The recent literature indicates that iAs biotransformation likely plays an important role in the relationship between iAs exposure and the epigenome, in addition to the sex and genetic background of individuals. The research also shows that relatively low to moderate exposure to iAs is associated with epigenetic effects. However, while it is well established that arsenicals can alter components of the epigenome, in many cases, the biological significance of these alterations remains unknown. The manner by which these and future studies may help inform the role of epigenetic modifications in the development of iAs-associated disease is evaluated and the need for functional validation emphasized.Inorganic arsenic (iAs) poses a major threat to worldwide human health, and yet the molecular mechanisms underlying the toxic effects associated with iAs exposure are not well understood. There is increasing experimental evidence indicating that epigenetic modifications may play a major role in the development of diseases associated with exposure to environmental toxicants. Research in the field has firmly established that iAs exposure is associated with epigenetic alterations including changes in DNA methylation, miRNA abundance, and post-translational histone modifications. Here, we summarize recent studies that have expanded the current knowledge of these relationships. These studies have pinpointed specific regions of the genome and genes that are targets of arsenical-induced epigenetic changes, including those associated with  Exposure to arsenic is a major global threat to human health. The most common route of exposure worldwide is through the consumption of drinking water contaminated with naturally-occurring, geologic sources of inorganic arsenic (iAs) . AlthougThe molecular mechanisms underlying the diverse health effects associated with iAs exposure are not well understood and are likely complex. Numerous toxic effects have been attributed to exposure to iAs and its reactive metabolites. For instance, the generation of reactive oxygen species (ROS) is believed to play a major role in mediating the toxic effects of various arsenicals by causing biomolecular damage and the activation of cellular signaling pathways that promote disease development . HoweverIn recent years, there has been increased recognition of the importance of the epigenome in maintaining cellular homeostasis, and therefore it has been hypothesized that environmentally-induced epigenetic perturbations may play an important role in disease development. Detailed descriptions of the various components of the epigenome are outside the scope and purpose of this work but are the subjects of reviews elsewhere , 9. Briein utero is associated with increased risk for chronic disease development later in life [in utero play a major role in the fetal origins of adult disease by causing permanent changes, or “reprogramming,” of the epigenome in the fetal environment that persist throughout life [The recognition that epigenetic changes may mediate the toxic effects of iAs stems in part from the observation that it is a potent carcinogen but not a point mutagen. The long-term health consequences of prenatal iAs exposure also support that iAs may exert adverse effects via epigenetic mechanisms. In both humans and animal models, exposure to iAs  in life –16. In a in life , 18. Obsout life , 19. Theout life , 21. Theout life .As the effects of arsenicals on the epigenome have been the subjects of several reviews in recent years –24, the In mammals, DNA is methylated almost exclusively at the cytosine residue of CpG-dinucleotides in which a single methyl group is enzymatically transferred from S-adenosyl methionine (SAM) to generate 5’-CpG-3’ . CpGs arin vitro studies, in vivo studies, and studies in human populations (as reviewed in [CDKN2A/ p16) or tumor protein 53 (TP53) . Takiewed in , 24). Iniewed in , has beein vitro , 36, 37.Aside from the examination of the promoter DNA methylation status of genes implicated in iAs-associated diseases, a major research focus has been the investigation of prenatal iAs exposure on DNA methylation patterns. There is considerable experimental evidence suggesting that iAs may act as a prenatal toxicant by altering the DNA methylome. This is supported by the observation that similar relationships between changes in DNA methylation patterns and altered disease risk has been observed with exposure to other prenatal toxicants or perturbed prenatal nutritional status –42.Another major area of interest has been the investigation of the relationship between iAs biotransformation and the DNA methylome. IAs biotransformation involves a series of alternating reduction and oxidative methylation steps that yields six major arsenicals found in the urine of humans and most mammals, namely trivalent and pentavalent forms of iAs and its monomethylated and dimethylated metabolites  –45 Fig.. These mIn recent years, examinations of the relationship between alterations in the DNA methylome and prenatal iAs exposure and/or iAs biotransformation have continued as major research thrusts Table . As in eOur laboratory was interested in identifying genome-wide gene-specific DNA methylation changes associated with chronic iAs exposure in humans. An examination of DNA methylation patterns in peripheral blood leukocytes (PBLs) from an exposed population in Zimapán, Mexico, identified a set of 183 genes with differentially methylated promoters in women with iAs-associated skin lesions vs. women with unlesioned skin •. Most oAS3MT) gene, which is the gene encoding the enzyme required for iAs biotransformation  methyltransferase  (MLH1), and the Long Interspersed Nuclear Element 1 (LINE-1) in the blood of chronically exposed women from the Argentinian Andes [AS3MT haplotype and p16 methylation. The concentration of total urinary arsenic  was associated with p16 and MLH1 methylation, urinary %iAs was positively associated with p16 methylation, and urinary %DMAs was negatively associated with p16 methylation. Interestingly, although increased urinary ratios of MMAs and/or high ratios of MMAs/DMAs are associated with increased risk for a variety of iAs-associated diseases [p16 promoter, at least in DNA isolated from blood. No significant relationship was observed between LINE-1, used as an indicator of global DNA methylation status, and urinary arsenical measurements [A separate study examined the relationship between the an Andes •. A signurements •.AS3MT promoter in Native Americans with low vs. moderate U-tAs [AS3MT promoter, a region was identified that contained 30 hypomethylated CpGs in the moderate U-tAs group compared to the low U-tAs group, but there were no associations observed between AS3MT promoter methylation levels and %MMAs or %DMAs in urine. This study also revealed that short-term exposure of peripheral blood mononuclear cells (PBMCs) to sodium arsenite in vitro induced AS3MT expression, which was accompanied by a decrease in DNA methylation of the AS3MT promoter. In addition, AS3MT knockdown inhibited arsenite-induced global DNA hypomethylation in PBMCs in vitro. While this study established a relationship between AS3MT promoter methylation status and arsenic exposure in humans, further studies will be necessary to determine if these DNA methylation alterations are associated with changes in AS3MT expression in human populations and how these changes impact iAs biotransformation and risk of iAs-associated disease development.One study investigated the promoter DNA methylation level of the te U-tAs . Out of in utero identified a positive relationship between arsenic exposure and TP53 promoter methylation status but not LINE-1 methylation status [TP53 and p16 promoters were examined in maternal and umbilical cord leukocytes isolated from iAs-exposed mother-child pairs in Bangladesh [p16 promoter. The data revealed evidence for a nonlinear dose-response relationship, as these effects were most evident in the middle tertiles of exposure. These data suggest that moderate levels of iAs, which are likely relevant to many areas endemic for arsenic poisoning, may have significant effects on DNA methylation patterning that could result in genomic alterations.Several studies have examined the DNA methylation status of targeted genes including tumor suppressors and/or global methylation of LINE-1 in the cord blood of exposed newborns. Analyses of cord blood from a cohort of newborns in Thailand exposed to varying levels of iAs n status . The relSupporting the impact of moderate levels of iAs exposure on the epigenome are changes in DNA methylation observed in a population exposed to relatively low levels of iAs. A genome-wide examination of cord blood isolated from newborns from New Hampshire revealed that while no global effects on DNA methylation were observed in relationship to maternal U-tAs levels, most of the U-tAs-associated effects were observed in CpG islands •. Of theThere are a few caveats to consider when interpreting the existing body of literature related to arsenical-associated changes in DNA methylation patterning. For example, although PBL DNA methylation profiles may serve as useful biomarkers for iAs exposure, disease state, and/or disease susceptibility, it is currently unknown whether PBL DNA methylation profiles reflect those that would be present in target cells of iAs toxicity such as liver, lung, and bladder. In addition, it is important to note that multiple white blood cell types are present in PBL preparations, and these cell populations may shift upon exposure to iAs or due to other environmental factors. It is also unknown if the gene-specific DNA methylation changes correspond to changes in mRNA or protein levels of their genes. Additionally, the relationship between DNA methylation and gene expression is more complex than often considered, where increased DNA methylation may be associated with increased gene expression. There is currently limited information on the stability of the DNA methylation changes over time. Also, in the case of prenatal iAs exposure, the studies presented thus far do not investigate true transgenerational effects, as iAs can readily pass through the placenta , 57, andHistones play an essential role in the compaction and organization of the genome and in the regulation of gene activity. Histones comprise the protein component of the nucleosome, which is the fundamental repeating unit of chromatin. The nucleosome contains ~146 bp of DNA wrapped around a histone octamer comprising two copies each of the four core histones H2A, H2B, H3, and H4 . Each ofin vitro, in which changes in both global and gene-specific histone PTM