Skeletal muscle has a remarkable capacity to regenerate by virtue of its resident stem cells (satellite cells). This capacity declines with aging, although whether this is due to extrinsic changes in the environment and/or to cell-intrinsic mechanisms associated to aging has been a matter of intense debate. Furthermore, while some groups support that satellite cell aging is reversible by a youthful environment, others support cell-autonomous irreversible changes, even in the presence of youthful factors. Indeed, whereas the parabiosis paradigm has unveiled the environment as responsible for the satellite cell functional decline, satellite cell transplantation studies support cell-intrinsic deficits with aging. In this review, we try to shed light on the potential causes underlying these discrepancies. We propose that the experimental paradigm used to interrogate intrinsic and extrinsic regulation of stem cell function may be a part of the problem. The assays deployed are not equivalent and may overburden specific cellular regulatory processes and thus probe different aspects of satellite cell properties. Finally, distinct subsets of satellite cells may be under different modes of molecular control and mobilized preferentially in one paradigm than in the other. A better understanding of how satellite cells molecularly adapt during aging and their context-dependent deployment during injury and transplantation will lead to the development of efficacious compensating strategies that maintain stem cell fitness and tissue homeostasis throughout life. Stem cells are essential for the maintenance and repair of many adult tissues during normal physiology or in response to damage. Operationally defined, stem cells produce daughter cells that differentiate to repair damaged tissue and self-renew to repopulate the stem cell pool. Although long lived, tissue resident stem cells do not retain their fitness and function indefinitely. The unique requirement of tissue resident stem cells to maintain themselves and form new specialized cells may explain why their decline has a greater detrimental impact than that of other cell types on tissue regeneration. Across different stem cell compartments, age-dependent changes that cause stem cell dysfunction are multifactorial, encompassing, systemic, local, and intrinsic factors .Adult stem cells have tissue-specific properties, related to the tissue they serve, such as distinct rates of turnover and specialized differentiation programs. Yet, they also have many common characteristics. They transit between quiescence and activation stages, their chromatin adopts bivalent states to facilitate rapid differentiation of self-renewal, they are capable of undergoing symmetric and asymmetric divisions, their metabolism is specialized to adapt to their particular needs, and they are located within microenvironments which influence their functions , 3. ThesDuring aging, many tissues undergo changes in stem cell number and function that impact tissue homeostasis. Optimal stem cell function necessitates appropriate extrinsic support from the local microenvironment (niche) and systemic environment (circulation). Hence, aging of the stem cell local and systemic environment is pertinent to stem cell demise. Since the original demonstration using parabiosis that muscle repair was under the control of soluble factors present in serum, alterations in the composition of the systemic environment has been the prevailing model to explain defects in skeletal muscle repair during aging , 5. WithSatellite cells constitute the principal stem cell pool of adult skeletal muscle. Genetic ablation studies and transplantation studies together confirm that Pax7+ satellite cells are sufficient and required for adult muscle repair –11. In rUnlike other types of stem cells, such as hematopoietic stem cells, not only the function but also the number of satellite cells declines with aging , 14, 20.At variance with other types of stem cells, information regarding the molecular pathways promoting aging of satellite cells is abundant. Parabiosis and whole muscle grafting experiments reveal that the mechanisms controlling the regeneration capacity of stem cells may be principally ascribed to changes in the aging environment , 21–23. In this review, we discuss what is known about the principal stem cells of skeletal muscle, focusing on the control of stem cell function from the extrinsic environment and intrinsic regulation. We specifically consider what degree of stem cell dysfunction can be ascribed to the age-associated changes in the environment, cell-autonomous changes, or both, what makes satellite cells more or less susceptible to aging; and whether this can shed light on potential rejuvenating strategies.2-induced muscle injury and observed that muscle fiber size did not return back to pre-injured levels in aged mice [The influence of the extrinsic environment on the efficiency of the regeneration process arouse from whole muscle transplantation experiments and parabiosis. Transplantation of whole muscle grafts of young and old mice showed that the regenerative ability of young and old muscles depended mainly on the age of the host, as young hosts allowed successful engraftment of both young and old muscles while both graft types failed to regenerate in old rats . Howeverged mice . PossiblParabiosis has been used to test the effects of circulatory or systemic factors of one animal on the other for over 100 years . AnastomRecently, the assessment of the expression and role of a circulating factor, growth differentiation factor 11 (GDF11), has provided conflicting results , 33. TheOverall, these findings demonstrate reversibility of satellite cell dysfunction in old animals through restoration by, or mimicking of, a young extrinsic environment. Due to the nature of the muscle injury assay, and the influence of many cell types to its outcome, it is not possible to determine if these stimulatory effects are acting at the level of the stem cell, myogenic progenitors, or different cell types. Indeed, direct evidence for non-stem cell-mediated control of muscle repair comes from studies investigating inflammation. A transient blood-mediated inflammatory response to muscle injury is key for successful regeneration , 36. ParIn more recent years, the direct assessment of muscle stem cell potential via transplantation has become more commonplace. Stem cell transplantation was pioneered by Till and Mcullough 55 years ago , 40. TodInjection of tractable adult satellite cells as a population, and at the single cell level, has provided definite evidence for their stem cell status, based on the ability to proliferate, differentiate (fuse to recipient pre-injured muscle), and self-renew (reoccupy the niche and express satellite cell markers). Comparisons between purified satellite cells from adult and aged mice transplanted into pre-injured adult muscle show a substantial decline in the number of aged satellite cells that repopulate the niche and contribute to muscle fiber differentiation Table . In agreOver the past few years, it has become apparent that functional heterogeneity exists within the satellite cell pool , 44–46. mdx mice or the blunted immune response of immune-compromised mice will exert opposite, albeit impactful effects on cell engraftment.The functional deficit of aged satellite cells engrafted in a young host in its strictest sense has been used to conclude that there is a non-reversible component to stem cell aging. However, parabiotic conditions provided only a partial recovery of muscle repair in aged mice exposed to a young systemic environment. One could consider this due to a partial dilution of aged blood; however, it could also be due to other factors outside the systemic environment, including the niche or intrinsic defects. To date, a direct comparison of aged satellite cells after engraftment in adult and aged recipients is lacking. In addition, using heterochronic parabionts to serve as the source of donor satellite cells or recipient muscle for transplantation experiments has not been examined. For example, if the functional deficit of aged satellite cells (versus adult satellite cells) engrafted into an aged host was twice that observed in a young host, the results from parabiosis, transplantation, and in vitro studies would be in general agreement: that alterations in stem cell function during aging is a product of intrinsic and extrinsic deregulation. Alternatively, if results from transplantation studies do not support an extrinsic component, in contrast to endogenous muscle cells after injury, this would highlight the context dependence of the assays employed. Another factor that limits direct comparison is the age of the mice examined. Parabiosis studies have classically been carried out on 18-month-old mice, whereas transplantation studies have predominantly used mice that are 20 months and older. As age-dependent phenotypes are progressive, this may impact the rejuvenation potential of satellite cells. Another distinction is the length of recovery after injury between the two types of studies. Acute repair processes have been studied predominantly after parabiosis, and longer recovery periods assessed in transplantation studies. Identifying the long-term injury response on parabionts would help to resolve this potential discrepancy. Finally, we cannot discard that the genotype and age of the recipient mice in cell engraftment experiments might also influence the regenerative outcome. Indeed, aged mice experience chronic inflammation, yet the inflammatory response in whole muscle grafts is less efficient with age, and this appears to impact new myofiber formation [Resolving the relative contribution of intrinsic and extrinsic control on stem cell function is critical, but it should also be considered that the assays deployed for determination are not equivalent and may overburden specific cellular regulatory processes and thus probe different aspects of satellite cell properties. The cellular experience of the stem cell pool is likely very different if activated by injury while in their resident niche, compared to the physical manipulations that take place to extract, purify, and inject satellite cells into a foreign muscle. It is likely that the isolation of satellite cells followed by their transplantation (sometimes with a prior cell culturing period) may provoke cellular changes  that influence satellite cell output and tissue contribution. On the other hand, parabiosis procedure-associated complications like limitation on mice mobility, chronic inflammation (which perturbs the environment), or hypoglycemia, may account for some of the observed effects .Intriguingly, in other stem cell systems, different stem cell subsets can dominate tissue contribution depending on the assay employed. Using genetic bar coding, which allowed individual hematopoietic stem cells (HSC) clones to be tracked over time, the Camargo lab found that HSC contribution during transplantation was derived from substantial expansion of rare clones . In contThe functional deficit of satellite cells as a feature of aging examined under transplantation suggests a strong cell-intrinsic and irreversible component that could include genomic instability, DNA and oxidative damage, and alterations in mitochondrial function. Indeed, satellite cells have an increased resistance to DNA damage and capacity to repair DNA lesions than their committed progeny . SatelliINK4a (a cell cycle inhibitor and marker of senescence) via ubiquitination of histone H2A, this repression is relieved in aged cells [INK4A and p21CIP1 loci, provoked by alterations in H3K4me3 and H3K27me3 chromatin marks, in aging satellite cells after injury [At the level of single genes, adult satellite cells epigenetically repress the INK4a locus that encodes p16ed cells . Furtherr injury .INK4a, which causes loss of the G0 reversible quiescent state. In response to regenerative pressure by injury, p16INK4a caused the de-phosphorylation of the retinoblastoma protein (Rb) and repression of E2F target genes in proliferation-promoting conditions, which accelerates entry into full