There has been a large confirmation over the last decades that stroke may produce cardiac changes . In ischemic stroke, systolic dysfunction is associated with a high risk of mortality during hospitalization. A recent study demonstrated that cardiac diastolic dysfunction could also accompany acute stroke besides the systolic dysfunction already pointed out by previous studies, being a predictive marker of acute cerebrovascular events. Increased sympathetic activity is contributory, inducing a reversible cardiac myocyte damage and cardiac enzyme surges. Some of the most frequent electrocardiographic abnormalities in stroke are ST segment abnormalities and various tachyarrhythmias  and bradyarrhythmias. One can infer the importance of careful and continuous electrocardiographic monitoring of the stroke patient in order to identify these quite frequent electrocardiographic alterations, as it is well known that death due to cardiac arrhythmias is common among acute stroke patients. In order to increase the diagnostic yield, a high level of NTproBNP  may be used as a discriminant for the patients with a higher probability of cardiac arrhythmias and mortality at presentation, during hospitalization and on the long term. In such patients, cardiac monitoring techniques are more likely to reveal abnormalities. A high BNP level may have potentially important management implications as it may signal a worse prognosis and may prompt the undertaking of certain therapeutic measures. This review summarizes the possible pathological mechanisms of heart-brain connections and their clinical and therapeutical implications. Abbreviations: AF = atrial fibrillation, ECG = electrocardiography, HRV = heart rate variability, cTn = cardiac troponin, SAH = subarachnoid hemorrhage, CK-MB = creatine kinase-MB, BNP = brain natriuretic peptide, NT-proBNP = N-terminal of the prohormone brain natriuretic peptide, ANP = atrial natriuretic peptide, mRS = modified Rankin Scale, NIHSS = the National Institutes of Health Stroke Scale.  More than half of the stroke patients are left with a major disability . Approximately 85% of strokes are ischaemic, the remainder 15% being haemorrhagic [4]. The most frequent causes of ischaemic stroke are atherosclerosis - 50%, [5] (- P262), cardioembolism - 20%, [5] (- P260) and microinfarcts (lacunar strokes) - 25% [5] (- P270) [3]. Rare causes (responsible for 5% of cases) are arterial dissection and patent foramen ovale [5] (- P355-358).Stroke is one of the most important causes of mortality and morbidity in industrialized countries. In Europe, it is the second mortality cause, accounting for approximately 10% of all death causes in men and 15% in women [Neuro-heart disease6]. Cerebrovascular diseases can induce electrocardiographic abnormalities and a variety of cardiac arrhythmias that can sometimes cause sudden death. These findings have major clinical and therapeutical implications in patients with cerebrovascular events [6]. In the last decades, the study of the connection between brain and heart has grown ever more important, the concept of neurocardiology being born. Literature revealed that the most frequent cause of the stroke is cardiac arrhythmia, most often AF. Beside the risk of cerebral embolism, AF also represents a risk factor for hippocampal atrophy and for cognitive dysfunction [7].The first findings about the neural control over the body as a whole were made about 200 years ago by Magendie [8]. When injured, the right insular cortex may be the origin of sympathetic discharges leading to the cardiac alterations typical for stroke, whereas parasympathetic activity may increase as a consequence of left insular cortex damage [9]. Our current knowledge in neuroanatomy and neurophysiology cannot entirely explain either these phenomena, or the lateralization mechanisms [8]. The “laterality hypothesis” can also be applied to emotions: negative emotions originate mainly in the right hemisphere, while positive emotions arise especially in the left hemisphere [10]. A similar lateralization of the autonomic activity exists in the heart (right- and left-sided nerves being distributed to the right and left ventricle respectively) [10]. The anterior cingulate cortex, amygdala, parabrachial nucleus, hypothalamus, periaqueductal grey matter, the anterior insula and some areas of the medulla have an important role in modulating the cardiac function. Through the sympathetic and parasympathetic nervous system, these cerebral structures are involved in cardiac activity (modulating the force of contraction and heart rate), in the responses to stressful and emotional events and in the homeostatic reflexes [Laboratory animal studiesMany of these studies have shown that some neurological disease can induce anatomical and functional cardiac alterations. 11]. The first report in this field was Openheimer’s and his coworkers’. They noted that the stimulation of a cortical region could induce cardiac rate alteration, without affecting other parameters. According to the laboratory studies, the insular cortex plays a crucial role in the management of the heart’s chronotropic activity. While tachycardia occurs by stimulating the rostral posterior insula, the stimulation of the caudal region of the posterior insula evokes brachycardia. It was shown that these cardiac rhythm variations could be stopped by administering atenolol, but not atropine, thereby proving the influence of the sympathetic nervous system over cardiac activity [12]. The tachycardia induced by stimulating the rostral insular cortex is exclusively accompanied by a noradrenaline  surge. By contrast, no change in the catecholamines level is produced by stimulating the caudal region of the posterior insula. The results demonstrate that tachycardia depends on the sympathetic nervous system, while brachycardia on the parasympathetic nervous system [13]. In support of these theories, Melville described that the stimulation of the anterior hypothalamus in cats produces a burst of parasympathetic activity while ensuing bradycardia. In contrast, the stimulation of the lateral hypothalamus increases heart rate and alters the ECG . Furthermore, that pathological stimulation of the insular cortex by an epileptic seizure, a stroke, or a severe emotional stress may provoke ECG abnormalities, cardiac arrhythmias, and even sudden death [Autonomic nervous system6].Autonomic systems  play an important role in the control of heart rate variability (HRV), which is an important risk factor for atherosclerosis, arrhythmias, heart failure, myocardial infarction, and sudden cardiac death [6]. Any agent that can increase brain acetylcholine level can protect the suprachiasmatic nucleus, keeping in check the sympathetic activity and thereby mitigating the risk of myocardial infarction and sudden death [6].While an increased sympathetic tone determines high levels of catecholamines, cortisol, serotonin, renin, angiotensin, aldosterone, and free radicals, enhanced parasympathetic activity is correlated with high levels of dopamine, acetylcholine, endorphins, nitric oxide, Q10 coenzyme, and other protective substances [16]. Laboratory studies have shown that, in stroke, catecholamines are discharged in large quantities, resulting in cardiac dysfunction and an increase in cTn concentration. High levels of catecholamines determine the release of intracellular calcium and reversible cardiac myocyte damage [17]. To back up this affirmation, another study noted that cardiac systolic dysfunction is correlated with surprisingly normal myocardial perfusion, but increased sympathetic activity [18].Enhanced parasympathetic activity has a protective role for the ventricles, while sympathetic stimulation produces arrhythmia. In contrast, in prolonged QT syndrome and Brugada syndrome, increased parasympathetic activity produces arrhythmias, although it is usually very difficult to tell enhanced vagal activity from decreased sympathetic activity [10]. Parasympathetic activity is increased by the stimulation of the posteroinferior parts of left ventricle, while the stimulation of the anterior left ventricle enhances the efferent sympathetic tone [10]. Clinical and experimental data suggest a correlation between sudden cardiac death and mental stress: interrupting the communication between the frontal cortex and brain stem pathways preventing stress-induced ventricular fibrillation, while vagal stimulation plays a protective role from stress-induced cardiac arrhythmias. Conversely, ventricular fibrillation is precipitated in long-QT syndrome by emotional arousal [Cardiac changesElectrocardiographic changes and cardiac arrhythmias19]. The first to describe electrocardiographic alterations in stroke were Bodechtel and Aschnebrenner (1938) and Byer& Co (in 1947). [20]. There has been a large confirmation over the last decades that stroke may produce cardiac changes . The incidence of these manifestations is higher in subarachnoid hemorrhage (SAH) (60-70% of patients) and its complications [20], than in ischemic stroke (15-20%) [19]. Cardiac arrhythmias are more frequent in right cerebral hemisphere strokes compared to those afflicting the left cerebral hemisphere [22]. These electrocardiographic (ECG) alterations in stroke can appear in the context of a preexistent cardiac disease, but also in its absence, in which case they have a neurologic origin , left axis deviation [19], prolonged QT [19], atrial fibrillation (AF)/ atrial flutter, sinus tachycardia, ventricular tachycardia, atrial and ventricular premature complexes, bradyarrhythmias  [24]. Some of the most frequent electrocardiographic abnormalities in stroke are: ST segment abnormalities, negative T waves, U waves [23]. Atrial fibrillation is the most common tachyarrhythmia in acute stroke and is associated with an increased risk of systemic thromboembolism [23]. A long QT can precipitate a ventricular polymorphic tachycardia and sudden cardiac death [23]. One cannot overemphasize the importance