patterns have been observed (as reviewed in [WNT5A), which is essential for the maintenance of the malignant phenotype [Most of the investigations into the effects of arsenicals on histone PTMs have been performed iewed in ). Of nothenotype .As observed in the case of DNA methylation, recent years have witnessed an increase in the number of studies that have examined histone PTMs in iAs-exposed populations Table . For insp16 mRNA in the livers of C57BL/6 mice, which was accompanied by an increase of the transcription-restrictive PTM H3K9me2 in the p16 promoter region but no change in promoter DNA methylation status [One study in mice indicated that changes in promoter PTMs are not always associated with changes in DNA methylation patterns. Long-term (6 months) exposure to iAs resulted in the downregulation of n status •.in vitro and in vivo studies have provided important groundwork for studying the relationship between iAs exposure, changes in histone PTMs, and potentially toxic effects, such as the transcriptional modulation of genes implicated in disease development . The relationship between these events and iAs-associated disease development must be addressed in future work. In addition, the studies highlighted here underscore the need to examine the interplay between multiple epigenetic components that likely work in unison to mediate the toxic effects of arsenicals.Together, these recent studies demonstrated that exposure to iAs is associated with genome-wide changes in histone PTMs in animals and humans and that some of these effects in humans may be sex-specific. Importantly, mechanistic miRNAs are non-coding RNAs 21–24 nucleotides in length that are best characterized as regulators of gene activity at the post-transcriptional level, although they have been shown to regulate gene expression at the transcriptional level as well , 67. At in vitro, and few have investigated the functional consequences of these perturbations [in vitro studies have continued to predominate, but many are mechanistic in nature, in which miRNAs have been implicated in mediating various toxic effects of arsenicals . Previorbations , 23. In PHLPP) gene, which has known tumor suppressor functions. Another study identified the upregulation of miR-21 as an important event in the iAs-mediated transformation of human embryo lung fibroblast cells, which was dependent on the activation of ROS-sensitive pathways involving extracellular signal-regulated kinase mitogen-activated protein kinase (ERK/MAPK) and transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [Some of these studies have focused on miRNAs as mediators of the apoptotic effects of arsenic trioxide (ATO) , 73, a c (NF-κB) •.Together, these studies indicate that miRNAs may be involved in mediating multiple toxic effects of arsenic species, including the modulation of pathways that control cell growth, differentiation, and apoptosis. The studies also provide insight regarding the involvement of epigenetic modifications and cellular signaling pathways in the modulation of miRNA levels in response to iAs exposure. Future research could elucidate the mechanisms by which iAs can perturb miRNA expression levels and assess whether such expression is altered in biological samples from humans. As with the other forms of epigenetic modifications, it will be critical to confirm the functional consequences of changes in the expression of miRNAs on the expression of their transcriptional targets and to determine whether these changes ultimately influence protein abundance and function.What is the relationship of the epigenetic changes to disease?-The significance of these arsenic-associated epigenetic effects on human disease development is likely dependent on a number of factors, including genotype, combined exposures to other contaminants, and other environmental influences such as nutrition; (2) How do the epigenetic changes influence disease directly?-While many associations have been established, it is largely unknown if there is a causal relationship between the epigenetic changes and arsenic-associated disease development, as well as the mechanisms by which these changes cause disease; (3) How stable are the epigenetic changes and are they functional?-It is important to consider that some epigenetic changes may be adaptive or stochastic responses that do not promote disease development. Likewise, global and/or gene-specific changes in DNA methylation patterning and histone PTMs may not be reflective of functional changes that are manifested at the protein level. Of note, it is important to consider that several major research trends have emerged in recent years. For instance, the DNA methylation studies were almost exclusively conducted on samples from human populations, whereas the miRNA studies were dominated by in vitro studies. These trends reveal gaps in research that must be closed in order to fully understand the molecular mechanisms that drive arsenical-associated epigenetic alterations, the consistency of these alterations across experimental platforms and study type , and ultimately, the relevance of these alterations to altered disease risk in exposed populations. Lastly, it will be increasingly important to examine the effects of arsenicals at a systems-wide level, where the impacts of both epigenetic and non-epigenetic alterations are examined. It is likely that the true biological picture of the epigenetic contributions to the mechanism of iAs-induced disease represents the complex interplay of a multitude of signaling events within the cell.In summary, current literature clearly indicates that arsenic exposure is associated with changes that include altered patterns of DNA methylation , modifications of histone PTMs, and altered expression levels of miRNAs. Importantly, answers to some key questions remain unknown: (1)


Congenital diaphragmatic hernia (CDH) occurs in 5-10% associated with chromosomal abnormalities like, Pallister Killian syndrome, Trisomy 18, and certain deletions.. Association of CDH with trisomy 13 (Patau syndromes) is very rare. Here, we report such an unusual association, where surgical repair was done, but eventually the case succumbed as a result of multiple fatal co-morbidities.  A 34 weeks male preterm weighing 1020 grams; an outcome of non-consanguineous marriage was delivered normally. He was born to a 35 years fourth gravida mother with one abortion in past, an old case of multi-drug resistant (MDR) tuberculosis. At birth, he was observed to have an aplasia cutis on scalp, wide fontanelle, bilateral post-axial polydactyly in hands, scaphoid abdomen and bilateral cryptorchidism. The face revealed low set ears, hazy corneas, and bilateral complete cleft lip with complete cleft palate . The baby had right sided heart sounds with a basal systolic murmur. In view of respiratory distress and hypoxia he was shifted to Neonatal Intensive Care Unit (NICU) on oxygen. In view of progressive respiratory acidosis ventilator support was initiated. A requirement of maximum oxygen index (OI) of 12 was suggestive of favorable outcome. An echocardiography revealed a small (3 mm) atrial septal defect (ASD) and an inlet Ventricular Septal Defect (VSD). A left sided congenital diaphragmatic hernia (CDH) was confirmed on postnatal chest X ray.The surgery was done on the baby on the next day. Intraoperative findings suggested an isolated 2cm x 3cm defect in the left diaphragm with herniating spleen, splenic flexure of colon and a part of left liver. On day two, the baby developed shock with bleeding from multiple sites with thrombocytopenia which, promptly responded to inotropes and platelet transfusion and he was extubated to oxygen hood at 84 hrs. On fifth day, he had a refractory severe apnea requiring ventilator care. The workup for apnea was non suggestive, except for a mild asymmetric bilateral ventriculomegaly (Left more than right) with 2 small sub-ependymal cysts. He had stormy course in the NICU with progressive thrombocytopenia, acute kidney injury (AKI) and dyselectrolytemia. On eighth day of life, he had another refractory apnea followed by a fatal cardiac arrest. The karyotype report received after 3 weeks was suggestive of trisomy 13.Patau is a rare syndrome first recognized in 1960 [1]. It commonly results from, non- disjunction of chromosome 13 and rarely as a result of deletions, duplications and translocation [1]. Advanced maternal age is often seen as an association [2]. They have wide spectrums of anomalies ranging from mid-face to complex heart and brain malformations. The resuscitation is abandoned in these babies considering the associated lethal anomalies [3]. The median survival in spite of the aggressive management is less than 2 weeks and the karyotype report is seldom available [4]. Only 2 to 3 % of the trisomies survive to birth [4]. Our case even though had a history of spontaneous abortion; the genetic workup of fetus was unavailable. More than half of them have central nervous system, hearing, eyes, mouth, auricles, skin, hands, and feet, skeletal, cardiac, and genital abnormalities [5]. Diagnosis is often difficult, due to overlapping clinical findings with other syndromes notably trisomy 18 (Edwards). However, in our case the characteristic constellation of the findings suggested a possibility of Patau [6]. The newer modalities for early diagnosis like Fluorescent in Situ Hybridization (FISH), array-based comparative genomic hybridization (CGH) are costly and not widely available. We could only send for the conventional karyotype estimation. It is very difficult to decide and discuss the management plans of these babies with parents, without a confirmed diagnosis in hand. The syndromes like the anencephaly, trisomy 18 and 13, are suggested for “non-initiation of resuscitation” by most of the resuscitation guidelines considering their high mortality and universal morbidity [7]. Our case did not need any resuscitation at birth, even though he had features suggestive of Patau, but this was not confirmed at birth. Considering the willingness of the parents and the promising clinical course, we performed the repair of the CDH. With the antenatal anomaly scans and the biochemical screening it is now possible to confirm and diagnose these cases antenatally [8]. In such cases, where the diagnosis is confirmed at birth an alternate decision may be taken unlike our case. It is also advisable at this point to consider each case individually and offer appropriate diagnostics and genetic counseling to the parents.Source of Support: NilConflict of Interest: Corresponding author is editor of the journal but he is not involved in decision making of the manuscript.


