Liver fibrosis is the result of a deregulated wound healing process characterized by the excessive deposition of extracellular matrix. Hepatic stellate cells (HSCs), which are activated in response to liver injury, are the major source of extracellular matrix and drive the wound healing process. However, chronic liver damage leads to perpetual HSC activation, progressive formation of pathological scar tissue and ultimately, cirrhosis and organ failure. HSC activation is triggered largely in response to mechanosignaling from the microenvironment, which induces a profibrotic nuclear transcription program that promotes HSC proliferation and extracellular matrix secretion thereby setting up a positive feedback loop leading to matrix stiffening and self-sustained, pathological, HSC activation. Despite the significant progress in our understanding of liver fibrosis, the molecular mechanisms through which the extracellular matrix promotes HSC activation are not well understood and no effective therapies have been approved to date that can target this early, reversible, stage in liver fibrosis. Several new lines of investigation now provide important insight into this area of study and identify two nuclear targets whose inhibition has the potential of reversing liver fibrosis by interfering with HSC activation: Yes-associated protein (YAP), a transcriptional co-activator and effector of the mechanosensitive Hippo pathway, and bromodomain-containing protein 4 (BRD4), an epigenetic regulator of gene expression. YAP and BRD4 activity is induced in response to mechanical stimulation of HSCs and each protein independently controls waves of early gene expression necessary for HSC activation. Significantly, inhibition of either protein can revert the chronic activation of HSCs and impede pathological progression of liver fibrosis in clinically relevant model systems. In this review we will discuss the roles of these nuclear co-activators in HSC activation, their mechanism of action in the fibrotic process in the liver and other organs, and the potential of targeting their activity with small molecule drugs for fibrosis reversal. Chronic fibroproliferative disease is a major cause of death in the industrialized world, placing a substantial burden on healthcare systems  and bromodomain-containing protein 4 (BRD4)  induced fibrosis, whereas portal fibroblasts are the major source of myofibroblasts in response to cholestatic injury (bile duct ligation) induced fibrosis  are the ultimate effectors of the Hippo pathway, a key growth control and tumor suppressor pathway originally identified by genetic studies in The Hippo pathway is primarily regulated by mechanical inputs from the microenvironment Figure , includiIn the mammalian liver, Hippo pathway signaling is involved in development and regeneration as well as pathological processes, such as cancer and fibrosis. During liver development, YAP is important for bile duct formation (tubulogenesis), although the molecular mechanism remains to be elucidated; no effect has been observed from YAP deletion in the adult liver to date  induced fibrosis in mice  prevents HSC activation in vitro and fibrogenesis in vivo  and Ctgf (connective tissue growth factor), are direct targets of YAP , α-SMA, and ankyrin repeat domain 1 (ANKRD1) staining; this HSC activation was reversed by pharmacologic inhibition of YAP , promoting cell-matrix adhesion and continued nuclear accumulation of YAP, thus resulting in a positive feedback loop by which YAP mechanotransduction leads to chronic fibrogenesis  family member BRD4. BRD4 is a double bromodomain-containing transcriptional enhancer that binds to acetylated chromatin during interphase and mitosis, thereby serving as a co-activator for transcription of MYC, E2F, and nuclear factor-κB (NF-κB) regulated cytokines to regulate the cell cycle by promoting G2 to M phase transition  expression and promoting profibrotic TGF-β signaling by applying force to collagen-binding integrin receptors leading to downstream mechanotransduction and crosstalk with SMAD proteins  midline carcinomas (reviewed in Filippakopoulos and Knapp, Col1A1 expression and extracellular matrix remodeling (Tang et al., Similarly to the mechanism by which VP inhibits YAP, the competitive bromodomain inhibitor JQ1 inhibits binding between Histone H4 and bomodomains, thereby preventing BRD4 from acting as a transcriptional co-activator of target genes (reviewed in Filippakopoulos and Knapp, in vivo models of liver fibrosis, targeting the fibrotic response at the transcriptional level presents an attractive avenue for further research.The activation of quiescent HSCs and their transition to a proliferative, contractile myofibroblast phenotype represents a critical control point between a normal and pathological tissue repair response. Few regulatory factors have been identified that regulate this transition in the liver and that can be targeted for liver fibrosis reversal. Recent progress has shown that mechanotransduction induced by increased contact between activated hepatic myofibroblasts due to pathologic accumulation of extracellular matrix components and increased focal adhesion formation is orchestrated, in part, by the transcriptional co-activators YAP and BRD4 in concert with their respective transcription factors. Traditionally, small molecule inhibitor development has focused on disrupting ligand-receptor interactions rather than transcriptional regulation. However, no therapies have been developed to date that can reverse liver fibrosis in patients, which has become a major clinical challenge in the industrialized world. More effective therapies are therefore critically needed to treat chronic fibroproliferative diseases. Given that VP and JQ1 have shown efficacy in BRD4 and YAP both regulate profibrotic gene expression that plays a central role in HSC activation, tissue remodeling and liver fibrosis. Nevertheless, these nuclear co-factors appear to regulate distinct sets of target genes suggesting that BRD4 and YAP represent independent pathways that are both critically involved in the regulation of early stages of fibrosis. Thus, there appear to be multiple positive feedback loops involving integrins, TGF-β, and other cytokines that contribute to HSC activation and fibrosis progression by regulating specific nuclear gene expression programs through YAP and BRD4. The studies by Mannaerts et al.  and DingLast, but not least, of particular interest will be the elucidation of the precise mechanisms by which mechanical signals from the environment are relayed across the membrane to modulate YAP and BRD4 function in the nucleus. Integrins are the primary mechanosensors and transducers of tissue stiffness and several integrins have been identified as key drivers of profibrotic signaling in various organs. Some of these integrins, in particular αv subunit integrins, such as αvβ1 and αvβ6, have been shown to control the activation of latent TGF-β1 complex and targeting these integrins with small molecule inhibitors can block or reverse fibrosis progression in mouse models (Henderson and Sheppard, Alternatively, integrins can control HSC activation through bi-directional “outside-in” and “inside-out” signaling mechanisms leading to changes in actin organization and actomyosin contractility and subsequent alterations in tissue remodeling. Indeed, several integrin-associated signaling proteins, including integrin-linked kinase (ILK, Zhang et al., In sum, mechanotransduction in myofibroblasts is clearly relevant for fibrotic disease and it is increasingly being realized that components of mechanosensitive signal transduction pathways are not only drivers of disease progression, but also promising targets for therapy. While additional work will be required to address the precise roles of YAP and BRD4 in fibrosis, overall the current findings suggest that YAP and BRD4 are key terminal effectors of mechanosensing signal transduction pathways that initiate and perpetuate fibrosis and that targeting YAP and BRD4 alone or in combination has great promise toward treating patients with organ fibrosis and reducing the burden of fibroproliferative diseases. A more refined understanding of the mechanisms underlying myofibroblast activation across different organ systems and better insight into the roles and regulation of YAP and BRD4 in this process could lead to more personalized treatment options that may slow and perhaps even reverse fibrosis progression in the future.AZ, AT, KK, LW, and JK have written this review. All authors have read the review and gave their agreement for submission.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.