of careful and continuous ECG monitoring of the stroke patient in order to identify these quite frequent electrocardiographic alterations, as it is well known that death due to cardiac arrhythmias is common among acute stroke patients . Rarely, other tachyarrhythmias, such as atrial tachycardia, can occur. Tachyarrhythmias can increase the extent and/ or severity of the cerebral damage during a stroke or in its aftermath  and can also increase the risk of ischemic cardiac lesions in patients with a history of heart disease [Echocardiographic changes in strokeSystolic dysfunction16]. Ventricular dysfunction is characterized by hypokinetic wall-motion changes on echocardiography. Previous studies demonstrated the absence of perfusion defects on radionuclide studies and of vascular lesions on coronary angiography in SAH cases. The transient character of the above-mentioned wall motion changes further proves their neurogenic nature [16]. Cardiac systolic dysfunction has been noted in all three types of stroke. Neurogenic stunned myocardium is not infrequent after SAH: between 2% to 30% of the patients are afflicted [26].Systolic dysfunction on echocardiography appears in 13-29% of the cases of ischemic stroke. These changes are associated with a high risk of mortality during hospitalization (and so are cardiac troponin (cTn) elevation and AF) [Diastolic dysfunction27], besides the systolic dysfunction already pointed out by previous studies [16]. A recent study demonstrated that cardiac diastolic dysfunction could also accompany acute stroke [27]. It has been demonstrated that the diastolic dysfunction  is a predictive marker of acute stroke [27]. Cardiac diastolic dysfunction results in elevated left ventricular end diastolic pressure and sympathetic arousal, leading to injury of endothelial cells and consequent hypercoagulability [Serum enzymatic changes in stroke28]. Later studies endorsed this original finding and described in detail the involvement of the sympathetic nervous system in these biohumoral alterations [29]. In 1979, Hachinski & Co described high levels of cardiac enzymes in acute stroke [Myoglobin36]. Although there is no unanimity regarding these correlations, Guerrero et al. showed that myoglobin level was not related to the stroke’s severity [31]. A recent prospective study has shown that myoglobin level  is higher in patients with ischemic stroke and a critical state than in those in a better condition . Creatine kinase-MB (CK-MB) does not hold the upper hand in specificity over the other cardiac markers, as it may increase in situations other than myocardial infarction, such as toxin or drug exposure, or in renal insufficiency. Although high CK-MB levels have been proposed as a humoral marker for stroke-related myocardial damage, it seems that CK-MB increases in some cases of cerebral infarction in the absence of a provable acute myocardial infarction. Troponin T is more sensitive and specific compared to CK-MB in detecting slight myocardial injury, and it has been shown not to increase after cerebral infarction. The fact that a normal cTn can coexist with a high CK-MB underlines that CK-MB should not be considered a biochemical marker for cardiac myocytolysis, as its elevation in acute stroke might not be of cardiac origin [29]. Clinical and experimental data suggest that cardiac abnormalities in stroke are the result of increased sympathetic activity secondary to insular cortical damage. Similarly to other studies, Butcher et al noted increased CK-MB in some stroke patients but, unlike previous studies, no correlation was found between the high CK-MB levels and sympathetic activity. The authors suggested that the origin of the elevated CK-MB might be non-cardiac in some patients and cardiac in others. An abnormal sympathetic activity may be one of the factors leading to cardiac enzyme changes .Myocardial infarction is the consequence of prolonged cardiac ischemia. cTn emerged as the most specific marker for cardiac lesions. Unfortunately, it is not only the most sensitive , but also too sensitive, as it fails both to differentiate necrosis from mere lesion , and to point out the underlying condition. Increased cTn levels can occur also in various other conditions : stroke, acute myocarditis, acute pericarditis , sepsis, pulmonary embolism, heart failure, renal insufficiency etc. .Meta-analyses have shown that more ECG changes are to be expected in patients with stroke and high level of cTn than in those with normal levels of cTn. Similarly, the risk of death was related to the cTn level, being higher in patients with increased cTn [16]. In conclusion, in the cases of ischemic stroke but without a history of ischemic heart disease, cTn is rarely increased, and when it is, its high levels are attributable to renal or cardiac insufficiency, rather than to myocardial infarction. Nevertheless, as previously mentioned, the prognosis of these patients is darkened by a higher risk of death in the next two years [38].A cardiovascular disease  in the history of the patient may be the reason for the increased level of cTn. However, what about patients with no known cardiac disease? Radionuclide studies comparing patients with high levels of cTn with those with normal levels of cTn and no proof of cardiac ischemia have shown no difference in the myocardial perfusion abnormalities found in the two categories of patients [Natriuretic peptide39].There are 3 types of natriuretic peptides: BNP , ANP , and C type of natriuretic peptide (the secretion of which is induced by the inflammation and lesion of the vascular endothelium) [40]. Nonetheless, as its name implies, this neurohormone is also secreted in the brain  . Its vasodilator and natriuretic properties rely on direct effects on the renal vessels and tubules, corroborated by neurohormonal actions including the inhibition of the sympathetic and renin-angiotensin-aldosterone systems [40]. BNP is a neurohormone primarily secreted in the ventricles in response to volume and pressure overload [40]. BNP is used to evaluate cardiac failure, but its increase in acute stroke can be not only the result of the ventricular systolic dysfunction, but also of enhanced sympathetic activity [43-47]. Recent studies showed that the BNP level is higher in patients with paroxysmal AF. An increased level of BNP may be a harbinger of AF [48]. On the other hand, plasma BNP levels are higher in patients with larger ischemic strokes, higher CHADS2 scores and higher mRS  scores [49].A first study of Nigro et al. evaluated the possibility of a connection between increased BNP or cTn levels and the risk for stroke recurrence, but failed to establish such a connection [Prognostic50].It is difficult to estimate the prognosis in stroke patients. A study on 122 patients with acute ischemic stroke attempted to find a correlation between BNP level and NIHSS scale . The higher mortality in patients which increases in both BNP values, and NIHHS scores at admission, endorsed the conclusion that the combination of elevated BNP and NIHHS score should be considered an indicator of grim prognosis in stroke patients [51]. As data on this subject are contradictory, García-Berrocoso endeavored to cast some light upon the putative correlation between BNP or NT-proBNP,  values and mortality in the aftermath of a stroke. Using MEDLINE and EMBASE databases, the authors conducted a search for relevant articles published until October 2012. The ensuing meta-analysis proved BNP to be a predictive marker for the post-stroke mortality, independent of the NIHHS score and demographic factors (sex and age) . An increased natriuretic BNP level in acute stroke is a predictor of mortality at presentation, during hospitalization, and after discharge on the long term [25] at admission were related to a bad prognosis in acute stroke patients . In addition, positive cTnI and ECG abnormalities  inducing approximately 12.500 cerebrovascular infarctions per year in patients with cardioembolism . The correct treatment for AF can reduce this number by about 4500 cases, and can prevent about 3000 stroke-related deaths annually. In these cases, oral anticoagulation is recommended for the secondary prevention .In 20 percent of the cases, ischemic stroke is caused by a cardiac disease, the major culprit being AF, which increases the risk of cerebrovascular events by about 5 times [55], therefore, no specific medication is warranted [40].In cryptogenic ischemic strokes, the cause may remain obscure  [43]. Cardiac-monitoring strategies  can detect AF and other arrhythmias, but only in a small percentage of patients. In order to increase the diagnostic yield, a high level of NTproBNP may be used as a discriminant for the patients with a higher probability of cardiac arrhythmias and therefore of positive results on cardiac monitoring techniques [43]. It has been demonstrated that higher BNP levels correlate with AF  . A high BNP level may have potentially (very) important management implications as it may signal a worse prognosis and may prompt the undertaking of certain therapeutic measures [58]. The explanation for the unpredictable and sudden cardiac changes accompanying acute stroke probably lies in the interplay between brain and heart, highlighting the need for a better understanding of and possibly, for new approaches to this matter, which may lead to novel therapeutics aimed at preventing sudden death in cerebral infarction patients [AcknowledgementsI would like to express the deepest appreciation to Academician Professor Constantin Popa, who has shown the attitude and the substance of a genius: he continually and persuasively conveyed a spirit of adventure concerning research and scholarship, and an excitement about teaching. Without his supervision and constant help, this review would not have been possible. Also my thanks and appreciations go to my colleague in developing the project, and people who have willingly helped me out with their abilities.DisclosuresNone.