senescence [INK4a has been shown to delay aging in several tissues of BubR1 progeroid mice, including skeletal muscle [INK4a knockdown or ablation of p16INK4a-expressing cells is restricted to senescence modulation exclusively.Despite the always attractive possibility for rejuvenation as indicated by parabiosis experiments, satellite cell aging proceeds to a limiting point where it essentially becomes irreversible, particularly at the late geriatric age , suggestnescence . Regardll muscle . It remaINK4a-induced senescence can contribute to loss of satellite cells, mice overexpressing p16INK4a, due to genetic loss of the polycomb repressor complex 1 (PRC1) protein Bmi1 [INK4a expression in the resting state, and pro-senescence phenotypes [Whether the pre-senescent state of satellite cells from geriatric mice is restricted to a subset or a feature of most satellite cells is not known. However, it is tempting to propose that, as for satellite cell apoptosis, senescence might be an alternative way to restrict unfit satellite cells of aged mice from undergoing myogenesis. Supporting the notion that p16ein Bmi1 , 61, or ein Bmi1 , showed enotypes . Interesenotypes , suggestSpry1 (encodes Sprouty1), an inhibitor of FGF signaling and upregulation of FGF2 in the niche. Genetic elimination of Spry1 in satellite cells enforced FGF signaling, which resulted in loss of quiescence and subsequent reduction in the satellite cell number. Spry1 elimination during adult muscle repair led to sustained activation of the ERK pathway, which impaired self-renewal and caused apoptosis in a subset of satellite cells [Spry1 in muscle stem cell regulation is not restricted to murine studies. Myogenic progenitors from adult and aged human biopsies show increased DNA methylation at the Spry1 locus coupled with a loss of ‘reserve’ cells to return back to a quiescent state [While intrinsic molecular effectors determine cell functionality, it is less clear whether they are autonomously driven or a consequence of altered extrinsic factors. The adult satellite cell pool is functionally heterogeneous, with subsets enriched for stem cell potential under transplantation conditions , 45, 64.te cells . Togethete cells . The crint state . DemethyThe normal asymmetric divisions that produce one stem and one committed progenitor in adult satellite cells are disrupted in aged satellite cells via chronic elevation of p38αβ MAPK signaling and desensitized FGFR1 activity. Instead, aged satellite cells preferentially undergo symmetric divisions that produce two committed daughters. This limits the expansion of aged satellite cells, favoring their permanent cell cycle exit , 16. WhiSimilarly, as indicated before, an increasingly active IL-6-JAK/STAT3 pathway in satellite cells with age appears to reduce their regenerative potential by limiting their proliferative capacity and affecting their symmetric versus asymmetric polarization , 18. ThiIt is widely appreciated that freshly isolated satellite cells from aged mice have a predisposition for apoptosis and senescence when placed in culture conditions , 16, 41.INK4a. Elevation of stress pathways beyond a certain threshold can promote cellular arrest and predispose to senescence or apoptosis fates, either by inducing mitochondrial dysfunction, increasing reactive oxygen species (ROS), or DNA damage . Theiewed in . Importaiewed in . In contiewed in . TherefoINK4a-induced senescence) can allow their functional rejuvenation upon engraftment.The satellite cell pool becomes more homogeneous with age, with reduced representation of cells having high regenerative potential, and increased proportion of cells with reduced clonal proliferation capacity . Thus, iParabiotic studies have demonstrated extrinsic components, whereas transplantation assays have confirmed intrinsic components that cause age-dependent muscle stem cell decline. At first glance, the results based on satellite cell transplantation appear to be in conflict with those from parabiosis. The main purpose of this review has been to highlight the potential causes of discrepancy among the different papers and discuss how these differences teach us new facets about muscle stem cell biology and aging. We believe that the procedure chosen to assess the capacity of the young environment to rejuvenate aged satellite cells greatly accounts for the differences. We propose that the isolation of satellite cells and their subsequent transplantation may exacerbate intrinsic changes that importantly alter their functions. Thus, stem cell potency is greatly influenced by their capacity to overcome the stress of engraftment. From this viewpoint, old satellite cells would be less resistant to this stress. Importantly, strategies that modulate stress pathways in old satellite cells constitute a robust method for functional rejuvenation. Noteworthy, acute inflammation and stress, induced by the surgical joining of two animals in parabiosis experiments, might cause systemic perturbations that could affect satellite cell functions in yet unpredictable ways. In addition, the choice of genotype and age of the recipient mice for engraftment assays might also influence the regenerative outcome.Another issue that may contribute to the apparently discrepant results between parabiosis and transplantation experiments relies on the molecular and functional diversity within the satellite cell population. Rejuvenation of aged satellite cells, in the context of transplantation, might derive from the augmented expansion of a small subpopulation of the fitter and more stress-resistant stem cells. Whether distinct satellite cell subsets are mobilized preferentially in the context of repair and transplantation or are differentially sensitive to intrinsic and extrinsic stressors warrants testing. A greater comprehension on the causative factors that drive satellite cell dysfunction during aging will lead to strategies that promote tissue rejuvenation and a beneficial impact on the quality of life of the elderly.


The thick-tufted layer 5 (TTL5) pyramidal neuron is one of the most extensively studied neuron types in the mammalian neocortex and has become a benchmark for understanding information processing in excitatory neurons. By virtue of having the widest local axonal and dendritic arborization, the TTL5 neuron encompasses various local neocortical neurons and thereby defines the dimensions of neocortical microcircuitry. The TTL5 neuron integrates input across all neocortical layers and is the principal output pathway funneling information flow to subcortical structures. Several studies over the past decades have investigated the anatomy, physiology, synaptology, and pathophysiology of the TTL5 neuron. This review summarizes key discoveries and identifies potential avenues of research to facilitate an integrated and unifying understanding on the role of a central neuron in the neocortex. The mammalian neocortex is endowed with a daunting diversity of neurons. These neurons are classified into excitatory pyramidal neurons and local circuit interneurons, which are predominantly inhibitory. Pyramidal neurons exist in all layers of the neocortex except layer 1, forming the most abundant neuron type. In particular, thick-tufted layer 5 (TTL5) neurons are one of the most extensively studied cell types in the neocortex Larkman, .in vitro patch-clamp recordings from brain slices using IR-DIC microscopy  and are characterized by a pyramid-like soma, and a stereotypical dendritic morphology with a prominent apical dendrite. In contrast to the other significant population of pyramidal neurons located in upper layer 5 (layer 5A) with slender apical dendrites, which project across hemispheres to the corpus callosum, TTL5 neurons mainly project to subcortical regions. Over the past two decades, The generic anatomy of TTL5 neurons in rodent somatosensory cortex is characterized by a triangular soma, two distinct dendritic domains emanating from the base and apex of the soma , and a single axon sending out several collateral branches that further bifurcate and ramify exuberantly within the neocortex. The apical dendrites further branch into oblique dendrites and terminate with a crown-like thick tuft of dendrites in layer 1. There is a lack of quantitative data comparing the time-scales of anatomical development of TTL5 neurons across different cortical regions and species. Most of our knowledge on the development of TTL5 neurons can be attributed to studies in the frontal, parietal, temporal, and occipital lobes, in particular the visual, auditory, and somatosensory areas.In sensory cortical areas, the anatomical development of TTL5 neurons is marked by three phases of growth; a fast growth phase of basal, oblique and apical branches from postnatal days 7 (P7) to 14 (P14), an intermediate localized growth phase of basal, apical and oblique branches growth from P14 onwards to postnatal day 21 (P21), and a slow growth phase from P21 to postnatal day 60 (P60) Nicolai,  We furthThe somata of TTL5 neurons have been subjectively classified according to triangular, round, and oval shapes  increases between P7 and P14 while the density of large boutons (diameters between 1.0 and 1.8 μm) significantly increases between P14 and P21  and muscarinic activated potassium (IM) conductances. The attenuation of dendritic responses measured at the soma  and somatic responses measured at dendrites  are similar, indicating that dendrites are electrotonically compact during this period , the resting potential at dendritic tufts is more depolarized than the soma. The dendritic input resistance is only slightly lower than at the soma. At P28, depolarizing step current injections at dendritic tufts are capable of eliciting either single APs or a burst of 2–3 APs at the soma Zhu, . The inca2+ Zhu, . Also, dh conductance. Dendritic regenerative potentials evoked by depolarizing current injections give rise to bursts with 2–4 APs at the soma. During this period, the regenerative potentials at dendritic tufts attain their final kinetics, implying that Ca2+ conductances could have reached their peak densities , the dendritic and somatic membrane properties are similar to those measured in the previous period. The depolarizing sag response to hyperpolarizing dendritic current injection is very conspicuous and indicates further increased levels in the dendritic Iies Zhu, . The atties Zhu, .