Aging is a complex, multi-factorial biological process shared by all living organisms. It is manifested by a gradual accumulation of molecular alterations that lead to the decline of normal physiological functions in a time-dependent fashion. The ultimate goal of aging research is to develop therapeutic means to extend human lifespan, while reducing susceptibility to many age-related diseases including cancer, as well as metabolic, cardiovascular and neurodegenerative disorders. However, this first requires elucidation of the causes of aging, which has been greatly facilitated by the use of model organisms. In particular, the budding yeast Saccharomyces cerevisiae has been invaluable in the identification of conserved molecular and cellular determinants of aging and for the development of approaches to manipulate these aging determinants to extend lifespan. Strikingly, where examined, virtually all means to experimentally extend lifespan result in the induction of cellular stress responses. This review describes growing evidence in yeast that activation of the integrated stress response contributes significantly to lifespan extension. These findings demonstrate that yeast remains a powerful model system for elucidating conserved mechanisms to achieve lifespan extension that are likely to drive therapeutic approaches to extend human lifespan and healthspan. The intent of lifespan-extending regimens is to prevent and treat the major diseases of society. However aging studies in humans and mammals are hampered by their very long lifespan. Thus, short-lived organisms have gained popularity as experimental models for aging studies. In particular, budding yeast is a highly informative model for aging studies with its short lifespan, genetic tools, and fully sequenced genome 1Budding yeast provides two separate, but partially overlapping paradigms for aging studies, replicative lifespan (RLS) and chronological lifespan (CLS) 5Strikingly, most interventions that extend lifespan are, or induce, limited amounts of stress that have a beneficial effect via the phenomenon of hormesis, because these stresses would be toxic or lethal at higher doses. These lifespan-extending hormetic stresses induce a protective cellular stress response. This conserved stress response in yeast was termed the General Amino Acid Control (GAAC) because it was initially identified as a response to amino acid depletion that upregulates the genes required for amino acid synthesis 1317iMet). This in turn reduces delivery of Met-tRNAiMet to the 40S ribosome, hampering initiation of global cap-dependent translation GCN4 mRNA leader occurs, followed by the ribosomes dissociating from the GCN4 mRNA, precluding initiation at the GCN4 start codon . However, under conditions where there are limiting amounts of 40S ribosome associated with Met-tRNAiMet, the ribosomes scan through the inhibitory uORFs within the GCN4 leader and initiate translation at the GCN4 start codon. The mammalian counterpart of Gcn4, ATF4, is induced by an analogous mode of translational control During the ISR in yeast, a variety of stressful conditions activate the kinase Gcn2, which phosphorylates eIF2α, the central regulation node of the ISR. Phosphorylation of eIF2α promotes a stress-resistant state by global attenuation of protein synthesis, which reduces the load of unfolded proteins on chaperone networks and diverts amino acids from energetically costly protein synthesis to other metabolic pathways. Although the mammalian ISR has three other eIF2α kinases in addition to Gcn2, the stress response pathways are otherwise very similar in yeast and mammals los1∆, also activate Gcn4 in the absence of Gcn2, presumably because there is insufficient amount of ternary complex formation 22iMet complex. Notably, amino acid depletion is a clear mechanism to extend both RLS and CLS, while reduced abundance of ribosomal subunits or deletion of the LOS1 gene required for tRNA export extend RLS in a manner dependent on Gcn4 but not Gcn2 2223Gcn4 can also be synthesized in the absence of Gcn2 activation, by conditions that directly reduce global cap-dependent translation . For example, Gcn4 is translated in yeast strains lacking Gcn2 upon rapid depletion of amino acids The Gcn4 transcriptional activator upregulates genes that allow adaptation to cellular stress We are still at an early stage in trying to understand which of the downstream stress response pathways induced by Gcn4 are key to the lifespan extension mediated by the ISR . Although not conclusive, some clues as to the relative importance of the different ISR-induced stress response pathways to lifespan can be provided by the phenotypes resulting from deletion of genes activated by the ISR. For example, it is unlikely that purine biosynthesis is key for lifespan extension, given that deletion of purine biosynthesis genes extends CLS 29Autophagy is a conserved catabolic housekeeping mechanism whereby bulk cytoplasm, including proteins and organelles, are sequestered into membrane-bound vesicles (autophagosomes) which subsequently fuse with vacuoles where their content is degraded by vacuolar proteases 29The yeast RLS assay utilizes the asymmetric division of mother and daughter yeast cells to track the replicative capacity of individual cells, and has been used to identify various types of factors that contribute to replicative senescence. Comparison of daughter cells and old mother cells has revealed that many factors selectively accumulate in the mother cells, and these so-called senescence factors have been suggested to drive the aging process. These senescence factors include extra-ribosomal DNA circles ATG genes or GCN4 has minimal effects on RLS 23There is currently no evidence to suggest that either Gcn4 or autophagy contribute to longevity under nutrient-rich growth conditions. This is consistent with the fact that deletion of most GCN2 or GCN4 blocked nitrogen starvation-induced macroautophagy, as determined by electron micrography of large autophagosomes GCN2 or GCN4 only partially diminished autophagic flux, but formed multiple small autophagosomes, rather than large autophagosomes ATG1 and ATG32 transcription upon nitrogen starvation ATG genes have Gcn4 binding sites in their promoters Gcn2 and Gcn4 were initially described as being required for the induction of autophagy upon nitrogen starvation. Specifically, deletion of Amino acid depletion is another common mechanism used to induce autophagy. Notably, amino acid depletion is not only a mechanism to extend yeast RLS 46Although Gcn2 is conventionally activated by tRNAs that are not bound to amino acids (uncharged tRNAs), Gcn4 synthesis is induced under many conditions besides amino acid depletion. These include starvation for purines 13GCN2TOR1 deletion was mediated at least partially by Gcn4 GCN4 deletion. The reason for the discrepancy between the two studies could reflect that, although Gcn2 is activated upon TOR inhibition, Gcn2 activation may not be essential for Gcn4 translation upon TOR inhibition. The reasoning for this is that there could be reduced translation of the uORFs in the GCN4 leader upon TOR inhibition or TOR1 deletion as a consequence of loss of TOR-mediated phosphorylation of the eukaryotic initiation factor eIF4E binding protein 4E-BP1 and accompanying down regulation of global protein synthesis. Alternatively, this could be due to potential changes in the small ribosomal subunit caused by decreased phosphorylation of ribosomal protein S6 (RpS6) by Sch9/S6K, which might interfere with 40S structure, changing its activity and increasing the leakiness of translation on the GCN4 mRNA. As discussed above, Gcn4 can be activated in the absence of Gcn2, in conditions where protein synthesis is reduced . There is also feedback between the two pathways at least in one direction because inhibition of TOR signaling induces activation of Gcn2 1348ATG genes . Whether Gcn4 synthesis per se is sufficient to extend RLS or induce autophagy remains untested.The TOR pathway and the ISR pathway appear to be interlinked, although this is somewhat controversial. One report showed that the activation of autophagy that is triggered by rapamycin-mediated inhibition of TOR was not affected by deletion of RER1 encodes a receptor that maintains ER compartmentalization, and upon deletion extended RLS by 64% RER1 resulted in ER stress, as it activated the UPR and autophagy, which are likely to mitigate an increase in ER load. In agreement with lifespan extension being caused by ER stress, the drug tunicamycin, which also activates ER stress, extends RLS. The autophagy induced by RER1 deletion was required for RLS extension, because deletion of genes encoding Atg8, or Atg5, the latter of which mediates the Atg8 lipidation required for expansion of autophagosomes, abolished the otherwise extended RLS in rer1∆ yeast. However, the UPR induced by ER stress was not required for RLS extension, because deletion of the IRE1 gene, encoding the principle sensor of the UPR, did not block RLS extension. The screen also recovered MNT3, encoding a Golgi glycosylase as being long-lived upon its deletion. Deletion of MNT3 led to high basal levels of UPR, elevated autophagy and extended mitotic lifespan Disruptions of the ER-Golgi apparatus can extend RLS by inducing autophagy. In fact, this provides the most compelling evidence to date for a role for autophagy in RLS extension. An innovative genetic screen for deletion mutants that maintain the transcriptional silencing that is usually lost in old cells identified novel gene deletions that extend RLS ATG7, ATG11, and ATG15, potentially through its influence on histone acetylation + antiporter Qdr3, is dependent on the transcription factor Gcn4 Polyamines, such as spermidine, play a role in longevity. Polyamine levels, including spermidine, decrease during aging. Notably, addition of spermidine extends yeast RLS when given to old cells, but not young cells, isolated by elutriation LOS1 deletion was shown to be in the same pathway as lifespan extension by dietary restriction (DR) via epistasis analyses LOS1 leads to the activation of Gcn4, reflected in the upregulation of many of its transcriptional targets, and seemingly in the absence of Gcn2 activation, because no global decrease in mRNA translation occurs iMet which would promote scanning through the Gcn4 upstream ORFs and Gcn4 synthesis  without the need for Gcn2 activation. It will be interesting to discern if RLS extension upon LOS1 deletion is dependent on autophagy or other pathways induced by Gcn4.Gcn4 is required for RLS extension that occurs upon nuclear retention of tRNAs. Deletion of the gene encoding the tRNA exporter Los1 was found to extend RLS in a systematic analysis of 4,698 single gene deletions for the effect on the yeast RLS AFG3 deletion reduced cellular mRNA translation Gcn4 is required for RLS extension that results from deletion of specific mitochondrial components. Yeast lacking the gene encoding the mitochondrial AAA protease Afg3 are long-lived TOR1, SCH9 or GCN2 blocked cryptotanshione from extending CLS GCN2, but independent of TOR1GCN4 blocks this CLS extension Investigations into the involvement of Gcn2/Gcn4 in yeast CLS extension to date are limited, but are consistent with a role for the ISR in CLS extension. The Chinese herbal compound cryptotanshione extends CLS, but only when there are ample essential amino acids, and this is seemingly through the amino acid sensing pathways Yeast aging models have been indispensable for elucidating mechanisms of cellular aging, especially with regards to nutrient signaling pathways. During an organism’s lifetime, aging cells undergo a multitude of changes and accumulate macromolecular damage. Decrease in high-nutrient PKA/Sch9/TOR signaling pathways leads to a slowing of the aging processes, likely due to the increased stress resistance and the regulation of proteostatic mechanisms. In this review we provide an overview of emerging evidence indicating the importance of the ISR as a parallel, yet opposing, nutrient-signaling mechanism to TOR signaling. Also, we highlight findings suggesting a fundamental role of the ISR as a target effector of nutrient signaling on yeast lifespan.Although growing evidence suggests that the ISR is required for lifespan extension, we do not yet know if the ISR is sufficient for lifespan extension. Furthermore, is Gcn4 activation sufficient to extend yeast RLS or CLS? If this were the case, it would suggest that the general inhibition of cap-dependent translation is not required for yeast lifespan extension: rather it is the resulting upregulation of stress response gene expression that is key. As such, increasing Gcn4/ATF4 protein levels may be a promising therapeutic intervention to extend lifespan and healthspan without the detrimental effects of reduced global protein synthesis and immunosuppression that occur in humans upon TOR inhibition by compounds such as rapamycin. Notably, it is not yet clear whether the autophagy that is induced by lifespan extension regimens is dependent on Gcn4, or other autophagy-inducing pathways. Thus, therapeutics to directly activate autophagy could be another promising approach to extend lifespan and healthspan.Gcn4 induces transcription of limiting components of multiple different stress response pathways . This raises the question: is autophagy the only downstream process activated by Gcn4 that is required for lifespan extension? This could be answered by testing whether induction of autophagy is sufficient to extend yeast lifespan. This is clearly the case in multicellular eukaryotes because overexpression of Atg5 in mice, which increases autophagic flux, is sufficient to extend lifespan The phenomenal pace of research and discoveries being made using the yeast model organism indicate that it will continue to be a central model for aging studies for the foreseeable future. Critically, since many of the components of longevity-regulating mechanisms are highly conserved from yeast to higher eukaryotes, the discovery of pharmaceutical and physiological regulators of these processes in yeast will likely have biological significance to human health and aging.


