network which typically consists of nodes representing biological entities  and edges representing the relationships among them . We first queried DTome using resveratrol as input. This resulted in output DB02709 categorizing resveratrol as antioxidants, platelet aggregation inhibitors, enzyme inhibitors, anti-carcinogenic agents, anti-neoplastic agents, phytogenic and anti-mutagenic agents. Moreover, using the grouping status of resveratrol as an experimental drug, query of DTome indicated that it is a chemical investigated for the treatment of herpes labialis infections (cold sores) Table . Table 2Helicobacter pylori infection (8 genes) and melanogenesis (9 genes) , cell cycle (15 genes), MAPK signaling 19 genes), Wnt signaling (15 genes), Neurotrophin signaling (12 genes), adherens junction (10 genes), prostate cancer (10 genes), p53 signaling (8 genes), epithelial cell signaling in 1 genes,  genes, WAlthough DTome proffers resveratrol as the choice of treatment for infections Table ; the funcBio portal also provides interactive analysis and construct networks to view genes that are altered in cancer. Here, we first show the network to contain all neighbors of 3 query genes, PTEN, TP53 and CDKN1A  combined with cBio portal . Analysis of resveratrol using these two Web-based resources identified 4 primary DPTs  Table  and 10 edis=35 nM) using affinity chromatography and X-ray analysis [Another aspect of FAN analysis relevant to its utility and implications for the role resveratrol plays in prostate cancer relates to the three primary targets of resveratrol: CSNK2A1, NQO2 and PTGS2  understanding of malignant disease mechanisms, (ii) facilitate early disease diagnosis and improve the accuracy of disease prognosis, and (iii) unravel critical roles of resveratrol in human cancers. This approach is superior to the manual integration of information on resveratrol and its target genes which can be challenging, labor-intensive, and error-prone, possibly because an extremely large amount of heterogeneous data sources must be considered, tested and assayed. Comparatively, FAN analysis is completely machine-readable integrated from heterogeneous sources: DTome, KEGG pathway and cBio portal. FAN analysis can be used to obtain unified resveratrol-relevant knowledge and bring insights into the regulation and control of cancer disease processes. By providing a deeper understanding of resveratrol's functions, FAN analysis will also assist resveratrol bio-curation and new biological experiment design and could significantly accelerate cancer biology research. Although generally configured for providing primary genomic results rather than clinical associations, FAN analysis is extensible and can be readily generalized to other biomedical areas. The candidate genes revealed can facilitate the interpretation of genomic results for non-computational biomedical researchers and other users.Lastly, one can surmise that FAN analysis may find application in the repurposing of drugs. Traditional drug development faces unique challenges mainly due to high cost and long duration for approval of use in clinical settings . AccordiSome difficulties remain in applying computational learning techniques to problems of predicting chemical/drug response. These challenges are appropriately summed up in one phrase: the intrinsic variability and difficulty of reproducibility of biological data. There is significant inherent nonrepeatability of experimental conditions and/or biological phenomena. Quality control on the extremely costly equipment used to make biological measurements can be a more insidious factor contributing to variability and lack of repeatability. To some degree, both factors can be in part counteracted by the FAN analysis.In summary, as advances in research on resveratrol using traditional experimental techniques and approaches continue, more resveratrol targets will unquestionably be identified; nevertheless, the determination as to whether a specific biological effect on resveratrol can be definitively assigned to individual or simultaneous modulation of its newly or established identified targets can remain obscure. We believe that some of the uncertainty in connecting resveratrol to its targets and subsequent phenotype expression can be offset using FAN analysis, and that this approach can be used to direct future studies with reasonable experimental feasibility for testing and validating rationalized hypotheses regarding the effects of resveratrol in cancer. For example, a functional network can be developed based on the core elements of FAN analysis to further evaluate the impact of resveratrol as gauged by the changes in altered gene sets shown for a specific cancer type or cancer in general by expanding the complicity of cBio portal altered gene network Figure . In addiDTome is a web-based bioinformatic tool able to query the information embedded in three open-source databases, respectively, DrugBank, PharmGSK, and PINA; the data compiled are analyzed, grouped, and used to construct a drug-target interactome . InteracThrough use of the DTome search function, drug-protein and second level protein-protein interactions data were first generated for resveratrol. The data were integrated into a