Prostate cancer is a common malignancy among men in Western countries. Recently the morbidity and mortality of prostate cancer increase dramatically in several oriental countries including China. Rapidly evolving technology in molecular biology such as high-throughput sequencing and integrative analysis of genomic and transcriptomic landscapes have enabled the identification of key oncogenic events for prostate cancer initiation, progression and resistance to hormonal therapy. These surging data of prostate cancer genome also provide insights on ethnic variation and the differences in histological subtype of this disease. In this review, differences in the incidence of prostate cancer and the prevalence of main genetic alterations between Asian and Western populations are discussed. We also review the recent findings on the mechanisms underlying neuroendocrine differentiation of prostate cancer and the development of small cell neuroendocrine carcinoma after androgen deprivation therapy. Prostate cancer is the second most commonly diagnosed nondermatologic cancer and the sixth leading cause of cancer-related deaths in men worldwide . The morIn contrast, the incidence and mortality rates of prostate cancer are much lower in Asian countries including China, India, Japan, Philippines, South Korea, Thailand and Vietnam ,4, althoTo identify genetic variants associated with prostate cancer risk, multiple genome-wide association studies (GWAS) have been carried out in populations of European descent, African-Americans, Japanese and Chinese. So far 77 single nucleotide polymorphisms (SNPs) associated with prostate cancer susceptibility have been identified ,6, incluHOXB13 and SPOP [HOXB13 gene (HOXB13 G84E) was reported to be highly associated with familial prostate cancer in Caucasians [HOXB13 was found to be associated with increased prostate cancer risk among Chinese men [SPOP, a gene encoding the substrate-binding subunit of a cullin-based E3 ubiquitin ligase. While wild-type SPOP protein interacts directly with steroid receptor coactivator SRC-3 and enhances its ubiquitin-dependent proteasomal degradation to control the transcriptional activity of androgen receptor (AR), it has been recently demonstrated that most prostate cancer-associated SPOP mutants lack the capacity to promote the turnover of SRC-3 protein and thus display attenuated tumor suppressor effects in prostate cancer cell lines [in vitro studies showed that forced expression of SPOP mutant or siRNA-mediated knockdown of SPOP led to increased invasion, but no significant changes in cell growth and viability [Of the 23 newly identified susceptibility loci for prostate cancer, rs11650494 is located at chromosome 17q21, a gene-dense locus that contains several prostate cancer candidate genes such as  HOXB13 G4E was renese men  as well nese men  describell lines . Consistiability , suggestERG, ETV1, ETV4 or ETV5) were found to be the most frequent genetic alterations in prostate cancer, which can be detected in as many as 50%–70% of prostate cancer samples [ETS gene fusions resulting from either interstitial deletion or chromosomal translocation lead to the generation of a handful of fusion transcripts that commonly contain 5′ regulatory elements from androgen-responsive genes, such as TMPRSS2, and the coding sequence of ETS transcription factors. The TMPRSS2-ERG fusion at chromosome 21q22 is the predominant subtype of ETS fusions [TMPRSS2-ERG fusion in prostate cancer appears to vary in different ethnic groups, with the highest frequencies of occurrence in Caucasians (~50%) [In the past decade, rapidly evolving technologies have revolutionized our understanding of the cellular and molecular basis for the development of prostate cancer. In addition to the aforementioned SNPs associated with prostate cancer susceptibility, a variety of genetic and epigenetic alterations have been found to be involved in prostate cancer initiation, progression, metastasis and drug resistance. Recurrent gene fusions involving several members of ETS transcription factor family  ,20, modes (~50%) ,20 and ms (~50%) ,21–23, iPTEN allele is frequently present in primary prostate cancer, and is correlated with disease progression to the metastatic stage [Similar ethnic differences have also been demonstrated in PTEN status, another common early event involved in prostate carcinogenesis. In Western countries, loss or alteration of at least one ic stage ,32. It hic stage ,31. By pic stage  revealedERG gene fusion and PTEN deficiency in Chinese patients with prostate cancer suggests that alternative molecular mechanisms may play important roles in the development of prostate cancer in Asian men. Using RNA-seq technology to profile the changes in the transcriptome of primary prostate cancer samples from China, Ren et al. [CTAGE5-KHDRBS3 and USP9Y-TTTY15, occurred at high frequencies (~35%) in Chinese patients [SDK1-AMACR and RAD50-PDLIM4 were also found with relatively lower prevalence (24%–28%) in this cohort of Chinese descent. Although the functional relevance and clinical significance of these novel gene fusions remain elusive, the high recurrence of these gene rearrangements indicates that unique genetic alterations in alternative pathways may affect prostate oncogenesis among Asian patients.The low prevalence of n et al.  reportedpatients . Two addBRAF and RAF1 occur at a comparable frequency between Chinese and Western samples, the prevalence of BRAF copy number gain in Chinese patients was significantly higher than that in patients for the United Kingdom (29% vs. 9.2%) [In addition, the differences in activated RAS-RAF-MAPK signaling pathway also have been characterized between Asian and Western patients with prostate cancer. Although constitutive activation of RAS-RAF-MAPK pathway occurs in a majority of advanced prostate tumors, the incidence of direct mutations of the upstream activators such as KRAS and BRAF are not commonly found in prostate cancer . Howevers. 9.2%) . Taken tAR gene, gain-of-function mutations and alternative splice variants of AR, changes in co-regulatory molecules, ligand-independent activation of AR signaling as well as other AR-independent pathways that facilitate cancer cell survival and growth under androgen-depleted conditions [PTEN, AR, RB1, TP53 and APC mutations, several novel somatic mutations in multiple chromatin/ histone modifiers including MLL2, UTX and ASXL1 as well as transcription factors FOXA1 and ETS2 were found in CRPC [Despite the considerable differences in epidemiology and etiology of prostate cancer between Asian and Western countries, prostate cancer patients of different ethnic groups are currently treated with the same modalities mainly based on tumor grade and stage. We know very well now that a large portion of these patients have indolent tumors that will impact neither quality of life nor life expectancy, so that active surveillance is a preferred option. Localized cancers are treated with surgery or radiation with similar efficacy. For advanced and metastatic prostate cancer, hormonal therapy that inhibits androgen production and/or blocks androgen receptor (AR) function is the first-line treatment. However, many patients experience only a short term disease regression, and nearly all of them will eventually recur with castration-resistant prostate cancer (CRPC). During the past several decades, the cellular and molecular basis underlying the development of CRPC has been intensively investigated. Diverse mechanisms have been proposed, including sustained intratumoral synthesis of androgen, amplification of nditions . Recentl in CRPC .In addition to these identified molecular alterations, a pathological characteristic referred as neuroendocrine differentiation of prostate cancer has also been demonstrated to be significantly associated with the development of CRPC –38. EpitERG gene fusion status in neuroendocrine tumor cells by FISH analysis consistently showed that despite the lack of ERG protein expression, the TMPRSS2-ERG fusion can be detected in neuroendocrine cells that intermingle with ERG rearrangement positive adenocarcinoma component within the same tumor foci [In prostate adenocarcinoma, the predominant histological subtype of human prostate cancer, the neoplastic glands are mainly composed of proliferating luminal type cancerous cells, and complete loss of basal cells. However, scattered or nests of neuroendocrine cells are also present in almost all cases of prostate adenocarcinoma , with vamor foci . These fInterestingly, neuroendocrine tumor cells also display several unique features distinct from luminal secretory-type cancer cells in prostate cancer. While highly proliferating luminal cancer cells are generally positive for AR and PSA, neuroendocrine cells in prostate adenocarcinoma are usually quiescent and lack expression of AR and PSA ,41. Due It has been increasingly recognized that neuroendocrine differentiation following hormonal therapy is associated with tumor progression and castration resistance ,45,46. HTo date, the cellular origin of neuroendocrine tumor cells in prostate adenocarcinoma is unclear. Given the pluripotency of prostate stem cells/progenitors that can give rise to basal, luminal and neuroendocrine cells in the regeneration assay –50, it iAURKA and N-myc gene (MYCN), indicating that N-myc and AURKA could play important roles in the development of SCNC of the prostate. Moreover, the preclinical studies with an AURKA inhibitor PHA-739358 demonstrated that this kinase antagonist has specific inhibitory effects on the growth of neuroendocrine tumor cells in cell culture and xenograft models [Using next-generation RNA sequencing and oligonucleotide arrays, Beltran et al.  showed tt models , suggestt models , to neurTP53 gene showed that five of seven cases of SCNC harbor a recurrent p53 mutation (D184N) [In contrast to the absence of proliferative activity of neuroendocrine cells in benign prostate tissues and prostate adenocarcinomas, neuroendocrine cells in primary (de novo) or secondary SCNC are highly proliferative, which results in almost all patients dying within one year following diagnosis ,57,58. T (D184N) , supportprobasin promoter [The identification of p53 protein as a key molecular determinant for the regulation of neuroendocrine cell proliferation and quiescence not only offers the critical link between the transformation of neuroendocrine cells and the development of prostate SCNC, but also provides a mechanistic explanation for the neuroendocrine phenotype of prostate tumors from the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The TRAMP transgenic mouse strain is engineered to express the SV40 virus large T and small t tumor antigens in prostate epithelial cells under the control of the AR-responsive rat promoter ,64. Thespromoter . It is wpromoter , indicatpromoter . These dIn addition to genetically engineered mouse models, the tissue recombination/transplantation model of prostate cancer is a very efficient approach to rapidly interrogate the functional consequences of various genetic alterations central to the initiation and progression of the human disease . By apprin vivo transformation assay using naïve human prostate epithelial cells directly harvested from patients [in vivo regeneration approach, we demonstrated that CD49fhiTrop2hi basal cells from primary benign human prostate tissue are able to initiate prostate cancer in immunodeficient mice. The synergistic effects of AKT activation, overexpression of ERG and AR in basal cells closely recapitulated the histological and molecular features of human prostate cancer, with loss of basal cells and expansion of malignant luminal cells expressing PSA and AMACR [Importantly, primary prostate epithelial cells freshly isolated from benign human prostate can be used as the starting material to study prostate cancer development in the tissue recombination model, which makes it possible to examine many fundamental differences in prostate cancer biology between mice and humans. Recently, we successfully established an patients ,75. By cnd AMACR . These fWith continuous improvement in technologies and the significant reduction in cost for high-throughput sequencing, remarkable advances have been made in molecular characterization of prostate cancer. These findings at the molecular level are being translated into valuable diagnostic biomarkers and prognostic predictors in the clinic, offering an opportunity for patient stratification and personalized therapy. However, several unique features of prostate cancer, including vastly different biology of different tumors, tumor multifocality, tumor heterogeneity and the preference of bone as metastatic sites, pose significant challenges for the acquisition and analyses of tumor samples. The exact roles of many newly identified genetic alterations in prostate carcinogenesis remain uncertain. In addition, the contribution of other factors, such as epigenetic changes, to ethnic variation and histological subtype differences in prostate cancer has not been extensively investigated. Despite these limitations and challenges, the recent molecular findings have provided strong candidates for the development of novel targeted therapeutic agents. Combination therapies targeting multiple molecules and pathways will likely substantially improve the clinical outcome of patients with advanced prostate cancer in the near future.