Previously, we have developed a method generating human serotonergic neurons from induced pluripotent stem cells (iPSCs). In this study, we analyzed the features of these human iPSCs-derived serotonergic neurons both in vitro and in vivo. We found that these human serotonergic neurons are sensitive to the selective neurotoxin 5, 7-Dihydroxytryptamine  in vitro. After being transplanted into newborn mice, the cells not only expressed their typical molecular markers, but also showed the migration and projection to the host’s cerebellum, hindbrain and spinal cord. The data demonstrate that these human iPSCs-derived neurons exhibit the typical features as the serotonergic neurons in the brain, which provides a solid foundation for studying on human serotonin system and its related disorders.In the brain, the serotonergic neurons located in the raphe nucleus are the unique resource of the neurotransmitter serotonin, which plays a pivotal role in the regulation of brain development and functions. Dysfunction of the serotonin system is present in many psychiatric disorders. Lack of  Although there are only about 300,000 serotonergic neurons in the human brain , serotonergic neurons, based on staining for serotonin, have been observed  and induced pluripotent stem cells (iPSCs) make it possible to establish an Recently, we have demonstrated the accurate timely regulation of WNT, SHH and FGF4 signaling pathways during the serotonergic neuron differentiation and generated an enriched population of serotonergic neurons from human ESCs and iPSCs  in vitro. After being transplanted into newborn mice, the cells not only expressed their typical molecular markers, but also showed the migration and projection to the cerebellum, hindbrain and spinal cord. The data demonstrate that these human iPSCs-derived neurons exhibit the typical features as the serotonergic neurons in the brain, which provides a solid foundation for studying on human serotonin system and its related disorders.In this study, we further analyzed the features of human iPSCs-derived serotonergic neurons both Human iPSCs  were maintained on mouse embryonic fibroblast (MEF) feeder in a stem cell growth medium  were injected 2 mm after lambda along the midline and 3 mm deep into the 4th ventricles of P1 newborn severe combined immunodeficiency (SCID)-beige (Taconic) mice. Three months after transplantation, animals were processed for histological analysis as described before Ma et al.  became neurons, indicated by Tuj1 and NeuN staining Figures ; few astin vitro (data not shown). This data indicates that most of the transplanted cells became serotonergic neurons and the host’s brain environment help them become mature and functional.Transplanted cells (in the human grafts around the 4th brain ventricle) were further identified with bio-markers of serotonergic neurons. It was observed that the transplanted human cells (labeled by either STEM121 or human specific MHC1) expressed serotonin neuron specific marker serotonin and TPH2, as well as other markers for serotonergic neurons, such as GATA2, GATA3, AADC, HTR1a and VMAT2 Figure , while iin vitro and in vivo.In this study, we characterized the features of human iPSCs-derived serotonergic neurons both in vitro electrophysiological features of serotonergic neurons were assayed on these neurons; in addition, because of the enriched population of serotonergic neurons, it makes possible to detect the serotonin neurotransmitter released in the medium via ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), which facilitates this culture system to serve as a platform to validate serotonin enhancers and selective serotonin reuptake inhibitor (SSRIs). Although systematically studied in our previous work, some important features of serotonergic neurons still need to be validated on human iPSCs-derived serotonergic neurons.As early as 17 years ago, it was reported that serotonergic neurons could be obtained from mouse ESCs .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. The handling Editor declared a shared affiliation, though no other collaboration with the authors and states that the process nevertheless met the standards of a fair and objective review.


Epigenetic studies have led to a more profound understanding of the mechanisms involved in chronic conditions. In the case of alcohol addiction, according to the National Institute on Alcohol Abuse and Alcoholism, 16 million adults suffer from Alcohol Use Disorders (AUDs). Even though therapeutic interventions like behavioral therapy and medications to prevent relapse are currently available, no robust cure exists, which stems from the lack of understanding the mechanisms of action of alcohol and the lack of development of precision medicine approaches to treat AUDs. Another common group of addictive substance, cannabinoids, have been studied extensively to reveal they work through cannabinoid receptors. Therapeutic applications have been found for the cannabinoids and a deeper understanding of the endocannabinoid system has been gained over the years. Recent reports of cannabinergic mechanisms in AUDs has opened an exciting realm of research that seeks to elucidate the molecular mechanisms of alcohol-induced end organ diseases and hopefully provide insight into new therapeutic strategies for the treatment of AUDs. To date, several epigenetic mechanisms have been associated with alcohol and cannabinoids independently. Therefore, the scope of this review is to compile the most recent literature regarding alcohol and cannabinoids in terms of a possible epigenetic connection between the endocannabinoid system and alcohol effects. First, we will provide an overview of epigenetics, followed by an overview of alcohol and epigenetic mechanisms with an emphasis on histone modifications and DNA methylations. Then, we will provide an overview of cannabinoids and epigenetic mechanisms. Lastly, we will discuss evidence of interactions between alcohol and cannabinergic pathways and possible insights into the novel epigenetic mechanisms underlying alcohol-cannabinergic pathway activity. Finalizing the review will be a discussion of future directions and therapeutic applications. Epigenetics, a term coined by Conrad Waddington in the early 1940’s . These eTo date, there is still a need to comprehend epigenetic regulation in cells to increase our understanding of mechanisms underlying several adverse chronic conditions. In one early review, it was shown that understanding the epigenetic effects involved in therapeutic drugs efficacy uncovered important epigenetic mechanisms underlying inflammatory bowel disease . The artAlcohol abuse remains a major issue in the United States and studies to elucidate the health risks and benefits of consuming alcohol, as well as the mechanisms underlying alcohol-induced end-organ effects and its addictive properties are still within the National Institute on Alcohol Abuse and Alcoholism (NIAAA) priorities. Even though several well established molecular pathways have been implicated in alcohol abuse, studying epigenetics, especially histone modifications and the genes they regulate can help identify positions of promoters and enhancers that mediate gene function and help us further understand the mechanism of action of alcohol in our body –22. TherAccording to NIAAA, Alcohol Use Disorders (AUDs) occur due to overuse or problem drinking of alcohol. According to NIAAA statistical report, in the year 2014, 16.3 million adults, age 18 or older had an AUD in the United States. Consumption of alcohol in levels that can be defined as problem drinking have been associated with morbidity and mortality ranging from car crashes due to Driving Under the Influence (DUI) to chronic diseases such as cancer and cardiomyopathy . AdverseThe current treatments available for heavy drinking include behavioral therapy directed towards identifying and changing behaviors that lead to heavy drinking  and pharin vitro with ethanol in a dose and time dependent manner. This study showed an increase in acetylation (ac) at 9th lysine (K9) of histone (H) 3 peaking following 24 h of ethanol exposure [One of the major targets of alcohol use is damage to the liver. One of the first studies to show epigenetic modulation due to the effect of alcohol was carried out by Park et al. using rat hepatocytes treated exposure . This acexposure  and FAS exposure , which aexposure .in-vivo administration of ethanol to rats, in liver, lung and spleen tissues [A modulation in H3K9ac was also observed due to acute  tissues . These a tissues . An impo tissues . When it tissues . In term tissues . The sam tissues .Following chronic alcohol exposure, it has been shown that there is an increase in H3K9ac in liver nuclear extracts of rats, and this effect was due to an increase in histone acetyl transferase p300 . HistoneApart from the hepatic region, alcohol related epigenetic effects have also been studied in the brain. An increase in acetylation in histone H3 and H4, CREB Binding Protein (CBP) and a corresponding decrease in HDAC activity was seen in rat amygdala due to acute alcohol exposure and these effects were reduced when treated with the HDAC inhibitor, trichostatin A during ethanol withdrawal . A studyIn the context of histone deacetylation, studies from our laboratory, have demonstrated acute alcohol increases HDAC 1 and 3 gene expression and HDAC activity in CNS cells along with an increase in serotonin receptor 5-HT3 activity . Moreovein-vitro in primary human Monocyte-Derived Dendritic Cells (MDDCs) treated acutely with alcohol and also in MDDCs from alcohol users [Acute vs. chronic histone modification related epigenetic effects in the immune system and other peripheral organs: Recently, even in the immune system, alcohol was shown to increase class I HDACs ol users . Also cool users .Apart from studies in liver, brain, and immune system, some studies done in other peripheral organs