They demonstrated that Sox2 helps maintain the cortical identity of NSC ex vivo as evidenced by the expression of Pax6 and the nestin-linked epitope RC2. Only the Sox2+ cells isolated from Soxβ−geo/+ neurospheres were capable of generating secondary neurospheres while the Sox2− fraction could not, confirming that only the Sox2+ cells can self-renew in vitro. Sox2+ neurospheres differentiated towards Tuj1+ cells with long axons like cortical neurons, while wild type and Soxβ−geo/+ neurospheres derived-neurons developed short axons, showing that these cells had distinct developmental and differentiation potential.In 2011, Remboutsika et al.  are multipotent stem cells generated during development when the neural plate folds to form the neural tube. NSCs give rise to all cells of the central nervous system. At the beginning of neurogenesis, neuroephitelial cells are replaced by radial glia, cells that can divide asymmetrically and differentiate into neurons, astrocytes and oligodendrocytes    are expressed widely in the central nervous system, and are implicated in neural development  by transducing Sox2 alone or in combination with other transcription factors  from human cord blood (CB) derived CD133The study by Remboutsika et al. was one of the first to demonstrate the importance of Sox2 in maintaining NSC undifferentiated, creating homogenous neurospheres, containing cells with the same spatiotemporal identity. This study and many others subsequently have detailed the role of Sox2 in establishing (through differentiation from ESC or de-differentiation from somatic cells) and maintaining cortical NSC features.Over the last 20 years, studies have been mostly focused on understanding which molecules and signaling pathways regulate differentiation, proliferation and migration of NSCs with an ultimate goal of applying cell replacement therapy for treatment of chronic neurologic diseases. The translational phase of this remarkable research has already begun. Several phase I/II clinical trials have been performed using purified NSCs for the treatment of amyothrophic lateral sclerosis, stroke, Batten disease, Pelizaeus-Merzbacher disease, high-grade gliomas (Trounson et al.,


We report a unique case of an adverse interaction between the oxazolidinone antibiotic linezolid, the tricyclic antidepressant amitriptyline and the opioid analgesic fentanyl in a 68-year-old woman with advanced ischemic peripheral arterial disease and sepsis, under empirical antibiotic treatment. We also summarize the current relevant literature as identified via PubMed, EMBASE, and PsycINFO as well as reference sections of selected articles. She was admitted to our hospital due to sepsis and possible diagnosis of infection of the ischemic left foot. Anamnestic history included advanced peripheral ischemic disease, diabetes mellitus type II, arterial hypertension, and major depression. The patient was receiving treatment with fentanyl transdermal patch 25 μg/h every 72 h since 10 days and amitriptyline 25 mg BD for depression. The low dose of amitriptyline 25 mg BD was maintained due to its antidepressant [Ms. B, a 68-year-old woman, presented at our outpatient clinic with intense lower foot pain and fever since a week. Clinical examination revealed the 3 first phalanges of the left foot painful, cyanotic, and swollen in the absence of palpable pulsus at the ventral and dorsal tibial arteries with concomitant fever 38.5°C) and tachycardia 110 bpm). A complete blood count showed elevated total number of white blood cells , a drug category that is believed to act through boosting of serotonin and norepinephrine neurotransmission via blockade of serotonin and norepinephrine reuptake pumps , as wellμ-opioid receptors (mu receptors) resulting in inhibition of pain neurotransmission [Fentanyl is an synthetic opioid analgesic, which is characterised by high lipid solubility and therefore it easily penetrates the central nervous system (CNS), where it acts through binding to smission –22. Fentsmission . The nonsmission . Interessmission , which hLinezolid is an oxazolidinone category antibiotic that is believed to act through early inhibition of protein synthesis via binding to the 23S portion of the 50S ribosomal bacterial rRNA subunit , 27 indu1 histamine receptor antagonist as well as a nonspecific serotonin receptor antagonist [Serotonin syndrome usually consisted of a constellation of neurological and mental state symptoms and commonly diagnosed according to the widely accepted criteria of Sternbach and/or Hunter , 17, 30,tagonist  which maThe development of the