Regenerative medicine, a multi-disciplinary approach that seeks to restore form and function to damaged or diseased tissues and organs, has evolved significantly during the past decade. By adapting and integrating fundamental knowledge from cell biology, polymer science, and engineering, coupled with an increasing understanding of the mechanisms which underlie the pathogenesis of specific diseases, regenerative medicine has the potential for innovative and transformative therapies for heretofore unmet medical needs. However, the translation of novel technologies from the benchtop to animal models and clinical settings is non-trivial and requires an understanding of the mechanisms by which the host will respond to these novel therapeutic approaches. The role of the innate immune system, especially the role of macrophages, in the host response to regenerative medicine based strategies has recently received considerable attention. Macrophage phenotype and function have been suggested as critical and determinant factors in downstream outcomes. The constructive and regulatory, and in fact essential, role of macrophages in positive outcomes represents a significant departure from the classical paradigms of host–biomaterial interactions, which typically consider activation of the host immune system as a detrimental event. It appears desirable that emerging regenerative medicine approaches should not only accommodate but also promote the involvement of the immune system to facilitate positive outcomes. Herein, we describe the current understanding of macrophage phenotype as it pertains to regenerative medicine and suggest that improvement of our understanding of context-dependent macrophage polarization will lead to concurrent improvement in outcomes. The macrophage has long been known to play an important role in the tissue remodeling response which occurs following injury. In brief, macrophages arrive at the site of tissue injury 24–48 h post-injury, serve as phagocytes clearing the wound bed and initiating the processes that lead to the default outcome of scar tissue formation , 2. HoweA correlation of macrophage phenotype with functional recovery in wound healing has been suggested for more than two decades . With thThe M1/M2 paradigm has been widely studied in the context of disease pathogenesis, particularly cancer, for more than two decades –13. The Regenerative medicine approaches to tissue reconstruction or organ replacement seek to restore the form and function of lost, damaged, or diseased tissues. These approaches logically rely upon our understanding of wound healing, development, and regeneration as guideposts for design. These approaches may incorporate one or more biomaterials, biologically active molecules, and/or cell sources. Recent advances in these areas have enabled highly innovative and promising therapies, but translation of such strategies, without exception, requires in depth investigation and understanding of the host response following delivery.The purpose of the present review is (1) to provide rationale for a macrophage centric approach to tissue reconstruction; and (2) to give an overview of the current state-of-the-understanding of the implications associated with host macrophage responses in regenerative medicine.in vivo reality. Though it is now clear that M1 and M2 macrophages each play distinct roles in tissue remodeling following injury, the inflammatory process which occurs following injury is dynamic both spatially and temporally and macrophages may express transitional phenotypes. Logically, these cells will also express functions such as phagocytosis, antigen presentation, and effector molecule production to differing degrees during the inflammation and remodeling process. For the purposes of simplicity and general discussion, and as the M1/M2 terminology are used ubiquitously throughout the literature, we describe macrophage phenotype as M1 and M2 in the below examples with further discussion of the spectrum of possible phenotypes and their potential roles in regenerative medicine thereafter.It should be noted that the description of macrophages as having either an M1 or M2 phenotype is a simplification of the There is evidence for both pathogenic and protective roles of macrophages in many biologic processes , 23. It The role of the M1/M2 paradigm during the skeletal muscle injury response is relatively well characterized. Skeletal muscle tissue possesses inherent regenerative capacity following acute injury such as exercise-induced trauma. The capacity of muscle tissue to regenerate relies heavily upon a population of normally quiescent muscle specific progenitor cells, referred to as “satellite cells,” and their interactions with inflammatory cells that infiltrate the injured muscle microenvironment , 25. An Following acute skeletal muscle injury, one of the earliest events is the infiltration of the damaged tissue by inflammatory cells. Neutrophils comprise the initial wave of cells to enter the damaged muscle tissue and reach elevated levels as soon as 2 h post-injury and maximum numbers after 6–24 h , 32, 33.Monocytes originate in the bone marrow and express chemokine receptors and adhesion molecules which facilitate their extravasation and migration from the blood to the injured skeletal muscle tissue site where they differentiate into macrophages , 35. TisAfter 2 days, macrophages participating in the remodeling of injured skeletal muscle show a transition from the pro-inflammatory M1 to the immunoregulatory and anti-inflammatory M2 phenotype. The mechanisms behind this M1 to M2 phenotypic switch remain only partially understood but include exposure of M1 macrophages to increased IL-10 concentrations from skeletal muscle at 48 h post-injury , 41; M1 This transition of the initial response dominated by M1 macrophages to a more M2 dominated population following acute muscle injury facilitates skeletal muscle remodeling and is required for efficient and compete functional restoration. Specifically, the pro-inflammatory products of M1 macrophages promote the activation and expansion of quiescent muscle satellite cells within the tissue injury site , 46, 47.The participation of a heterogeneous population macrophages following muscle injury is highly regulated. For example, the perturbation or prolongation of either the M1 or M2 macrophage population during the skeletal muscle injury response results in impaired skeletal muscle regeneration. Depletion of phagocytic leukocytes, including macrophages, prior to toxin induced skeletal muscle injury blocks the removal of cellular debris and impairs regeneration . DepletiThe participation of M2 macrophages is also required for the skeletal muscle injury response. As stated above, these immunomodulatory cells drive the late or differentiation stage of myogenesis. For example, when macrophages were deleted after 2 days post-injury, a time point consistent with the transition to an M2 macrophage response, myoblast differentiation, and subsequent regeneration was impaired . FurtherAdult mammalian cutaneous wound healing is another highly regulated process that follows a sequence of events comprising the following three interdependent and overlapping phases: (1) the inflammatory phase; (2) the granulation tissue formation and wound contraction phase; and (3) the matrix deposition and tissue remodeling phase , 57. MulThe onset of wound healing, designated the inflammatory phase, can be further sub-divided into an early and late inflammatory phase. Immediately following injury, hemostasis provides a provisional matrix for cell migration. During the early inflammatory phase, which occurs at 1–4 days post-injury, neutrophils and monocyte-derived macrophages respond to pro-inflammatory signals released from the wound microenvironment including growth factors, cytokines, damage associated molecular patterns (DAMPS), and pathogen associated molecular patterns (PAMPS) . These pThe late inflammatory phase, which occurs at 5–7 days post-injury, is marked by an accumulation of apoptotic as opposed to necrotic cells, which upon phagocytosis facilitate the polarization of macrophages toward the immunomodulatory M2 phenotype . During The matrix deposition and tissue remodeling phase, which occurs after 10 days post-injury, is marked by a decrease in macrophage numbers populating the wound site, along with an overall decrease in total cellularity. Granulation tissue formation reaches a plateau and tissue present within the wound site is partially remodeled into fibrotic scar tissue at this time , 57.The above stages of cutaneous wound healing are carefully regulated by the activity of responding macrophages. Similar to the M1/M2 macrophage paradigm associated with the skeletal muscle injury response, cutaneous wound healing is dependent upon a heterogeneous macrophage population and an M1 to M2 phenotypic transition. The pro-inflammatory activity of M1 macrophages during the early inflammatory phase is required for efficient pathogen control. Conversely, M2 macrophage activity during the late inflammatory phase is required for the resolution of inflammation and the recruitment of cells, which facilitate granulation tissue formation and wound re-epithelialization. Perturbation of the M1 macrophage phenotype during the early inflammatory phase, either by conditional depletion or due to impaired recruitment, results in delayed granulation tissue formation and wound closure , 64. SimThe role of M1 and M2 macrophages following injury in the central nervous system (CNS) is more ambiguous when compared to other tissues, and is made more complex by the presence of the blood–brain barrier. However, similarities to the macrophage heterogeneity associated with the injury response in other tissues are increasingly being reported.Central nervous system resident macrophages, referred to as microglia, have long been considered the primary responders to injury in the CNS with little to no role having been recognized for circulating cells until recently. Generally, microglia are recruited to, and form a dense barrier around, the lesion site immediately following spinal cord injury , 70. TheThe participation of M2 polarized macrophages in the CNS injury response is essential to the repair process. For example, the endogenous partial recovery which can be observed following spinal cord injury is abrogated when M2-like macrophages are depleted using antibodies or conditional ablation , 75. ConThe above studies support an emerging dogma of effective recovery from tissue injury in which initial responses consisting predominantly of M1 macrophages and secondary or later stages consisting predominantly of M2 macrophages drive functional remodeling outcomes. Furthermore, it is appears that M2 macrophages contribute to more than immunomodulation during the response which follows tissue injury. Several organ systems, in addition to the above examples of muscle, skin, and CNS tissue have now been shown to undergo similar responses following injury and are also characterized by heterogeneous and temporally shifting macrophage phenotypes.The observation of dichotomies in macrophage phenotype in disease pathogenesis as well as tissue remodeling following injury represents a departure from the classical understanding of the macrophage as a primarily phagocytic and pro-inflammatory cell. The foreign-body reaction (FBR) has been well studied over the last three decades . LogicalDuring a FBR persistent inflammatory stimuli, such as the presence of a non-degradable biomaterial, lead to chronic inflammation and the formation of multinucleated foreign-body giant cells . MultinuThe interpretation of the FBR as a negative occurrence in this context led to the development of materials with a focus on “inertness” and “biocompatibility” –86. ThisThe emergence of regenerative medicine and the need and desire for therapies which restore endogenous tissue function has led to a significant increase in our understanding of the role of stem cells in tissue repair as well as innovation and development of new biomaterials as stand alone therapies and/or delivery systems for cells or biologic factors. These materials are most often degradable in nature, and include engineered biologic cues or inherent bioactivity when derived from natural or tissue based sources. As such, the host response to these materials will be significantly different and more complex than the response to mono-component, synthetic or metallic implants. Further