+ dependent to Ca2+ dependent . The apical dendrites are not fully mature until P42. The dendritic tufts switch from being electrotonically close to the soma at P2 to electrotonically distant at P14  channels, a plethora of Ca2+ channels, and small and large conductance  Ca2+ dependent K+ channels, all of which influence the integration of synaptic input  and local dendritic spikes  and high  voltage-gated Ca2+ activated K+ channels  channels occur with a homogeneous density along the somato-dendritic axis of TTL5 neurons, and their activation reduces the occurrence of local dendritic Ca2+ spikes thus rendering dendrites less excitable. BK channels do not influence the temporal window to initiate back-propagating Ca2+ spikes, thus actively decoupling the axo-somatic and the dendritic AP initiation zones during high-frequency inputs  causing a burst of APs at the soma as a direct consequence  spines might serve to optimize the filling of a large number of afferent synapses onto a dendritic segment by increasing the available surface area  in the AIS of TTL5 neurons encode for the fast-inactivating transient (INaT) and slow-inactivating persistent (INaP) currents  and Cav2.2 (N-type) channels have been recently discovered in the AIS of TTL5 neurons , first observed in bullfrog sympathetic neurons  heteromultimers  by delivering precision to the temporal window  substantially increases the amplitude and shortens the latency of the disynaptic inhibitory response in neighboring TTL5 neurons  thalamus  and modulates global TTL5 function in vivo. Further in vivo experiments are essential to peel out the roles of specific populations of GABAergic interneurons that selectively innervate targeted parts of TTL5 dendrites, soma and axons to better understand their normative circuit function in behaving animals.Thus, it appears that the function of TTL5 neurons Donald Hebb’s pioneering theoretical contributions to learning and memory have exerted a continuing influence on neuroscience and psychology for more than half a century. Hebb postulated that when one neuron persistently drives another in its neighboring vicinity, then the connection between them is strengthened Hebb, . FuthermIn spite of a vast body of experimental evidence for Hebb’s postulates, several previous studies have indicated that Hebb’s postulate of “neurons that fire together wire together” would lead to saturation of synapses within assemblies of cells, curbing their dynamic range and contracting memory storage capacity , and acetylcholine (ACh) on neuronal excitability in the juvenile rodent neocortex  modulates the induction of LTD by suppressing CaIn vivo experiments measuring TTL5 excitability have thus far been exclusively carried out under the use of anesthetics, particularly urethane, barbiturate, or ketamine, which can profoundly impact the active properties of TTL5 dendrites and intrinsic firing properties  is crucial in maintaining low [Cl−]i resulting in hyperpolarizing GABA responses. A decrease in KCC2 after neuronal injuries resulted in increases in [Cl−]i and enhanced neuronal excitability in TTL5 neurons due to depolarizing GABA responses  caused acute epileptogenesis in pathophysiological TTL5 neurons  on TTL5 neurons of juvenile rat somatosensory cortex through whole cell patch-clamp recordings, and discovered that a single prenatal injection of VPA caused a significant enhancement of the local recurrent connectivity formed by TTL5 neurons (Rinaldi et al., We have endeavored to provide a unifying view of the dendritic, axonal, synaptic properties and plasticity of TTL5 neurons, and to a lesser extent the pathophysiology of TTL5 neurons from more than five decades of research. In this review, a degree of inevitable recurrence exists in the description of some key phenomena. A high level of interdependence is evident between dendritic excitability and synaptic plasticity and vice-versa, where on the one hand activity-dependent regulation of dendritic excitability induces synaptic plasticity, and synaptic plasticity controls dendritic computations on the other.in vivo, in vitro and in silico approaches for targeted experiments on TTL5 neurons, specific questions can be asked about their occurrence and generalization across different species and/or cortical regions, robustness and redundancy in local circuit organization, gene expression changes across developmental time scales and activity patterns, protein transport and localization across different morphological regions, gene and protein networks underlying specific functions, the biophysical role of spines, possible cell assemblies based on afferent input and efferent output, the neuromodulation of plasticity, how different parts of the dendrite participate in the emergence of receptive fields, integration of translaminar, transcolumnar and transregional synaptic input, to outline a few. Gaining a grasp of a how single TTL5 neuron is actively modulated by the myriad of excitatory and inhibitory synapses impinging onto different dendritic domains is fundamental to better understand the multimodal information processing capabilities of these neurons. The pursuit to unravel the anatomy and physiology of TTL5 neurons will increase our understanding of their pivotal roles in neuronal microcircuitry and why our neuronal network is so wired to make us who we are.Although we have learnt much more about the function of TTL5 neurons in the past several decades than ever before, this is still the tip of the iceberg. To reveal all anatomical and physiological aspects seems still far in the future. With the advent of newer 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.


Urological cancers consist of bladder, kidney, prostate, and testis cancers and they are generally silenced at their early stage, which leads to the loss of the best opportunity for early diagnosis and treatment. Desired biomarkers are scarce for urological cancers and current biomarkers are lack of specificity and sensitivity. Epigenetic alterations are characteristic of nearly all kinds of human malignances including DNA methylation, histone modification, and miRNA regulation. Besides, the detection of these epigenetic conditions is easily accessible especially for urine, best target for monitoring the diseases of urinary system. Here, we summarize some new progress about epigenetic biomarkers in urological cancers, hoping to provide new thoughts for the diagnosis, treatment, and prognosis of urological cancers. Urological cancers are comprised of bladder, prostate, renal, and testis cancers, which are among the 10 most frequent cancers in man except testis cancer. So far, the gold standard diagnosis of urological cancer is pathological diagnosis and the early screening methods are rare. Some existing biomarkers such as prostate-specific antigen may be useful in prostate cancer screening but it is invasive and short of specificity and is causing overdiagnosis and overtreatment, which limits its application , holdingDNA methylation, mediated by DNA methyltransferases (DNMTs), using S-adenosyl methionine (SAM) as the methyl supplier and adding methyl group to the 5-carbon of the cytosine, is the most well delved epigenetic modification in human diseases , 4. ThisHistones modification includes methylation, carbonylation, phosphorylation, ubiquitinylation, sumoylation, glycosylation, and acetylation, which are known as “the histone code,” together with DNA methylation regulating the expression of specific genes . The mecAnother epigenetic modification has been studied deeply, known as microRNAs (miRNAs). miRNAs, a class of noncoding RNAs, which are about 18–25 nucleotides at length, could be synthesized and processed in the nucleus. miRNAs alter the expression through binding to mRNAs; then RNA-induced silencing complex takes part and regulates protein translation , 15. It n = 117) and PCDH17  was related to the development of bladder cancer and it was an independent predictor of cancer-specific survival time [Bladder cancer is the most common male urology malignancy in China and the total number of cases in males for 2015 is approximately 62.1 thousand . Apart fval time , 24.With the deepening of research, it was thought that there was no single gene found to be methylated in vast majority of bladder tumors. More and more researches have focused on using gene panels to early detect bladder cancer and predict tumor recurrence, progression, and metastasis. A methylation analysis of PCDH17 and POU4F2 was developed recently in 148 individuals by qMSP using urine sediment. The combination of POU4F2/PCDH17 detection had sensitivity and specificity of 90.00% and 93.96% in the validation of 312 individuals . Renard Histone modifications were also found to relate to the pathogenesis of bladder cancer and regulation of cancer cell proliferation. A study in 2011 identified global histone H4K20 trimethylation that could predict cancer-specific survival in patients with muscle-invasive bladder cancer . A meta-miRNAs are synthesized and processed in the nucleus and released into cytoplasm, binding to specific mRNA and inducing gene silence . RecentlKidney cancer is the third most common urology malignancy in China and there were about 66.8 thousand people diagnosed with renal carcinoma in 2015. Among these patients, 43.2 thousand are males and females only account for 35% . BesidesHistone modifications in kidney cancer were found to have significant relevance with the prognosis of renal cell carcinoma (RCC). Patients with positive immunostaining of H3K4me2 and H3K18Ac but accounting for a lower proportion generally had a shorter 1-year survival probability than those with more histone modifications . This stmiRNAs are also involved in the development and progression of renal cell carcinoma. Zhang et al. reported that miRNA-126 can inhibit tumor cell invasion and metastasis by downregulating ROCK1 in renal cell carcinoma. They analyzed 128 pairs of ccRCC and adjacent normal tissue samples, measured miRNA-126 expression levels, and found the association between miRNA-126 and various clinicopathological parameters . Using oFindings on epigenetic biomarkers in kidney cancer are summarized in Screening of individuals without any cardinal symptom by the PSA test has been the focus of increasing criticism, primarily due to potential overtreatment and less comprehensive evaluation . CandidaMethylation levels of two genes, PCDH17 and TCF21, were quantified in a total of 12 cancer cell lines and 318 clinical samples. These two gene methylation levels provided a sensitivity rate of 96% for prostate cancer. The high exposing of PCDH17 and TCF21 methylation in prostate cancer cell lines was significantly different from primary tumor tissues. Furthermore, methylation levels were meaningfully lower in bladder and prostate nontumorous tissues, providing a biological influence as cancer biomarkers , 50. In It has been noticed that HOXB13 became overexpressed during malignant progression of the prostatic tissue and played an important role in the pathogenesis of the prostate gland as a novel biomarker for the prognosis of prostate cancer . ADAM19 Other promising RNA markers are the transcripts of fusion genes which between the androgen-regulated transmembrane protease serine 2 gene (TMPSS2) and ERG transcription factors through chromosomal rearrangements become stable and produce a viable mRNA during transcription. TMPSS2-ERG transcription factors have been identified as promising urinary novel biomarkers .Several potential microRNAs for prostate cancer have been identified as important biomarkers. Circulating miRNA-410-5p level was significantly higher in the prostate cancer patients than in normal patients . RecentlThe curiousness of CTCs for specific tumor characteristics has drawn major attention over the past few years. The genomic profiles of CTCs have been found to be largely comparable to primary tumors and/or metastatic tissue, indicating that CTCs are able to reflect tumor characteristics including the extent of intratumoral and biological heterogeneity. CTCs have been shown to be tumorigenic and capable of forming new metastases –67. AndrIn 1960, prostate-specific antigen (PSA) was first discovered by Rubin Flocks. Since the mid-80s, PSA has been the most commonly used biomarker for prostate cancer to judge current and future risk, detect response to treatments, and detect recurrence in all stages of the disease. Due to this work, the Food and Drug Administration (FDA), in USA, approved the use of PSA for monitoring recurrence after treatment. It was later known that it was human species-specific . ProstatRecent genomic studies have identified risk SNPs in testicular germ cell tumors (TGCT). Increased PDE11A, SPRY4, and BAK1 promoter methylation and decreased KITLG promoter methylation in familial TGCT cases versus healthy male family controls can be used to diagnose TGCT in the early time . It is rIn general, these findings provide new directions for detection, diagnosis, and prognosis of urological cancer which may revolutionize the clinical management of cancer patients. DNA methylation is the most explored modification in urological cancer and modifications in histone and miRNA are becoming a hot research topic. Nevertheless, the majority of these markers are single target discovered in a small amount of clinical cases and lack specificity; therefore a panel consisting of multiple targets is needed and this kind of research will become a trend. Besides, there is plethora of epigenetic biomarkers which has been identified, but only extremely rare biomarkers can be applied to clinical diagnosis and therapy targets. The selected biomarker has to be evaluated through rigid clinical trials in appropriate scale and determine the relevance between the biomarkers and clinical practice. We expect more novel publications about epigenetic biomarkers in urological malignances especially from large clinical cases.