serotonin syndrome has been reported as an interaction of linezolid plus almost every category of antidepressant medication. Interactions have been found with the selective serotonin reuptake inhibitors (SSRIs) as citalopram , 33–39, To the knowledge of the authors, this is the first case of serotonin syndrome reported in a patient receiving this common three-drug combination.Fentanyl is primarily metabolized via CYP-P450-3A4 isoenzyme system by oxidative dealkylation to the main metabolite norfentanyl which is inactive .Amitriptyline is metabolised via CYP-P450-1A2 isoenzyme by demethylation to form the active metabolite nortriptyline  and via Linezolide is metabolised via oxidation procedure in a way independent of cytochrome P450 (CYP-450); consequently there is no possible pharmacokinetic mechanism of interaction between linezolide and other medication metabolized through CYP450 pathways .Also, the serotonin syndrome pathophysiological mechanism does not include idiosyncratic, neither idiopathic nor pharmacokinetic drug reactions, but it is considered to be a predictable and preventable pharmacodynamic consequence of the excess of serotonergic agonism in CNS and peripheral serotonergic receptors .We suggest that physicians avoid use of linezolid in patients receiving combination of amitriptyline and fentanyl due to possible serotonin syndrome induction.


Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions  at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated  Recently, researchers have been focusing their efforts on the study of the non coding part of the human DNA and, in particular, on its predicted role in the regulation of gene expression cis-regulatory elements.A database of human and mouse noncoding fragments with a gene enhancer activity has been developed HBB) enhancers SHH) limb-enhancer point mutations can cause preaxial polydactyly RET proto-oncogene enhancer variant Chromosomal rearrangements or deletions may lead to a disturbance of long-range control and, as a consequence, to pathological conditions While impressive results have been obtained in the discovery and mapping of tissue specific enhancers Recently, Array-Based Comparative Genomic Hybridization (aCGH) has been found to be able to detect causative alterations in patients with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), and multiple congenital anomalies (MCA) in between 11% and 15% of examined cases Moreover, enhancers have been demonstrated to be located near genes active during development CNVs encompassing enhancer noncoding sequences could in this way affect target gene expression, causing human disorders.Enhancer Chip custom array that is described below.To characterize CNVs overlapping VISTA enhancer loci, we have compared the coordinates of human VISTA enhancer loci with CNVs deposited to the Database of Genomic Variants (DGV) and Indels  and with two highly polymorphic sets of deleted and duplicated regions. We have shown that highly polymorphic CNVs are under negative selection at VISTA enhancer loci, suggesting that copy number variable enhancers could represent functional variants. Array-CGH represents a reasonable, cost-effective instrument to investigate multiple DNA regions. To confirm the functional relevance of enhancers and to verify whether dysmorphic features or mental retardation could be associated with rare or private duplications and deletions in these elements, we have designed the www.enhancer.lbl.gov/) http://projects.tcag.ca/variation) Genomic coordinates of 1,275 human enhancer loci, 67,419 CNVs and 34,186 Indels were downloaded from Vista enhancer database (http://decipher.sanger.ac.uk) The coordinates of Genomic microduplication and microdeletion syndromes were downloaded from DECIPHER database v5.1  and searching for 24.3 million computationally validated CGH oligonucleotide probe database.Probe selection was performed using the web-based Agilent eArray database version 5.0  and the Human CGH 1K Agilent Replicate Probe Group  were also included into design.Enhancer Chip array was designed to provide redundancy with high sensitivity and specificity for detection of clinically significant unbalanced chromosomal abnormalities, while minimizing detection of non pathogenic CNVs or CNVs of uncertain significance. To this aim, we selected 322 diseases related to development delay or congenital physical anomalies, listed in Enhancer Chip we designed 25,000 probes to cover all the genome (backbone) with an average spacing of 100 Kb and a resolution of 300 Kb (a). We also added 18,000 probes to cover regions of