also show the epigenetic effects of alcohol. A recent study showed chronic alcohol reduced methylation at H3K4 and increased H3K27 trimethylation in a pancreatic cell line, modulating its ability to uptake ascorbic acid . AlcoholBesides histone modifications, alcohol has also been widely associated with modulation in DNA methylation. DNA methylation, which is mediated by DNMTs, transfer a methyl group from S-adenosyl methionine (SAM) to 5-methyl cytosine and produce S-adenosyl homocysteine, which is then broken down by SAH Hydrolase (SAHH) to form homocysteine ,70. StudIt was recently shown that binge ethanol exposure during adolescence causes altered genome-wide DNA methylation patterns in the hypothalamus of offspring who have never been exposed to alcohol . A studyA recent study that looked at a possible role of DNA methylation and its association with alcohol dependence found that alcohol dependent rats showed increased DNA methylation in medial pre-frontal cortex as well as decreased expression of proteins involved in synaptic neurotransmitter release . MoreoveEven though, micro RNA based epigenetic modulations have not been discussed in this review, it is of importance to mention that immense number of studies have shown specific micro RNA are modulated due to alcohol abuse as reviewed by Szabo and Bala . SpecifiOverall, according to the literature reviewed on alcohol and epigenetics, epigenetic mechanisms, including both DNA methylation and histone modifications have been implicated in both the acute and chronic effects of alcohol in different organs such as the brain, liver, and immune system. However, most of these are in animal models and there continues to be a need to elucidate alcohol’s epigenetic effects in humans. Our current laboratory efforts are focused on understanding epigenetic modifications induced by alcohol in human peripheral immune cells, with the hope of establishing certain epigenetic markers and drug targets for AUDs.9-tetrahydrocannabinol (THC) [According to the National Institute on Drug Abuse (NIDA), marijuana or cannabis is currently the most common illicit drug abused in the United States . While tol (THC) , there aol (THC) . THC alsol (THC) .1 receptor is primarily found in the brain, with lower levels in the periphery. A second cannabinoid receptor, CB2, is primarily found in immune cells and cells of the hematopoietic system [1/CB2 receptor, receptive to cannabinoids is GPR55, which is found in the human brain [1 receptor [Endocannabinoid signaling relies on G-protein-coupled receptors (GPCRs), which are the main receptors responsible for the action of cannabinoids . CB1 recc system  and appec system  and pancc system . Anotheran brain , liver aan brain ,94. It ian brain . Researcreceptor .1 and CB2 receptors also activate MAP kinases, ERK kinases, and nuclear factors like NF-κB downstream signaling pathways [As recently reviewed, when CB1 and CB2 receptors get activated they modulate adenylate cyclase and correspondingly production of the secondary messenger cAMP further affecting the protein kinase A pathway . CB1 andpathways . When stpathways . Other rpathways .in-vitro study, anandamide reduced differentiation of keratinocytes by inducing DNA methylation of the CB1 receptor [1 receptor mediated DNA methyltransferase activity, which was proposed to be involved with many more functions other than just differentiation of keratinocytes [Understanding epigenetic mechanisms by which cannabinoids bring about their effects may lead to a deeper understanding of their molecular mechanism of action. Although there is limited literature on the epigenetic mechanisms regulating cannabinoid signaling, one of the pioneering reports showed, in an receptor . Paradisinocytes . More reinocytes . Additioinocytes . Anotherinocytes . Additioinocytes  while ininocytes . Cannabiinocytes ,106, how1 receptor function, which may be due to constant stimulation by the endogenous cannabinoids, arachidonylethanolamide and 2-arachidonylglycerol, with this activity also modulated by chronic ethanol exposure [1 cannabinoid receptor and the nuclear peroxisome proliferator-activated receptor PPARα were found to be lower in spleens of intermittently alcohol-exposed adolescent rats compared to control rats [2 expression was higher, indicating that alcohol exposure even during adolescence was enough to cause difference in splenic expression of cannabinoid receptors [2 receptor knockout or apoptosis related gene ATG5 knockout mice were exposed to chronic ethanol followed by binge ethanol feeding showed that CB2 receptor activation in liver macrophages protects mice from alcohol-induced steatosis by blocking hepatic inflammation through an autophagy-dependent pathway [1 binding while chronic alcohol exposure caused a decrease in CB1 binding that is maintained even after abstinence for at least a month [Alcohol and its association with the endocannabinoid system, which is involved in reward signaling mechanism of the brain, has been studied quite extensively with cannabinoids and its receptors being explored as pharmacological drug targets for AUDs . The canexposure . The CB1rol rats . Converseceptors . Another pathway . In huma pathway . Exposur pathway . Further a month . The enh a month .2 and GPR55 in MDDCs from alcohol users, which was further validated in-vitro in primary human MDDCs treated with alcohol [Apart from the brain and liver, in a recent review comparing the differential effects of alcohol and cannabinoids on the immune system, marijuana abusers were shown to have reduced secretion of inflammatory cytokines from peripheral dendritic cells compared to alcohol users . Additio alcohol . All of Regarding studies examining epigenetic mechanisms underlying alcohol and cannabinoid activity, a recent study reported post-natal alcohol exposure induced neonatal neurodegeneration by enhancing CB1 exon1 activity through upregulated histone H4K8 acetylation along with downregulated H3K9 methylation . This apEven though there is a dearth of studies looking into the epigenetic mechanisms behind alcohol and cannabinoid activity, certain inferences can be drawn from the studies reviewed herein. Histone modification sites like H3K9, H3K4 and H4K8 have been commonly implicated in these studies. DNA methylation also seems to be significantly influenced by both alcohol and cannabinoids. It is important to isolate these epigenetic effects and understand their functionality. Interestingly, in 2004, the first histone Lysine Specific Demethylase 1 (LSD1) was identified by Dr. Shi Yang’s group . FurtherThe adverse effects of alcohol and cannabinoid abuse are widely known; however, with the legalization of marijuana in some states and availability of medical marijuana, the potential of endogenous cannabinoids and their receptors to be of therapeutic significance is immense. From the review above, we can see a vast existing literature showing the connection between effects of alcohol and cannabinoid activity, such that cannabinoid receptor agonists and/or antagonists may be able to reverse alcohol’s deleterious effects or reduce alcohol self-administration. Understanding epigenetics may help us ultimately understand the connection between alcohol and endocannabinoid signaling mechanisms. In summary, as shown by the schematic representation in Considering the similarities in effects that exist between the two at the cellular level, there may be possible overlap between these two pathways that can reveal therapeutic targets. As shown in After reviewing the literature, there are many questions that still remain to be elucidated  includin


The production of several viral vaccines depends on chicken embryo fibroblasts or embryonated chicken eggs. To replace this logistically demanding substrate, we created continuous anatine suspension cell lines (CR and CR.pIX), developed chemically-defined media, and established production processes for different vaccine viruses. One of the processes investigated in greater detail was developed for modified vaccinia virus Ankara (MVA). MVA is highly attenuated for human recipients and an efficient vector for reactogenic expression of foreign genes. Because direct cell-to-cell spread is one important mechanism for vaccinia virus replication, cultivation of MVA in bioreactors is facilitated if cell aggregates are induced after infection. This dependency may be the mechanism behind our observation that a novel viral genotype (MVA-CR) accumulates with serial passage in suspension cultures. Sequencing of a major part of the genomic DNA of the new strain revealed point mutations in three genes. We hypothesize that these changes confer an advantage because they may allow a greater fraction of MVA-CR viruses to escape the host cells for infection of distant targets. Production and purification of MVA-based vaccines may be simplified by this combination of designed avian cell line, chemically defined media and the novel virus strain. Although the wide spectrum of current vaccines provides one of the most successful medicinal interventions available to health systems ,2, infecWith MVA as an example in the center of a modern vaccine production process, this review summarizes the development from generation of a continuous cell line that is permissive for different vaccine viruses to the design of an efficient and scalable production process in chemically defined media. First results are also presented on what appears to be a genetic adaption of MVA to the highly controlled biopharmaceutical process conditions.Vectored vaccines are based on well-characterized infectious agents amenable to genetic manipulation and known to be stably attenuated ,9,10. PrEven closely related viruses (both as vectors and as pathogens) can differ substantially in their interaction with the immune system and the immune responses they elicit ,12. It cPoxviridae, subfamily Chordopoxvirinae . Poxvirus particles are enveloped and