Supplemental Digital Content is available in the text. Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries.PERP) and programmed cell death 2–like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow.First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites. Previous studies of the EC transcriptome revealed that flow alters the expression of hundreds of genes, but the function of the majority of them is unknown.19 Therefore, new strategies to identify the function of flow-modulated genes and their role in vascular physiology are urgently required. Screening gene function represents a powerful and unbiased approach used widely to study cellular responses to biochemical signals. However, to our knowledge, functional screening of cells exposed to mechanical force has not been reported.Endothelial cell (EC) responses to wall shear stress (WSS), a force exerted on the endothelium by flowing blood, play a crucial role in vascular homeostasis and also contribute to arterial disease. Plaque formation occurs at branches and bends exposed to disturbed blood flow that generates sites of hemodynamic stasis and WSS with low magnitude and variations in direction (oscillations).See cover image20 they have been used less frequently to study adult vasculature. Here, we tested the hypothesis that flow regulates EC physiology through mechanisms that are, at least in part, conserved between in adult mammalian arteries and developing zebrafish vasculature. If correct, then zebrafish embryos could provide a valuable model for studying the function of genes that are coexpressed in adult mammalian arteries. We tested this by examining whether zebrafish embryos can be used to identify genes that regulate apoptosis in adult mammalian arteries under different hemodynamic conditions. Our studies revealed that flow is a potent regulator of EC apoptosis in developing zebrafish vasculature. A panel of flow-sensitive input genes expressed in mammalian arteries was assembled by transcriptome profiling of ECs from high and low WSS regions of the porcine aorta. Gene silencing of this panel in zebrafish embryos led to the identification of 4 genes that regulated apoptosis: perp(p53-related protein) and pdcd2l(programmed cell death 2–like protein), which functioned as positive regulators of apoptosis under conditions of flow cessation, whereas cdh13 (cadherin 13) and angptl4 (angiopoietin-like 4) exerted antiapoptotic function. The regulation of PERP, PDCD2L, CDH13, and ANGPTL4 by shear stress was confirmed by en face staining of the murine endothelium and by using EC exposed to flow. The ability of PERP and CDH13 to regulate EC apoptosis was confirmed by gene silencing studies in cultured human EC. We conclude that mechanosensitive pathways that control EC apoptosis are partially conserved between zebrafish embryos and mammalian systems. Thus, zebrafish embryos may provide a useful model for functional screening of mechanosensitive pathways.The zebrafish is a unique vertebrate model that combines advantages characteristic of invertebrate models  with a high degree of evolutionary conservation with mammals. Thus zebrafish are invaluable not only for studying vertebrate development and physiology but also for modeling human diseases. Although zebrafish embryos have been used extensively to study the effects of flow on angiogenesis and other developmental processes,online-only Data Supplement.Materials and Methods are available in the silent heart (sih) antisense morpholino oligonucleotide (MO), which targets cardiac troponin T2 leading to a nonbeating heart21 or by treating embryos with the anesthetic tricaine to stop cardiac contraction. It should be noted that embryos lacking blood flow remain viable for up to 5 days because a sufficient supply of oxygen and nutrients is provided by diffusion.22 Consistent with this, we did not observe a hypoxic response in sih embryos during development using a hypoxia reporter line phd3:GFP23  and zebrafish embryos lacking blood flow did not upregulate hypoxia responsive genes.24We wished to know whether flow regulates EC apoptosis in the vasculature of zebrafish embryos. This was addressed by manipulating flow that normally commences with cardiac contraction at ≈24 hours post fertilization (hpf). To study hemodynamic responses in embryos, blood flow was blocked either by using flk1:EGFP-NLS embryos25 (green fluorescent protein+ EC nuclei) by active caspase-3 immunohistochemistry . Increased EC apoptosis was observed at 30 to 32 hpf in the aorta and caudal vein plexus of embryos lacking blood flow , whereas total EC numbers were comparable to controls . We conclude that the suppression of flow triggers a transient wave of EC apoptosis accompanied by EC loss and altered vascular remodeling. On the contrary, blood flow drives EC survival during zebrafish development.EC apoptosis was assessed in transgenic 26 we hypothesized that zebrafish embryos may be used for screening of mechanosensitive genes that regulate apoptosis in adult arteries. To test this, we generated a panel of candidate regulators of apoptosis by transcriptome profiling of ECs from low and high WSS regions of the porcine aorta. Healthy pigs aged 6 months were used to allow identification of genes that predispose low WSS sites to disease. Although pigs have been used extensively to study focal atherogenesis, WSS in the porcine aorta has not been defined previously. Therefore, we used magnetic resonance imaging and computational fluid dynamics modeling to characterize flow and WSS in the porcine aortic arch . Steady state simulations revealed velocity profiles skewed toward the outer wall and rotated toward the anterior wall leading to higher WSS on the outer wall compared with the inner wall . Unsteady state simulations were run over multiple cardiac cycles using a single geometry and periodicity was reached at the fourth cycle . Velocity profiles were computed at multiple locations of the aortic arch at four representative time points of the cardiac cycle . The curvature of the arch introduced a rotation of the velocity profile clearly visible in the deceleration and diastolic phase. At peak systole, the presence of the branches caused a reflection of the incoming high-velocity flow that was directed toward the inner wall of the arch. It is also important to note the presence of retrograde flow  starting at the late systole and reaching the maximum at peak diastole, as previously observed in human studies.28 Time-averaged WSS was similar to steady simulations with the inner curvature of the arch being exposed to WSS with low-magnitude and high oscillatory shear index .Given that enhanced EC apoptosis was observed in embryonic zebrafish vasculature exposed to static conditions Figure  and in aonline-only Data Supplement) and quantitative reverse transcriptase-PCR (qRT-PCR) revealed high expression of CD31 (EC marker) and negligible quantities of smooth muscle cell or macrophage (CD14) markers . RNA samples were labeled and hybridized against GeneChip Porcine Genome Arrays (Affymetrix), which revealed 867 genes to be shear responsive (Table I in the online-only Data Supplement). Functional annotation found that 494 genes have a known or putative function, and molecules with an inferred or known role in the regulation of apoptosis showed maximal enrichment (Table II in the online-only Data Supplement). The expression of putative apoptosis regulators at high and low WSS sites was visualized using a heat map  were excluded because they have no orthologues in zebrafish. By contrast, LGALS1 has 3 zebrafish orthologues (Table III in the online-only Data Supplement) and was excluded from further analysis because of possible redundancy between the 3 paralogues, which would make functional analysis difficult. One of the candidate genes, CD74, had 2 zebrafish orthologues, cd74a and cd74b, and both of these were included in the functional screening study. Therefore, a total of 12 zebrafish genes were selected for functional screening (Table III in the online-only Data Supplement). To test whether the candidate genes are expressed in the zebrafish endothelium, we isolated ECs from 26 hpf flk1:EGFP-NLS embryos using fluorescence-activated cell sorting . The vascular identity of purified green fluorescent protein+ cells was confirmed by enriched expression of the EC marker cdh5 , whereas sorted green fluorescent protein− cells were used as a control . We detected endothelial expression of all 12 candidate genes, with some genes being particularly abundant .We further selected genes for functional screening based on the existence of 1 to 2 orthologues in zebrafish. From the 14 candidate apoptotic regulators, 2 genes  resulted in embryonic abnormalities even at a relatively low MO dose . Alternatively spliced transcripts in MO-injected samples were observed as a band shift after gel electrophoresis of RT-PCR products , and these transcripts were confirmed by sequencing to contain a frameshift leading to a premature stop codon (data not shown). For angptl4, fadd, perp, and tnip1, a reduced level of the wild-type transcript was observed in the MO-injected samples, and qRT-PCR was consequently used to assess the efficiency