resveratrol-mediated network and analyzed using Cytoscape (version 2.8.0) . The biohttp://cbioportal.org) is an open platform for exploring multidimensional cancer genomics data by encapsulating molecular profiling data obtained from cancer tissues and cell lines into readily understandable genetic, epigenetic, gene expression, and proteomic events [The cBio Cancer Genomics Portal (c events . ComplexIn this study, the cBio Portal was used to explore the connectivity of resveratrol associated genes across all prostate cancer studies available in the databases. Through use of the portal search function, resveratrol associated genes in all samples of prostate cancer studies are classified as altered or not altered. The genomics datasets are then presented using OncoPrint as heatmaps — a visually appealing display of alterations in gene arrays across tumor samples . Another


In a Perspective, Klaus Pantel discusses prospects for use of cell-free DNA and circulating tumor cells in cancer detection. Analysis of circulating cell-free DNA (cfDNA) or circulating tumor cells (CTCs), also known as liquid biopsy, has received substantial attention in recent years owing to the potential benefits for detecting tumors and informing treatment . cfDNA iBesides CTCs and ctDNA, liquid biopsy analyses can also focus on microvesicles (particularly exosomes), cell-free microRNAs, and blood platelets, which also provide interesting information on molecular features of a tumor lesion and might, therefore, have the potential to contribute to early detection of cancer –10. MoreAs an example, Fernandez-Cuesta and colleagues  recentlyIn addition to the need for higher sensitivity, the specificity of cfDNA measurements also faces serious challenges. Recent reports have shown that cancer-associated mutations are not restricted to cancer patients. For example, TP53-mutated cfDNA fragments were observed in 11.4% of 123 matched noncancer controls . LikewisBRAF, RAS, EGFR, HER2, FGFR3, PIK3CA, TP53, CDKN2A, and NF1/2 genes, all of which are considered hallmark drivers of specific cancers, can also be identified in benign and premalignant conditions, occasionally at frequencies higher than in their malignant counterparts [BRAF V600E mutations are paradoxically more frequent in benign nevi (∼80%) than in dysplastic nevi (∼60%) or melanomas (∼40%–45%) [FGFR3 mutations in seborrheic keratosis [The findings of such studies appear to reflect general principles that challenge the promise of ctDNA analysis for early cancer detection. Genomic alterations, such as those in the terparts . For exa40%–45%) . Driver eratosis .Cancer patients harbor CTCs in their bloodstream that can be detected at very low concentrations using advanced technologies . After aAchieving acceptable levels of sensitivity and specificity for early cancer detection will require further technical advances. Extensive research is required to identify possible combinations of cancer-specific mutations and define potential quantitative thresholds to avoid overdiagnosis. Based on the findings to date, it can already be envisaged that DNA sequencing needs to be broad, so as to encompass considerable tumor heterogeneity, and deep, to detect minute amounts of ctDNA fragments in the milieu of extensive genetically normal cfDNA. Nonmalignant conditions that can lead to the death of normal cells may also lead to a further dilution of ctDNA molecules and hamper quantitative evaluations. Very sensitive technologies that allow the detection of less than 0.1% of ctDNA in blood plasma  have been developed and applied to cfDNA analyses in patients with various forms of cancer ,6,22, buThe potential use of CTCs for early cancer detection faces similar challenges of sensitivity and specificity. DNA sequencing of single CTCs has been successfully performed by several groups, but the whole genome step required to obtain sufficient amounts of DNA requires standardization . RNA seqStudies evaluating new approaches to early cancer detection usually begin with comparisons of small cohorts of cancer patients with controls . Subsequent investigations will typically involve larger study populations and extended follow-up times and require substantial financial resources. Focusing on population groups at an elevated risk of developing cancer  is a good strategy to speed up the process of testing and validation of new approaches and technologies. In contrast, large population-based screening efforts may reveal tumor lesions that cannot generally be cured by current therapies  or that have an indolent disease course in many patients  and where the need for therapy is not clear.Another important clinical application is early detection of minimal residual disease (MRD) in patients with resected primary tumors. Postsurgical surveillance of MRD by cfDNA analysis has enabled investigation of clonal evolution in colorectal cancer patients ,28. CurrFinally, verification of the findings of approaches involving liquid biopsy will call for acquisition of information on the putative location of the occult lesion to select the appropriate imaging modalities or other diagnostic means prior to planning appropriate therapies. In this context, the detection of tissue-specific gene expression in CTCs  might be