interest  with an average resolution of about 40 Kb (b and c). This design fulfills these suggestions. Moreover, 7,790 probes were designed to investigate VISTA enhancer loci. The number of probes on each element depends from enhancer length, with at least 3 probes for each enhancer and an average spacing of 238 nt (d). The residual free space on the array  was randomly filled with probes from the commercially available Agilent Human Genome 44K array CGH.For f 300 Kb a. We alut 40 Kb b and c.f 238 nt d. The rAnonymous blood samples were collected from healthy, unrelated Italian individuals. All subjects studied entered the diagnostic centers of Naples or Rome and signed an appropriate consent form for genetic testing as well as forms related to privacy of data. Approval for the study was obtained by the Seconda Università di Napoli Ethics Committee (prot. 862/08). Genomic DNA was extracted using standard procedures.After amelogenin-based sex confirmation by PCR, concentration and purity of DNA samples were assessed using Nanodrop ND 1000  by evaluating the A260/A280 and A260/A230 ratios to exclude contaminating proteins or other organic compounds. After dilution to a final concentration of 100 ng/µL, six sex-matched DNA samples were pooled together and used as male or female reference DNA samples in array CGH experiments.For validation experiments, we utilized DNA samples from patients in which genomic deletions or duplications were previously detected with alternative diagnostic methods. In addition, we also utilized DNA samples from patients with development delay and/or congenital anomalies but without a molecular diagnosis. Further DNA samples from healthy individuals were also analyzed.In particular, forty DNA samples were collected at Università Cattolica del Sacro Cuore (Rome) and seven at the Seconda Università di Napoli (Naples).For all DNA samples, sex was confirmed by amelogenin-based PCR assay. Concentration and purity of each DNA sample were assessed using Nanodrop ND 1000 . After dilution to a final concentration of 100 ng/µL, all DNA samples were blindly tested in array CGH experiments.http://projects.tcag.ca/variation/) and with the Decipher database (http://decipher.sanger.ac.uk/) to facilitate interpretation.Labeling and hybridization were performed according to the manufacturer’s specifications . Scanned array images were analyzed with Feature Extraction software . Graphical overview and analysis of data were obtained using DNA Analytics as part of Agilent Genomic Workbench software , evaluating the quality of each test with the quality control (QC) metrics generated with DNA Analytics software. For identifying duplications and deletions we used the standard set-up of the Aberration Detection Method 2 (ADM-2) algorithm for the data that passed QC metrics testing. The threshold applied to the algorithm was empirically chosen for each test. An aberration filter was set to select aberrant regions with at least 3 targets showing the same direction in copy-number change and to exclude aberrant regions if the average log2 ratio within the region was less than the value of Derivative Log Ratio spread (DLRSpread). Variants not known to be pathogenic were compared with the Database of Genomic Variants . The PCR conditions were the following: 96°C×1 min; 30 cycles of 96°C×30 s, 62°C×1 min and 68°C×4 min plus 5 s/cycle, with 72°C×12 min as final step.PCR products were double-strand sequenced using BigDye Terminator sequencing chemistry  and analyzed on an ABI 3130×L automatic DNA sequencer .http://projects.tcag.ca/variation). “Polymorphic-CNP” To verify whether the characterized CNVs overlap the VISTA enhancers, we compared the positions of their loci with three sets of CNVs: ‘DGV-deposited’  We identified 326 VISTA enhancer loci localized in “DGV-deposited” CNVs  and 3 inb and c) shows that the fractions of the enhancer loci (0.19% and 0.33%) covered by the two sets of “polymorphic” CNVs are at least five times lower than the fractions of the covered genome (1.05% and 2.10%). Also the 34,186 small Indels deposited in the DGV (with a genomic coverage of 0.3%) overlap the VISTA enhancer loci two-fold lower than expected  or whether the CNVs were underrepresented at these loci . To test these hypotheses, we compared fractions of the enhancer loci and fractions of the genome covered by the differentially defined CNV regions. These data demonstrate a negative selection of highly polymorphic CNVs and of small Indels at enhancer loci.The CNV purification effect is less strong if one compares the fraction of the enhancers (24%) covered