Stress impacts negatively on plant growth and crop productivity, caicultural production worldwide. Throughout their life, plants are often confronted with multiple types of stress that affect overall cellular energy status and activate energy-saving responses. The resulting low energy syndrome (LES) includes transcriptional, translational, and metabolic reprogramming and is essential for stress adaptation. The conserved kinases sucrose-non-fermenting-1-related protein kinase-1 (SnRK1) and target of rapamycin (TOR) play central roles in the regulation of LES in response to stress conditions, affecting cellular processes and leading to growth arrest and metabolic reprogramming. We review the current understanding of how TOR and SnRK1 are involved in regulating the response of plants to low energy conditions. The central role in the regulation of cellular processes, the reprogramming of metabolism, and the phenotypic consequences of these two kinases will be discussed in light of current knowledge and potential future developments. Figure 1). In this review we summarize recent advances in the understanding of LES and the different signaling pathway components from energy deficiency toward adaptation, focusing on Arabidopsis thaliana as a plant model.Suboptimal growth conditions related to temperature, light, water supply, and soil characteristics are among the most limiting factors for crop yield worldwide. Fruit and seeds constitute about 75% of world crop production . They reControlling energy homeostasis is a challenge for all organisms, as they must constantly sense and integrate internal and external signals to optimize growth and development, often under suboptimal conditions . This isThese massive alterations on all cellular levels that comprise LES occur during many different stress situations and are therefore discussed as central processes necessary for adaptation. Even though LES involves a collection of phenotypic outcomes, their interconnection and regulation remain to be fully described. Generally, stress adaptation involves both universal and stress specific reactions, indicating that plants perceive multiple stresses and transduces the signal through pathways which may cross-talk at various levels . A numbeSOS2 , involved in conferring salt tolerance . The non-catalytic subunits are also conserved among the SnRK1/SNF1/AMPK complex. They are likely involved in substrate recognition, subcellular localization, and regulation of the complex activity  were shown to complement a yeast triple kinase mutant by restoring SNF1 upstream kinase activity . The association between trehalose metabolism and sugar-sensing in plants has recently become more evident . Despitein vitro at physiological concentrations, causing expression changes of KIN10 marker genes consistent with an inactivation of SnRK1 . They belong to a large family of several classes in eukaryotes  transcription factors are probably the best described .Sucrose-non-fermenting-1-related protein kinase-1 also affects enzymes by direct phosphorylation. For example, it inhibits the activity of HMG-CoA reductase, the rate-limiting step in sterol synthesis . Two othFigure 3). Most of the affected genes correspond to genes related to ribosomal proteins (RPs) and translation, which is in accordance with the role of SnRK1 as a repressor of biosynthetic processes and as a modulator of energy metabolism  and TORC2, characterized by different interaction partners. Of these, only subunits of TORC1  are present in the apamycin  makes thapamycin . Alternaapamycin , modulatapamycin  and the apamycin .Arabidopsis TOR promoter is active in root and apical primary meristems, embryo and endosperm, but not in source leaves or differentiated cells  and massively affect the transcription of a number of genes. By comparing the expression of genes targeted by SnRK1/KIN10 . Amongst others, this leads to protein, lipid, and chlorophyll breakdown cellular localization could provide insights to their mode of action and possible interaction. Protein interaction studies may uncover novel interactions between known LES components or even unravel new components of the network. Their role in the metabolic reprogramming induced by energy deprivation may then be tested by metabolomics studies which directly give information about changes in the concentrations of central metabolite pools. A multidisciplinary approach on various levels of cellular and organismal organization is needed to be able to draw a comprehensive picture about low energy induced metabolic reprogramming.The use of systems biology is limited by the availability of data and requires generalization, simplification, and assumptions. However, it clearly has great potential to increasingly contribute to the understanding of biological networks, including the LES pathway, in combination with other approaches that involve cell biology, biochemistry, or genetics. TOR and SnRK1 are related in the processes they regulate and are activated under opposite conditions, but so far there is no indication in plants that they or their targets interact directly to optimize the activation and repression of certain processes. While in mammals AMPK phosphorylates the TSC2/TSC1 complex and Raptor, TSC2 and the AMPK phosphorylation site in Raptor are missing in Filipa Tomé is first author, Thomas Nägele is second author, Magdalena Gamm is last author, and all other authors contributed equally to the work.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.