resemble flattened barrels approximately 260 nm in length [MVA is derived from a vaccinia virus, a member of the n length . They enn length ,20,21,22Avipox genus of the Poxviridae) or have been adapted experimentally to avian cells . They are investigated in primer-boost regimes against infectious and neoplastic diseases that range from AIDS [Poxviruses that are highly attenuated for humans are very promising vaccine vectors that naturally infect only birds  . CVA is Safety of MVA for use in humans has been demonstrated in the final years of the smallpox eradication campaign of the World Health Organization and, more recently, in numerous clinical trials  are preferred for most vaccine production processes. If embryonated eggs of a vaccine-quality non-SPF status are used, additional adventitious agents testing may have to be performed during the vaccine development and production process. Maintenance of SPF status requires strict isolation of the flocks from environmental contaminants, personnel that showers and changes into protective clothing, and treatment of food and air prior to contact with the animals. Furthermore, animals are screened for at least eighteen pathogens and no medication that may interfere with the sensitivity of detection should be administered. Supply constraints have been reported [Contrary to the common human diploid finite cell lines, WI-38 and MRC-5 , no cellreported ,50 becauAnother disadvantage of production with primary cells is that an increased effort is necessary to perform scalable suspension processes. Because of the complex logistics associated with embryonated eggs, transportable processes for implementation at multiple sites and in newly industrialized countries may also be difficult to realize with such a substrate.Cairina moschata) explants [Anas genus has been sequenced [To advance production of vaccine strains adapted to replication in avian cells, we have generated a continuous cell line, AGE1.CR.pIX (CR.pIX thereafter in this text), derived from primary muscovy duck  selection. Immunofluorescence assays of cultures at various passage levels demonstrated that only cells that stably expressed the E1A and E1B genes survived beyond senescence, confirming the intended induced (as opposed to a spontaneous) immortalization. The final step in the cell line development program, adaptation to proliferation without anchorage, was performed by cultivation in commercial media designed for human suspension cell lines [Confidence in the defined-risks calculation is proportional to the level of knowledge about derivation and properties of a continuous cell line. To facilitate acceptance of the CR.pIX cell line, we chose to immortalize the primary cells with the E1 region of human adenovirus 5. As described previously , retina ll lines .Several advantages are associated with immortalization using the adenovirus E1 region.(1)There is precedence for this method in the HEK 293  and PER.(2)Human adenoviruses are both endemic and pandemic pathogens responsible for 2%–7% of upper respiratory infections in children. Although they have also the capacity to cause severe disease especially in immune-compromised patients  they are(3)Immortalization is the result of a concerted action of distinct and separate E1A and E1B gene products. This synergy necessitates concurrent events for experimental immortalization but also increases the probability against accidental tumor induction by a contamination.(4)INK4a/ARF gene locus, lead into activation of apoptosis [E1B 55K gene product for efficient adenovirus replication [E1B 19K gene product [E1A gene therapy for cancer patients [That many of the biochemical pathways leading to immortalization by the E1 region are known may further facilitate defined-risks estimates. Briefly, by relieving inhibition of the E2F transcription factors, the E1A protein promotes cell cycle progression. However, various parallel mechanisms, including induction of DNA damage responses and augmented expression of an alternate reading frame (ARF) of the poptosis . One prolication . Another product ,73. The  product ,74 is in product  and E1A patients ,77.(5)For certain applications there may also be a virological advantage if E1 proteins are expressed in a host cell. Some vaccine strains are attenuated via lesions in factors that allow the virus to mask itself against the innate immunity of the cell. E1A has also been implicated in the viral defense against antiviral responses ,79. AlthThe CR cell line was further modified by transfection of an expression plasmid for the adenovirus pIX structural protein to obtain the CR.pIX cell line. We hypothesize that some viruses, even if they are not related to adenoviruses, benefit from the presence of pIX because this protein may constitutively activate Hsp90, a central factor in heat shock responses . Replica8 pfu/mL within 48–72 h post infection, peak titers were variable and often below 107 pfu/mL in suspension cultures [8 pfu of MVA/mL at cell densities between 0.5 × 106 and 2.5 × 106 cells/mL and MOI between 0.01 and 0.1 [The adherent CR and CR.pIX cell lines were adapted to proliferation in suspension and tested for propagation of a wide spectrum of human and veterinary vaccine viruses ,81,82. Scultures ,66. Resucultures . For thi and 0.1 .Having developed a new cell line for production of vaccines in bioreactors, investigation of metabolic properties was an early research topic. One motivation for these studies was that although cells of avian origin are being used for derivation and production of vaccine strains since the 1930s ,85, someConversely, it is possible that CR.pIX cell lines show the metabolic characteristics expected of continuous vertebrate cell lines such as HEK 293 or CHO, irrespective of their non-mammalian origin. Such a property would facilitate medium and process optimization and may contribute to the study of evolutionary conserved biochemical pathways.Indeed, extensive analyses of CR.pIX cell cultures in different cultivation systems combined with metabolic flux analyses demonstrated that some metabolic properties of this duck-derived cell line are similar to previously characterized mammalian and even insect cell lines. For example, CR.pIX cells share an overflow metabolism fueled by glucose consumption and terminating predominantely in lactate production and release . The ene7 cells/mL). The aim of this development is to stabilize long term cultivation of infected cultures in bioreactors, which in turn may increase yields for some vaccines and can extend harvest intervals [Extracellular lactate concentrations remained typically below growth-limiting levels of 40 mM. However, for cultivations that either target high cell concentrations or long cultivation intervals pH control is required to compensate lactate release. Current metabolic and media optimization studies focus on lowering lactate consumption, on improving efficiency of the citric acid cycle and on reaching high cell densities . ThAfter recovery of a pure isolate of the new genotype, yields and replication kinetics of MVA-CR and wildtype MVA were compared in single-cell and aggregate suspension cultures in chemically defined media, and on adherent monolayers in basal medium supplemented with bovine serum . A largeWe hypothesized that mutations in MVA-CR may facilitate release of viruses with this genotype. Alternative explanations for this effect may be an improved infectivity of MVA-CR or faster maturation of viral particles. To study whether virions detach more easily from the host cell, we compared induction of syncitia in monolayers infected with MVA-CR19 or an earlier passage of an MVA population, MVA-CR3, where the majority of the viruses still is wildtype . SyncitiEGFP and DsRed1 reporter genes under control of a hybrid early/late MVA promoter into deletion site III of the different MVA instances [One limitation in the previous experiments is that three mutations have been detected concurrently in MVA-CR19. We do not know whether all three mutations need to cooperate to yield the observed phenotype, and whether the phenotype does indeed reflect enhanced escape of the infectious units from the host cell. We can also not be certain that additional mutations and genomic alterations that may have been missed in the sequence evaluation contribute to the observed properties of the new MVA strain. To address some of these questions, we currently assemble the three mutations in different combinations into the wildtype genome. For improved detection and quantification of the recombinant viruses, we also inserted nstances ,106.As shown in Another open question is whether reactogenicity and attenuation of MVA-CR viruses may have been modulated by the mutations. An important mechanism underlying the attenuation phenotype of MVA in human cells appears to be a block during assembly of infectious particles ,108. EarIn this review, we have focused on one approach for production of a promising group of viral vectors, poxviruses attenuated by host-restriction. Novel avian cell lines permissive for these poxviruses have been generated with focus on conformity with regulatory guidelines. The CR and CR.pIX cell lines of this program were adapted to proliferation in suspension cultures, and chemically-defined media were developed to minimize risks to vaccine recipients and to improve lot-to-lot consistency. A considerable fraction of infectious virions remain attached to the infected cell. To improve virus replication in suspension cultures, the process was refined to facilitate cell-to-cell spread of virions via induction of cell aggregate formation. In this system, a novel MVA genotype (MVA-CR) was discovered that we believe has advantageous properties for suspension cell culture-derived vaccine manufacturing. Production and purification of MVA independent of primary chicken embryo fibroblasts is associated with both advantages and significant challenges ,109,110.