of the knockdown .Antisense MOs were used to transiently knock down the expression of candidate genes to assess their function in zebrafish embryos. We initially performed dose–response experiments to determine an optimal dose of each MO for gene knockdown and to assess gross effects on embryogenesis. Knockdown of 3 genes  did not modify EC apoptosis  either in the presence or in the absence of blood flow.After knockdown of specific genes using MOs, EC apoptosis was assessed in embryos by staining of active caspase-3 in the presence (control MO) or in the absence of flow Figure . Knockdos Figure  or EC nucdh13 resulted in an increase in EC apoptosis in the absence of flow , whereas depletion of angptl4 increased apoptosis in the presence of flow . These data suggest that although cdh13 and angptl4 exert antiapoptotic effects, they function under different mechanical conditions. By contrast, depletion of perp or pdcd2l led to a profound decrease (>30%) in EC apoptosis in the absence of flow  suggesting that these genes are proapoptotic.Knockdown of online-only Data Supplement). The efficiency of the MO2–mediated knockdown was confirmed by RT-PCR , qRT-PCR  or by phenotypic analysis . Injection of the second, nonoverlapping angptl4 MO2 resulted in 2.8-fold increase in EC apoptosis in the presence of flow . Therefore, we tested whether the p53 pathway is involved in flow-regulated EC apoptosis in zebrafish embryos. Knockdown of p53 resulted in a decrease in EC apoptosis in the absence of flow that was comparable to perp or pdcd2l knockdown  by en face staining of the aorta using mice.online-only Data Supplement). In addition, we compared gene expression in porcine aortic ECs exposed to flow patterns that model the in vivo situation (high uniform and low oscillatory WSS) using 2 complementary flow systems, an orbital shaker and an ibidi pump system. In the orbital shaking platform, the cells were exposed to flow using 6 well-tissue culture plates that were orbited to generate high unidirectional shear stress in the periphery of the well, and low shear with greater variation in direction at the center.29 On the contrary, the ibidi pump system involved seeding cells onto specialized μ-slides and the generation of WSS using a computer-controlled syringe pump to generate flow of desired magnitude and frequency. CDH13 expression was enhanced in EC exposed to the high shear using the ibidi system but not in cells exposed to orbital flow  possibly reflecting differences in the mechanical conditions generated by these 2 systems.En face staining of the murine aorta using anti-Cdh13 antibodies revealed that Cdh13 was expressed at higher levels in the high shear (outer curvature) compared with the low shear (inner curvature) region Figure . A majoronline-only Data Supplement). Notably, gene silencing in cultured human umbilical vein endothelial cells using small interfering RNA revealed that knockdown of CDH13  resulted in significantly increased rates of apoptosis in EC exposed to the high or low shear stress . Knockdown of PERP  resulted in significant reduction in EC apoptosis  compared with the high shear (outer curvature) region Figure . Perp los Figure . These dANGPTL4 and PDCD2L were expressed at higher levels in cells exposed to the low compared with high shear stress using the orbital system , which is consistent with their enrichment at the low shear region of arteries  did not influence apoptosis in cultured EC exposed to the high or low WSS . Thus, although both of these molecules regulated apoptosis in zebrafish embryonic EC exposed to flow, they did not influence the viability of cultured mammalian EC, possibly reflecting differences in the developmental stage, mechanical environment, or species studied.Studies of cultured porcine aortic ECs revealed that s Figure . HoweverTaken together, our studies of murine arteries and cultured EC exposed to different shear stresses confirm observations made using zebrafish embryos that PERP and CDH13 function as shear-responsive regulators of endothelial apoptosis. By contrast, the effects of ANGPTL4 and PDCD2L on EC apoptosis observed in zebrafish embryos were not recapitulated in vitro. The study demonstrates the utility of our zebrafish platform for functional screening of shear-responsive genes and also emphasizes the potential divergent functions of some genes in in vivo and in vitro systems.32 has identified multiple mechanoresponsive genes, the majority of them have not been studied at a functional level. To address this challenge, we established a zebrafish platform to allow functional screening of flow-responsive genes. To our knowledge, this is the first in vivo system developed for screening of mechanosensitive genes.The molecular mechanisms underlying the effects of WSS on EC physiology and atherosclerosis are not fully understood but are known to involve transcriptional changes. Although transcriptome analysis of EC exposed to different flow parameters under in vitro or in vivo conditionsKLF2/klf2a was suppressed by static conditions in embryos and by low WSS conditions in adult vertebrates.33 In addition, EC cilia are present mainly in areas of low WSS in mammalian arteries and in static conditions in zebrafish embryos and were shown to disassemble on exposure to laminar shear stress in both systems.36 Finally, a recent study found considerable overlap in the transcriptome of EC exposed to low shear or static conditions.37 Because of these considerations, we hypothesized that manipulation of genes in developing zebrafish vessels may provide information on the pathways that regulate mammalian arterial physiology. Consistent with this notion, our study revealed that flow regulates EC apoptosis via mechanisms that are, at least in part, conserved between embryonic and adult arteries.The zebrafish embryo model that we used diverges from vascular response to flow in mammalian arteries in several important respects including species, scale, and hemodynamics. Nevertheless, there are many parallels between endothelium exposed to static conditions in zebrafish, on the one hand, and endothelium exposed to disturbed flow and low, oscillatory WSS in mammals, on the other. For example, the atheroprotective transcription factor 38 Notably, our study revealed multiple WSS-related genes that were not identified as differentially expressed previously. This discrepancy may be related to technological differences because the commercial porcine gene arrays used in the current study contained >10 000 genes more than the custom arrays used previously,16 and analysis of our data benefitted from sequencing and annotation of the porcine genome (http://www.ensembl.org/Sus_scrofa/Info/Index). More fundamentally, there is only a partial overlap between the anatomic sites studies by the Davies group and those studied currently. For example, whereas we used outer curvature of the aortic arch as the source of ECs exposed to atheroprotective flow, previous studies focused on the descending thoracic aorta,16 carotid artery, and renal artery.16 Of note, although only a proportion of genes were found to be consistently differentially expressed in the current and previous studies of porcine arteries, the majority of them showed a similar pattern of expression in the low and high shear areas (Table IV in the online-only Data Supplement). It is also interesting to compare the current study with elegant experiments that revealed a causal relationship between WSS and the EC transcriptome in the murine carotid artery.17 Here, WSS was modified in the left carotid artery by partial ligation before assessment of EC gene expression. This approach revealed that WSS regulates multiple genes in murine ECs including those involved in inflammation and immunity. Interestingly, although our current study of the porcine aorta identified several genes that were also regulated by WSS in the murine carotid artery,17 the relationship with WSS was not conserved between models (Table IV in the online-only Data Supplement). Several important biological differences can potentially explain this disparity including differences in vascular bed, variation in fluid dynamics, species, and the time that EC were exposed to disturbed flow. We also compared our microarray data to the list of >1600 shear-responsive genes obtained by meta-analysis of published microarray studies performed in vitro on human umbilical vein endothelial cells (Table V in the online-only Data Supplement).39 We found that <50% of genes shown to be mechanically regulated in both data sets exhibit the same relationship with WSS. This observation further emphasizes the sensitivity of EC to physiological and mechanical stimuli that vary between in vitro and in vivo systems.We assessed the ability of the zebrafish model to identify mechanosensitive genes that control EC function by focusing on apoptosis regulators that are controlled by flow. To generate a panel of putative flow-sensitive apoptosis genes, we performed microarray transcriptome analysis of the high and low shear stress regions of the porcine aorta. Functional annotation revealed apoptosis regulators as the most highly enriched group, which is consistent with the