complexity is seen when materials are used in combination with cells or other factors.It is now recognized that certain materials when used alone or in concert with a cellular component can provide an inductive template for constructive and functional tissue remodeling. That is, the provision of a bioactive material and/or cells leads to the formation of new, site-appropriate tissue. One example of such materials is biologic scaffolds composed of ECM , 88. TheDespite the distinct differences in long-term outcomes which have been observed with various ECM based scaffold materials, all ECM implants have been shown to elicit a histologically similar cellular response in the first week to month post-implantation , 91. ThiBased upon these disparate outcomes, it was hypothesized that, though the early host response to the materials was histologically similar , differences in the early macrophage phenotype to certain ECM scaffold materials might exist and that these differences may be related to downstream remodeling outcomes. Indeed, this hypothesis was shown to be correct with those ECM scaffolds which elicited constructive remodeling outcomes being associated with a timely transition from an M1 to an M2 macrophage phenotype , 92, 93.An endogenous host injury response, consisting of immunomodulation, including the participation of M2 type macrophages represents a necessary component of efficient and functional tissue repair. It is therefore logical that regenerative medicine strategies aimed at activating or augmenting endogenous repair mechanisms should utilize a similar strategy. Regenerative medicine strategies aimed at promoting M2 macrophage activation have included cell-therapy and the implementation of synthetic and biologic scaffold materials, among others.Cellular therapy is generically defined as the transplantation or delivery of exogenous cells to sites of injured or missing tissues. Stem and/or progenitor cells are often used in regenerative medicine applications because of their multi-lineage differentiation potential and well-recognized resistance to oxidative stress , 95.The cell source is most commonly autologous due to immune rejection considerations, although many studies are investigating the use of allogeneic sources. Cellular therapy based strategies aimed at promoting tissue remodeling have been used to treat injured tissues including the myocardium, the spinal cord, and skeletal muscle, among others. Despite moderate pre-clinical and clinical success, cell-therapy is associated with limitations including failure of the exogenous cells to engraft within host tissue –101. It Co-culture experiments have shown that the secreted products of different stem/progenitor cells directly promote an M2 macrophage phenotype –113. A lRegenerative medicine strategies aimed at promoting tissue reconstruction or replacement often employ the use of surgically implantable synthetic or biologic materials designed to serve as cellular support scaffolds. As described above, implantation of these materials following injury alters the default injury response. For example, following surgical placement, synthetic and/or biologic scaffold materials are able to affect the phenotype of infiltrating inflammatory cells, host progenitor cell activity, as well as fibrosis and fibrous capsule development , 122. ThAs stated above, the surgical placement of non-degradable synthetic scaffold materials is commonly associated with a FBR consisting of persistent M1 macrophage activity and an increased deposition of scar tissue , 123–125A recent series of studies has demonstrated the effects of material pore size upon integration of the material as well as macrophage phenotype , 128–131in vitro, with results suggesting that macrophage derived factors were at least in part the cause of the observed chemotaxis of Schwann cells.Another manner in which biomaterials can be tailored to promote shifts in macrophage phenotype is through the use of biologically active molecules such as growth factors and cytokines. Examples of these approaches are numerous and are commonly employed in regenerative medicine with resulting improvements in remodeling outcomes. A recent study investigated the effects of incorporation of either M1 (IFN-γ) or an M2 (IL-4) promoting cytokines within a polysulfone tube upon nerve growth across a gap defect when the tubes were used as guidance conduits . The resThe biologically derived scaffold materials used in regenerative medicine applications are sourced from a variety of natural sources including mammalian tissues as well as plant, insect, and bacterial sources. These materials offer the inherent advantage of the native ligand landscape and bioactivity resulting from their source material. This inherent bioactivity also leads to added complexity in the host response to these materials. Among these materials, scaffolds derived from mammalian tissues represent the most commonly used materials in pre-clinical and clinical regenerative medicine applications –135.Biologic scaffold materials composed of mammalian ECM have been used to promote constructive tissue remodeling in a variety of clinical applications including hernia repair, rotator cuff reconstruction, esophageal preservation, and skeletal muscle replacement, among others , 137. ECIt is now well recognized that the ability of these materials to promote constructive remodeling is tied to their ability to modulate the host macrophage response , 93, 135in vitro studies, further demonstrate the inherent immunomodulatory nature of ECM based biomaterials as well as their ability to improve remodeling outcomes (The necessity of degradation for the transition to an M2 phenotype suggests that breakdown products of the ECM scaffold material may possess immunomodulatory activity. Studies have shown that ECM bioscaffolds can be solubilized and the degradation products formed into a hydrogel under physiologic conditions , 141. Thoutcomes , 142.The above examples clearly illustrate an emerging paradigm in regenerative medicine. That is, strategies which are able to modulate the host response from an M1 to an M2 macrophage response are associated with better outcomes. However, these results should be interpreted with caution. Macrophage phenotypes have been described in many ways . “M1” anAdding further complexity to the definition of macrophage phenotypes in regenerative medicine applications is the variability in tissue resident macrophage populations. For example, microglia are the resident macrophages of the brain and derive from the embryonic yolk sac during development and persist in the brain thereafter, presumably through a process of local replication –146. As At present, studies investigating macrophage phenotype following exposure to biomaterial implants have largely relied upon single surface or gene expression markers as indicators of M1 and M2 polarization. It is now well established that macrophages possess highly complex and plastic phenotypes and that the use of multiple phenotypic markers is essential. Further, a better understanding of the functional implications of these phenotypes is needed to create a mechanistic understanding of the ways in which macrophages may direct tissue remodeling outcomes following biomaterial implantation or stem cell delivery. With this understanding, next generation therapies can be developed to target and modulate specific macrophage phenotypes with desirable characteristics for the given application.It should further be recognized that baseline macrophage polarization states may be affected by patient characteristics. As is mentioned above, there is now increasing evidence for changes in macrophage phenotype and response to activating stimuli with age and disease both acute and chronic , 149. CoMacrophage polarization has been clearly shown to be an important determinant of success in regenerative medicine strategies for tissue reconstruction. Macrophages can promote both positive and negative outcomes, which are dependent upon the context in which they are encountered, their phenotype, and function. However, at present, there remains much to be investigated and defined regarding macrophage phenotypes associated with biomaterials, stem cells, and regenerative medicine. Thus, context specific definitions and identification of beneficial phenotypes are needed. Similarly, the unique functions of these cells must also be clearly defined in order to better understand their true role in the remodeling process. Indeed, a focus upon macrophage function during the process of constructive remodeling may prove more useful than further characterization of complex phenotypic markers. Moving studies from correlative to causative and expanding the number of outcome metrics, both phenotypic and functional, will assist in defining both biomaterials and stem cell associated phenotypes and also provides targets for next generation regenerative medicine therapies, which seek to modulate macrophages as a means of promoting functional tissue recovery – a macrophage centric approach. It is increasingly clear that those strategies that adopt such an approach to regenerative medicine will meet with improved success.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.


In recent years, controversy has arisen regarding the risks and benefits of certain types of gain-of-function (GOF) studies involving avian influenza viruses. In this article, we provide specific examples of how different types of data, including information garnered from GOF studies, have helped to shape the influenza vaccine production process—from selection of candidate vaccine viruses (CVVs) to the manufacture and stockpiling of safe, high-yield prepandemic vaccines for the global community. The article is not written to support a specific pro- or anti-GOF stance but rather to inform the scientific community about factors involved in vaccine virus selection and the preparation of prepandemic influenza vaccines and the impact that some GOF information has had on this process. Influenza viruses are a major cause of morbidity and mortality worldwide. Each year, outbreaks of seasonal influenza viruses are estimated to result in 25 to 50 million cases in the United States alone, leading to over 200,000 hospitalizations and 30,000 to 40,000 deaths annually. Globally, the burden of disease is estimated to be as high as 1 billion cases annually , 7. In ahttp://www.hhs.gov/nvpo/nvac/meetings/pastmeetings/2014/barda-vaccines-february2014.pdf).The primary public health medical countermeasure against influenza is vaccination. Although the first inactivated influenza vaccines were introduced in the 1940s, it soon became evident that influenza viruses undergo antigenic changes that reduce vaccine efficacy. Surveillance networks were soon developed to identify the emergence of antigenically drifted viruses and identify a representative candidate vaccine virus (CVV) for use in vaccine manufacturing. Since 1952, the WHO has coordinated monitoring of the antigenic properties of influenza viruses in humans and, more recently, in animals within the Global Influenza Surveillance Network (GISN), which in May 2011 was renamed the Global Influenza Surveillance and Response System (GISRS). The GISRS is currently comprised of 6 WHO collaborating centers (CCs) , 4 essential regulatory laboratories (ERLs), 141 national influenza centers (NIC) in 111 WHO member states, and 13 H5 reference laboratories. GISRS also collaborates closely with the animal health sector, including the Food and Agriculture Organization (FAO), the World Organization for Animal Health (OIE), and the OFFLU . Throughout the year, samples are collected from patients with influenza-like illness or acute respiratory infection and sent for assessment of influenza infection and virus isolation. Isolated viruses undergo detailed characterization, including genetic, antigenic, antiviral drug susceptibility, and molecular analyses. Influenza surveillance is also conducted in the animal sector, including in healthy animal populations. Twice yearly, the WHO GISRS subject matter experts meet to review data in the context of CVV selection. Seasonal influenza viruses in circulation in the months leading up to September are reviewed to identify viruses with distinct antigenic characteristics and associated epidemiological features that would justify a change in the current recommended CVVs for use in the following year in the Southern Hemisphere, and those in circulation in the