While neurons have long been considered the major player in multiple brain functions such as perception, emotion, and memory, glial cells have been relegated to a far lesser position, acting as merely a “glue” to support neurons. Multiple lines of recent evidence, however, have revealed that glial cells such as oligodendrocytes, astrocytes, and microglia, substantially impact on neuronal function and activities and are significantly involved in the underlying pathobiology of psychiatric disorders. Indeed, a growing body of evidence indicates that glial cells interact extensively with neurons both chemically  and physically , supporting a role for these cells as likely significant modifiers not only of neural function in brain development but also disease pathobiology. Since questions have lingered as to whether glial dysfunction plays a primary role in the biology of neuropsychiatric disorders or a role related solely to their support of neuronal physiology in these diseases, informative and predictive animal models have been developed over the last decade. In this article, we review recent findings uncovered using glia-specific genetically modified mice with which we can evaluate both the causation of glia dysfunction and its potential role in neuropsychiatric disorders such as autism and schizophrenia. Glial cells are the non-excitable supporting cells of the central nervous system (CNS) and classified mainly as oligodendrocytes, astrocytes, and microglia. These cells are typically smaller in size, but can be far more numerous than neurons in certain brain regions such as the cerebral cortex. Overall, the ratio between neurons, and glial cells in the human CNS is approximately 4:1 , with ol+ ions to name but a few  gene where neuronal and glial biology have been extensively investigated (Table 1). Subsequently, we examine several other mouse models for schizophrenia (Table 2) and how glial cell function or dysfunction contributes both to the phenotype and pathophysiology.The focus of this review is to examine what is known regarding the relationship between altered glial cell function and the pathobiology of psychiatric disorders, and whether glial dysfunction can play a causative role. First, we examine the monogenic disorder Rett syndrome (RTT) and examine the mouse model harboring a mutant X-chromosome-linked gene MeCP2  identified phenotypes that resemble some of the symptoms of RTT-like phenotypes -cre transgene ; an established target of MeCP2 binding (p38 mitogen activated protein kinases (MARK) pathways are hyper-activated in this model irrespective of exposure to lipopolysaccharide.With the loss of MeCP2 expression in astrocytes, there are significant abnormalities in the expression of  binding . Interes binding , and it in vivo mouse model led to both significantly improved locomotion and anxiety levels as well as respiratory state , myelin oligodendrocyte glycoprotein (MOG), or myelin proteolipid protein (PLP)  .Activated microglia release a large amount of glutamate and this microglial-associated neurotoxicity is mediated primarily by NMDA receptor signaling . In addiAllogeneic transplantation of wild type bone marrow into irradiation-conditioned MeCP2 null mice led to the engraftment of wild type microglia into the MeCP2 null brain parenchyma. The lifespan of these mice was significantly extended as compared with MeCP2 null mice that received either an autologous bone marrow transplant or untreated MeCP2 null mice. Similarly, body and brain weights of MeCP2-null mice recipients of wild type bone marrow recovered approximately to the level seen in wild type mice. And while the overall appearance, tremor, and gait of MeCP2 null mice recipients of wild type bone marrow were improved, the hindlimb clasping phenotype was not changed. Additionally, these mice exhibited significantly reduced numbers of apneic episodes and greatly reduced respiratory irregularities. Interestingly, these benefits resulting from engraftment with wild-type microglia were diminished when phagocytic activity was inhibited pharmacologically by using annexin V which results in substantial blocking of phagocytic activities .neuregulin 1 (NRG1) knockout mice as models for schizophrenia initially focused on the role these proteins play in neurons, but more recently, their activities in glial cells have begun to be considered.Thus, the focus of RTT studies using animal models has begun to swing from only studying potential neuronal dysfunction to include interrogation of a role for glial dysfunction and the results of the above studies lends credence to the hypothesis that glial dysfunction may play a primary rather than a secondary role in the causation of RTT-like symptoms. Similarly, studies of DISC1 knockout mice and t balanced translocation, in a large Scottish pedigree, resulting in a cohort with several major mental illnesses such as schizophrenia, bipolar affective disorder, and recurrent major depression , expressed under the control of a promoter for αCaMKII, were generated  was used to induce mutations in exon 2 of mouse Disc1 gene, resulting in the occurrence of missense mutations such as Q31L (glutamine to leucine) or L100P (leucine to proline), causing an increase in depression-like behaviors in Q31L mice and schizophrenia-like behaviors including impaired prepulse inhibition (PPI) and latent inhibition in L100P mice  known to markers of myelin maturation were decreased following full length DISC1 overexpression. In contrast, the knockdown of endogenous DISC1 using RNA interference increased the expression of CNPase as well as the number of mature oligodendrocytes , also known as ZNF365 or Su48, is a CNS specific member of the DISC1 interactome and is a novel DISC1 binding protein with a predicted C2H2-type zinc-finger motif and coil domains . DBZ regin vitro . In DBZ in vitro . Althougin vitro .N-methyl-D-aspartic acid (NMDA) receptor antagonists phencyclidine and MK-801 induce behaviors that closely resemble those observed in schizophrenic patients, dysfunction of the NMDA receptor is regarded as a particularly strong candidate for being a component of the mechanism of schizophrenia  degrades the D-serine, modulating D-serine levels and thence NMDA receptor function (D-serine was clarified by the purification and molecular cloning of serine racemase (SR), which transforms L-serine to D-serine, and, interestingly, DISC1 binds to and stabilizes SR -like domain-containing proteins that have been strongly implicated in neural development . NRG proD, L-amphetamine, and anxiety is reduced in a zero maze test and an acoustic startle paradigm in these mice. These findings imply that these mice could be utilized as a model of neurodevelopmental disorder (Synaptic cell adhesion molecule 1 (SynCAM1) is a member of the immunoglobulin (Ig) superfamily, a large group of proteins involved in cell surface recognition . SynCAM1disorder .Neuregulin 1-ErbB receptor signaling appears to play a critical role in the ontogeny of psychiatric disorders and this hypothesis has been supported by the identification of altered expression levels and/or function of NRG1, ErbB3, and ErbB4 in patients with schizophrenia . MoreoveTransgenic mice expressing a dominant-negative ErbB4 receptor in oligodendrocytes exhibit thinner myelin and less complex oligodendrocyte morphology and show schizophrenia-like behaviors including high anxiety and an enhanced sensitization to amphetamine. This abnormal response to amphetamine might be due to altered dopamine signaling as aberrant expressions of dopamine transporter (DAT) and dopamine1-like receptor in the cortex, nucleus accumbens, and striatum are evident .Neuregulin 1-ErbB signaling plays, at least in part, a critical role in oligodendrocyte development and CNS myelination . In humaProteolipid protein 1, a major protein in CNS myelin , which are responsible for the degradation of heme to biliverdin/bilirubin, free iron and carbon monoxide (CO), has been strongly implicated in mammalian CNS aging and diseases . MammaliIn conclusion, studies leveraging different animal models for the enhanced biochemical and physiological understanding of mental disorders have moved from strictly targeting the biology of neurons to also include an examination of the glia, especially since glial cells such as oligodendrocytes, astrocytes, and microglia have emerged as critically important modifiers of both CNS development and function. Postmortem brain analyses have clearly indicated that glial cell abnormalities are present in the brains of patients with schizophrenia. However, it remains uncertain whether the dysregulation and symptoms seen are a primary result of alterations in glial cell biology or the deficits in glial function occur as a side product of neuronal dysfunction. Nonetheless, as we have described, continued use of cell-type specific conditional knockout mice will allow us to better dissect how glial cells are implicated in nervous system dysfunction and perhaps illuminate their role in the pathobiology of psychiatric diseases such as schizophrenia.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.


Our analysis predicted 15 compounds and 24 substructures of potential oncometabolites that could result from the loss-of-function and gain-of-function mutations of metabolic enzymes, respectively. These results suggest a substantial potential for discovering unidentified oncometabolites in various forms of cancers.Altered metabolism in cancer cells has been viewed as a passive response required for a malignant transformation. However, this view has changed through the recently described metabolic oncogenic factors: mutated isocitrate dehydrogenases (IDH), succinate dehydrogenase (SDH), and fumarate hydratase (FH) that produce oncometabolites that competitively inhibit epigenetic regulation. In this study, we demonstrate  Cancer and metabolism have been considered to be associated for a long period since Otto Warburg observed that tumor cells consume glucose and convert most of it to lactate, despite the presence of oxygen. However, the role of the Warburg effect in oncogenesis had been under doubt because no solid links between genetic variations in metabolic genes and cancer had been observed until recent days. When with the development of sequencing technologies, researchers found mutations in IDH1, IDH2 (isocitrate dehydrogenase) in medium-grade glioma and acute leukemia. As these mutated metabolic genes initiate unexpected enzymatic reactions, cancer cells show altered concentration of particular metabolites, here called “oncometabolites”. The oncometabolites regulate the epigenetic controls of cell differentiations. In this study, we predict potential oncometabolites that might originate from loss or gain-of-function mutations in nine types of cancer from massive scale cancer mutation data with a systems biology approach. Otto Warburg observed that cancer cells convert most of their consumed glucose into lactate, despite the presence of sufficient oxygen ; left). Surprisingly, 2-HG shows a 100-fold increased concentration in glioma and acute myeloid leukemia's (AML) patients with IDH1 or IDH2 missense mutations. This increased concentration of 2-HG competitively inhibits α-ketoglutarate (α-KG) binding to histone demethylases, thus blocking differentiation of cells ; middle, right) The notion of passive cancer metabolism is being challenged by recent studies. It was shown that altered metabolism can by itself be a driver for oncogenic Metabolism represents a complex network of biochemical reactions and it may be hard to decipher phenotypic consequences based on single reaction alterations. Annotated genomes and biochemical legacy data, however, have enabled the construction of genome-scale models (GEMs) of metabolism in silico GEMs et al. dissected underlying principles of elevated glycolysis through the simulation of biomass production rates using GEMs et al. identified drug targets for cancers based on synthetic lethal gene pair analysis using a generic GEM of cancer With the rapid development of high-throughput experimental methods, recent integrative studies using disparate omics data types have deciphered characteristics of cancer metabolism using In this study, we predict putative oncometabolites by incorporating genetic mutation information on a massive scale collected from more than 1,700 cancer genomes into context-specific GEMs of metabolism for nine cancer types. We reconstructed context-specific cancer and matching metabolic models from corresponding normal tissue for nine cancer types using gene expression profiles of primary cancer cells and site-matched normal cells. By integrating the exome mutation data source with the reconstructed GEM, we predict potential oncometabolites that could show altered concentration in cancer cells due to the loss- or gain-of-function mutations on enzymes (http://cancergenome.nih.gov/), Cancer Cell Line Encyclopedia (CCLE) http://www.ncbi.nlm.nih.gov/geo/)  Table 1.Table S1 in Text S1, Figure S1 in Text S1).To identify potential oncometabolites that originated from genomic variations in metabolic enzymes, we determined the total number of mutations in enzymatic genes across nine cancer types. The metabolic genes were identified from the global human metabolic network Recon 2 , File S1).In order to predict oncometabolites that could originate from mutations, we selected metabolic genes that are recurrently found to be mutated in more than 5% of samples. Transporters were removed from our analysis, as they usually do not represent canonical metabolic transformations. As shown in the examples of IDH and FH, mutations could change the enzyme activity such that it gains a new function (gain-of-function) or loses its original function (loss-of-function). In this study, we divided the potential oncometabolites into two categories: (i) native oncometabolites that could change concentration due to the loss-of-function (LoF) mutations, and (ii) promiscuous oncometabolites that could change concentration due to the gain-of-function (GoF) mutations. Therefore, for this oncometabolites analysis, we classified mutations into two classes. First, we adopted a definition for LoF mutations to be correlated with loss of function: nonsense (stop codon introducing), splice site indels&SNP (splice site disrupting mutations), and frame shift indels (disrupting reading frame) , Figure S2 in File S1). Notably, we observed mutation recurrence in LoF SDHB that is one of the previously identified oncometabolite-producing enzymatic genes .From the 96 recurrently mutated metabolic genes, we next selected genes that could have significant functional