by the “DGV-deposited” CNVs with the fraction of the genome covered by those CNVs (30%)a. As alFinally, not only common CNVs but also CNVs implicated in specific diseases can affect enhancer loci and thus can play an important role in pathogenesis. We have identified 77 enhancers  located Enhancer Chip validation, we analyzed 47 blind DNA samples (Enhancer Chip and two (27 and 28) from healthy individuals were also included as negative controls.For the  samples . These iEnhancer Chip detected no clinically relevantgenomic imbalances in any of the 4 negative controls and confirmed the molecular diagnosis in 31 out of 31 samples, that had previously been analyzed (KCNQ1gene (MIM 607542) and sample 20 showed a heterozygous intragenic deletion of the FGF14 gene (MIM 601515). These variations, undetectable by Agilent 44K array, had been identified by using a 244k Agilent array and confirmed by using the Enhancer Chip. We also revealed a NF1 deletion (118) detected by MLPA and a 6p duplication (IM and IF) detected by G-banding. In these cases our array proved to be a valuable tool to define breakpoint boundaries and the extension of the aberrations (data not shown), demonstrating that it is a good alternative to commercial all-genome platforms.The analyzed . Sample Enhancer Chip array was able to detect new small copy-number changes, classified as variants of uncertain clinical significance (In 2 out of 12 patients (17–21) with only a clinical diagnosis, the ificance .Enhancer Chip detected 3 deletions  and neural tube . None ofTo genotype these 3 new deletions, we developed a triplex PCR assay d. The f“Polymorphic” CNVs show some purifying effects at VISTA enhancer loci, as already seen for miRNA genes PAX6 transcription unit SOX9 gene, leading to campomelic dysplasia Although it is very difficult to predict how many highly polymorphic CNV-enhancers are present in the human genome, they are potential functional variants and could represent candidate loci, especially if located in regions implicated in diseases by linkage or association studies. A previous study The lack of precise data on CNV-enhancers, their polymorphisms and their putative pathogenic role is mostly due to the absence of appropriate methods for their identification and characterization in a large number of samples. A simple and inexpensive method that enables an accurate characterization of several CNVs of interest has never been proposed up to now, hampering the analyses of CNVs and their correlation with the phenotype.Enhancer Chip, a straightforward and cost-effective assay with research purposes. A custom-designed array represents an important diagnostic instrument Enhancer Chip has allowed the detection of three new and supposedly rare deletions covering enhancers. These three enhancers have been demonstrated to be active during embryonic development even if nothing is known about their gene targets. Our samples show heterozygous deletions on these elements. It would be interesting to evaluate the expression levels of their putative targets, once identified. Probably none of the three alterations is directly responsible for the observed phenotypes in these patients. Further studies on a large population could help to identify the phenotypic effects of copy variable enhancers.In order to characterize all CNV-enhancers and eventually identify cryptic disease-associated deletions or duplications, we have developed our Other recent papers have demonstrated the utility of an exon-targeted oligonucleotide array  to detect intragenic copy-number changes in patients with various clinical phenotypes Although we have focused on distant-acting enhancers, there are other categories of functional elements in the non-coding portion of the genome , which are also crucial targets for a large scale study of regulatory elements in the human genome. To identify deletions or duplications in all these elements, we are going to develop a new customized CGH-array, to investigate these regulatory regions.Figure S1Enhancer Chip design.Probe selection strategy for (DOC)Click here for additional data file.Table S1Bioinformatic data analysed.(DOC)Click here for additional data file.Table S2Enhancer Chip array.Disease loci investigated by (DOC)Click here for additional data file.Table S3DVG variations.VISTA enhancer loci localized in (DOC)Click here for additional data file.Table S4VISTA enhancer loci localized in Decipher Syndromes Regions.(DOC)Click here for additional data file.Table S5Enhancer Chip.CNVs of uncertain significance detected by (DOC)Click here for additional data file.