months leading up to February are reviewed for the recommendation of viruses to be included in vaccines for the following winter in the Northern Hemisphere. At the same meetings, an expanded group including animal health experts also reviews comprehensive data about zoonotic influenza infections, outbreaks of influenza in animals, and virologic characteristics of corresponding influenza viruses to determine if one or more CVVs should be developed for pandemic preparedness. It should be noted that CVVs for zoonotic viruses are recommended primarily if these subclades have been associated with human infections. For seasonal influenza vaccines, from the time these recommendations are made, manufacturers require 6 months to produce vaccines and begin distribution. An overview of the annual production cycle for influenza vaccine can be found in reference The remainder of this article will focus on how information garnered from gain-of-function (GOF) studies helps to inform the influenza vaccine strategy for pandemic preparedness, from selection of candidate vaccine viruses and development of high-yield seeds to manufacture of safe vaccines for the global community; the needed information includes mutations identified by virulence and transmission studies with A/H5N1 viruses, identification of important antigenic sites through vaccine escape studies, basic research on the hemagglutinin (HA) region associated with virulence in poultry, and ways to enhance growth in culture systems.To understand how information from GOF studies has aided the decision-making process, we will focus on data from zoonotic viruses, specifically HPAI H5N1 viruses. Since emerging in 1997, the HPAI H5N1 viruses have spread geographically and become endemic in poultry in several parts of the world where zoonotic infections continue to occur. As of today, the HA genes of these viruses have evolved from the original A/goose/Guangdong/1/96 lineage, resulting in 10 unique first-order HA clades, with most containing numerous subclades comprised of genetically and antigenically distinct viruses . To keepAt the twice-yearly WHO Consultation on the Composition of Influenza Vaccines meeting, genetic and antigenic information from H5Nx viruses (and other zoonotic subtypes) isolated from infected animals and humans is analyzed to determine if new CVVs are required. Specifically, the HA sequences of related viruses are aligned and compared to available CVVs  to deter15 toIn a recent example, HA gene mutations were detected in clade 1 H5N1 viruses causing human infections in Cambodia that were associated with alteration of receptor-binding specificity and increased respiratory droplet transmission in ferrets . Amino a20–The great majority of influenza vaccines are prepared from viruses propagated in embryonated chicken eggs, which are then inactivated with either formaldehyde or β-propiolactone and either split by detergent or subjected to surface antigen purification in accordance with WHO recommendations. Producing vaccines from egg-propagated wild-type HPAI viruses is problematic because the rapid killing of chicken embryos leads to low vaccine yields, poor-quality harvests, and biosafety issues. Work with HPAI viruses requires enhanced containment facilities, which very few vaccine manufacturers possess, and moreover, production staff would be immunologically naive to such viruses , 26. ConCurrently, HPAI and many other zoonotic CVVs are produced using reverse genetics (RG) based on the WHO-approved A/Puerto Rico/8/34 (PR8) virus. PR8 was isolated from a human and has been propagated extensively in eggs and other laboratory systems to yield a virus that is highly attenuated for humans but grows to high titers in eggs . We knowThe ability of the HA protein to evade antibodies through “antigenic drift” drives the need to update seasonal influenza vaccines. Antigenic drift is caused by amino acid substitutions in HA epitopes recognized by antibodies that neutralize viral infectivity by blocking HA interaction with host cell sialic acid receptors. Much of our understanding of the HA alterations associated with evasion of host antibody responses has come from characterizing vaccine escape mutant viruses generated in the laboratory, a precursor of GOF work that began in earnest during the 1970s along with genetic sequencing. These studies have provided invaluable information on the importance of the antigenic sites on HA recognized by neutralizing antibodies, specific amino acid alterations associated with loss of antigenicity, and the importance of glycosylation, and they have demonstrated that viruses with such changes frequently circulate in nature –36. Duri–Among the most important GOF work for the vaccine production process is the generation of high-growth or high-yield reassortant (HGR or HYR) CVVs . GeneralIn summary, information garnered from basic influenza virus research, including GOF studies, focused on specific viral traits, such as receptor binding, transmissibility, pathogenicity, antigenicity, and antigen yield in eggs, has benefited public health and the vaccine production process in numerous ways. These studies help to define molecular markers associated with mammalian infection, evasion of neutralizing antibodies, and attenuation and/or replication in various hosts, thereby informing vaccine recommendations to aid in vaccine design and production. Frequently, this knowledge has been derived from basic research studies that relied on GOF to test a hypothesis; however, the insights that ultimately benefitted public health often were not appreciated until much later. With the continual evolution of influenza viruses and the potential for zoonotic events leading to pandemics, we must remain vigilant. For this reason, care must be taken in considering the regulation of GOF studies of influenza viruses to avoid unforeseen consequences for global public health and to ensure the safe and ongoing provision of essential vaccines and public health countermeasures.


Although many of the genes that encode HSPG core proteins and the biosynthetic enzymes that generate and modify HSPG sugar chains have not yet been analyzed by genetics in vertebrates, recent studies have shown that HSPGs do indeed mediate a wide range of functions in early vertebrate development, for example during left-right patterning and in cardiovascular and neural development. Here, we provide a comprehensive overview of the various roles of HSPGs in these systems and explore the concept of an instructive heparan sulfate sugar code for modulating vertebrate development.Heparan sulfate proteoglycans (HSPGs) have long been implicated in a wide range of cell-cell signaling and cell-matrix interactions, both  Summary: This Review article examines the role of heparan sulfate proteoglycans in vertebrate development and explores the concept of an instructive 'sugar code' for modulating development. Vertebrate development – from a fertilized egg to a whole organism – is a complex process orchestrated by many signaling pathways. The generation of various cell types and organs from a single cell is achieved through successive developmental programs that require the spatial and temporal coordination of developmental signaling molecules, including morphogens and growth factors. Evidence accumulated over the past twenty years has shown that most, if not all, of these developmental factors are regulated by heparan sulfate proteoglycans (HSPGs).Table S1). HS chains are synthesized in the Golgi apparatus in a multi-step process that involves several enzymes (Table S2). Exostosin (Ext) enzymes elongate HS chains by adding alternating glucuronic acid and N-acetylglucosamine residues, whereas N-deacetylase/N-sulfotransferase (Ndst) enzymes, C5 epimerase and 2-O-, 3-O-, and 6-O-sulfotransferases modify them by catalyzing deacetylation, epimerization and sulfations at different positions  chains are covalently linked. Syndecans (Sdc) and glypicans (Gpc), which are among the main classes of core protein, are anchored to the cell surface by a transmembrane domain or a glycosylphosphatidylinositol anchor, respectively, and can be released into the extracellular space following cleavage by various enzymes . Other HGiven their various HS chains and localization (i.e. at the cell surface or in the ECM), HSPGs have been proposed to regulate signaling pathways in many different ways . They caCaenorhabditis elegans have strongly supported the sugar code hypothesis . In the absence of genetic mutants, the roles of HSPGs in zebrafish have mostly been assessed based on MO phenotypes, and these studies have provided some interesting insights into the specific roles of HSPG core proteins and biosynthetic enzymes in embryonic morphogenesis. There are, however, increasing concerns about potential MO artifacts and the possibility that mutants can be functionally compensated by other family members or other regulatory events  cause drastically different phenotypes in mice versus zebrafish or Xenopus. It is thus likely that a fuller understanding of HSPG functions will require the combination of multiple mutants, transgenic and other techniques to test lineage-specific roles of HSPG core proteins and biosynthetic pathways in vertebrate development.The functions of HSPGs in development have been assessed using various approaches, including targeted mutants in mice, genetic mutants in zebrafish and morpholino (MO) knockdowns in zebrafish and y events . As suchAlthough studies in pothesis , evidencvivo see . In thisExt1 or Ext2 in mice are embryonic lethal, exhibiting defects in gastrulation (ext2 (dackel) or extl3 (boxer) mutants undergo normal gastrulation owing to a maternal contribution of both enzymes, but later develop cartilage and limb defects and display impaired FGF, Wnt and Shh signaling , suggesting functional redundancy between multiple gene family members that can contribute to specific biochemical steps in HSPG synthesis. Analyzing combinations of mutants will be required to uncover the early developmental functions of HSPGs.The first stages of development following egg fertilization  establish the different body axes and overall architecture of the embryo. Studies of mutants that are defective for genes encoding HSPG biosynthetic enzymes promote the view that HS sugar chains regulate multiple steps throughout these early developmental processes A. HS charulation . Zebrafiignaling . HS chai in mice , and epiHS chain . Interesgpc4 (knypek) mutants exhibit defects in Wnt11-dependent convergent-extension cell movements during gastrulation, suggesting that the potentiation of Wnt11 signaling is dependent on HSPG function , including a wide range of specific neurodevelopmental defects (discussed below), that often result in perinatal death.Studies of mutants that are defective for individual core proteins also provide evidence for early developmental roles for HSPGs. In zebrafish, function . Gastruling gpc4 . By contormation . Other rKupffer's vesicle (KV) is a ciliated organ that controls left-right (LR) patterning in the zebrafish embryo. During normal development, KV cells migrate and undergo a mesoderm-to-epithelial transition to form a vesicle. As the vesicle is inflated, each KV cell grows a specialized apical cilium that is motile and moves fluid asymmetrically in the KV, thereby establishing LR patterning throughout the embryo. A number of studies have implicated a role for HSPGs during this process of LR patterning B.sdc2 in zebrafish KV cells with MOs results in disrupted KV morphogenesis and shortened cilia, giving rise to altered LR patterning throughout the embryo  and hs3st3l  in the zebrafish KV results in normal KV morphogenesis, but causes LR patterning defects that appear to be due to distinct perturbations in KV cilia length and cilia motility  have also been implicated in LR patterning. The MO-based lineage-specific targeting of motility . This stependent .sdc2 reveals a role for this HSPG core protein in the non-cell-autonomous regulation of ECM formation, which is required for cardiac and gut mesoderm migration. In particular, the disruption of sdc2 function in embryonic yolk cells leads to altered ECM formation throughout the embryo and impedes the migration of both cardiac and gut precursor cells, resulting in cardia bifida (two separate heart tubes) and bifid gut tubes . Furthermore, various phenotypes have been reported for mice lacking Hs6st1  exhibit normal LR patterning and early development but show defects in contraction