hypothesis that multiple genes regulate EC apoptosis at regions of disturbed flow. Our data build on pioneering studies from Peter Davies’ group revealing enrichment of proinflammatory and stress response molecules at the inner curvature of the porcine aorta.40 Therefore, not all human genes can be studied in zebrafish, which was the case for CCL2 and CSF2. In addition, because of a whole-genome duplication that occurred early during the evolution of ray-finned fishes, many human genes have >1 orthologue in the zebrafish genome. Because functional redundancy between zebrafish paralogues can present a challenge when studying zebrafish physiological processes, we excluded from our study genes with >2 orthologues. Gene function in zebrafish has been studied using transient knockdown via MOs or by targeted gene disruption and generation of mutants. For functional screening of a large number of genes, generation of mutants is costly, labor intensive, and time consuming (zebrafish and mice have similar generation times). Therefore, our approach was to use MOs for the functional screening. One drawback of using MOs is the lack of spatiotemporal control because they are injected into embryos at the 1-cell stage and therefore reduce expression in all embryonic cells. Because of this, genes important for early embryonic development cannot be studied using this model. This was the case for 3 genes in our study, fadd, igf1, and tnfsf10, where knockdown caused gross morphological defects. Nevertheless, we were able to study vascular responses to flow after knockdown of 75% genes where embryonic morphology was normal. Overall, of 12 genes studied, we detected 2 antiapoptotic (angptl4 and cdh13) and 2 proapoptotic molecules (pdcd2l and perp) that were enriched at mechanically distinct regions of the aorta. To confirm the specificity of the MO-mediated knockdown phenotype, we used 2 separate, nonoverlapping MOs for each gene of interest.After our EC transcriptome analysis, we selected the putative apoptosis regulators that displayed the greatest differential expression for functional screening in zebrafish. Comparison between human and zebrafish reference genomes shows that ≈70% of human genes have at least 1 obvious zebrafish orthologue.CDH13, PERP, PDCD2L, and ANGPTL4 respond to WSS in cultured ECs. We used 2 complementary platforms, an orbital shaker and an ibidi pump system. In the orbital shaker system, the cells in the center of the well are exposed to WSS with a constant low mean magnitude and rapid changes in direction, whereas in the periphery of the well the WSS magnitude is relatively high and with relatively uniform flow direction.29 This contrasts with the ibidi parallel-plate system that uses a syringe pump to generate high uniform and low oscillatory  WSS. Two genes, PERP and PDCD2L, were expressed at higher levels in EC exposed to low WSS conditions in both in vitro systems, suggesting that their focal expression in vivo is maintained by local hemodynamics. On the other hand, CDH13 expression was enriched by high WSS only in the ibidi system, whereas ANGPTL4 upregulation under low WSS was only observed in the orbital system. These observations may reflect differences in the mechanical environment of the orbital and ibidi systems, for example, the presence of secondary flows that are generated exclusively by the orbital platform,29 and they emphasize the importance of studying mechanosensitive gene function using multiple experimental models. Second, we assessed the effects of silencing our genes of interest in cultured EC exposed to flow. This study confirmed that PERP is a positive regulator of EC apoptosis under low WSS conditions and that CDH13 has protective effects . Interestingly, although CDH13 reduced apoptosis in cultured EC exposed to low or high WSS, its protective effects in zebrafish embryos were only observed in EC exposed to static conditions. This discrepancy may arise from compensatory mechanisms present in embryonic but not in adult vessels exposed to flow. Further studies including genetic deletion of Cdh13 in mice are therefore required to determine whether CDH13 exerts protective effects under low and high WSS conditions in adult mammalian arteries. Our study also indicated that although angptl4 and pdcd2l regulate apoptosis in zebrafish endothelium, they did not influence EC viability in cultured human cells exposed to low or high WSS. Therefore, these molecules may regulate apoptosis specifically in developing EC, or their function may require anatomic or physiological features that are found in vivo but not in cultured cells, or there may be species-based differences. Further work is required to distinguish between these possibilities. Although we provide the first demonstration that perp, cdh13, angptl4, and pdcd2l are involved in hemodynamic control of apoptosis, they have been linked previously with apoptosis in other systems. PERP was previously identified as a p53 target gene by subtractive cloning of mouse embryonic fibroblasts and was shown to be expressed exclusively in apoptotic cells.30 Interestingly, PERP can feed back to p53 to promote its activity41 which is consistent with our observation that PERP and p53 cooperate to promote apoptosis in ECs. Our findings also resonate with previous studies that revealed post-translational modifications of p53 playing a role in disturbed flow-mediated EC apoptosis and contributing to atherosclerotic plaque formation.15 CDH13 has been shown to protect ECs from oxidative stress–induced apoptosis,42 and ANGPTL4 acts as a survival factor in the endothelium.43 Finally, although little is known about the function of PDCD2L, its paralogue PDCD2 promotes apoptosis in many human and mammalian cell lines and tissues.44To confirm our observations from functional screening in zebrafish embryos, we performed a series of experiments using cultured EC exposed to flow. First, to understand their mechanism of differential expression, we examined whether online-only Data Supplement). This discrepancy may be attributable to differences in oxygen transport and inflammation between these 2 systems. In the porcine aorta, oxygen delivery to luminal EC relies on transport of red blood cells through the vessel lumen. Computational modeling and empirical measurements suggest that this process can be influenced by secondary flows, which convect oxygen away from the vessel wall to generate regions with reduced oxygen levels.46 By contrast, oxygenation of zebrafish embryos (up to 72 hpf) does not rely on circulation because diffusion is sufficient because of their small size.24 Thus, although sites of disturbed flow in the aorta may have lower oxygen tensions leading to enhanced hypoxia-inducible factor 1α activation, this pathway is not activated in zebrafish in response to flow cessation because diffusion prevents the development of a hypoxic environment. Moreover, hypoxia-inducible factor 1α can also be induced by inflammatory signaling pathways47 that are known to be activated constitutively at the inner curvature of the aortic arch47 but have an uncertain role in zebrafish embryo responses to flow. Thus, we conclude that although zebrafish embryos provide a useful in vivo model to assess vascular responses to WSS, other systems are required to study the effects of altered oxygen transport on the vasculature.Although zebrafish embryos recapitulate some of the pathways that are regulated by flow in adult arteries, there are also limitations. One notable example is the hypoxia-inducible factor 1α pathway that was enriched at the inner curvature of the porcine aorta Figure  but was In summary, we have established a zebrafish-based model for functional screening of flow-sensitive genes and have used this system to identify novel regulators of EC apoptosis in response to disturbed flow. The study provides an additional mechanism to explain the focal distribution of EC injury and dysfunction in arteries and suggests that this involves p53-PERP–mediated EC apoptosis at atheroprone sites and CDH13-mediated EC survival at atheroprotected sites.This study was supported by the British Heart Foundation (Programme Grant RG/13/1/30042).None.