We recently reported a novel neurological syndrome characterized by a unique NREM and REM parasomnia with sleep apnea and stridor, accompanied by bulbar dysfunction and specific association with antibodies against the neuronal cell-adhesion protein IgLON5. All patients had the HLA-DRB1*1001 and HLA-DQB1*0501 alleles. Neuropathological findings in two patients revealed a novel tauopathy restricted to neurons and predominantly involving the hypothalamus and tegmentum of the brainstem. The aim of the current study is to describe the neuropathological features of the anti-IgLON5 syndrome and to provide diagnostic levels of certainty based on the presence of associated clinical and immunological data. The brains of six patients were examined and the features required for the neuropathological diagnosis were established by consensus. Additional clinical and immunological criteria were used to define “definite”, “probable” and “possible” diagnostic categories. The brains of all patients showed remarkably similar features consistent with a neurodegenerative disease with neuronal loss and gliosis and absence of inflammatory infiltrates. The most relevant finding was the neuronal accumulation of hyperphosphorylated tau composed of both three-repeat (3R) and four-repeat (4R) tau isoforms, preferentially involving the hypothalamus, and more severely the tegmental nuclei of the brainstem with a cranio-caudal gradient of severity until the upper cervical cord. A “definite” diagnosis of anti-IgLON5-related tauopathy is established when these neuropathological features are present along with the detection of serum or CSF IgLON5 antibodies. When the antibody status is unknown, a “probable” diagnosis requires neuropathological findings along with a compatible clinical history or confirmation of possession of HLA-DRB1*1001 and HLA-DQB1*0501 alleles. A “possible” diagnosis should be considered in cases with compatible neuropathology but without information about a relevant clinical presentation and immunological status. These criteria should help to identify undiagnosed cases among archival tissue, and will assist future clinicopathological studies of this novel disorder.The online version of this article (doi:10.1007/s00401-016-1591-8) contains supplementary material, which is available to authorized users. An intriguing progressive neurological disorder at the crossroads of autoimmunity and neurodegeneration was recently described in eight patients affected by a previously unknown disorder characterized by non-REM sleep (NREM) parasomnias, sleep apnea, and stridor . All patWhile these clinical, immunological, and genetic features were being established, results of the post-mortem neuropathological examination of a third patient became available confirming the initial findings of a possible novel tauopathy. Therefore, a Task Force was organized to describe in detail the neuropathological features of the disorder with the aim of developing neuropathological diagnostic criteria which should also help to identify this disease in retrospective studies of archival tissue from patients whose diagnosis was not obtained during life.The present study includes the neuropathological study of the brain of six patients. It comprises three previously reported cases, two of them from the archives of the IDIBAPS Neurological Tissue Bank, Barcelona, Spain  and one In order to establish the neuropathological diagnostic criteria a meeting was held by four neuropathologists , who were familiar with all of the six cases. Using a multi-header microscope the cases were re-evaluated and the histological features required for the neuropathological diagnosis of this novel tauopathy were agreed on by consensus. Subsequently, these neuropathological criteria along with clinical, immunological, and HLA haplotype findings were used by all the investigators [neuropathologists and clinicians ] to define three diagnostic levels of disease: “possible”, “probable”, and “definite”.All six brains were processed according to standard neuropathological procedures. Formalin-fixed and paraffin-embedded tissue blocks representing frontal, temporal, parietal and occipital cortices, caudate, lenticular nucleus, hypothalamus, thalamus, hippocampus, parahippocampal gyrus, amygdala, midbrain, pons, medulla oblongata, spinal cord, and cerebellum were retrieved from the archives of the four centers and were cut as 5 μm thick sections. Sections were stained with haematoxylin and eosin method and in selected cases and areas tissue sections were also stained with Bielschowsky’s or Gallyas silver impregnation and Luxol-fast-blue/cresyl violet method. For immunohistochemistry a panel of primary antibodies was used in each laboratory Table . Cases 1The main clinical and immunological characteristics of the six patients are summarized in Table The clinical history of the three patients in whom IgLON5 antibodies were not tested was dominated by bulbar dysfunction and repetitive episodes of respiratory insufficiency that required tracheostomy or multiple admissions to ICU. Other symptoms included gait instability, frequent falls, dysphagia, gaze palsies, central hypoventilation, dysautonomia, and chorea. These symptoms can suggest other diagnoses such as progressive supranuclear palsy or multiple system atrophy, although no parkinsonian signs were present. In none of these patients was the sleep formally studied, but in two of the cases symptoms of excessive daytime sleepiness, stridor, sleep apnea or parasomnia were documented Table .Table Detailed clinical information of the disease course has been previously reported  and is sA 48-year-old man developed recurrent vertigo and marked involuntary fidgety movements. At the age of 53 he developed breathing and swallowing difficulties. Multiple episodes of aspiration pneumonia and respiratory arrests warranted four admissions to the intensive care unit and, on two occasions, intubation for ventilator support was required. Due to recurrent aspiration, a gastrostomy tube was inserted for feeding. One year later, he developed progressive difficulties with his balance and intermittent diplopia. He had lateral and upgaze nystagmus with limitation of vertical and abduction eye movements. There was cerebellar dysarthria, finger-nose dysmetria, and he was unable to tandem walk. Choreiform movements were present in all limbs. Deterioration in excessive daytime somnolence caused him to fall asleep even when he was standing. His wife reported that his breathing pattern changed as soon as he was asleep but no abnormal movements were noted. During a hospital admission to investigate his sleep attacks, he had a respiratory arrest and died at the age of 60.Neuropathological examination was performed after appropriate written informed consent from the next of kin had been obtained. Consent for the use of this case for research purposes was also given. The brain was macroscopically unremarkable. Microscopic studies showed an increase in number of reactive astrocytes in the striatum and globus pallidus. There was some loss of neurons with accompanying astrogliosis in the dorsolateral aspect of the subthalamic nucleus while the substantia nigra was well populated with pigmented and non-pigmented neurons. There was some reduction of the height of the pontine tegmentum, which was marked in the medulla oblongata. Tau immunohistochemistry using the AT8 antibody demonstrated only occasional NFT and pretangles in neocortical areas. All hippocampal subregions were affected by neuronal tau pathology; there were moderate numbers of pretangles and NFT in the CA1 hippocampal subregion, which were sparse in the CA3 and CA4 subregions. There were frequent pretangles in CA2 and also in the granule cells of the dentate gyrus, which were ubiquitin and p62-negative. Fine neuropil threads were present in all hippocampal subregions. The tau pathology was similar, but mild in the subiculum while moderate numbers of Gallyas-positive NFT and neuropil threads were seen in the entorhinal cortex. There were scattered tau-positive neuropil threads, pretangles and occasional NFT in the striatum. Similar, but slightly more severe pTau pathology was seen in the globus pallidus. The subthalamic nucleus contained moderate numbers of pretangles, NFT and neuropil threads. A conspicuous feature was the severe tau deposition in the posterior hypothalamus and brainstem structures. In the midbrain a significant proportion of the substantia nigra pars compacta neurons showed granular or dot-like tau positivity, which was also seen in neuronal processes. Similar tau pathology was seen in the midbrain tegmentum, periaqueductal gray matter and tectum. The majority of the neurons of the otherwise well-populated locus coeruleus contained similar neuronal tau-positive inclusions and the pontine tegmentum and base were also extensively affected by similar tau pathology. The tau deposition was particularly severe in the tegmentum of the medulla including the dorsal motor nucleus of the vagus nerve, nucleus tractus solitarius, nucleus ambiguus and neurons of the magnocellular nucleus were severely affected. In the inferior olive neurons were often surrounded by a meshwork of fine tau-positive processes and dot-like tau positivity. There were scattered pretangles and NFT, neuropil threads and clusters of fine tau-positive dots in the cerebellar dentate nucleus. There was rather widespread granular tau positivity in the cerebellar cortex and an occasional Purkinje cell showed fine, dot-like tau positivity. Differential tau immunohistochemistry confirmed that the pTau deposits were composed of both 3R-tau and 4R-tau isoforms.For clinical history and the distribution of the NFT pathology see Lidov et al. . Tau immThe neuropathological findings in the six patients showed remarkable similarity consistent with a neurodegenerative disease with neuronal loss and gliosis, without the presence of inflammatory infiltrates. The most relevant finding was a prominent neuronal accumulation of pTau, comprising both 3R-tau and 4R-tau isoforms, involving the hypothalamus and more severely the tegmental brainstem nuclei, with a cranio-caudal gradient of severity, reaching the upper cervical cord. The cerebral cortex, basal ganglia, thalamus and subthalamic nucleus were mostly unaffected or mildly affected. In five cases the entorhinal cortex and hippocampus were also affected including dentate gyrus and CA4 to CA1 sectors, with variable involvement