of the cardiac ventricle. This myocardium contraction defect can be rescued by the transgenic expression of hs3st7 in the endocardium tissue that lines the cardiac tube, but not in the myocardium, indicating that the regulation of myocardium contraction by hs3st7 is non-cell-autonomous, implicating cell-cell signaling. Strikingly, genetic reduction of bone morphogenetic protein (BMP) signaling in the heart also rescues contraction in hs3st7 morphants  mice , in which HS synthesis is abolished in neural stem cells (NSCs) and, consequently, in the three lineages originating from them: neurons, astrocytes and oligodendrocytes  lining the ventricles. Neurogenesis is regulated by several morphogens and growth factors, including Fgf2, which plays a dominant role  and requype mice . Consequype mice .Gpc1 exhibit a reduced brain size as a result of decreased proliferation (by 20%) from embryonic day (E) 8.5 to E9.5  does indeed suggest that Gpc1 and Gpc4 might have overlapping functions in regulating brain size   . This is3/Mapk1) , supportSdc3 KO mice show a reduced migration response to EGF and pleiotrophin in vitro  disrupts neuronal migration  or between neurons and muscles . Neurotransmitters are released from presynaptic axonal termini into the synaptic cleft and bind to their receptors on the postsynaptic membranes of dendrites or muscles fibers. In the central nervous system, post-synaptic membranes are organized on dendrites into small protrusions of various shapes called spines. These spines evolve from actin-rich thin filopodial protrusions and develop into mushroom-like or stubby shapes during their formation and maturation.in utero  on developing skeletal muscle fibers in culture , is secrin utero . Similarin utero . In addiin utero . Synapsein utero .Sdc2 in rat hippocampal neurons accelerates spine formation, whereas inhibiting its expression prevents the development of dendritic protrusions  as an Sdc2-interacting partner . Overexptrusions . The abitrusions . The C1 trusions . Phosphotrusions . The C2 in vitro identified Gpcs (most notably Gpc4) as pre-synaptic partners of the leucine-rich repeat transmembrane neuronal protein Lrrtm4, which is located at the post-synaptic membrane . Genetic functional analysis is likely to be complicated by partially overlapping functional redundancies, for example within the same biosynthetic enzyme family, between some members of the same core protein family, and perhaps between different core families. Although it is clear that some core proteins have distinct functions (Table S1), those functions could be independent of HS attachments to that core protein. Conversely, one could speculate that an Sdc core protein and Gpc core protein might be able to perform some of the same functions as long as they can acquire the same modified sugar chains (sugar code) and deliver that information to the cell surface in a particular cell lineage during development. Similarly, it might be possible for multiple members of the same biosynthetic gene family, for example the seven or eight members of the Hs3st family in vertebrates, to participate in generating the same sugar code in a particular cell lineage. Thus, it might be necessary to generate double mutants and beyond, as has been done for a few biosynthetic enzyme genes (Table S2). Despite these challenges, genetic analysis will be essential for testing the functions of HSPGs in development.One key aim is to use genetics, transgenics and other functional assays to discern the roles of HSPG core proteins and the biosynthetic pathways that generate modified sugar chains. Although mutant analyses of some of the genes have yielded exciting results, most genes (including most of the multigene Hs3st family) have not yet been tested (in vitro, that some HS-protein interactions are relatively non-selective, as long as a specific type of sulfation (It should also be noted that the sugar code hypothesis is exciting but controversial. A number of key findings are in support of the hypothesis: distinct cells can generate unique compositions of HS; distinct HS structural motifs are associated with defined developmental events, especially in invertebrates; and some HS-protein interactions show specificity and selectivity. Of particular interest, it has been shown that 3-O-sulfation enzymes contribute to a very small level of HS modification and yet are represented by the largest gene family in the pathway , suggestulfation  or an ovWhen considering genetic functional analyses of individual enzyme family members, it should also be noted that a phenotype could arise not because other enzymes in the same family cannot compensate for function, but because those enzymes are not expressed in the affected tissue. One of the great challenges is that, in contrast to RNA synthesis, HS synthesis is not template driven. Thus, if there is a sugar code, building it is likely to be dependent on the innate properties of the biosynthetic enzymes and their complexes, which are perhaps dependent on subtle substrate specificities as they decide where to add modifications to HS chains in the context of other modifications on that chain.in vitro (in vivo development remains a real challenge. In analogy to advances in RNA expression profiling, one would ideally like to be able to analyze the ‘sequence’ of HS modifications, from one end of individual chains to the other, and to do so from a very heterogeneous mix of HS chains derived from single cells obtained from developing organisms. With these many challenges, there is clearly much work to be done to understand the roles of heparan sulfate proteoglycans in development.Finally, although powerful methods have been developed to study HS-protein interactions in vitro , with ne


A successful transition from the mitotic cycle to the meiotic program in sporogenous cells is crucial for sexual reproduction. By contrast, when meiosis is bypassed or a mitosis-like division occurs to produce unreduced cells, followed by the development of an embryo sac, clonal seeds can be produced by apomixis, an asexual reproduction pathway found in 400 species of flowering plants. An understanding of the regulation of entry into meiosis and molecular mechanisms of apomictic pathway will provide vital insight into reproduction for plant breeding. Recent findings suggest that AM1/SWI1 may be the key gene for entry into meiosis, and increasing evidence has shown that the apomictic pathway is epigenetically controlled. However, the mechanism for the initiation of meiosis during sexual reproduction or for its omission in the apomictic pathway still remains largely unknown. Here we review the current understanding of meiosis initiation and the apomictic pathway and raised several questions that are awaiting further investigation.Meiosis, a specialized cell division to produce haploid cells, marks the transition from a sporophytic to a gametophytic generation in the life cycle of plants. In angiosperms, meiosis takes place in sporogenous cells that develop  Meiosis is an extremely important step in sexual reproduction. It is widely accepted that it evolved from mitosis and shares certain features with mitosis . Yet at The cellular events during meiosis are evolutionarily conserved among species; however, the mechanisms controlling the initiation of meiosis are diverse . The molSaccharomyces cerevisiae, starvation induces expression of the Initiator of Meiosis I (IME1) gene, which encodes a transcription factor responsible for activating early meiotic genes , a vertebrate-specific gene, is then induced by RA and is required for the transition to meiosis . During anther development, after primary sporogenous cells  are differentiated, they first undergo several rounds of mitosis to proliferate, and then meiosis occurs synchronously in all the PMCs of each anther  mutant by am1 mutant allele does not undergo meiosis; instead mitosis-like divisions take place in well-developed meiocytes in both female and male organs  can enter meiosis but cells arrest in the leptotene/zygotene stage, resembling the phenotype of the rice am1 mutant that also carries an amino acid substitution (R360W) in a conserved region , exhibit abnormal meiosis with sister chromatid cohesion defects in male meiocytes, and the mitosis-like division was only observed in female meiocytes  and am1-praI  and their fertile siblings. In 1-mm anthers when meiosis is about to start in the wild-type, 484 genes were missing and 1208 genes were ectopically expressed in am1-489 anthers. These genes are considered to contribute to the initiation of meiosis or the suppression of mitosis. In 1.5-mm anthers, during prophase I in the wild-type, 3700 transcripts were missing and another 3107 genes were differentially expressed in am1-489 anthers. Nearly 60% of transcriptome changes, regardless of stage, were genes enriched in PMCs and many putative meiosis-related genes were found among them. However, none of the meiosis-related genes were regulated in an absolute On/Off pattern on the am1-489 allele, somewhat surprising given that the am1-489 PMCs perform mitosis instead of abnormal meiosis. These results redefine the role of AM1 in the modulation of transcript accumulation for many meiotic genes rather than simply switching them on or off  found about 2500 genes specific or enriched in germinal initials . SurprisMEL2, named for its “meiosis arrested at leptotene” phenotype. MEL2 encodes an RNA-recognition-motif (RRM) protein, and it is required for regulating the premeiotic G1/S-phase transition of male and female germ cells  mutants exhibit multiple MMCs compared to a single MMC in the wild-type ovule, and additional MMCs in the mutant are able to initiate gametogenesis without undergoing meiosis, resembling apospory  and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes required for siRNA biogenesis, also lead to an identical defect to that in ago9 mutants . Additionally, AGO104 is required for heterochromatic CHG and CHH methylation. Consistent with the idea that epigenetics regulates apomixis, mutations of two DNA methyltransferases, DMT102 and DMT103 in maize, also exhibit apomictic development (Arabidopsis ago9 mutant) or lead to apomixis in correctly specified MMCs (seen in maize ago104 mutant). Interestingly, both AGO9 in Arabidopsis and AGO104 in maize are specifically expressed in surrounding somatic nucellar cells, and not in the reproductive cells, implying that both genes control the apomictic pathway in a non-cell-autonomous manner. siRNAs produced from somatic cells may move to germinal cells to regulate the chromatin state by suppressing transposable elements. Indeed, many transposable elements are silenced in Arabidopsis wild-type ovules, in an AGO9-dependent manner (Apomixis is a type of asexual reproduction through seeds that avoid both meiosis and fertilization. In the apomictic pathway, differentiated MMCs or other somatic cells in ovules that gain germinal cell fate are able to bypass meiosis or undergo an abnormal meiosis to produce unreduced spores that further divide mitotically to form an embryo sac ( mutants . Similarenotypes . The maielopment . Thus, lt manner . These rt manner .MEL1. It encodes an AGO5 protein that is required for maintaining germ cell identity and normal meiosis progression. Interestingly, the mel1 mutant also shows defective chromosome condensation with abnormal pericentromere histone modification (Another gene belonging to the ARGONAUTE family with meiotic phenotypes is rice fication . Recentlfication . Furtherswi1 mutant (Over the past few years, the identification of mutants has shed light on genetic control of epigenetic mechanisms involved in apomixis. However, it is still not clear how the RdDM-dependent process affects cell fate specification, meiosis, and gametophyte development? Why is there a need for transposes-derived siRNA in the germ line? Is it possible that RdDM resets cell fate in the germ line, a role also demonstrated for the animal PIWI pathway ? Perhaps1 mutant , raisingUnderstanding the initiation of meiosis and apomixis in plants will be enlightening, and may have many potential applications for plant breeding and in agriculture including developing a strategy for acquiring apomixis in crops, and allowing manipulation of the meiotic cell cycle. It will be crucial to identify more participants in the mitosis–meiosis decision and the apomictic pathway and to explore their molecular functions.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.