Influenza A virus (IAV) infection is an important cause of respiratory disease in humans. The original reservoirs of IAV are wild waterfowl and shorebirds, where virus infection causes limited, if any, disease. Both in humans and in wild waterbirds, epithelial cells are the main target of infection. However, influenza virus can spread from wild bird species to terrestrial poultry. Here, the virus can evolve into highly pathogenic avian influenza (HPAI). Part of this evolution involves increased viral tropism for endothelial cells. HPAI virus infections not only cause severe disease in chickens and other terrestrial poultry species but can also spread to humans and back to wild bird populations. Here, we review the role of the endothelium in the pathogenesis of influenza virus infection in wild birds, terrestrial poultry and humans with a particular focus on HPAI viruses. We demonstrate that whilst the endothelium is an important target of virus infection in terrestrial poultry and some wild bird species, in humans the endothelium is more important in controlling the local inflammatory milieu. Thus, the endothelium plays an important, but species-specific, role in the pathogenesis of influenza virus infection. Orthomyxoviridae. IAVs can be classified into different subtypes based on antigenic differences in the two surface glycoproteins of the virus, the hemagglutinin (HA) and neuraminidase (NA). Each year, antigenic changes (or “drift”) in the HA and NA result in a seasonal outbreak of IAV in the human population. However, when there is a dramatic change in the HA and/or NA, often originating from the avian reservoir, global pandemics can result. In the last 100 years there have been four IAV pandemics in the human population: the 1918 H1N1 pandemic, the 1957 H2N2 pandemic, the 1968 H3N2 pandemic and the 2009 H1N1 pandemic. These pandemics have all caused significant morbidity and mortality. Indeed, the 1918 H1N1 virus was so severe that life expectancy in the U.S.A. dropped by 11.8 years from 1917 to 1918  is a negative-sense RNA virus of the Family The pathogenesis of IAV infection differs markedly between wild waterbirds, terrestrial poultry and humans. In all three host groups, endothelial cells play a key role in disease pathogenesis—either as the primary cellular target of viral infection or as orchestrators of the anti-viral immune response. Here, we review the currently available literature on the role of the endothelium in the pathogenesis of IAV (in particular HPAI H5N1 viruses) in terrestrial poultry, wild birds and humans. Specifically, we will compare the ability of IAV to infect and/or “activate” the endothelium across these different host groups.Typically, terrestrial poultry infected with LPAI viruses display limited clinical signs and with no evidence of endothelial cell infection   of black swans succumbed to disease within 2–3 days  (Brown et al., In wild birds, LPAI viruses normally present as a sub-clinical infection with little involvement of the endothelium in disease pathogenesis (Webster et al., in vitro studies using primary human lung microvascular endothelial cells demonstrated that endothelial cells can be infected by seasonal H3N2 IAV, with infection ultimately resulting in increased endothelial cell permeability (Armstrong et al., in vitro studies, there is limited evidence suggesting that IAV infection of human endothelial cells occurs in vivo. Post mortem analysis of patients who succumbed to H5N1 did not demonstrate the presence of virus in pulmonary endothelial cells (Gu et al., In humans, the primary cellular targets of IAV are epithelial cells in the respiratory tract. Seasonal IAVs, which are adapted to and circulate in the human population, typically infect ciliated cells in the upper respiratory tract, trachea and bronchi (van Riel et al., in vivo, endothelial cells may still play an important role in the pathogenesis of IAV in humans. During IAV infection pulmonary endothelial cells are thought to be the most important source of cytokines in the lung (Teijaro et al., Although human endothelial cells are not infected by IAV Upon the initial emergence of H5N1 viruses, mortality in cats was observed in areas where the viruses were spreading in wild and domestic birds. This suggested that cats were susceptible to infection. This was unusual as cats have long been considered to be refractory to IAV infection. H5N1 viruses administered to cats intratracheally resulted in productive infection of many organs, including the respiratory tract, with parenchymal and epithelial cells as the primary targets for viral replication (Rimmelzwaan et al., In addition to mediating cytokine production, endothelial cells may also indirectly control the inflammatory response in the lung during IAV infection via the expression of adhesion molecules, such as E-selectin, P-selectin, ICAM1, and VCAM1, on their apical surface. These adhesion molecules can bind to various leukocytes and mediate their extravasation to the infected lung. The increased expression of E/P-selectin expression on human endothelial cells following exposure to H5N1 (Perrone et al., in vivo remains to be determined. It is likely that as research and the availability of reagents for studying avian species continues to grow new roles for endothelial cells in pathogenesis of IAV will be discovered. However, what is clear at present is that endothelial cells contribute to the severity of IAV infections across multiple different species.Endothelial cells play important but distinct roles in the pathogenesis of IAV in wild birds, poultry and humans. Whilst endothelial cells are infected by HPAI viruses in chickens and swans, in humans they are more important in driving and controlling the inflammatory response in the lung. It is important to note that endothelial cells in both chickens and swans may also influence the inflammatory response to IAV. It has already been suggested that the overwhelming endothelial tropism of H5N1 viruses in poultry may disrupt the innate immune response (Suzuki et al., 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.


N-methyl-D-aspartate receptor (NMDAR). Previous studies have shown that the NMDAR is altered in varying epilepsy syndromes and notably in the cortical tubers found in TSC. The aim of this paper is to examine how felbamate monotherapy was able to achieve such robust antiepileptic effects in a unique patient and possibly offer a novel therapeutic approach to patients suffering from TSC- and NF-related epilepsy.A 15-year-old girl with maternal inheritance of neurofibromatosis type 1 (NF1) and paternal inheritance of tuberous sclerosis complex (TSC) developed intractable epilepsy at age 5. Her seizures were refractory to adequate doses of four antiepileptic medications until felbamate was initiated at age 7. She has since remained seizure-free on felbamate monotherapy. Although felbamate has multiple mechanisms of action, it is thought to have its most potent antiepileptic effects through inhibition of the  Tuberous sclerosis complex (TSC) and neurofibromatosis type 1 (NF1) are the two most common neurocutaneous disorders . Both TSOf the neurocutaneous syndromes, population-based studies suggest that the occurrence of epilepsy in TSC is 78% (up to 96% in clinic-based studies)  and 3–6% N-methyl-D-aspartate receptor (NMDAR). NMDARs are glutamate-gated cation channels that cause excitatory synaptic transmission and are critical for the development of the central nervous system, learning, memory, and neuroplasticity. Abnormal expression levels and altered NMDAR function have been implicated in numerous neurological disorders, including Alzheimer's disease, Parkinson's disease, neuropathic pain syndromes, psychiatric disorders, and epilepsy [Among other proposed mechanisms of action of felbamate, its most potent antiepileptic effects are through inhibition of theepilepsy . Focusinepilepsy .We report the case of a 15-year-old patient with TSC, NF1, and intractable epilepsy who only achieved seizure freedom with felbamate monotherapy and hypothesize a novel therapeutic approach to NMDAR-activated epilepsy syndromes.The patient was born at 39 weeks of gestation to a mother with NF1 and a father with TSC . PhysicaAt 5 years of age the patient developed seizures characterized by staring and atonic falls. Her initial electroencephalogram (EEG) showed left frontal and anterior temporal spikes and sharp waves as well as intermittent focal temporal slowing. She was first treated with carbamazepine (CBZ) 200 milligrams (mg) twice daily, but over the next 5 months she continued to have breakthrough seizures as well as new seizures characterized by rhythmic head movements and eye deviation. She was changed to valproic acid (VPA) (125 mg three times daily) and clonazepam (CLN) (0.5 mg twice daily) but after 3 months required the addition of levetiracetam (LEV) as well. She continued to have breakthrough seizures so the following year at age 7, VPA and CLN were replaced by felbamate 120 mg three times daily. She remained on dual therapy LEV (up to 750 mg twice daily) and felbamate for the next 2.5 years until she was able to wean off of LEV and transition to felbamate monotherapy at 240 mg twice daily. Her most recent EEG at age 13 showed rare left parietal and bilateral frontal sharp waves  as well NF1 is an autosomal dominant disorder caused by a mutation on chromosome 17q11.2, an area that codes for the tumor suppressor gene neurofibromin. Neurofibromin loss leads to upregulation of the renin-angiotensin system and hyperactivation of the mammalian target of rapamycin (mTOR) pathway . This inAmong seizures, cognitive impairment, and neurobehavioral abnormalities, epilepsy is the most common neurologic manifestation of TSC, occurring in 70–90% of patients . ApproxiThere are a number of proposed mechanisms by which epilepsy develops as a consequence of TSC, though cortical tubers are thought to be the largest determinant . AbnormaClassically, vigabatrin, an irreversible inhibitor of GABA-transaminase (the enzyme responsible for catabolism of GABA), has been used as the first line for TSC-associated infantile spasms. After a period of remission from spasms, focal and then generalized seizures usually begin to appear and can become quite severe and unremitting, requiring combinations of multiple antiepileptic medications . Other tNeurological manifestations of NF1 mainly include intracranial tumors such as optic gliomas, focal areas of T2-hyperintensity (UBOs), and intellectual and learning disabilities . The pre2+ and Na+ are conducted across the channel, resulting in excitatory postsynaptic potentials. Mg2+ plays an important role in blocking the channel and preventing ion permeation [The NMDAR is fundamental to excitatory neurotransmission and is critical for normal CNS function. The receptor is a glutamate-gated cationic channel composed of four polypeptide subunits around a central pore, including at least two obligatory NR1 subunits with variable expression of two of the four types of NR2 subunits (NR2A-D) . The varrmeation .2+ and Na+ are conducted in excess, there is resultant neuronal excitotoxicity and subsequent seizure discharges [Aberrant NMDAR function is implicated in a wide range of CNS disorders, including acute and chronic pain syndromes, stroke, head trauma, dementias, and