Thermotogales and Clostridiales adapted to living on hydrocarbons and complex refractory organic matter. However, as seawater and nitrate injection (used for secondary oil production) delivered oxidants, the microbial community composition progressively changed to fast growing opportunists such as members of the Deferribacteres, Delta-, Epsilon- and Gammaproteobacteria, with energetically more favorable metabolism . This perturbation has profound consequences for understanding the microbial ecology of the system and is of considerable practical importance as it promotes detrimental processes such as reservoir souring and metal corrosion. These findings provide a new conceptual framework for understanding the petroleum reservoir biosphere and have consequences for developing strategies to manage microbiological problems in the oil industry.Subsurface petroleum reservoirs are an important component of the deep biosphere where indigenous microorganisms live under extreme conditions and in isolation from the Earth’s surface for millions of years. However, unlike the bulk of the deep biosphere, the petroleum reservoir deep biosphere is subject to extreme anthropogenic perturbation, with the introduction of new electron acceptors, donors and exogenous microbes during oil exploration and production. Despite the fundamental and practical significance of this perturbation, there has never been a systematic evaluation of the ecological changes that occur over the production lifetime of an active offshore petroleum production system. Analysis of the entire Halfdan oil field in the North Sea (32 producing wells in production for 1–15 years) using quantitative PCR, multigenic sequencing, comparative metagenomic and genomic bins reconstruction revealed systematic shifts in microbial community composition and metabolic potential, as well as changing ecological strategies in response to anthropogenic perturbation of the oil field ecosystem, related to length of time in production. The microbial communities were initially dominated by slow growing anaerobes such as members of the  Despite significant efforts to reduce our dependence on energy from fossil fuels, oil, gas and coal still meet around 80% of global energy demand, and International Energy Authority projections indicate that fossil fuels will likely continue to be the dominant sources of energy and transportation fuels for many years .Petroleum reservoirs are an important component of the deep biosphere where autochthonous microorganisms live under extreme temperature and pressure conditions. However, this slow, diffusion-driven deep biosphere environment, which has been relatively stable and isolated over millions of years, is transformed, during oil exploration and production, into an advection-driven system where new electron acceptors, donors and exogenous microbes are introduced to the formerly static environment. Indeed, following primary recovery of oil from a reservoir, where oil and formation water are pushed to the surface by the natural pressure within the reservoir, water injection is used to maintain pressure of the reservoir and achieve higher levels of oil production (secondary recovery). Water injection, however, can have detrimental consequences, notably in offshore facilities when seawater is injected, stimulating deleterious microbial activities such as hydrogen sulfide production (souring), corrosion and reservoir plugging, which in turn lead to increased refining costs and health risks . Microbi−1 of nitrate before injection as a strategy for the prevention of souring and metal corrosion. Indeed, continuous nitrate injection was demonstrated to efficiently limit corrosion problems in the early years of production in the Halfdan oil field  in the North and formation water B (FWB) in the South, East and West of the field). In addition, the field includes a gas reservoir (Ekofisk) . This teAnalysis of an unrivaled sample set representing different production stages of the Halfdan oil field, ranging from early primary, through to late secondary oil production, has allowed us to assess emergent microbial community properties across the 32 producing wells over the 15 year operational history of the field. In combination with physicochemical data available for each of the wells, this study provided a unique opportunity to understand not only the interactions between reservoir conditions and microbial community composition, but also the interplay among the measured physicochemical parameters.2) were collected in December 2014  from 32 producing wells, drilled at different times since 1999 and covering the entire Halfdan oil field  with four different primer sets  genes from mer sets . Additiomer sets . Amplifimer sets .mcrA and dsrAB genes were also amplified in triplicate from a single DNA extract per sample as described in M then pooled together. After final dilution, 4 pM libraries containing 5% phiX V3 control DNA (Illumina) were loaded on Illumina MiSeq V3 cartridges (Illumina) for sequencing.16S rRNA genes were amplified in duplicate from two independent DNA extracts (* and ¤) as presented in the Metagenomes for eight produced fluid samples, selected based on their contrasted community composition, were