Diabetes mellitus is a metabolic disease that is defined by relative or absolute deficiency of insulin secretion. Diabetic kidney disease seems to be one of the most frequent complications of diabetes mellitus. Based on evidence, increased free-radical formation and/or diminished antioxidant defenses induce oxidative stress that is implicated in the pathogenesis of diabetic kidney disease. It is evident that diabetic state induces oxidative stress through different signaling pathways as well as reactive oxygen species (ROS) formation that attributes to the activation of various downstream signaling cascade leading to structural the way to structural and functional changes in kidney. It is evident that diabetic state induces oxidative stress through different signaling pathways as well as reactive oxygen species (ROS) formation that attributes to the activation of various downstream signaling cascade leading to the way to structural and functional changes in kidney.Diabetes mellitus is a metabolic disease that is defined by relative or absolute deficiency of insulin secretion. Today, diabetes is known to be one of the primary causes of mortality and morbidity in the world -3. ComplFor this review, we used a variety of sources by searching through Web of Science, PubMed, EMBASE, Scopus and directory of open access journals (DOAJ). The search was performed using combinations of the following key words and or their equivalents; diabetic nephropathy, reactive oxygen species, diabetic kidney disease, chronic kidney disease, microalbuminuria and advanced glycation end products.Diabetic kidney disease seems to be one of the most frequent complications of diabetes mellitus. Based on evidence, increased free-radical formation and/or diminished antioxidant defenses induce oxidative stress that is implicated in the pathogenesis of diabetic kidney disease -12. Anti2−), hydroxyl (∙HO), peroxyl (∙RO2−), hydroperoxyl (∙HRO2−), and non-radical of ROS include hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl). Hyperglycemia-induced oxidative stress can inhibit the secretion of insulin in pancreatic beta cell through the activation of an uncoupling protein-2 (UCP-2) which lowers the ATP/ADP ratio by leaking protons in the β cell. Also, ROS can leak into cell membranes and damage pancreatic β cells. Some researchers showed overproduction of free radicals such as superoxide anion in β cells leading to activation of stress-signaling processes which are able to induce downstream effectors such as NF-𝜅B which may lead to β cell apoptosis and dysfunction in the end reducing insulin secretion. Also, high concentrations of hydrogen peroxide directly with the phosphatidylinositol-3-kinase dependent pathway induce insulin signaling leading to insulin resistance prior to the onset of diabetes. Some reports have detected that in presence of hyperglycemia, hyperlipidemia can generate more ROS imposing β cell dysfunction. Likewise, high free fatty acids affect ROS overproduction leading to mitochondrial DNA damage and pancreatic β cell dysfunction  thickness, and mesangial expansion and also accumulation of extracellular proteins comprising laminin, collagens and fibronectin ,18. It i2O2), nitric oxide (NO), nitrous oxide (HNO2), hydrochlorous acid (HOCl) and nitrogen dioxide (NO2). Also, the evidence show that oxidative stress during diabetic kidney disease eventually results to the knocking down of normal kidney homeostasis and its proper functioning. It is well established that augmented ROS construction in kidneys of both type 1 and type 2 DM can be affected individuals through both enzymatic and non-enzymatic pathways  is counteracted. High glucose level, either due to resistance of cells to insulin or lack of insulin synthesis, plays an important role in production of ROS through the activation of various cellular responses thereby leading to deleterious effects like diabetic kidney disease. There are several factors which are involved in generation of oxidative stress during diabetes such as peroxyl, hydroxyl, superoxide and many other reactive compounds hydrogen peroxide . Then, PKC creates ROS which finally cause activation of PKC thus causing enhancement of mesangial expansion, GBM thickening and dysfunction of endothelial cells running to diabetic kidney disease. Furthermore, high glucose levels induce isoforms of nicotinamide adenine dinucleotide phosphate oxidases (NOXs), especially NOX-4 which results to endothelial dysfunction, inflammation and consequently apoptosis. Also, the others pathways of glucose auto-oxidation such as increased formation of AGEs, polyol pathway flux and glycation of proteins are involved in causing direct and indirect kidney injury by producing extensive proportion of ROS. Accordingly, diabetic kidney disease is also associated with an intensification in RAS activity, as well as aldosterone production which play an important and direct role in the creation and activation of ROS resulting to kidney injury. On the other hand, high glucose level leads to the induction of transforming growth factor-(TGF-β) presentation in the glomerular and tubulointerstitial compartments that enhances the level of TGF-β leading to kidney enlargement, glomerulosclerosis and tubulointerstitial fibrosis in diabetic kidney disease -20. UndeAntioxidant compounds are important for two main reasons. First, they can reduce the harmful effects of free radicals not only by preventing their formation but also by scavenging and inactivating free radicals or improving natural defense systems by inducing the activities of antioxidant enzymes and regenerating other proteins involved in antioxidant pathways. There are several strategies employed in the use of different antioxidants to contrast diabetic nephropathy. The choice of strategy depends on the type, structure, and concentration of the antioxidants. In addition, the stage, severity and prevalence of the disease are very important ,22.Vitamin E, a lipid-soluble vitamin, includes the stereoisomers a-tocopherol, b-tocopherol, g-tocopherol, d–tocopherol and d–tocotrienol that tocopherol and tocotrienol stereoisomers and their metabolites appear to have potent biological properties. Generally, vitamin E detoxifies free radicals directly and also interacts with recycling processes to create reduced forms of A and C vitamins. Vitamin E also has a number of biological activities such as immune stimulatory and preventive activity of genetic changes by inhibition of DNA damage. Vitamin E primarily serves as a chain-breaking antioxidant to prevent lipid peroxidation. Some literatures have shown that tocopherol can modulate cell signaling pathways by inhibiting PKC. Tocopherols also have gene regulatory functions because they potentially serve as ligands for the transcription factor peroxisome proliferators-activated receptor-γ (PPAR-γ). The tocopherol concentrations of plasma are lower in diabetics compared to controls and appear to be even lower in diabetics with complications like microangiopathy than diabetics without complications .Coenzyme Q10 (COQ10), an endogenous lipid-soluble micronutrient, is found in most living cells in the body that is a key component in the electron transport chain of the mitochondria, serving in the process of adenosine triphosphate (ATP) synthesis. Also, CoQ10 is a powerful antioxidant that scavenges free radicals and provides protection to cells in oxidative stress condition. Based on evidence, individuals with diabetic kidney disease often have deficiency in CoQ10 plasma levels compared to normal healthy subjects. Because of its antioxidant property, it is accepted that CoQ10 deficiency may impair the body’s defensive mechanisms against oxidative stress induced by hyperglycemia . TherefoAlpha-lipoic acid (ALA), one of the most successful antioxidants in clinical trials, is the only antioxidant capable of dissolving in both water and fats. It can be biosynthesized in plants and animals then metabolized to dihydrolipoic acid (DHLA) upon uptake into cells. Based on evidence, ALA and DHLA are powerful free radical scavengers that are also involved in the regeneration of vitamins C and E and oxidized glutathione within the cells. Also, ALA is a cofactor for a number of mitochondrial enzymes that can decrease lipid peroxidation, reduce oxidative stress, and improve blood flow in kidney. Some studies have shown that ALA reduces oxidative stress by inhibiting hexosamine and AGEs pathways .3] levels or prevalence of diabetes mellitus and its complications. Also, some previous studies showed an improvement of diabetic kidney disease symptoms after vitamin D supplementation. It has been demonstrated that vitamin D might reverse the progression of diabetic kidney disease for instance improving glucose metabolism, minimizing RAS activation, and finally reducing fibrosis  Dfibrosis . A studytabolism ,30.Flavonoids, the largest and the most important group of polyphenolic compounds in plants, are found in fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine. Flavonoids are made up of several subclasses that can scavenge free radicals and chelate metals for example; pro-anthocyanidin and luteolin have been shown to possess antioxidant activities which protect against diabetic kidney disease .The advantage of knowledge of powerful antioxidants is finding the modalities for greater insight in the treatment of diabetic kidney disease. In this review, we focused on understanding of diabetes induced oxidative stress through different signaling pathways as well as ROS formation that attributes to the activation of various downstream signaling cascade leading the way to structural and functional changes in kidney. We discussed that the administration of the anti-oxidative agents was able to restore the antioxidant defense system thereby preventing ROS mediated injuries.SB and LM searched the articles. EA prepared primary draft. SB and LM performed first edition. MRK did final edition. All authors wrote and signed the final paper.The authors declared no competing interests.Ethical issues  have been completely observed by the authors.The authors received no financial support for the research, authorship, and/or publication of this article.