epilepsies . In cerescharges .2+ blockade in children with cortical dysplasias and adults with temporal lobe issues [2+ in vitro models of epilepsy whereby decreased NMDAR blockade induces epileptiform activity [2+ and Na+ which further potentiates epileptogenicity. Additionally, anti-NMDA receptor antibody encephalitis is considered a relatively new entity in which antibodies are formed against the NR1 subunit of the NMDAR, subsequently causing a decrease in cell surface NMDAR expression and impaired glutamate regulation [Multiple investigators have shown that the NMDAR is altered in patients with epilepsy. One study demonstrated that increased phosphorylation of the NR2B receptor in vivo resulted in epileptic discharges . Anothere issues , 21. Furactivity , 23. In activity ; as glutgulation . This regulation .2+ inhibition, thus promoting neuronal excitability [In reference to TSC, one study by White et al.  examinedtability . These fThere are no studies to date that specifically examine alterations in the NMDAR in NF1. Neurofibromin has, however, been documented to interact with a number of molecules including NMDARs whereby NMDAR activation precedes neurofibromin inactivation of Ras, subsequently allowing for synaptic plasticity, structural plasticity , and new spine formation , 30. LosNMDAR antagonists were first described to possess anticonvulsant activity in 1982 . This haHowever, in clinical trials, NMDAR antagonists have demonstrated serious and unacceptable neurobehavioral side effects at therapeutic doses , includiFelbamate  was synthesized in 1955 but was first approved in 1993 as a novel antiepileptic to be used as monotherapy and as an adjunctive therapy to treat partial seizures with and without generalization in adults and as an adjunctive therapy for partial and generalized seizures associated with Lennox-Gastaut syndrome , 36.  FeDespite its favorable efficacy, there has been restricted approval of felbamate to only adjunctive therapy in patients with Lennox-Gastaut syndrome because of postmarketing experience suggesting felbamate-related idiosyncratic aplastic anemia and hepatotoxicity . In the Although the exact mechanism of action is unclear, the major antiepileptic effect of felbamate is at least partly due to inhibition of the NMDAR, which decreases excitatory neurotransmission . FelbamaAt the NMDAR, therapeutic concentrations of felbamate have an inhibitory effect on NMDA currents . However2+ inhibits the channel [The exact site of felbamate action on the NMDAR is still unknown, but a number of studies have offered hypotheses, including selective blockade of specific subunits , modific channel . Though  channel , 43. Thi channel . This al channel .As described, the majority of patients with TSC develop epilepsy whereas the prevalence of epilepsy in NF1 is only slightly above the 1-2% value reported for the general population. The patient described in this paper carries a diagnosis of both TSC and NF1. Presumably it was the TSC that primarily generated her refractory epilepsy, but the NF1 likely potentiated her risk. She was tried on a number of antiepileptic medications, both as monotherapy and in combination, but failed to gain seizure control until felbamate was initiated.There are a number of proposed mechanisms by which TSC causes epilepsy, of which upregulation of excitatory mechanisms such as NMDAR expression is an interesting theory in the context of this patient. Multiple studies have demonstrated that the NMDAR is altered in epilepsy syndromes including hippocampal sclerosis, cortical dysplasia, temporal lobe epilepsy, refractory status epilepticus, and anti-NMDA receptor antibody encephalitis.Of particular interest is the study by White et al.  that fouThe patient described had been tried on carbamazepine (CBZ), valproic acid (VPA), clonazepam (CLN), and levetiracetam (LEV) but did not achieve seizure freedom until felbamate was added. Though felbamate functions through similar mechanisms of action as these other drugs including inhibition of voltage-gated sodium channels , increasing GABA , and blocking calcium channels (VPA), the only mechanism of action exclusive to felbamate is its inhibition of the NMDAR . PerhapsIn both TSC and NF1, chromosomal mutations cause abnormal formation of mTOR inhibitory proteins and hyperactivation of the pathway. Abnormal cell growth and proliferation due to mTOR hyperactivation promote seizures by indirectly affecting the excitability of circuits through alterations in neurotransmitter and ion channels and neuronal and synaptic organization , 48. BotThe structure and function of the NMDAR were explained above in detail; however one of the faces of the NMDAR is as an important regulator of the mTOR pathway. The receptor works upstream of mTOR to downregulate the pathway and, thus, protein synthesis responsible for cellular growth and function . There hThese unexpected outcomes raise questions as to how NMDA inhibitors such as felbamate can have such potent antiepileptic effects if NMDA inhibition is thought to augment the mTOR pathway and thus promote epileptogenesis. As discussed above, felbamate has a number of mechanisms of action  and, at Although our findings are based on the experience of only one patient, the robust response and overlap between the mechanical underpinnings of the disease and the drug suggest that perhaps felbamate should be considered in the treatment of TSC-related epilepsy. Vigabatrin has proven to be effective in infantile spasms in TSC by upregulating GABA. Perhaps the addition of felbamate can help treat the succeeding refractory seizures in TSC by targeting the NMDAR as well. Though less is known about the cause of seizures and the role of the NMDAR in NF1, it has been documented that neurofibromin and the NMDAR do in fact interact. Interestingly, there is one case report on the use of ketamine, an NMDA antagonist, for a patient with severe chronic pain secondary to NF1 that was refractory to multiple therapies but responded quite dramatically to ketamine . PerhapsClearly large clinical study would be ideal to address our hypothesis, but further publications of clinical reports or case studies can begin to add depth to our proposal. As a final point, more research is also needed about the potential role of felbamate or other NMDAR antagonists in the varying epilepsy syndromes described above that have also been documented to have alterations in the NMDAR.


Hypertrophic cardiomyopathy (HCM) has been recently recognized as the most common inherited cardiovascular disorder, affecting 1 in 500 adults worldwide. HCM is characterized by myocyte hypertrophy resulting in thickening of the ventricular wall, myocyte disarray, interstitial and/or replacement fibrosis, decreased ventricular cavity volume and diastolic dysfunction. HCM is also the most common cause of sudden death in the young. A large proportion of patients diagnosed with HCM have mutations in sarcomeric proteins. However, it is unclear how these mutations lead to the cardiac phenotype, which is variable even in patients carrying the same causal mutation. Abnormalities in calcium cycling, oxidative stress, mitochondrial dysfunction and energetic deficiency have been described constituting the basis of therapies in experimental models of HCM and HCM patients. This review focuses on evidence supporting the role of cellular metabolism and mitochondria in HCM. Hypertrophic cardiomyopathy (HCM) was first recognized as a clinical entity, approximately 55 years ago Brock, . It is tAsymmetric ventricular hypertrophy and left ventricular outflow tract obstruction with normal or hyperdynamic systolic function are common morphologic manifestations of HCM   , which promotes mitochondrial Ca2+ efflux and decreases the mitochondria's ability to accumulate Ca2+ during conditions of high demand . In high work conditions, ADP accumulation is sensed by CK, which catalyzes phosphoryl group transfer from phosphocreatine to regenerate ATP  and Ca2+ pumps like SERCA, leading to systolic and/or diastolic dysfunction, reduction or blunted increase in stroke volume, increased levels of cytosolic Na+, Ca2+ and arrhythmias under conditions of high work load, such as intense exercise , hydrogen peroxide (H2O2) and hydroxyl radicals (.OH)- and reactive nitrogen species, including nitric oxide (NO) and peroxynitrite (ONOO−) . It has been hypothesized that HCM mutations may increase “Ca2+ trapping,” and through altered on–off kinetics may lead to altered Ca2+ signaling and arrhythmogenesis    and whether it leads to mitochondrial dysfunction in any/all HCM mutations, or whether high levels of cytosolic Na+ occur after the onset of myocyte hypertrophy and/or symptoms. Most importantly, none of the agents tested clinically have been demonstrated to change disease course in symptomatic patients  model (that simulates increased afterload caused by hypertension)  technology permit derivation of all HCM patients and whether mitochondrial dysfunction precedes development of energetic deficits. Further investigation of mitochondrial function, metabolism and its relationship to cardiac function and electrophysiology in animal models of HCM and/or patient-derived myocytes is needed to clarify the molecular mechanisms underlying the cardiac phenotype in HCM and to design therapies that prevent, arrest and reverse the disease phenotype.HCM is caused by mutations in sarcomeric proteins in 50–60% of patients. These mutations have been shown to increase the energetic cost of tension development. However, it is unclear whether energetic deficits are involved in generation of the cardiac phenotype in 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.


PLOS Genetics, Palian et al. describe new insights into the regulation of Maf1 and its function in mammalian systems Maf1 is a nutrient- and stress-sensitive global repressor of transcription by RNA polymerase (pol) III Studies on Maf1 regulation up until now have focused on posttranslational mechanisms, notably phosphorylation, which controls Maf1 localization (in yeast) and its interaction with the polymerase (in yeast and humans) In addition to its regulation by nutrients and stress, pol III transcription is inhibited by tumor suppressors, increased by oncogenic activation and cell transformation, regulated during the cell cycle, and targeted by viruses and other pathogens MAF1 in yeast affects the expression of gluconeogenic genes Maf1 knockdown in the fat body of Drosophila. In this case, increased organismal growth and accelerated larval development resulted from a diffusible signal, generated in the fat body, that affected systemic signaling by insulin-like peptides synthesized in the brain Fasn promoter. Clearly, genome-wide transcriptional profiling in cells with altered Maf1 expression is necessary to better appreciate the function of this global regulator.Beyond its role in the pol III system, previous work by Johnson et al. Altogether, the new study identifies an important mechanism of Maf1 regulation along with new Maf1-regulated protein-coding genes that impact a novel biological function. Since research on Maf1 has only just scratched the surface for a limited number of model organisms, it seems certain that additional regulatory targets and biological functions have yet to be discovered in your favorite eukaryote.