Cdon mutant mice have a largely subthreshold defect in SHH signaling, rendering them sensitive to a wide spectrum of HPE phenotypes by additional hits that are themselves insufficient to produce HPE, including transient in utero exposure to ethanol. These variable HPE phenotypes may arise in embryos that fail to reach a threshold level of SHH signaling at a specific developmental stage. To provide evidence for this possibility, here we tested the effect of removing one copy of the negative regulator Ptch1 from −/−Cdon embryos and compared their response to ethanol with that of −/−;Ptch1+/+Cdon embryos. Ptch1 heterozygosity decreased the penetrance of HPE in this system by >75%. The major effect of reduced Ptch1 gene dosage was on penetrance, as those −/−;Ptch1+/−Cdon embryos that displayed HPE did not show major differences in phenotype from −/−;Ptch1+/+Cdon embryos with ethanol-induced HPE. Our findings are consistent with the notion that even in an etiologically complex model of HPE, the level of SHH pathway activity is rate-limiting. Furthermore, the clinical outcome of an individual carrying a SHH pathway mutation will likely reflect the sum effect of both deleterious and protective modifier alleles and their interaction with non-genetic risk factors like fetal alcohol exposure.Holoprosencephaly (HPE) is a commonly occurring developmental defect in which midline patterning of the forebrain and midface is disrupted. Sonic hedgehog (SHH) signaling is required during multiple stages of rostroventral midline development, and heterozygous mutations in SHH pathway components are associated with HPE. However, clinical presentation of HPE is highly variable, and carriers of heterozygous mutations often lack apparent defects. It is therefore thought that such mutations must interact with more common modifiers, genetic and/or environmental. We have modeled this scenario in mice.  Holoprosencephaly (HPE), a common developmental defect of the forebrain and midface, results from disruption in patterning of the rostroventral midline Shh expression in a successive midline structure. SHH produced by the prechordal mesendoderm (PCM) is a key early step in development of the midline of the forebrain and midface Shh itself) in the rostral diencephalon ventral midline of the developing forebrain, which in turn leads to Shh expression in the ventral telencephalon Shh in the ectoderm of the frontonasal and maxillary processes of the developing face, helping to pattern the craniofacial midline Experiments with mice and other vertebrate model organisms indicate that development of the rostroventral midline occurs by a progressive patterning mechanism whereby SHH produced by one midline structure induces Clinical presentation of HPE is extremely variable, and the range of defects falls within a continuum known as the HPE spectrum Cdon mutant mice as such a model. Mice lacking CDON on a 129S6 genetic background have a largely subthreshold defect in SHH signaling that renders them sensitive to induction of HPE by second hits that are, themselves, insufficient to produce HPE  A full spectrum of HPE phenotypes is seen both in sporadic and familial cases −/−Cdon embryos −/−Cdon mice ranged from alobar HPE with an undivided eye field, to lobar HPE with single nostril, to microform HPE and defects in palatogenesis −/−Cdon mice is as follows: ethanol initiates defects in midline patterning specifically in genetically sensitized embryos; the variable severity of the HPE phenotype may arise as a consequence of individual embryos failing to reach a threshold level of SHH signaling at specific developmental stages, the stage of the deficit occurring stochastically and dictating phenotypic severity Ptch1 from 129S6.−/−Cdon embryos and assessed ethanol-induced production of HPE. As predicted by the model, Ptch1 heterozygosity effectively rescued such embryos.We recently reported use of these mice to develop a model of gene-environment interaction in HPE. In utero ethanol exposure at E7.0 had little or no effect on wild type 129S6 embryos, but produced a wide range of HPE phenotypes with >75% penetrance in Cdon or in utero ethanol exposure in 129S6 mice gave little or no phenotype individually, but together produced defects in midline patterning, inhibition of SHH signaling in the developing forebrain, and a broad spectrum of HPE phenotypes −/−Cdon embryos displayed severe phenotypes that included alobar HPE and failure to divide the eye field. These embryos died in utero and were resorbed by E11.0. When analyzed at E14.0, ∼70% of 129S6.