of transentorhinal region. Interestingly, CA2 sector was consistently involved. There was a lack of glial tau pathology except for the presence of isolated coiled bodies and granular fuzzy astrocytes in the hypothalamus and amygdala in cases 3 and 4. A detailed semiquantitative assessment of pTau pathology in different brain areas of all cases is represented in Table There were no other abnormal deposits of proteins, or if present these were mild and localized in a few areas suggesting the co-existence of other neurodegenerative or age-related pathologies. Case 2 showed isolated Bunina bodies and TDP43 protein aggregates in few brainstem and motor neurons in the spinal cord. A few neuritic plaques were observed in cases 2, 4, and 6, along with cerebral amyloid angiopathy in cases 2 and 4. Case 4 additionally showed scattered alpha-synuclein positive Lewy bodies and Lewy neurites in the substantia nigra and dorsal nucleus of the vagus nerve and argyrophilic grain pathology, and case 5 showed sparse neuronal cytoplasmic TDP43 protein inclusions in the granule cells of the dentate gyrus.definite anti-IgLON5-related tauopathy” should be considered. In contrast, when the information of the anti-IgLON5 antibodies status is missing but the clinical history is compatible or the patient harbored the HLA-DRB1*1001 and HLA-DQB1*0501 alleles, then the diagnostic category of “probable anti-IgLON5-related tauopathy” should be applied. The category of “possible anti-IgLON5-related tauopathy” should be reserved for those cases where clinical data are not available  and ancillary tests  could not be performed. Note that both the HLA genotyping and the anti-IgLON5 antibody assay could be done post-mortem from DNA samples and serum or ventricular CSF, respectively. No exclusion criteria have been proposed as the whole spectrum of pathological changes and possible concomitant pathologies are yet unknown.Based on the similarities of these cases we propose research criteria for the neuropathological diagnosis of the tauopathy underlying the anti-IgLON5 syndrome Table  and recoWe report here the main neuropathological features underlying the anti-IgLON5 syndrome that are different from those of other tauopathies , 11. TheWe propose that the tauopathy underlying the anti-IgLON5 syndrome can be diagnosed with three levels of probability: definite, probable, and possible. Accordingly, for example the previously reported case by Kaphan et al.  would fuThis condition has distinct microscopic appearances allowing its differentiation from other ‘primary’ tauopathies, although the topographical distribution of the tau pathology is, to some extent, reminiscent of that seen in progressive supranuclear palsy (PSP). Indeed, one post-mortem case has been previously reported as “early” PSP  and anotAs in Alzheimer’s disease, the tau filaments seen in the anti-IgLON5 syndrome ultrastructurally appear as paired helical filaments  and the In addition to the prominent brainstem involvement, five of the six patients had neuronal tau pathology in the hippocampus with frequent pretangles in CA4 and CA2 sector, and NFT in CA1 sector and entorhinal cortex, with variable involvement of transentorhinal region, and ring-shaped pretangles in the granule cells of the dentate gyrus. Selective involvement of CA2 sector has been reported to occur more frequently in 4R-tauopathies . The tauThe most intriguing aspect of the disease is the association of neuropathological findings suggesting a non-inflammatory degenerative process with the presence of highly specific antibodies against IgLON5  and a strong association with the HLA system. A possible mechanism that could explain tau hyperphosphorylation and accumulation in neurons might relate to one of the functions of IgLON family as glucosyl-phosphatidyl-inositol anchored proteins and its role in membrane stabilization . An antiHowever, the intriguing coexistence of neuropathological and immunological findings and the precise mechanism of the cross-talk between these two potential pathogenic mechanisms need to be elucidated.In summary, since the publication of two cases of a novel, brainstem accentuated neuronal 3R and 4R tauopathy associated with IgLON5 autoantibodies in patients with apnea and NREM parasomnia, a new case has been confirmed in Austria and three additional cases, one from Slovenia and two from the UK have been retrospectively identified on the basis of their nearly identical neuropathological phenotype. We suggest including these cases under the umbrella term of a single clinicopathological entity termed “anti-IgLON5-related tauopathy”. To facilitate communication with clinicians and researchers and to provide a framework for future clinicopathological and research studies we propose here research criteria for the neuropathological diagnosis of this unique tauopathy and recommend a protocol for tissue sampling. This information could also be of value in the re-evaluation of archival cases. Clearly, the recognition of more cases will be of utmost importance to identify the entire spectrum of the clinicopathological presentation and to elucidate the role of IgLON5 antibodies in initiating a cellular cascade finally resulting in abnormal hyperphosphorylation, aggregation and accumulation of tau. Only a better understanding of this interplay can result in the design of appropriate treatment strategies that can modify disease progression and consequently mitigate the risk of sudden death during sleep.Supplementary Figure 1: Simplified schematic representation of neuroanatomical pathways connecting different brainstem nuclei upwards and downwards by which a trans-synaptic propagation of IgLON5-related pathology could be postulated. Gray shaded boxes indicate nearly non-affected areas. (TIFF 23576 kb)Below is the link to the electronic supplementary material.


Addictions are associated with decision making impairments. The present study explores decision making in Substance use disorder (SUD), Gambling disorder (GD) and Obesity (OB) when assessed by Iowa Gambling Task (IGT) and compares them with healthy controls (HC).For the aims of this study, 591 participants  were assessed according to DSM criteria, completed a sociodemographic interview and conducted the IGT.SUD, GD and OB present impaired decision making when compared to the HC in the overall task and task learning, however no differences are found for the overall performance in the IGT among the clinical groups. Results also reveal some specific learning across the task patterns within the clinical groups: OB maintains negative scores until the third set where learning starts but with a less extend to HC, SUD presents an early learning followed by a progressive although slow improvement and GD presents more random choices with no learning.Decision making impairments are present in the studied clinical samples and they display individual differences in the task learning. Results can help understanding the underlying mechanisms of OB and addiction behaviors as well as improve current clinical treatments. Evidence based neurocognitive models of addiction propose that addiction-related behaviors are the result of an imbalance of three neural systems: an impulsive neural system that promotes habitual and salient behaviors, interoceptive processes that are involved in uncertain risk and reward, and a reflective neural system for inhibitory control and decision-making 2]. Deci. Deci2].Complementary, obesity (OB) is an increasing worldwide problem that shares similar patterns to addictions. IndividDirect comparisons of decision making profiles among SUD, GD and OB groups have yet to be conducted. However, direct comparisons can provide a valuable insight into the similarities and/or singularities of different addictive related behaviours.The present study aims to further explore the decision making profiles of Substance use disorder, Gambling disorder and Obesity when assessed by the Iowa gambling task and compares them with healthy controls (HC). The specific aims of the study are the following: (1) compare the overall performance of the three clinical groups and the healthy controls, (2) observe and compare specific patterns of learning across the task in the three clinical conditions and the healthy controls. It is hypothesized that the clinical samples will present poorer performances on the IGT when compared to the HC group. Also, specific patterns of learning across the task will be observed in the four studied samples. The results have the potential of improving our understanding of the specific executive profiles (namely decision making) underlying the association between obesity and addictive behaviours which in turn can also help improving current obesity treatments.The final sample consists of 591 participants  distributed as follow: 194 HC, 178 GD, 113 OB and 106 SUD individuals. GD and SUD diagnostic criterion were assessed by an experienced clinician (according to the DSM-IV-TR), by means of SCID-I .Seven centers, all involved in the CIBERobn Spanish Research Network, participated in the study: the Eating Disorders Unit , the Department of Endocrinology ; the Department of Diabetes, Endocrinology and Nutrition ; the Department of Endocrinology and Nutrition ; the Diabetes, Endocrinology and Nutrition Department, ; the Clinical Research Unit  and the Department of Basic Psychology, Clinic and Psychobiology . The GD, SUD and OB participants were patients that were consecutively referred to the clinics mentioned above. Recruitment of the controls took place by means of word-of-mouth and advertisements at local universities.. All participants gave written signed informed consent and received no additional compensation for being part of the study. In accordance with the Helsinki Declaration of 1975 as revised in 1983, the Ethics Committee of all the institutions involved in the project approved the study: Comissió Deontológica de la Universitat Jaume I, Subcomisión Clínica del Hospital Universitario “Virgen de la Victoria”, Málaga, Comite Etic de Investigacio Clinica Hospital Universitari de Girona Doctor Josep Trueta (048/10), Comite Etico de Investigacion Clinica del Consorci Mar Parc de Salut de Barcelona-Parc de Salut Mar (2010/3914/I), Comité de Etica de la Investigación Universidad de Navarra (110/2010) and Comité Etico de Investigación Clínica del Hospital Universitari de Bellvitge (307/06)]. Exclusion criterion were: (1) history of chronic medical illness or