Kisspeptin (Kp) is synthesized in the arcuate nucleus and preoptic area of the hypothalamus and is a regulator of gonadotropin releasing hormone in the hypothalamus. In addition, Kp may regulate additional functions such as increased neuropeptide Y gene expression and reduced proopiomelanocortin (POMC) gene expression in sheep. Other studies have found a role for Kp to release growth hormone (GH), prolactin and luteinizing hormone (LH) from cattle, rat and monkey pituitary cells. Intravenous injection of Kp stimulated release LH, GH, prolactin and follicle stimulating hormone in some experiments in cattle and sheep, but other studies have failed to find an effect of peripheral injection of Kp on GH release. Recent studies indicate that Kp can stimulate GH release after intracerebroventricular injection in sheep at doses that do not release GH after intravenous injection. These studies suggest that Kp may have a role in regulation of both reproduction and metabolism in sheep. Since GH plays a role in luteal development, it is tempting to speculate that the ability of Kp to release GH and LH is related to normal control of reproduction. Kisspeptin (Kp), also known as metastin, was first discovered and noted for its role in the inhibition of cancer cell metastasis. However, it was discovered to also stimulate gonadotropin releasing hormone (GnRH) release and subsequent secretion of luteinizing hormone. Neuroendocrine control of reproduction in ruminants culminates in the secretion of luteinizing hormone (LH) from the anterior pituitary. There has been much interest in Kp’s role in regulation of reproduction in a number of species including ruminants. More recently, Kp has been implicated in the integration of metabolic control of reproduction. This review will briefly summarize KP action on reproduction in ruminants and focus on recent discoveries of Kp action beyond reproduction control in ruminants.Kiss1 gene were associated with increased litter size in goats [Kisspeptin clearly stimulates a release of GnRH and subsequent secretion of LH. Intravenous administration of Kp-10 to ovariectomized (OVX) ewes stimulated increased circulating concentrations of LH and increased GnRH concentrations in the cerebrospinal fluid . Additioin goats .Kisspeptin appears to have a role in generation of the LH surge to stimulate ovulation. Constant iv infusion of Kp-10 for 8 h beginning 30 h after progesterone withdrawal stimulated an earlier LH surge and an earlier increase in circulating concentrations of progesterone than in ewes treated with vehicle [The action of Kp on LH in sheep appears to be via an effect on GnRH release from the hypothalamus and not direct action of Kp on pituitary gonadotropes. Cultured ovine pituitary cells can respond to Kp treatment with increased release of LH, but hypothalamo-pituitary-disconnected ewes do not respond to Kp-10 treatment with increased circulating concentrations of LH nor do hypophysial portal concentrations of Kp correspond with LH pulsatility and the LH surge . FurtherThe Kp response to steroid and nonsteroid cues is altered during the nonbreeding season to result in seasonal anestrous in sheep. In ewes, infusion of Kp-10 IV stimulated ovulation during the nonbreeding season . The numKiss 1 mRNA expression in cells in the rostral ARC [Tac2 mRNA, encoding for neurokinin B, was readily detectable in cells with Kiss 1 mRNA, but was decreased in rostral ARC cells following ram introduction [Introduction of a ram to seasonally anestrous ewes isolated from rams for at least one month will induce pulsatile LH secretion and can cause ovulation outside of the breeding season . Kisspeptral ARC . Interesoduction .Kiss 1 positive cells in the POA increased from 25 to 35 weeks of age in ewe lambs, although the number of Kiss 1 positive cells did not appear to be related to increases in LH pulse frequency. However, Redmond et al. [Kiss 1 positive cells in the middle ARC that was associated with increased frequency of LH pulses. Furthermore, the percentage of GnRH neurons in the POA with Kp positive close contacts was higher in post puberty in ewes and increased following ovariectomy in prepubertal ewes [Puberty in the ruminant is initiated by a decrease in negative feedback inhibition of LH by estradiol. Kisspeptin and neurokinin B, which is co-expressed with Kp in a number of hypothalamic cells, may play a role in initiation of puberty in ruminants. Injection of senktide, a neurokinin B agonist, in prepubertal ewes was immediately followed by a LH pulse . Howeverd et al.  did repotal ewes . Hourly tal ewes . Thus, K++ that occurred in 10 % of cultured cells. Moreover, in nonhuman primate pituitary cell culture, Kp-10 stimulated both GH and LH release through extracellular Ca++ entry, phospholipase C, protein kinase C, MAPK, and by additional intracellular Ca++ mobilization [There is circumstantial evidence of possible actions of Kp at the level of the pituitary in sheep, possibly actions not oriented towards regulation of LH. For example, in sheep the median eminence contains neuron terminals with specific staining for Kp . Kisspeplization . These din vitro, as well as release GH after intravenous injection under some conditions, it can be concluded that Kp has the potential to modify GH through actions at the pituitary, but is probably not a major direct regulator of GH release.Peripheral administration of Kp-10 to prepubertal heifers increased circulating concentrations of GH , suggestInfusion of Kp-10 via the lateral ventricle of sheep resulted in an increase in ARC neuropeptide Y (NPY) gene expression and a decrease in proopiomelanocortin gene expression . Since lAt present there have been no published findings regarding mechanism or the physiological significance for Kp in regulating GH. The link between Kp and NPY  and the These studies confirm a link between Kp and metabolic regulatory systems. Since adequate nutrition and GH are both needed for reproductive success, the connection of Kp to metabolic and GH systems may be a critical component of normal reproduction and should be examined in more depth.


Recent studies suggest that periodontal disease and type 2 diabetes mellitus are bi-directionally associated. Identification of a molecular signature for periodontitis using unbiased metabolic profiling could allow identification of biomarkers to assist in the diagnosis and monitoring of both diabetes and periodontal disease. This cross-sectional study identified plasma and salivary metabolic products associated with periodontitis and/or diabetes in order to discover biomarkers that may differentiate or demonstrate an interaction of these diseases. Saliva and plasma samples were analyzed from 161 diabetic and non-diabetic human subjects with a healthy periodontium, gingivitis and periodontitis. Metabolite profiling was performed using Metabolon's platform technology. A total of 772 metabolites were found in plasma and 475 in saliva. Diabetics had significantly higher levels of glucose and α-hydroxybutyrate, the established markers of diabetes, for all periodontal groups of subjects. Comparison of healthy, gingivitis and periodontitis saliva samples within the non-diabetic group confirmed findings from previous studies that included increased levels of markers of cellular energetic stress, increased purine degradation and glutathione metabolism through increased levels of oxidized glutathione and cysteine-glutathione disulfide, markers of oxidative stress, including increased purine degradation metabolites (e.g. guanosine and inosine), increased amino acid levels suggesting protein degradation, and increased ω-3 (docosapentaenoate) and ω-6 fatty acid (linoleate and arachidonate) signatures. Differences in saliva between diabetic and non-diabetic cohorts showed altered signatures of carbohydrate, lipid and oxidative stress exist in the diabetic samples. Global untargeted metabolic profiling of human saliva in diabetics replicated the metabolite signature of periodontal disease progression in non-diabetic patients and revealed unique metabolic signatures associated with periodontal disease in diabetics. The metabolites identified in this study that discriminated the periodontal groups may be useful for developing diagnostics and therapeutics tailored to the diabetic population. Periodontal disease is a chronic bacterial infection causing persistent gingival inflammation, and in some cases connective tissue destruction and bone resorption around the teeth. It is also characterized by pocket formation and recession. Although advances in dental care has resulted in improved periodontal status in certain populations, disparities persist as severe periodontitis is often found to be exaggerated in certain segments of the population, for example those from a low socio-economic background The gingival epithelium forms a crevice around each tooth that provides a protected space for bacterial colonization and proliferation Diabetic patients have a high prevalence of gingivitis, periodontitis, oral candidiasis, and xerostomia, and the severity of these diseases are correlated with the duration of diabetes and degree of glycemic control Saliva is a complex secretory fluid that contains trace metals, metabolites, biochemicals, proteins, glycoproteins, lipds, etc., that serve a spectrum of physiological needs. Saliva is a critical source of tissue lubricants, tooth mineralizing factors, acid buffers, toxin neutralizers, and antimicrobial components Metabolomic profiling is a rapidly evolving technology that has been increasingly used to discover early markers of disease This cross-sectional clinical study enrolled adult male and female subjects from the Buffalo, New York area and was conducted in accordance with the Helsinki Declaration. The Institutional Review Board at the