impacts on their catalytic activities due to the mutations. First, isoenzymes were filtered out because an unexpected mutational malfunction of one isoenzyme could be substituted by functions of other isoenzymes having duplicated catalytic activities. Second, mutated genes having smaller functional impact were removed. The impact of a mutation in the recurrently mutated genes was assessed by using the functional impact score (FIS) In order to predict oncometabolites originating from LoF mutations on enzymes, we simulated flux changes of reactions in a cancer cell using GEMs. For the reconstruction of cancer specific and matched normal metabolic networks, gene expression data sets of cancer and matched normal were used. Here, the specific characteristics of cancer and normal metabolic models were represented by the rerouted network structure based upon the presence or absence of an enzymatic reaction in the intracellular environment of cancer and normal cells. This presence or absence of reactions was determined by the present/absent (P/A) calls of enzymatic genes . This inconsistency is in accordance with previous finding that the expression differences of individual genes vary from cancer to cancer Figure S3B in Text S1). A pathway is considered to be significantly altered if the pathway is found to have a significantly higher number of genes with P/A alterations than the corresponding number found in a random set of genes (see Text S1 for details). Specifically, reactions transporting  substances inside and outside of the cell were frequently altered across nine types of cancer. Furthermore, we confirmed that these altered pathways had significant over representation of mutations . We reconstructed nine cancer and normal matching models using P/A gene expression calls the Gene Inactivity Moderated by Metabolism and Expression (GIMME) algorithm .With the result of P/A calls of enzyme-encoding genes in nine cancer and normal gene expression data sets, we evaluated the relevance of P/A call results to the cancer specific metabolism. Here, we first assessed gene-wise P/A alterations in cancer vs. normal across nine cancer types. In this analysis, we confirmed that the P/A alterations in cancer vs. normal of enzymatic genes were not consistent across different cancer types  how well the structure of the reconstructed network represents gene expression data, and (ii) how well the simulated fluxes predict metabolic states of cancer and normal cells.Figure S7 in Text S1). Thus, the reaction content of the cancer and normal models represent the gene expression data sets well.Since the characteristics of the reconstructed model are mainly determined by the result of gene expression P/A calls, we first evaluated how well the network structure of the model represents the gene expression P/A calls. Since a gene can be associated with multiple reactions, or vice versa, the correlation between P/A calls and the network structure was calculated in two steps. The final correlation was determined by correlations of two vectors of Pearson's correlation coefficient (PCC) calculated from pairwise correlations of presence/absence of gene expression and pairwise correlations of presence/absence of reactions between cancer types Second, we tested the accuracy of the simulated flux states of the reconstructed models by evaluating whether the flux states correctly predict metabolic states of cancer and normal cells. We hypothesized that if the flux through a reaction was predicted to increase (or decrease) its magnitude in cancer compared to normal, then the expression level and/or abundance of the associated active enzyme in cancer will increase (or decrease) in order to meet the change in flux. With this hypothesis, we compared the changing pattern (increase or decrease) between flux and gene expression for the reactions with a significant flux change. Flux changes were calculated using the Markov chain Monte Carlo (MCMC)  .To predict LoF oncometabolites, we first modified a reconstructed cancer model into mutated enzyme deficient models. For example, as shown in Table S7 in Text S1) Once the deficient models were built, we simulated flux changes that result from the enzyme deficiency by employing an MCMC sampling method to the enzyme deficient models and matching normal (no cancer with non-enzyme deficient) models. Substrates or products of reactions with significantly changed flux for deficient models as compared to normal models were chosen as potential oncometabolites. Finally, 15 unique metabolites catalyzed by the mutated enzymes and surrounded by significantly changing flux were predicted as context-specific LoF oncometabolites . Finally, from these reaction pairs, structural features of oncometabolites were predicted.To predict candidate oncometabolites resulting from promiscuous activities of mutated enzymes, a systems framework was developed to determine new enzyme functionalities due to mutated enzymes. First, each substrate and product present in the mutated enzyme reactions were compare against the Human Metabolome Database (HMDB) Figure S11 in Text S1 for details). The final GoF mutated enzymatic gene list for this analysis contains seven unique genes. Among the reactions associated with the seven mutants, reactions catalyzing large molecules that could yield more than 30,000 promiscuous activities due to the compounds complexity were excluded from the analysis, and finally 33 reaction associations were identified from the enzymatic list. Notably, cofactors were not taken into account for the compound list generation.Among the 17 genes with recurrent GoF mutations, seven genes could have catalytic activity in cancer .A summary of mutated enzymes associated reactions and their predicted promiscuity catalytic activities is given in Table S8 in Table S8 in Text S1). The compound structure shown in the Table S8 in In most cases promiscuous substrates show similarities with the native substrate in silico analysis should be targeted. As in the example of IDH, IDH gained an unexpected catalytic activity that is result from missense mutations. Therefore, in this study, we focused on the possible metabolic perturbation of a GoF mutation in an enzyme can raise. Note that, assuming a mutation to be GoF is different from asserting the mutation is GoF. Using the aforementioned criteria, we predicted 15 oncometabolites resulting from the LoF mutations, and 24 substructures of oncometabolites resulting from the GoF mutations. These predictions can be used as a guide to examine select mutant enzymes and generation of oncometabolites. Notably, in our reconstructed models, several cancer and/or normal models do not uptake glucose or secrete CO2 in their optimal flux states . In order to prevent this problem, we allowed small amounts of uptakes to important vitamins exchange reactions (Table S4 in Text S1). Also, we constrained models to uptake glucose, oxygen and secrete CO2, biomass. With the updates, now most of models produce presumably reasonable uptake and secrete flux states . Although still several models do not secrete any CO2 in the optimal flux states, this problem is not a critical problem in our study since the representative flux states in our results were determined from the sampling points within the suboptimal solution space  that is the flux variation ranges shown in the Table S5 and S6 in In this study, we predicted potential oncometabolites in nine types of cancer by analyzing the massive scale genetic variants integrated with cancer and normal GEMs. We first predicted potential oncometabolites that could result from LoF mutations by simulating flux changes in the metabolic network due to the LoF of mutated enzymes. Second, we predicted oncometabolites that could result from GoF mutations by inferring promiscuous catalytic activities of enzymes resulting from their GoF mutations. Setting aside the generally accepted LoF criterion , we rarely have information about the mutational directionality of a given missense mutation whether it functions as GoF or LoF. Specifically in the oncometabolite analysis, enzymes can be disrupted in both ways. Whereas an LoF of an enzyme gives a predictable malfunctions (e.g. an accumulation of a target product), a GoF mutation is much more unpredictable and is the very place in which an Altered energy metabolism in cancer cells is accepted as a hallmark of cancer Oncometabolites could be used to develop therapies and identify biomarkers associated with cancer. Recent studies showed that inhibition of mutant IDH1 delays growth of glioma cells and induces cellular differentiation in leukemia, which shows possibility of a potential application as a therapy for cancer In order to reconstruct cancer type specific metabolic models and their paired normal models, we used data sets of gene expression experiments collected from TCGA consortium and GEO Database which are composed of expression profiles of primary cancer cells and site-matched normal cells Table 1.The P/A call results of gene expression are then incorporated to the reactional space using Gene Inactivity Moderated by Metabolism and Expression (GIMME) algorithm implemented in COBRA Toolbox v2.0 Formula 1). However, normal cells may have different objectives depending on the growth signals Formula 2).As cancer cells are known to maximize their proliferation rate, the biomass formation was maximized in all cancer models Formula 2). Each reaction flux in the objective function was scaled by its maximum achievable flux in the given growth condition. The contribution of each component may be weighted with an additional coefficient (a and b) with a value of 0 denoting no contribution to the objective. Here, coefficient a and b are set to be 1 and 1, respectively .Because normal cells have different, or multiple, physiological objectives , we constructed an objective function that was a linear combination of these physiological functions (The distribution of feasible fluxes in the models used in this study was determined using Markov chain Monte Carlo (MCMC) sampling MCMC sampling was used to obtain thousands of feasible flux distributions using the artificially centered hit-and-run algorithm with slight modifications, as described previously For each reaction, a distribution of feasible steady-state flux values was acquired from the uniformly sampled points, subject to the network topology and model constraints. Similar measures were taken for all other models in this work.Formula 3).To simulate changes in reaction flux occurring in a shift between cancer and normal, the sampled fluxes for each reaction were compared between cancer and normal as follows. First, reactions that carried no flux in both conditions or that were involved in loops Once the flux values were normalized, the changes of fluxes between two conditions were determined as previously described Formula 4). The significance of the accuracy is demonstrated by a comparison with the background distribution using 10,000 random accuracy tests. The approach presented here extends the pre-published method called Flux Space Shift analysis (FSS) Accuracy of reconstructed cancer specific models was assessed using experimentally determined essential genes. We collected a list of cancer essential genes from in silico single gene deletion tests on each model, and genes whose deletion effect reduces the maximum objective reaction by more than 10% were determined to be in silico essential genes. Finally, as the total number of predicted in silico essential genes varies according to the models, the accuracy of the models were evaluated with P-values from the hypergeometric enrichment tests of experimental essential genes against the in silico essential genes.We performed Mutated enzymes are able to carry out new catalytic functions by simultaneously changing the native reaction mechanism and catalyzing different substrates. The changes could be measured and compared using a chemoinformatic approach. Previous studies have successfully shown the efficacy of the chemoinformatic approach for enzyme promiscuity predictions For compound and reaction representation we used MDL Molfiles. A Molfile contains information about the atoms, bonds, connectivities, and coordinates of a molecule. The Molfile consists of some header information, the Connection Table containing atom info, then bond connections and types, followed by sections for more complex information.i), in which Fi refers to a molecular fragment with real occurrences of a molecule. Fi is obtained by molecular fragmentation method. Each Fi in the fingerprint is represented in bit string where each position of the sequence is represented by ‘1’ or ‘0’ digits, depending on the presence or absence of the structural pattern predefined by Fi. Previous studies have shown good results by using linear fragments from 5 up to 6 bonds As stated before, substrates are represented by chemical fingerprints. The chemical fingerprint (CFP) of a molecule is defined as CFP  =  :The comparison of two fingerprints involves the calculation of the TC. Values of this metric are non-negative numbers. Here, we used the TC dissimilarity (TCdiss) metric. A zero dissimilarity value indicates that the two fingerprints are identical, and the larger the value of the dissimilarity coefficient the higher the difference between the two structures. In its original form, Tanimoto metrics it is a similarity metric  is the number of 1 bits in any binary fingerprint x:Formula 6):From that it is straightforward to obtain a dissimilarity measure  that has been used in previous studies, mostly for prediction purposes for metabolic engineering For each mutated enzyme, Maximum Common Substructure (MCS) analysis was used in order to identify the most common chemical patter from all predicted products and substrates. The idea is to identify from a range of chemical structures, the largest substructure common in all of them.www.chemaxon.com)].For all calculations, with regard to handling compounds, building reaction, substrate fingerprint generation, reaction fingerprint generation, TC dissimilarity calculations, BROs simulations, and MCS analysis we used MATLAB linked with ChemAxon's package libraries, specifically Marvin, JChem Base, Standardizer and Reactor [Click here for additional data file.File S2All detailed information of predicted promiscuity reactions for GoF analysis.(XLSX)Click here for additional data file.File S318 cancer and normal genome-scale metabolic models in SBML.(ZIP)Click here for additional data file.Text S1Supporting results including supporting figures, tables, and text.(PDF)Click here for additional data file.