These mice are mutated in the TBCE gene encoding the cis-Golgi localized tubulin-binding cofactor E, one of five chaperones that assist in tubulin folding and microtubule polymerization. Loss of TBCE leads to alterations in Golgi microtubules, which in turn impedes on the maintenance of the Golgi architecture. This is due to down-regulation of COPI coat components, dispersion of Golgi tethers and strong accumulation of ER-Golgi SNAREs. These effects are partially rescued by the GTPase ARF1 through recruitment of TBCE to the Golgi. We hypothesize that defects in COPI vesicles, microtubules and their interaction may also underlie Golgi fragmentation in human ALS linked to other mutations, spinal muscular atrophy (SMA), and related motor neuron diseases. We also discuss the functional relevance of pathological Golgi alterations, in particular their potential causative, contributory, or compensatory role in the degeneration of motor neuron cell bodies, axons and synapses.Pathological alterations of the Golgi apparatus, such as its fragmentation represent an early pre-clinical feature of many neurodegenerative diseases and have been widely studied in the motor neuron disease amyotrophic lateral sclerosis (ALS). Yet, the underlying molecular mechanisms have remained cryptic. In principle, Golgi fragmentation may result from defects in three major classes of proteins: structural Golgi proteins, cytoskeletal proteins and molecular motors, as well as proteins mediating transport to and through the Golgi. Here, we present the different mechanisms that may underlie Golgi fragmentation in animal and cellular models of ALS linked to mutations in SOD1, TARDBP (TDP-43), VAPB, and C9Orf72 and we propose a novel one based on findings in progressive motor neuronopathy  Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disease characterized by progressive degeneration of motor neurons in spinal cord, brainstem and cerebral cortex and of their corresponding axons in the corticospinal tract and in peripheral nerves. Degeneration of motor axons and loss of neuromuscular synapses leads to denervation of skeletal muscle fibers which causes progressive muscle weakness and paralysis and becomes fatal when it reaches critical muscle groups, usually within 2–5 years after disease onset , FUS and C9ORF72, or manifest as apparently sporadic form. In ALS motor neurons, many cellular functions are altered as illustrated by defects in nucleocytoplasmic transport  and sorting of proteins from their site of synthesis in the endoplasmic reticulum to their final destination, plasma membrane, extracellular space and endosomal/lysosomal compartments  and MT-associated motor proteins; the structural Golgi proteins; and the proteins of the Golgi transport machinery. Of note, most of the knowledge on these alterations comes from studies in mitotic non-neuronal cells and only few data are available from motor neurons.It has been known for 30 years that microtubule depolymerization (for instance with nocodazole or colchicine) alters the Golgi morphology of mammalian cells. Instead of the single copy organelle capping the nucleus in non-treated cells, the Golgi ribbon appears fragmented leading to single stacks or group of stacks , docking  is localized to the Golgi , and VAP-B.Schematically, four mechanisms of Golgi fragmentation can be distinguished: The first one involves defective microtubules or microtubule-dependent transporters due to mutations in SOD1 , and related motor neuron diseases.Mutations in the human SOD1 (Cu/Zn superoxide dismutase 1) gene account for ~5% of familial forms of ALS  DNA-binding protein of 43-kDa  and late-onset SMA have been associated with a dominant missense mutation (P56S) in the VAPB gene -Associated Protein) family of ER C-tail anchored proteins. This class of proteins has been shown to interact with the FFAT motif  characteristic of lipid transfer proteins enriched at membrane contact sites between the ER and the Golgi  and a consequent reduced level of the functional protein  stimulates recruitment of remaining TBCE to the Golgi, polymerization of microtubules and rescue of the Golgi defects  mice mutated in the Scyl1 gene. Scyl1 encodes a protein that normally interacts with α-COP and β-COP  gene  hexanucleotide repeat expansions in the first intron of the C9Orf72 gene  domains in C9Orf72  that causes severe proximal axonopathy does not induce any morphometric changes of the Golgi in rat motor neurons (Mourelatos et al., Conversely, mounting evidence suggests that Golgi alterations may contribute to, or in some cases even cause, aspects of motor neuron degeneration. In particular, alterations in the microtubule/COPI cross talk may impact on the normal function of the Golgi apparatus in the transport of proteins, lipids and RNAs that are essential for axon and synapse maintenance. The COPI subunits α-, β-, and γ-COP for instance bind numerous mRNAs (Bi et al., in vitro or in vivo, and to subsequently assess the specific consequences on axon degeneration, loss of synaptic vesicles and synaptic dysfunction. To achieve this, we need to gain deeper insights into the mechanisms driving this fragmentation.In conclusion, more work is needed to understand the relevance of Golgi fragmentation for onset and progression of motor neuron degeneration. One way forward is to experimentally inhibit Golgi fragmentation in model systems, whether Defects in the cross talk between the Golgi microtubule network and the Golgi transport COPI machinery seem an early common mechanism shared by human ALS, SMA, and other motor neuron diseases and thus appear as exciting new molecular targets for the development of biomarkers and pharmacological or gene-based therapies in these severe and currently incurable disorders.GH and CR prepared and wrote this 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.


Cell Death Discovery.1Research from our group has demonstrated a novel mechanism of cancer cell death by mTOR (mechanistic target of rapamycin) inhibitors, often used in hepatocellular (HCC) and renal cell carcinomas (RCC) therapy, by augmenting the gluconeogenesis pathway. Our results further suggest that upregulation of the gluconeogenic pathway creates a metabolic stress where bulk of the glucose is exported outside the cells creating a 'futile' cycle for glucose that in turn detrimentally affects the cell survival. The work is in-press and is scheduled to be published in the new NPG journal 3 An expanding compendium of metabolic pathways associated with oncogenesis, tumor progression and metastasis have been and being discovered. Otto Warburg's seminal observation of proliferating ascites tumor cells converting the bulk of glucose carbon to lactate, even in normoxia, was among the early experimental evidence of rewiring of glucose metabolism in cancer cells.4 Thus bypassing the mitochondrial respiration in oxygen-rich conditions albeit enhanced rate of glycolysis, now termed aerobic glycolysis, is one of the key features of metabolic reprogramming intrinsic to cancer cells. Indeed, alteration in the multiple metabolic shuts including pentose phosphate pathway, serine-glycine pathway and several control points in the glycolytic pathway such as hexokinase 2, pyruvate kinase M2 and lactate dehydrogenase stimulate glycolysis and provide prosurvival cues. Blockade of pyruvate flux into mitochondria via mitochondrial pyruvate carrier in parallel further accentuates the metabolic rewiring. ‘Glucose addiction' thus endows cancer cells with enhanced metabolic flexibility for their anabolic requirements to support growth, proliferation and survival.6Metabolic reprogramming, now considered a hallmark of cancer, has become a major research area in cancer biology in the past decade.7 Gluconeogenesis is defined metabolically as the production of glucose from non-carbohydrate carbon sources such as pyruvate, lactate, glycerol and gluconeogenic amino acids. Thus, it is imperative that concomitant activation or downregulation of glycolysis and gluconeogenesis would cause a metabolic stress, which might in turn disrupt the metabolic rewiring of cancer cells. Targeting glycolysis/gluconeogenesis pathways as a consequence of metabolic reprogramming therefore is an attractive therapeutic strategy because it is central to the growth and survival of cancer cells.8Although an in-depth understanding of glycolytic switch in cancer metabolism has grown in recent years, however gluconeogenesis, which drives the metabolic flux in parallel and opposite to glycolysis, has got little attention.10 Hyper-activation of mTOR signaling pathway is often associated with cancer cell growth and survival including HCC and RCCand the use of mTORC1 inhibitors rapamycin and its analogshas been approved for the treatment of advanced RCC, albeit with modest success.On the other hand, ATP competitive mTOR inhibitors that fully inhibit both the mTOR complexes exhibit stronger antitumor effects suggesting a potential contribution of mTORC2 in tumor progression.12The objective of our work was to elucidate the role of mTOR-mediated metabolic reprogramming and its consequences in proliferation and survival of human cancer cells.The mTOR kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, which serves as a central regulator for promoting cell growth, proliferation and survival.G6PC and PCK1, the two rate-limiting enzymes of gluconeogenesis, forkhead box O serves as a strong inducer of glucose production. Surprisingly, instead of glucose consumption being reduced, blocking mTOR led to an increase in glucose consumption and lactate production. Subsequent examination of mitochondrial metabolites showed that the increased glucose consumption was not leading to increased mitochondrial oxidative phosphorylation, but instead these metabolites were being shunted off into gluconeogenesis. Thus, mTOR inhibition arguably creates a 'futile' cycle where glucose consumption is increased, only to have it be broken down and then recycled through gluconeogenesis (PCK1 but not G6PC after mTOR inhibition could prevent gluconeogenesis from increasing, preventing this futile cycle from initiating, and substantially reduce apoptosis after mTOR inhibition. Finally, transcriptomic analysis was conducted for metabolic genes involved in gluconeogenesis, across multiple tumor types, and HCC and RCC was found to have reduced expression of genes for multiple enzymes found in this pathway. Interestingly, mTOR inhibition could only enhance cell death in HCC and RCC cell lines whereas in cell lines where mTOR inhibition could not lead to the activation of gluconeogenesis did not undergo significant cell death. Our findings thus uncover an important role of mTOR in cancer biology and possibly explain the phenomenon as to why mTOR inhibitors are effective in certain cancer types only.In this context, we took both pharmacological as well as RNAi approaches to block specifically mTORC1, mTORC2 or both followed by biochemical analysis of metabolic ramifications of inhibition mTOR in cancer cells, specifically HCC and RCC. Initial experiments suggested a potential sterol regulatory element-binding protein-independent downregulation of lipogenesis, a well established mTOR-dependent metabolic pathway. Wenext focused on transcriptional activation of forkhead box O following mTOR inhibition as a potential mediator of metabolic changes. By upregulating ogenesis . The det