Colorectal cancer is among the leading causes of cancer death worldwide. Despite numerous molecular characterizations of the phenomenon, the exact dynamics of its onset and progression remain elusive. Colorectal cancer onset has been characterized by changes in DNA methylation profiles, that, owing to the stability of their patterns, are promising candidates to shed light on the molecular events laying at the base of this phenomenon.To exploit this stability and reinforce it, we conducted a meta-analysis on publicly available DNA methylation datasets generated on: normal colorectal, adenoma (ADE) and adenocarcinoma (CRC) samples using the Illumina 450k array, in the systems medicine frame, searching for tumor gene episignatures, to produce a carefully selected list of potential drivers, markers and targets of the disease. The analysis proceeds from a differential meta-analysis of the methylation profiles using an analytical pipeline recently developed by our group , throughThese findings were finally validated in an independent cohort from The Cancer Genome Atlas (TCGA). Colorectal cancer is one of the main causes of death from cancer worldwide . The devEvidences accumulated in the past two decades indicate that profound genetic and epigenetic changes occur in colon epithelial cells during colorectal tumorigenesis’ onset and progression –6. MicroIn humans, DNA methylation consists of the covalent addition of a methyl group at the 5-carbon of the cytosine ring and occurs mainly in CpG dinucleotides . The disCACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 [Colorectal cancer, like other solid tumors, is characterized by a profound remodeling of normal DNA methylation patterns , 10. Widnd SOCS1 . CIMP-pond SOCS1 , 17–20, nd SOCS1 –26, highnd SOCS1 , 28 and Despite the progresses made in characterizing the epigenomic landscape of colorectal cancer, little is still known on the pathways affected by aberrant methylation and that could be deregulated in the progression of the disease. To contribute to this research, we first selected a number of robust DMRs by aggregating 3 original studies on colorectal cancer-referred hereafter as the Naumov , Luo 2626 and Tisystems medicine [To this aim we adopted a recently proposed analytical pipeline specifically tailored on the HM450 architecture , based omedicine , using nThe starting point of our meta-analysis is the collection of three original studies on colorectal cancer, referred as the Naumov , Luo 2626 and TiBumphunter algorithm to identify large (median length on the order of hundreds of kb) DMRs distinctive of solid tumors, including colon cancer and normal tissues, thus demonstrating that large hypomethylated blocks are a universal feature of solid tumors appearing early in tumor progression.In Naumov et al.  the idenTable In the N1xADE comparison we identified 2657 DMRs mapping on 2180 genes that were common to the Luo and Timp datasets. Unsupervised hierarchical clustering shows a clear separation of ADE versus N1 samples , cyclin-dependent kinase inhibitor 1A (CDKN1A) catenin beta-1 (CTNNB1) and cyclin D1 (CCND1), crucially involved in cell proliferation and differentiation, relevant in both tumor onset and progression.The first step highlights the hubs of the network, to identify biological key players . Owing tThe second analysis aims at the identification of distinctive functions characterizing each phase. For this, enrichment analysis on the nodes unique to each network  was performed . No conce.g. NOTCH1 and SOX2. Following PANTHER protein class classification, we observed that most of the identified DM hub genes codify for transcription factors, signaling molecules, and cell adhesion proteins  encodes for a structural protein expressed in epithelial tissues and has been already suggested as a colorectal cancer marker [ignaling . The genr marker , 37, henRELA) complexes with Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B-Cells 1 (NFKB1) and forms the NFKB transcription factor. Aberrant NFKB signaling plays a role in colorectal cancer initiation and progression [The hypermethylated gene V-Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A (gression  as well TP73 and SOX2) and signaling molecules . All these genes have been known to act in deregulating important pathways driving cells to tumorigenesis, although the exact correlation between the methylation and the biological effect remains to be elucidated [IGFBP3 and IGFBP7 areupregulated in 68 human CRC samples, an observation further refined by Hinoue et al. [BRAFV600E (with BRAF being a DM hub in our MET network) implicated in oncogene-induced senescence in melanomas and colorectal cancers [All the DM hub genes in the N1xCRC comparison are hypermethylated. They include genes that codify for transcriptional factors  in an independent cohort from the TCGA repository including colon (COAD) and rectum tumor samples (READ).In particular, since the TGCA dataset offered information on the age of the subjects  we performed the analysis corrected by age, to guarantee that the results of the differential analysis were not affected by this parameter, known to impact on methylation .q-value < 0.001) we obtained a list of 14578 DMRs, mapping to 9156 genes. Of the 2185 DMRs resulting from the N1xCRC GEO meta-analysis, 2030 were confirmed in the TCGA analysis. Further, 122/125 network DM nodes and all 10/10 DMH were validated in the TCGA results  with respect to the age of the TCGA subjects and no interaction between disease status and age was evident .To deepen our understanding on the biological meaning and potential clinical role of the identified features (DMR and DMH), we used data mining techniques to explore how samples tend to cluster when similarity is guided by all DMR features or only by DMH.The DAPC analysis Figure  shows thInterestingly, this distinction is not only preserved, but mildly improved when guided only by the 10 DMH. This indicates that the additional systemic analysis run on top of the differential analysis allows to isolate a handful of features whose biological meaning is indeed able to capture the essence of the full list of DMR, and further to remove some of the noise, allowing a clearer distinction of the four stages.BRAF and KRAS genes  we also evaluated the DNA methylation of the 10 DM hub genes according to the mutational status of AS genes . No statOverall, in this study, we observe and confirm the high reproducibility of methylation data, and hence the importance of this approach in identifying stable markers, drivers, targets of the disease. Finally, we highlight an exemplar case of methylated key molecule, emerging from the systemic approach we used, embodied by NFKBIA, a well-known genetic risk factor for colorectal cancer, here also emerging as in important epigenotype.GPL13534 (identifier of the Infinium 450k platform) and colorectal cancer as search terms. As to January 1st 2016 we identified, based on these terms, 3 studies from colonic fresh-frozen tissues  project , includiwse.org, ): they iwse.org, .q-value < 0.001, [DMRs were defined and selected according to the multivariate approach proposed by Bacalini et al.  to compa< 0.001, ).To guarantee robustness while avoiding loss of information we requested that the retained candidates show unanimous statistical significance across all datasets, without further limitations on the absolute value of the differential methylation (delta), owing to the recent findings on the relevance of minor changes in DNA methylation levels in terms of phenotypical consequences . Delta dThree pairwise comparisons  were run. In the TCGA validation cohort, the 45 N2 samples were compared with 373 CRC samples. In this last comparison, associations were corrected for the age of the subjects, an information that was not available for the 3 GEO datasets. In the following, if not explicitly stated, DMRs always refer to significantly differentially methylated BOPs.For each comparison, we identified robust results by selecting across all 3 datasets only common and concordant (coherently hyper- or hypo-methylated) DMRs .Tissues & Cell Lines parameter here set to large intestine and colon cancer cells. To guarantee proper reconstruction, Qiagen recommends to output from each meaningful list of molecules a maximum of 10 networks with a maximum of 70 nodes each. Successively, all networks sharing nodes (interconnected) were merged in one large interconnected map, leading to one network per comparison.Network analysis was adopted to gain understanding on the biological interconnections occurring among the DMRs identified in each meta-list: DMRs genes were set as nodes of the network, and connecting biochemical relations were reconstructed importing the gene lists into Ingenuity Pathway Analysis  in each network was filtered to select the genes exclusive to each tumor stage, leading to the final discussion on genes of topological and functional relevance, characterized by an aberrant methylation state in the disease.adegenet [Statistical analyses were performed using the computing environment R. Discriminant Analysis of Principal Componentes (DAPC) was performed using the R package adegenet .


Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research. Candida albicans, Candida glabrata and Cryptococcus neoformans in immunocompromised individuals. This socioeconomic burden is further amplified by the unprecedented rise in fungal diseases that are affecting plants and animals Yeast, a fungus that predominantly lives as a unicellular organism, has had an extraordinary influence on humanity throughout millennia, from its usage for baking and brewing to the potential of some species to cause life-threatening human diseases. The cultural, industrial, biotechnological, and medical impact of this organism remains unparalleled. The use of yeast fermentation to produce alcoholic beverages and to leaven bread coincided with the rise of ancient civilizations and has persisted until our days. Importantly, the continued development of yeast strains as vehicles for the development of new technology, for example in bioethanol, drug, and enzyme production, as well as the implementation of unconventional yeast species in industrial processes, highlights the ever increasing importance of yeast now and in the future 1Saccharomyces cerevisiae (the budding yeast) being one of the most thoroughly studied eukaryotes at the cellular and molecular levels. Indeed, yeast continues to be one of the preferred model organisms to explore eukaryotic cell biology, both due to its technical advantages in devising/sophisticating molecular tool kits to study cellular biology, and to a high degree of functional conservation 1011121314161718202223242729313334Yeasts have served as a successful research tool for the last century, In addition, studies on yeast have shed light on human diseases, providing a cellular platform to examine, for instance, prion biology, virus-host interactions, metabolic diseases, neurodegenerative disorders, cancer, or aging 373839404142434445464748495051525354555657585960216263646563697072de facto behave as a multicellular entity of communicating individuals rather than a group of isolated cells that do not interact with each other. In fact, a given yeast population originates from a single clone, and the ultimate biological goal of that population is the survival of the genetic information representing that very clone. Thus, under certain circumstances, the death of unfit or damaged yeast cells promotes the survival of the population as a whole. A number of physiological scenarios have been described that corroborate this teleological explanation for a cellular suicide program in yeast, including antagonistic interactions between yeasts, aging, mating, or colony formation 5461757677787980818283848687888990In multicellular organisms, the controlled suicide of single cells is crucial for development and homeostasis, providing a system that eliminates superfluous cells. The presence of such a mechanism also allows for the removal of damaged cells that might compromise organismal fitness. In a single-celled organism like yeast, this paradigm does not seem to apply at first sight, since - in this case - cellular suicide entails the death of the whole organism. However, in a way, a population of yeast cells Figure 1). In this paper we thus attempt for the first time to formulate a series of recommendations and caveats with respect to cell death-related results obtained in yeast. To this aim, we have followed the directions of the Nomenclature Committee on Cell Death (NCCD) 929394Saccharomyces cerevisiae, which we think can be extended to other yeast species and to multicellular filamentous fungi. Our goal is to frame a uniform set of guidelines that facilitate the communication among yeast cell death researchers, ultimately supporting and accelerating scientific advance (Box 1). In that respect, the nomenclature presented herein will likely need to be revised and updated as the field of yeast cell death moves forward and even more precise definitions are required.Even though it is now clear that yeast can indeed undergo cellular suicide, the corresponding terminology to describe this multifaceted process remains heterogenous and potentially misleading. Thus, we believe that there is timely need for a more precise and consistent nomenclature that clearly defines the concept of “yeast cell death”, considering morphological, enzymological, and functional aspects. Such standardization seems of importance, given that the field of yeast cell death is continuously expanding with significant progress being made at the phenotypical and mechanistic levels, including the finding that, akin to higher eukaryotes, yeast can also engage in distinct cell death modalities (in vivo is to use propidium iodide (PI). PI is a fluorescent nucleic acid intercalator that can only enter cells with a ruptured cell membrane, and can be routinely employed in both low and high throughput formats 969799100A crucial issue that demands a clear definition is the question of cell death itself. When is a cell dead? According to the NCCD guidelines this is only the case upon irreversible plasma membrane breakdown or complete cellular fragmentation, because only then the cell is factually disintegrated, irrespectively of which upstream pathway or routine has been engaged per se are still alive . Additional caveats include the possibility that live cells at the point of plating might die before forming a colony and/or that the plating procedure itself might drive (a fraction of) cells into death, which would be indistinguishable from cell senescence. Nonetheless, the literature suggests clonogenic capacity as a very good correlate to cell death in a plethora of different settings 6996104106Assessing clonogenicity with plating assays is the most commonly used method to quantify cell viability 62A further technique to assess yeast viability follows the growth rate of a given culture, which may decrease as a consequence of increased cell death. For this purpose, an aliquot is inoculated into fresh liquid medium and the growth is monitored, for instance, via photometric measurement of optical densities over a specific period of time 1086(3)) 120., viability or vitality) cannot be placed on a par with an increase in cell death. In conclusion, as mentioned above, the term cell death should be used only upon observing breakdown of the plasma membrane and thus loss of cell integrity . In addition, we suggest to strengthen this observation by simultaneously assessing clonogenic capacity , since (i) it represents the best-established output to accurately monitor overall cellular viability and (ii) it empirically correlates very strongly with actual cell death markers. Importantly, both methods are easy, quick and relatively inexpensive. The use of additional dyes/stainings/assays provides valuable complementary information, but cannot be used alone to unequivocally define a cell as dead.One possibility to evaluate yeast cell vitality is to directly assess the activity of specific enzymes directly. Although this is not widely employed in yeast cell death research, it represents an avenue to assay the physiological state of a metabolic pathway within the cell 1001121141002-DCF-DA) are converted by a broad range of other ROS, but only poorly by superoxides 127129130131133134131136137138Yeast cell death is often accompanied by oxidative damage and thus, a widely employed method in the field is the detection of reactive oxygen species (ROS) 96122123124Cellular demise in yeast may occur in two mutually exclusive variants: either as an accidental event or through a regulated pathway. Accidental cell death (ACD) occurs upon exposure to severe conditions, resulting in a rapid, uncontrollable and unavoidable form of death. ACD may follow a series of extreme stimuli, including physical conditions, such as very high temperatures or pressures, severe chemical insults like strong detergents and high concentrations of acids or bases as well as mechanical challenges, for instance, vigorous shearing or ultrasonic treatment. The immediate nature of ACD, which is characterized by a virtually immediate structural breakdown of cells, allows no form of pharmacologic or genetic inhibition. Thus, this form of cell death does not constitute a direct target for modulation or prevention. However, it remains unclear whether yeast cells undergoing ACD may release endogenous, bioactive molecules to the extracellular space 75140141142143versus regulated) modalities of yeast cell death manifesting with a necrotic morphology (see below).ACD is often equated with necrosis, which in yeast is usually identified as a cellular condition of early plasma membrane permeabilization in the absence of typical apoptotic markers and of complete disintegration of subcellular structures ., upon exposure to mild stress or as a consequence of mutations. Cell death occurring in the former scenario is termed "programmed cell death" (PCD), while the expression "regulated cell death" (RCD) encompasses both PCD as well as all other instances of cell death that depend on a molecular machinery 145146147That said, many lethal stimuli result in a form of yeast cell death that - at odds with ACD - is executed by a genetically encoded, dedicated molecular machinery. In higher eukaryotes, a distinction is made between such a controlled form of cell death when it occurs (i) in the framework of a purely physiological program, e.g., during (post-) embryonic development or tissue homeostasis, or (ii) as a response to either a perturbation of intracellular or extracellular homeostasis, e.gde facto representing an instance of PCD. During yeast chronological aging, the cellular community maintains homeostasis thanks to the programmed death of dysfunctional or old cells, which spares and provides nutrients to the fitter individuals 75151For yeast cell death, many authors have used the term PCD to interchangeably refer to all types of cellular demise that are not accidental . However, emerging evidence is confirming that a yeast population, be it a liquid culture or a solid colony, bears a degree of complexity reminiscent of multicellular organisms that demands a revision of this terminology. For instance, during yeast gametogenesis (or sporulation), immature meiotic products as well as the mother cell itself succumb via activation of vacuolar rupture 149Importantly, since RCD depends on a defined molecular machinery, it can be modulated with pharmacologic or genetic means. The extent of such modulation depends on the progression of the process across a hitherto poorly defined point-of-no-return. According to the NCCD, the processes preceding such point are part of cellular stress responses, while those following it belong to actual cell death signaling Figure 2). Finally, it should be noted that in cell death research, it is generally advisable to determine the kinetics of the parameters under scrutiny Table 1). Beyond the specificities outlined below, a number of general issues and notes of caution also need to be considered (Box 2).Yeast RCD may follow different subroutines (see below) that can be differentiated from each other by a series of morphological and biochemical features. To precisely characterize the lethal phenotype, we recommend (i) to first determine if cell death actually occurs , (ii) if it does, to then examine the subroutine(s) involved via morphological and biochemical observations, using at least two different detection methods Saccharomyces cerevisiae. This includes the first observation of an apoptotic phenotype in yeast, specifically in a strain with a point mutation in the gene coding for the cell cycle protein Cdc48 de novo protein synthesis, e.g. by cycloheximide Most studies on RCD in yeast have been conducted in the budding yeast One of the events most commonly associated with apoptosis is the exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane 931836396104ab initio" 185We believe that the second (Annexin V positive) and third (PI positive) subpopulations can be readily interpreted as apoptotic and primary necrotic, respectively, provided that at least one more assay is performed to validate this assumption. For the fourth subpopulation (Annexin V/PI double positive cells), we favor the following interpretation: unlike multicellular animals, a yeast population presumably does not eliminate apoptotic cells via the phagocytic activity of other