constructed using a Nextera XT library Kit (Illumina) according to the manufacturer’s recommendations with 1 ng of genomic DNA. An equal volume (2.5 μl) of DNA replicate (*) and (¤) were pooled and used as DNA template. Fragmentation and indexing were checked using a High Sensitivity DNA chip on an Agilent Bioanalyzer 2100  then libraries were diluted to 14.3 pM and sequenced using Illumina Miseq V3 kit (Illumina).dsrAB gene database  , similarity percentages breakdown procedure (SIMPER), analysis of similarity tests (ANOSIM)) were carried out according to recommendations of the Guide to Statistical Analysis in Microbial Ecology , using R2+, K+) and sulfate  and sulfate , higher sulfate (up to 4787 mg l–1), nitrate (up to 43 mg l–1) and nitrite (up to 7.6 mg l–1) concentrations were detected in cases of seawater breakthrough, whereas higher temperature (80 °C), higher ammonium (up to 52 mg l–1) and volatile fatty acids  of injected seawater in produced fluids were observed depending on the age of the wells and the progression of the water front through the oil reservoir . Wells wmcrA and dsrAB) to determine the microbial community composition and abundance in all produced fluids. In addition, shotgun metagenome sequencing of eight samples, selected based on their community composition and produced fluid geochemistry was performed. 16S rRNA genes recovered from the metagenome data were extracted and analyzed to confirm the microbial community composition observed by amplicon sequencing.DNA extraction from the 32 produced fluid samples was performed in duplicate and followed by PCR amplification, amplicon sequencing and quantitative PCR of several genes  community of comparable cell density to seawater was detected in the produced fluids  (P>0.4) between each of the replicates  . No signplicates . The micArchaea represented 1–38% of the microbial community composition based on both qPCR and metagenomic data . Although archaeal abundance was significantly lower in samples with seawater breakthrough , archaeal communities were similar in all producing wells and were dominated by thermophiles from the families Archaeoglobales and Thermococcales  were infrequently detected by 16S rRNA and mcrA gene sequencing , Clostridia  or Deferribacteres  (Deltaproteobacteria) were detected in all producing wells by 16S rRNA and dsrAB genes sequencing  were detected in the produced fluids . These wnalysis)  and 2a. quencing .Deferribacteres/Flexistipes lineages  and metagenome  analysis. In addition, the Bacteria to Archaea ratio increased by more than sixfold  in those wells, mainly because of a significantly lower archaeal abundance  on bacterial community composition. Strong correlation was detected between the relative proportion of bundance .Epsilionproteobacteria  and metagenome data sets , bacterial communities were significantly different  depending on the formation water composition (Clostridia (13±16% s.d.) (Firmicutes), Pelobacter (11±10% s.d.) (Deltaproteobacteria) and Thermovirga (8±13% s.d.) (Synergistes) were predominant. By contrast, in samples from the second oil accumulation with formation water FWB, and where produced fluids, comprised <10% seawater, the bacterial communities were dominated by members of the genera Petrotoga (36±22% s.d.) (Thermotogae) and Desulfotomaculum (16±13% s.d.) (Firmicutes). However, wells HBA-09 and HBA-25 from FWB seem to diverge from this general pattern with a community composition more similar to communities observed in produced fluids from FWA. These discrepancies may be because of unrecognized connectivity between these FWB wells and regions of the reservoir associated with FWA.RDA also revealed a strong influence of the formation water composition on bacterial community composition. Producing wells were clustered according to their formation water  was generally congruent with the clustering of the wells based on geochemical and operational data , and (poly)-saccharides , and for acetogenesis  was apparent in wells with a low proportion (below 10%) of injected seawater (In producing wells with a high injection water fraction (68% and above), there was evidence for considerable potential for nitrate reduction by members of the teraceae . Similarseawater .Genomic bins corresponding to representatives from a number of microbial lineages were assembled from the combined metagenomic data sets . MetabolbssA) for activation of alkyl-substituted aromatic hydrocarbons, were detected in most of the deltaproteobacterial genomic bins, in two bins from Clostridiales (Thermicola and Eubacterium), in Draconibacterium and in the most complete bin from a Thermovirga sp.. Gene clusters encoding the pathway for anaerobic degradation of monoaromatic compounds (bad genes) were identified in Desulfobacteraceae, Thioalkalivibrio (Gammaproteobacteria) and Flexistipes (Deferribacteraceae) bins.Overall, the reconstructed genomic bins suggested a predominance of heterotrophy. The bacterial bins revealed a large repertoire of pathways for organic matter metabolism  including fermentation  . HydrocaEpsilon- and Gammaproteobacteria bins. Sulfate reduction potential  was identified in Archaeoglobus, Desulfotomaculum (Firmicutes) and Deltaproteobacteria genomic bins, but these genes