University at Buffalo approved the protocol (ORB0650111E), including the clinical examination, collection of saliva and plasma samples. All subjects read and signed a written informed consent before their enrollment into the study. The inclusion criteria were: age between 18 and 65 years, in good general health (n = 81), or subjects diagnosed with diabetes (n = 80); and a minimum of 20 natural teeth (excluding third molars) present. Exclusion criteria included: inability or unwillingness to sign the informed consent form, diagnosis of a medical condition which required pre-medication prior to dental visits/procedures, 5 or more decayed untreated teeth at screening (cavities), diagnosis of other diseases of the hard or soft oral tissues, impaired salivary function, use of antibiotics or antimicrobial drugs within 30 days prior to the first study visit, a history of uncharacterized systemic disease, pregnant or nursing women, participation in any other clinical study within 1 week prior to enrollment into this study, present use of tobacco products, subjects who required dental treatment during the duration of the study, immune compromised individuals , or subjects who received periodontal treatment within the previous 30 days.Subjects were evaluated by a single dental examiner (SC) for inclusion/exclusion criteria. At the first visit, participants received a tube of standard, non-anti-microbial fluoridated toothpaste with instructions for use, in order to minimize variations in results that might be driven by the use of different toothpastes. A fasting blood sample was then collected from subjects self-identifying as diabetic to confirm diabetic status. During the second visit, subjects gave an update on their medical and medication history, contributed saliva and blood samples, had an assessment of their oral tissues to assess periodontal status, to monitor adverse events since the first visit and answer questions regarding their compliance as to the use of the standard toothpaste. A minimum of 0.5 ml of unstimulated saliva and 10 ml of blood was collected. Subjects were asked to refrain from eating or drinking (excluding water) from 11:00 PM the previous night; to brush their teeth and entire mouth the evening before the saliva donation, but not the morning of collection. Blood samples were obtained after collection of the saliva. Saliva was collected into sterile polypropylene tubes and immediately frozen in a dry ice bath and stored at −80 degrees C until shipped to Metabolon. No preservatives were added to the saliva samplesWithin the diabetic or non-diabetic cohorts, three sub-groups were established: periodontal health, gingivitis and periodontitis. Subjects were stratified according to the gingivitis index: Healthy: These subjects had an average full mouth Modified Gingival Index (MGI) For this study, diabetes status was established by self-reported history. Subjects who reported a history of diabetes were asked to confirm diabetic status by providing blood samples to measure HbA1c values. An individual was defined with Type 2 diabetes if hemoglobin A1C≥6.5%. Due to financial limitations, HbA1c levels were not assessed for those not reporting a history of diabetes. As a recent study suggested, the prevalence of diabetes (as measured by HbA1c>6.4) is likely low (extrapolated to be 5.0% with diabetes and 9.5% with pre-diabetes) in dental patients unaware of their diabetes status Saliva was collected in 50 mL polypropylene tubes and plasma was collected in Vacutainer tubes and anti-coagulated with EDTA. Samples were immediately frozen in a dry ice bath and stored at −80°C.Metabolomic profiling was performed as described previously t tests to compare data obtained from the healthy, gingivitis, and periodontitis samples. Multiple comparisons were accounted for with the false discovery (FDR) rate method, and each FDR was estimated by q-values.After normalization and imputation, the data were log-transformed. We then performed ANOVA and A total of 174 subjects were enrolled in this study . The meaAn untargeted metabolomic profiling approach was used to assess the chemical milieu of saliva and plasma. In total, 161 individual saliva samples were collected, 80 from subjects with diabetes and 81 from subjects without diabetes. We detected 475 and 772 metabolites in saliva and plasma, respectively, of which 370 and 430 could be mapped to known chemical structures  and S2.t tests and ANOVA identified statistically significant differences in these saliva samples between non-diabetic subjects and those with diabetes. Approximately 14% of the detected metabolites (69 of 475) in saliva showed altered levels (p≤0.05) among the healthy samples when compared to the diabetic cohort, suggesting that these may be markers of diabetic status in humans (t tests (data not shown).Welch's two sample n humans . AlternaGlycemic control can be monitored by changes in 1,5-anhydroglucitol . 1,5-AG is reabsorbed in the renal tubules, a process competitively inhibited by glucosuria, which leads to a reduction in its level in plasma. Thus, the greater the degree of glucosuria, the lower the amount of 1,5-AG present in the plasma α-hydroxybutyrate (AHB) is an early marker for insulin resistance in humans The analysis of each periodontal cohort (diabetic and non-diabetic) separately resulted in the identification of several metabolites that were associated with the progression of gingival status . The mosBoth periodontal status and diabetes status of the subjects were major driving factors in the biochemical changes observed. For the majority of metabolites with altered concentrations in the non-diabetic cohort, the levels at gingivitis samples were found to be intermediate between those levels found in healthy and periodontitis samples . This suBarnes et al. found differences in the biochemical signatures in saliva between non-diabetic subjects with healthy gingiva or periodontitis 2 to activate xanthine oxidase 2- and H2O2. Urate is known to function in redox balance as a radical scavenger or antioxidant under conditions of oxidative stress and in the process converted to other products . In the context of gingival disease, increased oxidative stress would result in higher levels of purine degradation metabolites, reflected in increased levels of hypoxanthine and xanthine. The results of this study are consistent with the results from previous studies Purine degradation is regulated, in part, by oxidative stress The levels of cysteine-glutathione disulfide and oxidized glutathione increased with gingival disease in non-diabetics and diabetics . GlutathSeveral lipid inflammatory mediators increased significantly in saliva from both the non-diabetic and diabetic groups during periodontal disease progression . In thisWe used metabolomic profiling technology to analyze saliva from subjects with and without diabetes who had a healthy gingiva, gingivitis, or periodontitis. In addition to verifying previously published observations in saliva Redox balance is vital for maintaining cellular homeostasis. Oxidative stress, caused by over production of reactive oxygen species and insufficient cellular anti-oxidant capabilities, can lead to cell damage. Glutathione and other anti-oxidant molecules serve to protect cellular proteins and structures from oxidative stress caused by reactive oxygen species. Glutathione is a thiol-containing tripeptide that plays a central role in cellular defense against free radicals and xenobiotics 2 enzymes ultimately resulting in the production of linoleic and arachidonic acid-derived eicosanoids which help to mediate the inflammatory response Several lipid compounds are important for the initiation and propagation of inflammatory signaling in cells and tissues. Linoleic acid and arachidonic acid give rise to many of these metabolites. These lipid signaling inflammatory mediators are liberated from the plasma membrane phospholipids by phospholipase APorphyromonas gingivalis is known to induce and alter sphingomyelin and ceramide signaling during infection Mobilization of lipid signaling pathways results in the synthesis of ceramides or the liberation of ceramides from intracellular stores. The activity of ceramidases results in the formation of sphingosine and sphingomyelin molecules that function in an array of intracellular signaling or maintenance of cell membrane architecture including apoptosis Prediction of risk and accurate diagnosis of current disease activity may facilitate effective prevention and treatment of gingival diseases. GCF and saliva have been extensively examined in attempts to assess the oral disease status. The markers identified in this study may be leveraged to monitor the effectiveness of interventions as well as identify the mechanisms of action for these interventions. Several different approaches have confirmed that identifying a single marker characteristic of a disease is unlikely; rather, a combination of biomarkers would likely constitute an effective clinical test. For example, insulin resistance is a feature if the pre-diabetes stage in humans prior to the development of diabetes. Untargeted biochemical profiling to identify biomarkers of insulin resistance following the development of targeted assays and validation of the targeted assays in a clinical cohort resulted in the development of a diagnostic that monitors insulin sensitivity status in humans Table S1Saliva Metabolic Profiling and Pathway Analysis. Statistical analyses for each biochemical detected in saliva between each of the gingival and diabetic cohorts.(XLSX)Click here for additional data file.Table S2Plasma Metabolic Profiling and Pathway Analysis. Statistical analyses for each biochemical detected in plasma between each of the gingival and diabetic cohorts.(XLSX)Click here for additional data file.