yeast cells. In the absence of such clearance by scavengers, an apoptotic cell eventually undergoes a metabolic collapse that results in breakdown of the plasma membrane integrity and thus a necrotic phenotype. This phenomenon is termed "secondary necrosis" to discriminate it from "primary necrosis", which describes the phenotype of "cellular necrosis occurring Still, these cells might also have undergone secondary necrosis following other cell death subroutines, but at this point there is no evidence for this possibility, which should be evaluated earlier in the cascade of events leading to cellular demise. Importantly, the phenotypical shift from apoptosis to secondary necrosis might reflect defined mo-ecular events and thus be experimentally distinguishable from ACD with necrotic features and primary necrosis also at the functional level 187189190191192In vitro, cultured metazoan cells that are left to finalize the apoptotic process without interruption  eventually succumb with features of secondary necrosis 186194In vivo, secondary necrosis may occur in multicellular animals, for example, when apoptotic cells are shed into the lumina of hollow organs with low probability to encounter scavengers or when apoptotic cell death occurs at a pace that surpasses the local scavenging capacity 186196In multicellular animals, clearance of apoptotic cells is a central physiological feature for maintenance of organismal homeostasis. Still, secondary necrosis does occur under certain circumstances 0/G1) has been previously employed as an alternative to assess apoptotic DNA degradation Besides PS externalization, apoptotic cells exhibit chromatin condensation, which can be readily assessed by nuclear staining with dies such as 4',6-diamidino-2-phenylindole (DAPI) followed by microscopic inspection 6363961049610415719920020120220379ψm) 96159160162163164209c release, which depends on the ADP/ATP carrier Apoptotic cell death often follows mitochondrial outer membrane permeabilization (MOMP), which culminates with the release of pro-apoptotic proteins from the intermembrane space and irreversible loss of mitochondrial transmembrane potential , endonuclease G (Nuc1) and the yeast metacaspase (Yca1), exert both lethal and vital functions 626996159160213214215A large number of apoptotic regulators and executors have been identified in yeast so far yca1 knockout strains may point towards an apoptotic mechanism. However, under certain conditions, apoptosis is not executed via Yca1, but instead relies on other factors, including Aif1, Nuc1, the human cyclophilin D ortholog Cpr3, the BH3-only protein Ybh3 or ceramides 96160217218219220221222YCA1), it is possible that other proteases might functionally substitute for metacaspases 22422570227For exploring a putative apoptotic mechanism in a given cell death scenario, the deletion strains of known apoptotic regulators should be harnessed, since distinct apoptotic subroutines exist that rely on different factors that may act independently from each other to orchestrate cellular demise. For instance, the yeast metacaspase Yca1 is involved in many apoptotic RCD and PCD settings 6269In human cells, extrinsic apoptosis defines a caspase-dependent cell death subroutine that is induced by extracellular lethal ligands. These ligands are sensed and transmitted either via specific transmembrane death receptors or through so-called ‘dependence receptors'. Dependence receptors can trigger two opposite signaling pathways: in the presence of ligand, they elicit signals involved in cell survival, migration and differentiation, but in the absence of ligand, they promote apoptotic RCD. Thus, dependence receptors only exert lethal functions when the concentration of their specific ligands falls below a critical threshold level 8223023123279236237In dying yeast, necrotic characteristics may appear in the frame of a primary or secondary necrotic process. While secondary necrosis is probably a consequence of apoptosis in most if not all cases (see above), a primary necrotic phenotype (which occurs without any preceding apoptotic traits) may result from two cell death modalities: ACD or RCD. Indeed, yeast primary necrosis can not only be the outcome of severe insults , but also develop as an event orchestrated by a genetically controlled machinery (regulated necrosis) bona fide primary necrosis in yeast. In addition, viability should be measured with at least one assay (preferably by assessing clonogenic capacity) to corroborate cellular demise. Finally, we strongly recommend to exclude the presence of apoptotic death indicators, and most importantly to differentiate the observed phenotype from secondary necrosis.Necrosis first leads to a gain in cell volume and organelle swelling (oncosis), which may be observed, for instance, using fluorescent microscopy of GFP-fused proteins that mark organellar membranes 103104157165144bona fide instance of ACD. This is in line with the concept that not only the type but also the intensity of a given perturbation determines the form of death 91As in higher eukaryotic cells, in yeast, ACD may be triggered upon the challenge to extremely detrimental conditions. Thus, agents like hydrogen peroxide, acetic acid, amphotericin B, or several metals that are pro-apoptotic at low doses may induce necrosis at high concentrations 125218241As mentioned above, yeast can undergo regulated necrosis, reminiscent of the RCD instances detected in human cells 104246247248249m dissipation 2511491652540 strain . However, as previously mentioned, mitochondria are the main executors of apoptotic cell death. Thus, mitochondrial dependence cannot be used as a sole determinant to characterize regulated necrosis and must be accompanied by a set of other assays that demonstrate the primary necrotic nature of cell death. Of note, several known mammalian mediators of regulated necrosis have homologs in yeast, including cathepsins, cyclophilin D, calpains, Hsp90, or protein kinase A, among others 103bona fide regulated necrosis. Similarly, it remains to be seen whether known inhibitors of regulated necrosis in mammals also interfere with some cell death scenarios in yeast as well Under certain circumstances, regulated necrosis in mammalian cells may be mechanistically linked to primary ΔψA number of questions remain to be answered with regard to the actual existence of a necrotic RCD subroutine in yeast. In mammals, regulated necrosis plays a number of key roles, most prominently due to its immunogenic nature, for instance upon pathogen infection 6175104In human cells, different types of regulated necrosis have been defined, with MPT-driven regulated necrosis and necroptosis among the most extensively studied forms 258Drosophila melanogaster and human cells 243262263264265D. melanogaster) 267In mammalian cells, a series of other RCD modalities have been defined. Macroautophagy (hereafter referred to as autophagy) is a conserved catabolic process that orchestrates the digestion of intracellular material  in the vacuole. During autophagy, double-membraned vesicles  form and engulf cytoplasmic components, followed by the fusion of autophagosomes with the vacuole, where the cargo is degraded and the resulting macromolecules are released into the cytoplasm for reuse 16In yeast, the term ATCD has been used to describe cellular demise occurring under specific external stress conditions like zinc-induced cell death 37272274275277A number of microscopic, biochemical and enzymatic assays are available and established 175279175166167168169118., by deleting PEP4 or PRB1) or pharmacologically  as well as by neutralizing the vacuolar pH  175262The causative implication of autophagy in cell death may be explored by deletion of autophagy-related (ATG) genes, which are the key orchestrators of the process 178179180175The expression "mitotic catastrophe" (MC) was first employed to illustrate the lethal phenotype of a temperature-sensitive fission yeast mutant strain that enters mitosis prematurely without effectively completing it 28228328463286287bona fide cell death executioner mechanism by itself In mammalian cells, death following mitotic aberrations can, indeed, be either apoptotic or necrotic 94A series of other cell death subroutines have been defined in human cells that, however, are restricted to specific cell types and thus do not apply to yeast.S. cerevisiae. However, other yeast species have been shown to share similar cell death characteristics and also bear a set of comparable cell death subroutines. Thus, we propose to extend the above-described recommendations formulated above to all yeast species.As previously mentioned, yeast cell death has been most extensively studied in Schizosaccharomyces pombe (fission yeast) has been shown to express an RCD machinery that responds to various stimuli. These include physiological triggers such as aging, defects like the abnormal metabolism of intracellular lipids 291292293292S. pombe (yet). Among the described S. pombe apoptosis executors are the chaperone Cnx1  and the metacaspase Pca1 295295S. pombe apoptosis is expected to involve additional players, as there is evidence for the presence of different factors in fission yeast that are homologous to effectors of S. cerevisiae apoptosis, including the protease Nma111 Candida albicans, which has become a molecular genetics model to study pathogenicity, virulence and fungal development 302242304305Candida biofilms, which are highly tolerant to standard antimycotics and hence difficult to eradicate. Exploiting the apoptosis machinery in cells constituting biofilms may pave the way to their effective eradication, and hence limit the incidence of indwelling device-associated infections (IDAIs) 307308C. albicans also harbors a gene encoding a metacaspase (CaMCA1) 311316Candida species, e.g., Candida glabrataCandida kruseiCandida dubliniensisCandida tropicalisCandida parapsilosis232The major opportunistic human pathogen Cryptococcus neoformans, an important pathogen of immunocompromised and immunocompetent patients, also undergoes apoptosis 323Cryptococcus genus member, Cryptococcus laurentii, has also been shown to respond to some stimuli with apoptotic RCD ., Kluyveromyces lactis326Pichia pastorisRhodotorula glutinis  Zygosaccharomyces bailii330332Aspergillus fumigatus, which has also been demonstrated to undergo apoptosis under certain conditions 334A. fumigatus codes for two metacaspases (CasA and CasB), whose relative contribution to cell death seems to depend on the scenario 335336Aspergillus nidulans is another member of the Aspergillus spp. that has been demonstrated to undergo RCD 338A. nidulans appears to code for an apoptotic machinery with relevant players like apoptosis-inducing factor and two putative