All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3’ to 5’ direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae.RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined  Understanding the patterns and mechanisms of genome evolution is one of the most important, yet least understood, aspects of RNA virus biology. The evolutionary challenge faced by RNA viruses is to maximize functional diversity within severe constraints on genome size. Here we show that rhabdoviruses, a family of RNA viruses that infect hosts as diverse as plants, insects and vertebrates, have an unusual capacity for genomic plasticity. By analysing the complete or near-complete genome sequences of 99 animal rhabdoviruses, we show that genome expansion and contraction has likely occurred frequently throughout the evolution of the family. Genomic plasticity has been associated with the evolution of alternative, overlapping and consecutive ORFs within the major structural protein genes, as well as the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. This has resulted in remarkable diversity in genome organisation and gene expression strategies that is reflective of the broad ecological diversity of rhabdoviruses. We conclude that genomic plasticity in rhabdoviruses may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome and propose a general model that accounts for both gains and losses in genome size and complexity. Nidovirales  ssRNA viruses. We demonstrate that remarkable changes in genome size and complexity have occurred in rhabdoviruses in a clade-specific manner, primarily by extension and insertion of additional transcriptional units in the structural protein gene junctions, followed by occasional losses. We also show that rhabdoviruses have evolved a large number of accessory proteins and that the use of non-canonical gene expression strategies appears to be common, particularly amongst vector-borne rhabdoviruses.de novo  employed in some viruses. The TTP motif G[U]7 was also conserved, with the variation A[U]7 occurring only in several genes of one virus (CHOV).Our data set comprised the complete or near-complete genome sequences of 99 animal rhabdoviruses, including 45 viruses isolated from various vertebrates and arthropods for which we determined the sequences de novo . Incomplde novo . These ade novo . The addNS, α1, α2, β and γ proteins), all ORFs ≥180 nt were assigned names according to the following rules: i) each additional transcriptional unit was designated U (unknown) followed by a number as they appeared in order in the genome presented in positive polarity ; ii) the first ORF within each transcriptional unit was assigned the same designation as the transcriptional unit; and iii) each subsequent ORF within any transcriptional unit  was designated by letter   . AlternaLyssavirus, Vesiculovirus, Perhabdovirus, Sigmavirus, Ephemerovirus, Tibrovirus, Tupavirus and Sprivivirus) and we assigned a further seven clades as proposed new genera . The taxonomic assignment of the two remaining clades was considered to be ambiguous  ,26 Fig.. All butmbiguous . For simLyssavirus), flies (Sigmavirus) and fish  , fish  or ungulates . Vector-borne rhabdoviruses were present in 12 of the 17 groups, dominating the dimarhabdovirus supergroup, but were largely absent from clades associated with bats (vivirus) . The excCuriovirus comprises a monophyletic group of four viruses isolated from biting midges (Culicoides sp.), sandflies (Lutzomyia spp.) and mosquitoes (Coqillettidia and Trichoprosopon spp.) from the forests of South America and the Caribbean  was proposed [Each of the seven newly proposed rhabdovirus genera formed an independent, well-supported monophyletic group in the L protein phylogeny (BSP ≥ 85), and comprised viruses with similar genome organization ; Fig. 2.aribbean . The genproposed . HoweverEphemerovirus clade, (BSP ≥ 85) and possessed the characteristic genome organization of ephemeroviruses, including a non-structural glycoprotein gene (GNS) followed by a viroporin (α1) and several other small proteins (Culicoides insignis), SWBV and BAV, clustered within the genus Tibrovirus (BSP ≥ 85) and exhibited the conserved N-P-M-U1-U2-G-U3-L genome organisation (Sweetwater Branch virus), but BAV is closely related to TIBV and may be regarded as the same species (Tibrogargan virus). Finally, a novel tupavirus (KLAV) identified from two species of vole (Microtus and Clethrionomys spp.), clustered with the TUPV and DURV clade in the L protein phylogeny (Five of the novel viruses (comprising four putative new species) identified in this study were assigned to established genera. Two of these, KOOLV and YATV, clustered within the existing proteins ; Fig. 2.nisation . SWBV wahylogeny .A more detailed rationale for the assignment of viruses to existing and proposed new genera is provided as supplementary text.Ledantevirus; 10,863 nt) to the largest in our data set . All genomes, including those for which extreme terminal sequences were unresolved, appeared to fall within this range. Variations in genome size were associated with: i) variation in the length of intergenic regions (IGRs) between transcriptional units; ii) variation in the length of 3’ and 5’ untranslated regions (UTRs) within individual transcriptional units; iii) the presence of additional transcriptional units containing long ORFs; and iv) the presence of overlapping or consecutive long ORFs within individual transcriptional units. An examination of genome size across the phylogeny revealed a general trend towards larger genomes in the lower third of the tree, which is comprised of the hapaviruses, curioviruses, tibroviruses and ephemeroviruses, as well as several unassigned viruses  to 6.4% (LJV). Similarly, in the genus Ephemerovirus the proportion of untranslated sequences varied from 1.2% in the smallest genome (YATV) to 9.6% in the largest genome (KOOLV). The presence of long stretches of untranslated sequence, which occurred primarily within transcriptional units, suggests these regions may be functional. However, it is unclear at this time why they are present in some rhabdoviruses and not in others.Whilst the genome architecture in some viruses was highly compact, others featured long stretches of sequence with non-ascribed function that occurred primarily as 5’UTRs and 3’UTRs within transcriptional units . The proGene duplication. Previous studies have provided evidence of gene duplication in the Rhabdoviridae, involving the G and GNS genes [NS genes ,21 and tNS genes  in the eNS genes ,19,20. TPutative accessory genes were found to be abundant and varied greatly in number and location in each genome . A complViroporins. Viroporins are small hydrophobic proteins that oligomerize in host cell membranes to form hydrophilic pores, disrupting various cellular processes and promoting virus replication [NS) gene (lication . ORFs enNS) gene . ORFs enNS) gene . SeveralOther small transmembrane proteins. Small proteins with a predicted central transmembrane domain but lacking other characteristics of class 1A viroporins were identified in several other rhabdoviruses  proteins. Small highly hydrophobic proteins (6.8–10.8 kD) lacking predicted transmembrane domains are encoded in all tupaviruses  and sripuviruses (as overlapping ORFs within the M gene) ( M gene) . All havLarge class I transmembrane glycoproteins. All ephemeroviruses encode a class I transmembrane glycoprotein (GNS) in the ORF following the G gene [Hapavirus) also encodes a GNS protein with similar structural characteristics [NS gene is likely to have arisen by an independent duplication event of the upstream G gene with which it shares amino acid sequence identity. ORF U1 immediately following the MCOV G gene (genus Hapavirus) also encodes a large class I transmembrane glycoprotein but lacks the set of conserved cysteine residues that are characteristic of G and GNS proteins, and our homology searches failed to identify similarity with any known protein  or separated by a short stretch of nucleotides were common in several groups of rhabdoviruses  sites. Overlapping ORFs are common in rhabdovirus genomes and represent a second common architectural arrangement requiring non-canonical gene expression. Overlapping ORFs occur within the N transcriptional unit , the G transcriptional unit  or within additional transcriptional units between the P and M genes  or the M and G genes . Expression of the second ORFs in these arrangements would require either internal initiation in an alternative reading frame or another mechanism such as RNA editing or a ribosomal frame-shift (RFS) to extend the first ORF. Use of alternative initiation codons has been reported in the M and P genes of VSV and the P gene of RABV, and RNA editing has been described in the P gene of paramyxoviruses [UUUUUUCA  or CCNUUUUUUGA  followed by a predicted stem-loop structure  are expressed from an alternative ORF within the P gene [e P gene ,24. On te P gene . AlthougRhabdoviridae. Previous studies have surveyed known rhabdoviruses for biological and genomic diversity, revealed phylogenetic relationships, and considered factors that may have determined their rates of evolution [We have conducted a detailed analysis of the structural organisation and genome evolution of a family of negative-sense RNA viruses—the volution ,15,47,48The most remarkable aspect of this analysis is the number and variety of additional ORFs identified in rhabdovirus genomes, which provides a very different perspective of the family and its evolution than had been obtained