neurological condition that might affect cognitive function; (2) head trauma, learning disability or intellectual disabilities; (3) individuals who have suffered a lifetime mental disorder according to DSM-IV-TR other than the specific disorder of study ; (4) age under 18 or over 65 ; (5) having diabetes mellitus. There was one extra exclusion criteria for all groups except from the SUD group: (6) history of substance abuse in the previous 3 months and use of psychoactive medication or drugs. The SUD individuals had not taken any drugs during the last 72 hours prior to explorations (assessed by urine drug testing). Finally, in addition to the already mentioned criterion, for the OB group (7) patients with obesity who had comorbid binge eating disorder (DSM-IV-TR) were also excluded.For the purpose of this study all individuals were assessed with the IGT . This co2) tests compared categorical variables between the diagnostic subtypes. Analysis of Variance (ANOVA), adjusted by the covariates age and years of education, compared the means for the cognitive measures (IGT scores). The ANOVA procedures included the between-subjects factors sex (two levels: men versus women) and diagnostic subtype , as well as the interaction group-by-sex to explore the potential moderator effect of sex into the relationships between diagnosis and cognition outcomes. The Holm-Bonferroni method, which is one of the Familywise error rate stepwise procedures that offers more powerful tests than the classical Bonferroni-correction, was used to control Type-I error due to multiple comparisons[Analyses were carried out with SPSS20 for Windows. Chi-square  and a HC. Results display impairments in the overall performance and in the task learning process in all clinical samples when individually compared to the HC.Results are in agreement with previous studies reporting impaired decision making in SUD and GD individuals ,14 as weResults also display no sex influence among groups. However, some differences are observed when comparing the IGT performance between men and women in the whole sample. Specifically, men present a better performance on the IGT. Previous studies report similar results and suggest that men tend to have a better performance than women in general population and in SOur data adds to the current literature more evidence of decision making patterns in SUD, OB and GD by comparing the three clinical samples. Importantly, in the analyses conducted none of the differences among the groups could be due to sex or other sociodemographic variables, which adds extra value to the findings reported here. Our study supports the hypothesis that OB shares specific cognitive and neurobiological patterns with SUD and GD which are known to suffer from impairments in dopaminergic pathways that regulate neural systems associated with reward sensitivity and incentive motivation. AccordiThe present study has some limitations. Firstly, there are sociodemographic differences concerning education, sex and age across the groups which are representative of the studied disorders. This is an expected result when working with a consecutive clinical sample referred to GD, SUD and OB treatments. However, we have controlled these variables in all the statistical analysis. Although in this study we have paid specific attention to one of the most relevant neuropsychological factors (namely decision making), other cognitive functioning variables or intelligence measures not assessed here may better explain specific differences  among the clinical groups. Finally, although participants did not present withdrawal symptoms or presented any life time mood-anxiety disorder or mental disorder that could hinder the assessment, the analyses conducted do not explicitly control for the participants’ hunger, anxiety or sadness feelings and these could also play a role in the participants’ performance on the task.To our knowledge, we present the first study that compares decision making in substance and behavioral related addictions, obese individuals and healthy controls. Results show similar impairments in decision making in the three clinical groups. These impairments are statistically significant when compared to the healthy control group but not different among the three clinical groups. Finally, the clinical groups present significant difficulties in learning across the task when compared to the healthy controls and also some specific differences when comparing clinical groups.


HSP70 genes, but also stabilization of the HSP70 transcripts, so that HSP70 protein expression can be sustained for longer period and at high level.1 Thus stabilization of HSP transcripts is vital to mount a potent stress response and cell survival. Few factors that were earlier suggested to participate in HSP70 transcript stability include eEF1A1 and miR-378*.3 However, a clear picture about how HSP mRNA is stabilized during heat shock yet to emerge. In our recent work published in Cell Death Discovery, we reported that protein arginylation plays an important role in this process as cells devoid of protein arginylation failed to stabilize HSP70 and HSP40 transcripts and became susceptible to heat stress.4Cellular stress responses are protective mechanism against diverse environmental and physiological factors, which cause macromolecular damage in cells. Different stress conditions, including heat stress, oxidative stress, and ER stress, are associated with denatured or unfolded proteins, which are recognized by molecular chaperons like heat shock proteins (HSPs). Interaction of unfolded proteins with chaperones lead to induction of expression and recruitment of more molecular chaperons, which reestablish the structural integrity of unfolded proteins. For long time, it was known that the level of cytosolic HSP70 during heat stress was boosted not only by induction of gene expression from the inducible isoforms of 5 Posttranslationally added arginine can be further modified by methylation leading to a unique double modification on the same site of a protein.6 Because of distinct positive charge on arginine side chain, it is thought to change the surface property of a protein and can have diverse functionality from destabilization and degradation of a protein to regulation of protein activity and interaction.8 Thus arginylation is emerging as a global regulator of cellular physiology by regulating cell survival to cell death.10 Cell survival in stressful conditions mainly depends on stress response pathways and involvement of arginylation has been reported in diverse stress conditions, including nitrosative stress, ER stress, and cytosolic misfolded protein stress. Oxidation of Cys residues upon nitrosative or oxidative stress and subsequent arginylation of oxidized Cys is reported to be a key regulatory mechanism during nitrosative and oxidative stress response.11 ER residing proteins and chaperon-like calreticulin and GRP78, and other molecular chaperons namely, chaperonin, HSPA8, ribophorin I, HSP90β, and HSP90α have been reported as substrate for arginylation.12 Although ER stress-induced arginylation of calreticulin helps these protein to dimerize and recruited in stress granules,13 cytosolic misfolded protein stress induces arginylation of GRP78 leading to its interaction with autophagic adapters.14 Large number of reports indicated importance of arginylation during cellular stress responses. However, our current understanding on this aspect probably is at a nascent stage and requires many more years of work to unfold fully.Arginylation is a protein modification in which proteins are modified by addition of arginine at the N-terminal amino group or side chains of reactive amino acids by enzyme arginyltransferase (Ate1).1 We hypothesized that loss of arginylation may have affected the stability of HSP transcripts thus dampening the stress response leading to induction of apoptosis. When we tested the stability of HSP transcripts in WT and Ate1 KO cells, we found that loss of arginylation (Ate1 KO) indeed reduced the stability of all three HSP mRNAs. Considering this, it is highly possible that KO cells could not accumulate enough HSP proteins to mount a potent stress response due to faster degradation of HSP mRNAs. Apart from protein folding, HSPs also inhibit apoptosis during heat stress by inhibiting release of pro-apoptotic proteins from mitochondria. Taken together these observations, it was proposed that arginylation-dependent stabilization of HSP transcripts help cells to attain high amount of HSP proteins, which not only resolve structural integrity of proteins, but also inhibit apoptosis (Our recent study to understand the role of protein arginylation in heat stress response showed that Ate1 KO mouse embryonic fibroblasts (MEFs) (KO cells) are more susceptible to heat stress compared with its wild type (WT) counterparts, a phenotype that can be rescued by stable expression of Ate1 in KO MEFs. Although at the given heat stress condition WT MEFs were protected, apoptosis was induced in KO MEFs. A loss of mitochondrial membrane integrity was also observed in heat stressed Ate1 KO cells. Gene expression analysis of inducible heat shock proteins, HSP70.1, HSP70.3, and HSP40, showed induction in KO MEFs during shorter period of heat shock. However, expressions of these genes are drastically diminished in KO MEFs upon longer period of heat shock, which were recovered by expression of Ate1-1 in KO cells. This raised the question that, why Ate1 KO cells are not protected in longer period of heat stress in spite of induction of HSPs. Earlier reports indicated that apart from induction of transcription, stabilization of the transcripts are also vital to achieve required protein levels of HSPs during heat stress conditions.poptosis . On the poptosis .HSPs play important roles in diverse disease conditions. Although overexpression of HSPs shown to have protective effect in several neurodegenerative disorders, HSP90 is been tested as an anticancer target due to its higher expression in many cancer cells where it help to fold many oncogenic proteins. Identification of arginylation as a regulator of HSP protein expression opens an exciting direction of research to explore its involvement in diverse disease conditions and search for its targets. Another exiting direction of investigation will be newly found function of arginylation in regulating mRNA stability, understanding its mechanism and implications in cellular physiology.