Better familiarity with this type of liver injury is important for early recognition of drug hepatotoxicity and crucial for preventing severe forms of liver injury and cirrhosis. Moreover, understanding the mechanisms leading to drug induced hepatic steatosis may provide much needed clues to the mechanism and potential prevention of the more common form of metabolic steatohepatitis.Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver disease in developed countries. Its frequency is increasing in the general population mostly due to the widespread occurrence of obesity and the metabolic syndrome. Although drugs and dietary supplements are viewed as a major cause of acute liver injury, drug induced steatosis and steatohepatitis are considered a rare form of drug induced liver injury (DILI). The complex mechanism leading to hepatic steatosis caused by commonly used drugs such as amiodarone, methotrexate, tamoxifen, valproic acid, glucocorticoids, and others is not fully understood. It relates not only to induction of the metabolic syndrome by some drugs but also to their impact on important molecular pathways including increased hepatocytes lipogenesis, decreased secretion of fatty acids, and interruption of mitochondrial  Drug induced liver injury (DILI) is a leading cause of acute liver failure and transplantation in western countries; and although rare, represents a serious clinical problem due to its unpredictable nature and possibly fatal course . The livNonalcoholic fatty liver disease (NAFLD) is recognized as a leading cause of liver disease in the western world and has increased in frequency over recent decades . The preThe diagnosis of DILI is challenging largely due to the fact that it remains a clinical diagnosis of exclusion. A reliable diagnosis requires demonstration of close correlation between the patient history and clinical, laboratory, and histological data. The initial presentation is typically elevated liver enzymes with or without jaundice, with a pattern that may be hepatocellular, cholestatic, or mixed. The differential diagnosis is wide including viral, autoimmune, metabolic, and genetic disorders, and clinical presentation may range from complete lack of symptoms to mild constitutional signs  to severe liver injury and even fulminant hepatitis. The ability to reach a definitive diagnosis is further complicated by the lack of objective diagnostic tests and the extensive list of drugs and herbal or dietary supplements that are recognized as potential causes of DILI. Consequently, a meticulous patient history and appropriate laboratory and imaging studies are required in order to exclude other etiologies before a reliable diagnosis of DILI can be established. A crucial element in establishing a diagnosis of DILI is the temporal relationship between drug exposure to development of liver injury and resolution following discontinuation of the drug. The manifestations of liver toxicity typically occur within days or weeks but, in some cases, may appear after months or even years of use and may even occur after treatment with the drug has been stopped. Moreover, liver enzyme elevations can persist for several months after the drug was discontinued.Several scoring systems were developed in an attempt to standardize the process of causality assessment in DILI such as The Council for International Organizations of Medical Sciences/Roussel Uclaf Causality Assessment Method (CIOMS/RUCAM) scale , 10, theThe principal histological feature of hepatic steatosis is defined as deposition of fat, principally triglycerides, within the hepatocyte. A minimum of 5% steatosis is required for histological definition of NAFLD. NASH is defined as the presence of hepatic steatosis along with inflammation and hepatocyte injury , with or without fibrosis . SeveralLiver biopsy is not considered a routine part of the clinical evaluation in cases of suspected DILI or NASH. However, it may offer further information in an attempt to evaluate a patient's liver injury in clinically complex situations, as well as defining the degree of injury. A correlation between the histological pattern and history of suspected drug use may strengthen the diagnosis of DILI based on formally recognized injury patterns and may also help exclude other potential etiologies of liver injury. The five most common histological patterns observed in 83% of DILI cases are acute and chronic hepatitis, acute and chronic cholestasis, and mixed cholestatic hepatitis . SteatohMany drugs may cause steatosis or steatohepatitis with pathological features resembling those of alcoholic fatty liver disease or NAFLD. The steatosis caused by drugs can be further characterized based on the predominant steatotic feature .Macrovesicular steatosis is described as the presence of small to large lipid droplets in the hepatocyte cytoplasm, with peripheral displacement of the cell nucleus [ nucleus –21. This nucleus , methotr nucleus  may alsoSteatohepatitis is characterized by steatosis, necroinflammation, hepatocellular ballooning, with or without Mallory hyaline bodies, and in some cases perisinusoidal fibrosis. Drugs associated with steatohepatitis are amiodarone, MTX, tamoxifen, and IRI.Microvesicular steatosis presents as accumulation of numerous very small droplets in the hepatocyte cytoplasm, without peripheral displacement of the nucleus [ nucleus . This is nucleus , aspirin nucleus , nucleos nucleus , nonster nucleus , and coc nucleus .β-oxidation or via secretion (Hepatic steatosis is characterized by accumulation of intrahepatocytes triglycerides-esters derived from glycerol and free fatty acids (FFA). The increased content of liver FFA may be caused by increased uptake , increased de novo lipogenesis within the hepatocytes, or reduced utilization either through ecretion .The process of lipogenesis, by which acetyl-CoA is converted to fatty acids in the hepatocyte, is transcriptionally regulated by the membrane-bound transcription factor sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c activates genes required for lipogenesis such as ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase (SCD). Peroxisome proliferator-activated receptors (PPARs) are other major transcription factors that participate in regulation of fatty acid homeostasis by downstream gene regulation of targets responsible for lipid synthesis and oxidation , 30. IncAnother mechanism that may lead to increased hepatocyte FFA accumulation is decreased incorporation of FFA into very low-density lipoprotein (VLDL) or attenuated secretion of the latter. This mechanism was demonstrated in tamoxifen-induced steatosis . The lipβ-oxidation, subsequently causing microvesicular steatosis and necrosis [Mitochondria play a pivotal role in lipid metabolism and ATP synthesis. As such they have been extensively investigated as targets for DILI. Studies have shown that drugs such as perhexiline, amiodarone, and tamoxifen can accumulate in mitochondria and interfere with the mitochondrial electron transport chain and necrosis –38. In fnecrosis –46. The necrosis –50.Steatosis is an exceedingly common finding in liver biopsies and the possibility that NAFLD and not DIS is the cause of an underlying liver disease should always be considered in the context of the specific patient . FeatureThe current concept of NAFLD and NASH is of a clinical spectrum of disease, resulting from a “multiple-hit” process , 55. TheThe number of drugs associated with liver lipotoxicity is large. In the following section we describe several key drugs for which the evidence of steatosis induction is stronger and the mechanism of injury has been at least partially elucidated.Amiodarone chlorhydrate is a di-iodinated benzofuran derivative, class III antiarrhythmic drug, used for both ventricular and atrial arrhythmias. Amiodarone is associated with many adverse effects, such as thyroid dysfunction, corneal microdeposits, optic neuropathy, peripheral neuropathy, and pulmonary and hepatic toxicities .Amiodarone hepatotoxicity usually occurs in two distinct clinical settings: the rapid intravenous infusion in acute settings, and the long-term chronic oral therapy. In the case of severe acute hepatitis following introduction of intravenous amiodarone, liver enzyme abnormalities occur within hours to days of treatment initiation and although most published cases were reversible after discontinuation of the treatment, acute liver failure and death have been reported. In some cases, a rechallenge with oral amiodarone was successful, leading some authors to ascribe the acute reaction to the effect of polysorbate 80 used as a solvent in the intravenous preparation –67.With chronic amiodarone therapy, asymptomatic elevation of aminotransferases, usually up to 3 times the upper limit of normal (ULN), was reported in up to half of the patients, although in more recent studies the numbers are considerably lower , 68–71. Symptomatic hepatic dysfunction may occur in 1–3% of patients using amiodarone , 68–70. Several mechanisms account for potential liver injury following amiodarone use. In this population of patients liver enzymes abnormality may be due to right-sided heart failure, MS, NASH, and other drugs ; howeverAmiodarone and its principal lipophilic metabolite, desethylamiodarone, are highly concentrated in the liver in a manner that appears to be related more closely to the total dose rather than to plasma concentrations . On compα, was observed, resulting in the increase of lipids in the liver. With amiodarone treatment, both PPARα and PPARγ targets were enriched suggesting a constant competition between increased lipid synthesis and the counter response of increased fatty acid oxidation [In animal models the hepatotoxic effect of amoidarone appears to be dose-dependent and consistent with increased production of cholesterol and accumulation of triglycerides in hepatocytes. In mouse studies, several genes were found to be regulated by amiodarone. Target enrichment of two nuclear receptors, androgen receptor and HNF4xidation . In vitrxidation .