metacaspases 339Podospora anserina, is used as an aging model that incorporates crucial apoptotic factors, including two metacaspases (PaMCA1 and PaMCA2) and at least five Aif members, of which only mitochondrial (but not cytosolic) isoforms seem to be relevant for aging-driven RCD 341P. anserina cyclophilin D ortholog in RCD 343344P. anserina346306Paracoccidioides brasiliensis Colletotrichum gloeosporioides Fusarium oxysporum Fusarium graminearum Mucor racemosus Botrytis cinerea Penicillium expansum Rhizopus oryzae Scedosporium prolificans Neurospora crassa 358. As multicellular organisms, filamentous fungi have developed programs that are reminiscent of organismal RCD. For instance, several putative homologs of factors relevant for animal apoptotic control that are not found in unicellular yeast are present in the genomes of filamentous species It should be noted that a growing body of work is addressing RCD in multicellular fungi. A major human pathogenic fungus that causes life-threatening disease is The impact of yeast  on our lives at multiple socioeconomic, scientific and medical levels emphasizes the importance of decoding the mechanisms that determine its survival and control its demise. Therefore, the molecular comprehension and potential manipulation of yeast cell death hold major promise for biotechnological and biomedical applications. We anticipate that numerous fields might benefit from the possibility to modulate yeast cell death. For instance, the productivity of yeast during large-scale processes in the pharmaceutical and industrial arenas largely depends on its viability and ultimately on its tolerance to stress and its demise in stationary cultures. Also, novel pharmacological approaches that specifically target the RCD machinery of yeast pathogens may bypass the ever-increasing resistance to classical antimycotics, which is an emerging public health problem. Other medical manipulations of yeast RCD are also conceivable, e.g., strategies to intervene on pathogenic deviations of the mycobiome. Finally, yeast will continue to help the community in deciphering eukaryotic cell death pathways as it serves as an important model for human disease. Given its power to study the relationship between genotype and phenotype, we expect to gain further insights from yeast to identify actionable targets that may be subjected to pharmacological (drug discovery) or genetical manipulation.For all these reasons, it is now imperative to set the standards for defining and studying cell death in yeast. That said, we want to emphasize that the present set of recommendations should be taken - as any scientific overview - as a snapshot of the current knowledge, rather than as a definitive compilation. Indeed, as research continues, we surmise that the present guidelines will have to be extended and revised. For instance, other nuanced changes to - or even novel types of - RCD may emerge from continued efforts to characterize the multicellular character of yeast populations, including but not restricted to uncovering intercellular communication, interaction between populations or cellular differentiation within colonies and biofilms. Still, neologisms should be introduced with care and only when the characterization of a lethal process that bears new functional and biochemical aspects requires it. Otherwise, new expressions should be avoided to limit confusion.Another crucial point is to acknowledge the inherent complexity and dynamic nature of RCD in general and its different subroutines in particular. In fact, it is the crosstalk between pro-life and pro-death signals that determines cellular fate, and the activation of pro-survival pathways (such as autophagy) may often accompany lethal signals. Also, stress conditions may activate different RCD subroutines that can be interconnected or may occur independently, sequentially, or in parallel. Indeed, the inhibition of one specific RCD modality might trigger backup mechanisms that still ensure cell death execution. It is thus important to keep these points in mind when classifying a lethal phenotype.Altogether, the present guidelines attempt to unify the nomenclature and definition of yeast cell death modalities and - in our opinion - will help other fields of unicellular research  to establish their set of recommendations using the present one as a basis. We are convinced that some degree of linguistic and experimental standardization is necessary for facilitating communication among researchers, especially at a point where the existence of yeast RCD is scientifically accepted and its socioeconomical impact is ever growing.


BRCA1 and BRCA2. Among them, a subset has hereditary susceptibility to cancer and requires further testing. We sought to identify specific groups who remain at high risk and evaluate whether they should be offered multi-gene panel testing.Many women with an elevated risk of hereditary breast and ovarian cancer have previously tested negative for pathogenic mutations in BRCA1 and BRCA2 either by limited or comprehensive sequencing. Additionally, they met one of the following criteria: (i) personal history of bilateral breast cancer, (ii) personal history of breast cancer and a first or second degree relative with ovarian cancer, and (iii) personal history of ovarian, fallopian tube, or peritoneal carcinoma.We tested 300 women on a multi-gene panel who were previously enrolled in a long-term study at UCSF. As part of their long-term care, all previously tested negative for mutations in BRCA1 and BRCA2. Ashkenazi Jewish and Hispanic women had elevated pathogenic mutation rates. In addition, two women harbored pathogenic mutations in more than one hereditary predisposition gene.Across the three groups, 26 women (9%) had a total of 28 pathogenic mutations associated with hereditary cancer susceptibility, and 23 women (8%) had mutations in genes other than BRCA1 and BRCA2 mutations, we identified three groups of women who should be considered for subsequent multi-gene panel testing. The identification of women with multiple pathogenic mutations has important implications for family testing.Among women at high risk of breast and ovarian cancer who have previously tested negative for pathogenic The online version of this article (doi:10.1007/s10549-017-4181-0) contains supplementary material, which is available to authorized users. The advent of next-generation sequencing (NGS) technology has revolutionized the clinical approach to genetic testing across many areas of medicine including medical oncology. Instead of single gene testing, interrogating a panel of multiple genes provides clinicians information about one or more disorders in a single test –4. AdditBRCA1 and BRCA2 mutations, but not a broader panel BRCA1 andBRCA1 and BRCA2 (6–7%) [BRCA1 and BRCA2 genetic testing for all women with ovarian cancer, and parallel re-testing of all of these women using multi-gene panels may be warranted due to the observed mutation rates in this group.In addition to these groups, there likely exist broader groups of the previously tested individuals who harbor pathogenic mutations that warrant re-testing using multi-gene panels. For instance, the high rate of mutations we observed in women who met NCCN criteria and also had personal history of ovarian cancer 8%) could indicate that all women with ovarian cancer may benefit from multi-gene panel testing. Supporting this hypothesis, previous studies have demonstrated elevated rates of pathogenic mutations in genes other than 2 (6–7%) , 8 among% could iBRCA1 and BRCA2 testing were previously used by other laboratories: Ashkenazi founder mutation testing, Hispanic founder mutation testing, and gene testing without analysis of large deletions and duplications. Multi-gene panel testing by Color panel identified three mutations in the BRCA1 gene (12% of mutations in the study cohort) that were not identified by the previous limited testing. One pathogenic BRCA1 mutation was identified in a woman who had testing limited to Ashkenazi Jewish and Hispanic mutations [BRCA1 and BRCA2 without assessment of large deletions and duplications.In the study cohort, three types of limited utations  due to hAll three of these mutations were missed in individuals of partial or full Hispanic origin. Given the increase in frequency of large deletions and duplications in the Hispanic population –29  inherited separately, whether that member tests positive or negative for the previously discovered family mutation.Pathogenic mutations in multiple genes in the same individual have been observed in approximately 3% of patients in larger cohorts who received breast, ovarian, colon, and general hereditary cancer risk testing [Following identification of a pathogenic mutation, a carrier’s family members are typically offered targeted single-site testing (or cascade testing) specific to the identified pathogenic mutation in order to determine each member’s risk of hereditary cancer. The identification of pathogenic mutations in two different genes in a single individual, present in both our validation set and study cohort, indicates that such testing may be inadequate to clinically determine the risk of hereditary cancer for two reasons: testing , similarBRCA1 and BRCA2 test results, in certain cases, clinicians often remain suspicious of another hereditary cancer syndrome due to the family history of cancer. Nevertheless, efforts to obtain additional genetic testing are often limited due to lack of insurance coverage, resulting in prohibitively high costs for patients. With the rise of more affordable testing options, clinicians and their patients now have greater access to multi-gene panel testing, both as a follow-on test for those with previously incomplete testing and as a first-line approach.Despite negative This study was enriched for individuals at high risk of breast and ovarian cancer as defined by the NCCN criteria. Consequently, this study cohort likely represents individuals with higher than average breast and ovarian cancer risks and is not representative of those patients with mild to moderate cancer risks nor is it representative of the general population. Larger cohorts will be required to determine more accurate rates of pathogenic mutations in women with previously negative hereditary cancer testing.The study population represented a cohort of primarily Caucasian women, which is not generalizable to the population at large. Furthermore, a large proportion (17%) of patients in the cohort were of Ashkenazi Jewish descent. While other studies have found no increased detection rate despite enrichment for Ashkenazi Jewish participants , 13, theSupplementary material 1 (DOCX 33 kb)Below is the link to the electronic supplementary material.