−/−Cdon embryos displayed phenotypes ranging from lobar through microform HPE, and variously included single nostril, defective palatogenesis, diminished nasal septal cartilage, and rudimentary vomeronasal organs. In asking whether removal of one copy of Ptch1 could rescue HPE in these animals, we analyzed E14.0 embryos to take advantage of the high penetrance of the phenotypes observed at this stage.Loss of Cdon mutation +/−Ptch1 line by back-crossing. These mice were bred with 129S6.+/−Cdon mice to generate double heterozygotes. Various offspring of intercrosses of these animals were further crossed, and pregnant females received IP injections of ethanol or saline control at E7.0 and four hours later . As expected −/−;Ptch1+/+Cdon embryos displayed lobar HPE, whereas one of six ethanol-treated −/−;Ptch1+/−Cdon embryos did so -stained sections of E14.0 embryos were examined to identify defects in forebrain and midline craniofacial structures. We compared four ethanol-treated s did so , Table 1s did so , Table 1ructures , Table 1l tissue , Table 1henotype , Table 1−/−;Ptch1+/−Cdon embryos, relative to similarly treated −/−;Ptch1+/+Cdon embryos. However, in most cases, embryos of either genotype that displayed external HPE features showed roughly similar defects in forebrain and midfacial regions. These results suggest that the major effect of removing one copy of Ptch1 is to decrease the penetrance of HPE in ethanol-treated −/−Cdon mice, rather than to selectively alter the severity of some HPE phenotypes and not others.Taken together, there may be a small diminution in HPE phenotypic expressivity in ethanol-treated Shh and a direct SHH pathway target gene, Nkx2.1, in the forebrain by whole-mount in situ hybridization at E10.25. We previously found that about half of the ethanol-treated −/−Cdon embryos at this stage had significantly reduced expression of these genes in the developing ventral telencephalon −/−;Ptch1+/+Cdon embryos had an obvious diminution in Shh expression and five of nine embryos of this genotype had an obvious reduction in Nkx2.1 expression  (We next tested the expression of pression . In cont (n = 7)  and S3.SHH expression or pathway activity in the early midline or forebrain HPE is etiologically complex, involving interactions between the timing and strength of developmental signaling pathways, genetic variation, and exposure to environmental agents −/−Cdon animals have only a substhreshold defect in SHH signaling that renders them sensitive to induction of HPE by additional insults, genetic or environmental, that are themselves insufficient to produce HPE Mouse models of HPE provide support for this model. We have shown that mice lacking the SHH co-receptor CDON display HPE with strain-dependent penetrance and severity, consistent with an important role for silent modifier genes in grading the spectrum of phenotypes −/−Cdon embryos, but is not teratogenic to 129S6.+/+Cdon embryos Epidemiological evidence suggests that fetal alcohol exposure raises the likelihood of HPE Ptch1 from −/−Cdon embryos and compared their response to ethanol-induced HPE with that of −/−;Ptch1+/+Cdon embryos. As predicted, reduced gene dosage of Ptch1 substantially reduced the penetrance of HPE in this system, from ∼65% in −/−;Ptch1+/+Cdon embryos to ∼15% in −/−;Ptch1+/−Cdon embryos, a reduction of >75%. The major effect of Ptch1 heterozygosity in this system was on penetrance, as those −/−;Ptch1+/−Cdon embryos that displayed HPE did not show major differences in phenotypic spectrum or selectivity from −/−;Ptch1+/+Cdon embryos with ethanol-induced HPE.This hypothesis argues that the level of SHH pathway signaling activity is an important determinant of phenotype. To provide experimental evidence for this possibility we tested the effect of removing