from studies of the traditional prototype members (VSIV and RABV). As many of these ORFs occur as additional transcriptional units complete with conserved transcriptional control sequences, there is a high likelihood that they would be expressed in infected cells. Expression of ORFs located in additional transcriptional units has been demonstrated previously for several ephemeroviruses and for the hapavirus WONV ,31,36,49de novo, rather than by recombination or lateral gene transfer, and that they play an ‘accessory’ role in viral pathogenicity or transmission instead of having functions in virion structure or replication [Notably, very few of the additional ORFs detected in this analysis encode proteins with identifiable sequence similarity to other known proteins. Sequence similarity, when detected, occurred only between closely related viruses assigned to a genus and, although some accessory protein families were identified, these were more commonly related by shared structural characteristics, such as charged or transmembrane domains, than by sequence. This has been observed previously for so-called orphan (‘ORFan’) proteins in other viruses and bacteria. It has been suggested that the uniqueness of orphan proteins, or their restriction to a single species or genus, is the result of creation lication –52. It hlication . As suchin vitro [–6 subs/nucleotide/replication), it is impossible to assess whether the increases in genome size observed here have been associated with concomitant reductions in mutation rate [Substantial variation in genome size and complexity was also observed in many rhabdovirus genera, suggesting that the length of the genome is not heavily constrained in all members of the family. Indeed, the presence of new ORFs and/or very long stretches of non-coding sequence within or between transcriptional units was noted frequently. Previous observations have demonstrated that foreign genes of up to ~6 kb can be inserted into the VSIV genome without significant disruption to viral replication in vitro ,54. Expain vitro ,8, and ain vitro . Howeverion rate .in vivo, such as stabilising RNA secondary structure, serving as a source of, or targets for, micro RNAs, or attenuating transcription of downstream genes to achieve the most effective balance of gene expression. Indeed, an analysis of patterns and rates of sequence evolution in the Rhabdoviridae and other families in the Mononegavirales revealed that, although non-coding regions are less conserved than those that encode proteins, their evolutionary rates are associated with relative genomic position, suggesting that they impact on gene expression [It is also striking that while some rhabdovirus genomes appear to have undergone major changes in length and complexity, others contain only the 3’ and 5’ promoter regions and five canonical transcriptional units with minimal 5’ and 3’UTRs. This suggests that the acquisition and loss of new genes and intergenic regions may be a regular feature of rhabdovirus evolution. Previous studies of RNA viruses have concluded that constraints on genome size imposed by polymerase error have led to various strategies to minimize genome size while increasing functional complexity, such as gene overlaps and protein multi-functionality ,56. Givepression .Additional ORFs and non-coding sequences occurred at all junctions of the canonical structural protein genes , although there was variation in both the frequency of insertion and the extent of expansion. Notably, insertions at the N-P junction are rare, with a single additional ORF present in the closely related sripuviruses CHOV and SMV, and short overlapping ORFs present within the N gene transcriptional unit in some hapaviruses. It has been reported previously in a study of VSIV recombinants that only the N-P gene junction was refractory to the stable expression of an inserted transcriptional unit, and resulted in a virus with significantly reduced replication efficiency . In contde novo in the absence of the evolutionary constraints imposed on alternative or overlapping ORFs. This is most likely to occur when ORFs are present in transcribed non-coding regions (UTRs) such as the ψ region of WCBV in which, uniquely amongst lyssaviruses, an ORF of 180 nt has been identified [de novo in non-transcribed IGRs, such as those present in the G-L gene junctions of LJV, KOTV and KOOLV, almost certainly would require prior or simultaneous evolution of new or modified transcriptional control sequences to allow their expression.The relationships, locations and contexts of additional ORFs in various viruses lead us to propose a general model for rhabdovirus genome plasticity, which can account for both gains and losses in genome size and complexity . In eachentified . The creWe recognise that other mechanisms of genome expansion are also possible. In Central American isolates of VSIV, for example, imprecise reiterative insertions of up to 300 nt in the 5’-UTR of the G-gene (variations of 3’-UUUUUAA-5’) have been attributed to non-templated extension by polymerase stutter at the TTP sequence ,67. AlthRhabdoviridae, similar mechanisms of genome expansion appear to occur in other families of non-segmented (-) ssRNA viruses . For example, amongst the Paramyxoviridae genome length varies by 46.5% from human metapneumovirus  to Beilong virus , and paramyxoviruses also contain novel accessory genes in transcriptional units inserted at various gene junctions [Although our analysis was limited to the unctions . The appRhabdoviridae would likely require a larger set of sequenced genomes at lower taxonomic levels [Rhabdoviridae has been based primarily on the identification of well-supported monophyletic groups using unambiguously aligned regions of the L gene, together with a consideration of common features of genome organisation and known aspects of viral ecology. Genome organisation has proven here to be a useful taxonomic marker as similar arrangements of accessory genes and other conserved elements of genome architecture appear to be the result of significant evolutionary events that provide resolution between the family and species levels. For some of the new genera, host and/or vector associations have also been relatively informative but in many cases, only single isolates of a species are available and else little is known of their ecology. It is likely that the proposed assignments of viruses to genera and the placement of the proposed unassigned species will evolve into a more complete taxonomic description as more viruses are discovered and as ecological data accumulates.Finally, this study has also provided an important advance in rhabdovirus taxonomy, allowing the assignment of six new species to existing genera and the assignment of 37 species to seven proposed new genera as well as the identification of six new unassigned species. There are currently no formal criteria for genus demarcation in rhabdoviruses. A system of genetic classification (DEmARC) that allows demarcation of viral taxa based on pairwise evolutionary distances has been proposed and, for picornaviruses, was shown to be comparable to expert-based taxonomic classification ,74. Howec levels , and wouc levels . Our assDetails of the viruses included in this study, including taxonomic status, sources and dates of isolation, and GenBank accession numbers of genome sequences are given in de novo assembly program ABySS [Viruses sequenced in this study were prepared as described previously . With tham ABySS  was usedam ABySS  and visuam ABySS  to verifhttp://www.expasy.org/).Assembly of full genome sequences was performed as previously described  and predTo quantify the location and extent of variation in genome size in our data set, we compared the average length of each genomic region within and between rhabdovirus genera. For all viruses, we normalized the length of each gene region  and intergenic region by dividing by the length of the corresponding L gene, which varied least across the data set . As there was substantial variability in the proportion of the 5’ and 3’ UTRs that were included in the sequence data set, we considered each genome to begin at the first TI sequence and end at the final TTP sequence for this analysis.Cytorhabdovirus and Nucleorhabdovirus) were excluded as their sequences were highly divergent. We therefore utilized four members of the genus Novirhabdovirus  as outgroups. Unfortunately, these novirhabdovirus sequences were also far too divergent  to establish a reliable rooting for our data set, as three different basal groups were identified using different models of amino acid substitution, although overall tree topologies were similar among substitution models (results available on request). In addition, the use of the novirhabdoviruses as outgroups resulted in excessive numbers of residues being removed following Gblocks pruning (see below). Based on the observation that most known rhabdoviruses are either insect viruses or replicate in insect vectors, it has been reasonably argued that plant and animal rhabdoviruses may have origins in insects [To infer evolutionary relationships among animal rhabdoviruses, we compiled sequences of the L (RNA-dependent RNA polymerase) protein, as this was the most highly conserved protein across the data set. We initially attempted to root the tree using a standard outgroup method. Members of the rhabdovirus genera that infect plants  was comprised of amino acid sequences aligned using the MUSCLE program , with amS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.