were not found in bins from members of the Desulfuromonadales (Desulfuromonadaceae and Pelobacter). Genes involved in thiosulfate reduction were detected in Desulfobulbaceae, Dethiosulfovibrio (Firmicutes) and in Alcanivoracaceae and Thioalkalivibrio (Gammaproteobacteria) bins. Polysulfur reduction potential was detected in bins affiliated to Eubacterium, Kosmotoga, Candidate division OP9 and Geoglobus lineages. Hydrogen sulfide- (Sox) and sulfur- (Oxi-dsr) oxidation genes were identified in Thioalkalivibrio and Thiotrichales genomes and sulfide:quinone reductase gene encoding SQR for the oxidation of sulfide to polysulfide were detected in both Epsilon- (Sulfurovum and Sulfurospirillum) and Gammaproteobacteria  genomics bins. Genes for nitrate or nitrite reduction were identified in Delta- Epsilon- and Gammaproteobacteria bins. Nitrate reduction potential was also detected in a Flexistipes (Deferribacteraceae) bin. The capacity for iron reduction via extracellular multiheme cytochromes (mtrA/omcB) was also detected in Flexistipes as well as in Pelobacter and Desulfuromonadaceae genomic bins. Methane (pmoA) or methanol (mxaF) oxidation genes were detected in gammaproteobacterial Thiotrichales and Methylophaga bins respectively, whereas an autotrophic methanogenesis pathway was identified in Methanoculleus and Methermicoccus bins (Various high-energy yielding metabolic pathways were identified. The capacity for aerobic respiration at low oxygen concentration (high-affinity cbb3 cytochrome oxidase) was found in cus bins .There have been very few studies of the geochemistry and microbial communities across an entire oil field. A series of studies of the onshore, Medicine Hat Glauconitic C (MHGC) field provide a notable exception (mcrA and dsrAB genes) and metagenomic analysis of the produced fluids from 32 wells in the Halfdan oil field revealed considerable variation in terms of microbial community composition, abundance and in situ metabolic potential. Our data clearly demonstrated that contrasting microbiomes identified in different wells from a single oil field were related to the production history of the wells and hence associated geochemistry. Depending on the proportion of injected seawater in producing fluids, reflecting the progression of oil through the reservoir and the different periods in the production lifecycle of the oil field, microbial community composition was dominated either by members of the Thermotogales, Clostridia, Deferribacteres or Proteobacteria. The metabolic capacities of these microbial communities were respectively centered either on degradation and fermentation of detrital organic matter (cellulose and other complex glycans such as laminarin and fucose), acetogenesis and aromatic hydrocarbon degradation, or, nitrate reduction and oxidative processes such as hydrogen sulfide, sulfide or sulfur oxidation, as demonstrated by metagenomic mining and genomic bin reconstruction. An important consequence of such differences in community composition and function from a single oil field is that community composition or metagenomic reconstruction from a single well, are not representative of the entire reservoir but rather relate to a specific stage in an oil field production cycle. This finding has serious implications for microbial monitoring and control practices for field operators.Although we do not present a conventional longitudinal study, in that a single well was not sampled over time to assess succession in the microbial communities, we have used the sampling of wells from the same field that have been in production for different periods, as a proxy for time. Given the sampling constraints in an operating oil field, and that the provenance of the produced waters from the different formations in the field is relatively well constrained we consider this to be a pragmatic and defensible approach. Quantitative PCR, sequencing of single genes , influence of the seawater injection appears to be negligible and the microorganisms identified could be considered primarily as indigenous to the oil field. In these producing wells, statistical analyses  indicated that microbial community composition and metabolic potential was shaped by the produced water composition , which iArchaeoglobales and Thermococcales), commonly observed in hot oil reservoirs (>70 °C) (dsrAB) indicated a strong potential for hydrogen sulfide production in these Archaea. Cultivated Thermococcales gain energy by fermentation using peptides as the carbon source and most of them require elemental sulfur as an electron acceptor (hydBG) involved in elemental sulfur/polysulfide reduction in Thermococcus were identified. Capacity for hydrocarbon degradation coupled to sulfate reduction in members of the Archaeoglobales was also detected in the Halfdan oil field, supporting previous genomic analysis of the Archaeoglobus lineage (mcrA genes sequencing) was dependent on the formation water associated with the sampled wells. Ninety percent (9/10) of the wells associated with FWA harbored detectable methanogens, whereas they were detected in <50% (9/19) of the wells associated with FWB. This suggests that formation water composition has an important influence on the occurrence of methanogens in petroleum