one copy of the negative regulator Ptch1 haploinsufficiency occur? The PCM is required for induction of the ventral forebrain and subsequent partitioning of the forebrain and eye field, as well as patterning of the midfacial midline Cdon is expressed in the PCM but not in the ventral forebrain, even though the latter structure is affected in −/−Cdon mice; this observation and additional results argue that loss of Cdon function in the PCM is the most likely cause of HPE in such mice Cdon, Ptch1 is expressed in the PCM −/−;Ptch1+/−Cdon embryos may occur as a consequence of enhanced SHH signaling function in the PCM itself. Alternatively, enhanced responsiveness to PCM-derived SHH may occur in the +/−Ptch1 ventral forebrain, exclusive of co-expression of Cdon and Ptch1. Finally, Cdon and Ptch1 are also co-expressed in the developing facial mesenchyme, and restoration of SHH signaling there may contribute to rescue of facial midline phenotypes. These possibilities are not mutually exclusive, and conditional mutagenesis will be required to address this issue. The mechanism whereby ethanol synergizes with Cdon mutation to inhibit SHH signaling is also not fully resolved but must involve early patterning events between E7.0 – E8.0, as ethanol is no longer detectable by E8.0 At what developmental stage(s) might the rescue of HPE by Ptch1 rescues HPE phenotypes in ethanol-treated −/−Cdon embryos is consistent with PTCH1 functioning as a negative regulator of SHH signaling. However, heterozygous, germline missense mutations in PTCH1 have been identified in HPE patients PTCH1 mutations found in HPE are complete loss-of-function mutations. In fact, heterozygous, germline loss-of-function mutations in PTCH1 result in a different disorder, Basal Cell Nevus Syndrome  Δloop2 variant Studies on individuals with HPE and animal models conclusively demonstrate that HPE is associated with loss of SHH pathway function. The fact that haploinsufficiency for Our findings are consistent with the notion that even in an etiologically complex animal model of HPE (one that models the complexities of human HPE), the level of SHH pathway activity is rate-limiting. These findings are highly relevant in the emerging era of human genomics and soon-to-be widespread personal access to genome sequences. Systematic analyses of common variants in SHH pathway components may reveal not only alleles that predispose offspring of heterozygous HPE mutation carriers to severe phenotypes but also alleles with protective effects. With a core database of variants – which will need to be functionally validated – it may be possible to predict the sum effect of deleterious and beneficial alleles on penetrance and expressivity of bona fide mutations and, conceivably, their interaction with non-genetic risk factors like fetal alcohol exposure.All animal work was approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC). Our animal facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).tm1Mps/JPtch1 mice +/−Ptch1 mice were crossed with 129S6.+/−Cdon animals to generate 129S6.+/−;Ptch1+/−Cdon double mutant mice. These animals were then used to generate −/−;Ptch1+/−Cdon, +/−;Ptch1+/−Cdon, and +/−;Ptch1+/+Cdon mice, which were then variously crossed for ethanol treatment as previously described STOCK.Embryos were processed and analyzed as previously described Figure S1In utero exposure to saline does not induce external or forebrain features of HPE. (A–D) Frontal views of E14.0 embryos. (E–H) H&E-stained coronal sections of E14.0 embryos. This figure serves as a control for (TIF)Click here for additional data file.Figure S2In utero exposure to saline does not induce defects in palatogenesis or development of the midfacial midline (A–L) H&E-stained coronal sections of E14.0 embryos. This figure serves as a control for (TIF)Click here for additional data file.Figure S3In utero exposure to saline does not alter expression of Shh or Nkx2.1. Whole mount in situ hybridization analyses of Shh (A–D) and Nkx2.1 (E–H) expression in E10.25 (31–35 somites) saline-treated embryos of the indicated genotype. This figure serves as a control for (TIF)Click here for additional data file.