β-oxidation and increased production of ROS. Thus, hepatotoxicity associated with amiodarone can at least, in part, be explained by mitochondrial β-oxidation of fatty acids and the subsequent production of microvesicular steatosis and induction of apoptosis and necrosis [Another important mechanism, indicated by the microvesicular injury pattern, is mitochondrial dysfunction; indeed, amiodarone and its metabolite are concentrated in the hepatic mitochondria and have been shown to inhibit electron transport and uncoupled oxidative phosphorylation. In animal models amiodarone caused decreased mitochondrial necrosis , 83.Dronedarone (Multaq), a new class III antiarrhythmic agent, is a noniodinated amiodarone derivative associated with fewer adverse effects and reduced toxicity . Reportsβ-oxidation is a pivotal mechanism of dronedarone induced hepatotoxicity. In an in vivo study comparing mechanisms of hepatotoxicity of dronedarone and amiodarone, Felser et al. found that both caused cytotoxicity and apoptosis, in addition to reduced cellular ATP content compatible with impaired mitochondrial function. Both drugs caused uncoupling and inhibition of the mitochondrial respiratory chain and inhibition of mitochondrial β-oxidation leading to accumulation of ROS and intracellular lipids [β-oxidation resulting from reduced activity of carnitine palmitoyltransferase I (CPT I) in liver mitochondria, without effecting the activity of the respiratory chain ex vivo [As in the case of amiodarone, inhibition of mitochondrial r lipids . Interes ex vivo .2) to low dose (<50 mg/m2) MTX over a short time period, treatment of autoimmune diseases usually involves low doses of MTX over long periods of time. The mechanism of action may differ according to the treatment dose. MTX competitively inhibits the enzyme dihydrofolate reductase (DHFR) interfering with purine and pyrimidine biosynthesis and consequently decreasing DNA and RNA synthesis.Methotrexate is a folate antagonist that is used in the treatment of malignancies and autoimmune diseases. While treatment of malignancies may involve administration of high dose , alcohol consumption, obesity, total cholesterol, and diabetes , 97.The underlying mechanism of MTX hepatotoxicity is not fully understood. Oxidative stress has been implicated as an important cause through both increased production of ROS and decreased defense mechanisms. In vivo studies have demonstrated an increase of reactive oxygen metabolites such as myeloperoxidase (MPO), lipid peroxidation product malondialdehyde (MDA), and thiobarbituric acid reactive substances (TBARS) following MTX administration. Furthermore, a reduction in the levels of the antioxidant glutathione (GSH) was also demonstrated. However, increased levels of antioxidant enzymes such as catalase (CAT) and glutathione-S-transferase (GST) may suggest a possible adaptive mechanism of the liver –42. In aTamoxifen is a selective estrogen receptor modulator (SERM) with both agonist and antagonist effect in different tissues. Tamoxifen induced hepatotocixity manifesting as elevated liver enzymes may be found in up to 43% of the patients; however, normalization of liver enzymes is usually complete within 6 months of treatment discontinuation , 57. SevFactors associated with the development of tamoxifen induced liver injury are shared with traditional NAFLD risk factors and include glucose intolerance and diabetes, obesity, hyperlipidemia, and hypertension . GeneticIn vitro HepG2 human hepatoma cell line treated with tamoxifen showed induced hepatocyte steatosis and increased hepatocyte triglyceride concentration. A possible mechanism of tamoxifen induced hepatic steatosis was via enhancement of fatty acid synthesis through the upregulation of SREBP-1c and its downstream lipogenesis target genes. Furthermore triglyceride accumulation stimulated microsomal triglyceride transfer protein (MTP) expression that was associated with VLDL assembly and secretion . In vivoβ-oxidation and respiration in part by inhibiting CPT I, the rate-limiting enzyme of mitochondrial fatty acid β-oxidation, and also inhibits topoisomerases thus progressively depleting hepatic mitochondrial DNA [The role of the mitochondria in tamoxifen induced hapatotoxicity is controversial. Rodent studies have indicated that tamoxifen accumulates in mitochondria where it impairs rial DNA . In contrial DNA . Likewisrial DNA , 34. Oxirial DNA , 44.Valproic acid (2-n-propylpentanoic acid) is widely used as a broad-spectrum antiepileptic drug. A wide range of adverse effects have been reported in connection with VPA, among which its potential hepatotoxicity is a major concern. Serum aminotransferases are commonly elevated in up to 44% of patients on VPA therapy; however, this elevation is generally mild (1–3x ULN) and does not involve a rise in serum bilirubin. Patients are mostly asymptomatic or suffer mild symptoms . This presentation of VPA hepatotoxicity is frequently transient and resolves with dose reduction or drug discontinuation . Incidenβ-oxidation [In recent years the association between VPA treatment and NAFLD has been further studied and it is well recognized that long-term use of this drug can induce features of the MS consistent of substantial weight gain, insulin resistance, and lipid abnormalities . Hepaticxidation .β-oxidation, which typically accounts for about 40% of its metabolism [β-oxidation plays a key role in VPA induced hepatic toxicity. The two main mechanisms involve mitochondrial dysfunction and oxidative stress. Formation of valproyl-CoA causes depletion of intramitochondrial CoA affecting fatty acid β-oxidation, impairment of ATP production, and inhibition of CPT1 as well as depletion of carnitine stores [Other mechanisms of VPA induced hepatic steatosis are possibly related to the drug's metabolism, as VPA is primarily metabolized in the liver. One of the major metabolic pathways, accounting for 30–50% of VPA metabolism, is conjugation, predominantly with glucuronic acid but also other minor conjugation reactions with glutathione, carnitine, coenzyme A, and other amino acids, while the other main metabolic pathway is mitochondrial oxidative reactions, especially tabolism . As in oe stores , 115. Ane stores , 46. Inte stores .Antiretroviral therapy (ART) has markedly increased life expectancy in patients with the human immunodeficiency virus (HIV). ART regimens usually consist of NRTIs, usually as paired agents, with at least one other antiretroviral drug with a different mechanism of action such as a protease inhibitor (PI), and/or a nonnucleoside reverse transcriptase inhibitor (NNRTI).Elevated liver enzymes are common among HIV patients receiving ART with an estimated prevalence of 20–40%. In patients with HBV/HCV coinfection it can be above 60%, with severe hepatotoxicity in about 10% of the patients . All antγ, the enzyme responsible for the replication of mitochondrial DNA (mtDNA); impairment of mitochondrial function causes the release of cellular lactate and defective liver fatty acid β-oxidation and may result in a spectrum of injuries ranging from potentially fatal hepatotoxicity and lactic acidosis to various degrees of hepatic marcovesicular and microvesicular steatosis [Steatohepatits related to ART results from multiple mechanisms. The occurrence of NAFLD among these patients may be attributed to metabolic abnormalities commonly found among HIV patients receiving ART, in particular NRTI-PI combinations, and include subcutaneous lipoatrophy and accumulation of central fat, dyslipidemia, and insulin resistance . Other mteatosis , 124. Miteatosis , 126. Anteatosis . Hepatocteatosis .Regimens containing FOLFOX  and FOLFIRI (5-FU + folinic acid + IRI) are commonly used treatments for colorectal cancer . Chemothβ-oxidation and production of ROS [5-FU is a fluoropyrimidine antimetabolite that causes irreversible inhibition of thymidylate synthase. Although little is known about the mechanisms of hepatotoxicity associated with 5-FU, it is well recognized that mild and reversible hepatic enzymes abnormalities are common in patients treated with 5-FU and may be found in up to 40% of the patients . Studiesn of ROS .IRI is a topoisomerase-1 inhibitor and a very active antineoplastic drug. Its use is associated with 5 times increased risk of CASH, found in 20% of treated patients, and 10 times increased 90-day mortality, specifically death from postoperative hepatic failure . It has Other metabolic risk factors may play an important role in the development of steatosis or steatohepatitis in these patients, both as an underling condition or as part of the “multiple-hit” process. This notion is strengthened by findings of 208 patients with colorectal liver metastasis that underwent resection in whom BMI was the strongest predictor of steatosis and steatohepatitis, independent of the use of preoperative chemotherapy. Furthermore, among patients receiving preoperative chemotherapy, BMI was a predictor of chemotherapy liver injury with a 39% rate of steatosis or steatohepatitis among obese patients . InteresThe data regarding the prognostic effect of chemotherapy-induced steatosis or steatohepatitis is conflicting. While some studies have demonstrated reduced survival in patients with CASH , others Hepatic steatosis is a rare form of DILI. DIS/DISH may present as macrovesicular steatosis, microvesicular steatosis, and steatohepatitis depending on the particular mechanism of a specific lipotoxic drug and may show variable latency prior to the occurrence of a clinically apparent injury. The pathogenesis of DISH from most drugs is complex involving major biological pathways of hepatocyte lipid metabolism. The relationship between DIS and “primary” NAFLD is of particular importance, as some drugs  clearly precipitate the induction of traditional NAFLD risk factors via their metabolic effect, while on the other hand it appears that preexisting steatosis may render the hepatocytes vulnerable to drug insults, may alter hepatic drug metabolism, and may eventually exacerbate existing damage.The diagnosis of DIS/DISH requires a high index of suspicion because it represents a rare cause of steatosis. Thus a patient with DISH may erroneously be diagnosed as having “primary” NAFLD.The potential reversibility of the initial liver injury, preventing the development of severe liver injury and ultimately liver fibrosis, underlines the importance of early recognition of this unique hepatotoxic effect. Better understanding of the molecular mechanisms involved and potential risk factors, as well as the intricate relationship with metabolic NAFLD, may offer potential clues in identifying patients more susceptible to this type of drug injury and better understanding of the mechanisms leading to metabolic steatohepatitis.


The West African Ebola (EBV) outbreak that spanned two years infected 30,000 people with case mortality rate of 36%. Of the 11,000 deaths, 500 (4.5%) were Health Care Workers (HCWs). Majority of HCWs deaths occurred in the initial months of the outbreak depleting the active HCWs workforce in affected countries . RapiditThis outbreak had significant public health impact because for the first time (despite EBV recurring in African countries since the early seventies), the outbreak occurred in an urban setting leading to the rapid dissemination. Factors documented from previous studies that contributed to the epidemic include environmental features, poor human resources, infrastructure deficits, and residence in areas of high poverty . MultinaIn Africa, the health infrastructure is fragile (except in a few countries) and generally is not capable of handling an unexpected or sudden increase in surge capacity such as experienced in the EBV epidemic. The combination of high African HCWs EBV related mortality and previous low estimates of HCW density  and 12.4 nurses and midwives per 10,000 population (range of 1.7-2.7 in the affected countries))  validateThe recently released report of the Harvard-LSHTM Independent Panel on the Global Response to Ebola by Suerie Moon et al. illuminates several essential reforms necessary to prevent future epidemics and consequently pandemics These reforms target the following three areas: 1) Prevention, 2) Response of major disease outbreaks, and 3) Research which encompasses production and sharing of data, knowledge and technology . AbilityAs health care professionals from developing countries, we ask our governments to invest in the core components of disaster preparedness and capacity building so that countries are self-sufficient and prepared to respond promptly in future responses instead of waiting for external help. The return of investment (ROI) in public health infrastructure strengthening include accelerated economic development, prompt response capability, improvement in life expectancy, increase economic activity and growth, saving of lives of millions of Africans and prevention of disabilities. It is documented that healthy individuals are more productive, earn more, save more, invest more, consume more, and work longer, leading to a positive impact on GDP of a nation. These ROI factors should motivate African governments to start planning and re-enforcing health systems now.