reservoirs. Genomic bins of two methanogens affiliated to Methermicoccus and Methanoculleus indicated that H2 and CO2 were the predominant substrates for methanogenesis  for activation of alkyl-substituted aromatic hydrocarbons were detected in the most complete Thermovirga bin as well as in metagenomic reads , suggesting the possibility that Thermovirga spp. potentially have a role in aromatic hydrocarbons degradation. Similarly, genomic bins of Clostridia and Pelobacter highlighted considerable potential for peptide degradation and production of volatile fatty acids through fermentation. This is consistent with the significant correlations observed between relative abundance of 16S rRNA genes from Clostridia and Pelobacter, and propionate , as well as acetate  concentrations in produced fluids. Together this indicated that in low sulfate, and low-nutrient formation waters, indigenous microbial communities were driven by fermentation of complex carbon sources and anaerobic hydrocarbon degradation potentially linked to archaeal hydrogenotrophic methanogenesis as previously suggested by crude-oil biodegradation experiments , frequently found in thermophilic lineages . These catabolic properties are commonly observed in thermophilic microorganisms isolated from oil reservoirs (bssA gene) was detected in a genomic bin of a Desulfotomaculum-related bacterium, an observation consistent with previous survey of benzylsuccinate synthase genes  were dominated by lineages . Geneticservoirs . Peptideservoirs  or by thThese data indicated that petroleum reservoir formation water composition is an important factor shaping the potential activity and community composition of the indigenous microbes, which in turn gradually modify the formation water composition by production of metabolic products . The low sulfate concentration in produced fluids, compared with formation water samples, from wells, which exhibit little influence of seawater injection , may be dsrAB genes n=11; 9 Deltaproteobacteria, 1 Desulfotomaculum, 1 Archaeoglobus; SimpsondsrAB gene amplicons: 0.52±0.16), confirming that microbially driven souring is at least in part attributed to sulfate-reducing Deltaproteobacteria, or Archaeoglobales, as reported for other high temperature reservoirs  may have in hydrogen sulfide production in high temperature petroleum reservoirs  and the effect of nitrate dosing for souring mitigation was evident in the microbial communities, as previously reported (dsrAB) and sequences from sulfate-reducing microorganisms in 16S rRNA gene libraries from samples influenced by seawater injection. By contrast, abundant 16S rRNA and nitrate reduction genes recovered from members of the Deferribacteres lineages were detected, indicating a major role for Deferribacteres in nitrate reduction in these seawater-rich produced fluids  and can utilize nitrate, iron and elemental sulfur as electron acceptors . These very long-term seawater-injected producing wells exhibit a further shift in microbial community composition. Consistent with the cooling of the reservoir, the relative abundance of thermophilic Archaea decreases whereas mesophilic Epsilon- and Gammaproteobacteria, previously identified in the seawater and injected fluids of this and other oil facilities . Substrates to support the metabolic repertoire of these microorganisms were presumably furnished by the large amount of injected seawater and/or produced by the microbial communities that were dominant during the earlier stages of the reservoir’s production history .In producing wells with the longest history of seawater exposure (>12 years in service as of 2014), produced fluids, comprised >95% seawater, leading to a decrease of the Deferribacteres, which were prevalent in higher temperature wells and when lower levels of seawater breakthrough were apparent. However, since the rapid growth of exogenous microorganisms may lead to plugging of porosity in the reservoir, and the elemental sulfur generated by sulfide oxidation is as corrosive as hydrogen sulfide  in the make-up of indigenous reservoir microbial communities and that multiple native reservoir communities can co-occur in a single oil reservoir when different oil accumulations are present. Third, our results provided evidence that anthropogenic interventions for oil production are significant drivers of the oil field microbiome and engender considerable spatial and temporal variability of oil field microbial communities regardless of the indigenous microbiome. When secondary production begins, injected fluids cool the reservoir and deliver exogenous oxidants, changing the microbial communities considerably as the energetic potential of the environment increases over the production lifetime of the field. Anthropogenic perturbation of the oil field ecosystem may appear to effectively limit the souring capacity of the oil reservoir microbiome. However, extended water flooding may open new avenues to metal corrosion and plugging when the temperature becomes permissive and the proportion of exogenous bacteria outcompete the indigenous communities. Microbial monitoring and control programs therefore, should account for a diverse and dynamic system ensuring adequate sampling coverage and frequency.