The nonreceptor protein tyrosine kinase Src plays a crucial role in the signal transduction pathways involved in cell division, motility, adhesion, and survival in both normal and cancer cells. Although the Src family kinases (SFKs) are activated in various types of cancers, the exact mechanisms through which they contribute to the progression of individual tumors remain to be defined. The activation of Src in human cancers may occur through a variety of mechanisms that include domain interaction and structural remodeling in response to various activators or upstream kinases and phosphatastes. Because of Src's prominent roles in invasion and tumor progression, epithelial-to-mesenchymal transition, angiogenesis, and the development of metastasis, Src is a promising target for cancer therapy. Several small molecule inhibitors of Src are currently being investigated in clinical trials. In this article, we will summarize the mechanisms regulating Src kinase activity in normal and cancer cells and discuss the status of Src inhibitor development against various types of cancers. Following phosphorylation by Src, Cbp can bind to the SH2 domain of Csk, thus allowing its recruitment to the plasma membrane where active Src resides. This creates a negative regulatory loop in which Cbp mediates the cross-linking of active Src with its suppressor, Csk . An indeα,  PTPγ, SHP-1 and -2, and PTP1B. PTPα is ubiquitously expressed and enriched in brain tissue [α-overexpressing cells [α also can dephosphorylate Src in A431 cell lines and cause enhancements in cell adhesion [α acts as an activator or repressor of Src molecules. Antisense studies of PTPα in 3T3-L1 adipocytes [α−/− murine studies [α  protein in cells. Several protein tyrosine phosphatases (PTPs) are able to dephosphorylate Src Tyr530 and are responsible for the regulation of its kinase activity, such as PTPn tissue –42 and ing cells , 43. Oveadhesion , 45. A gipocytes  and PTPα studies , 48 showγ was first identified from chicken brain tissue as a homolog of CD45 capable of dephosphorylating the SFK Lck [γ can modulate Src activity in osteoclast precursor cells treated with 1,25-dihydroxyvitamine D3; there was a dramatic increase in Src kinase activity without an increase in total protein levels. This change was accompanied by a decrease in phosphorylation at Tyr530 Interestingly both PTPγ mRNA and PTPγ protein levels were upregulated upon 1,25-dihydroxyvitamine D3 treatment suggesting the possibility that PTPg might be responsible for elevated Src kinase activity [PTP SFK Lck . It is eactivity . SHP1 is another member of the protein tyrosine phosphatase protein family that is also known as PTP-1c. It is a cytosolic two-SH2 domain containing PTP expressed in epithelial and hematopoetic cells . Somani SHP2 is a cytoplasmic SH2 domain containing PTP, which is also able to dephosphorylate Tyr530 .in vitro and in vivo activation of Src kinase activity as a result of its specificity towards tyrosine residues at the C-terminal tail. Human melanocyte [Another tyrosine phosphatase known as PTP-1B  was first identified by Charbonneau et al. and first cloned and purified from human placenta –59. Latelanocyte  and sevelanocyte  have elelanocyte . Src that are truncated at codon 531 have been reported in some cases of advanced colon cancer patients [Src 531 mutation resulted in the production of a stop at codon 531, one residue beyond the regulatory Tyr530. Due to the lack of a C-terminal regulatory region, phosphorylation of Tyr530 did not result in a closed conformation and the mutated Src remained constitutively active. Rare activating mutations in patients . The SrcSrc can acts as an upstream or downstream modulator of several receptor molecules, as well as nonreceptor tyrosine kinases, which are responsible for the robustness and persistence of RTK signaling . Src actα [A variety of Src-binding proteins have been detected that compete for binding to the protein's SH domains and disturb the intramolecular interactions that allow the activation of Src kinase. v-Src cellular counterpart (c-Src) forms activated dimerized receptors via its SH2 domain binding to specific phosphotyrosine residues in the platelet-derived growth factor receptor (PDGFR) juxtamembrane region . Other rα . Recentlα . Nef [86α  are examin vivo. Selective inhibition of Src and Fyn limited EGFR-dependent tumor cell motility. Src inhibition combined with an anti-EGFR monoclonal antibody further inhibited tumor growth and increased survival in an orthotopic glioblastoma mouser model [in vitro immunocomplex kinase assay system, which suggests that Src-dependent cell survival is also regulated by c-Met receptor activation, at least in head and neck cancer cells [There is also evidence to suggest that Src cooperates with EGFR in growth signaling , 89. Srcer model . Src is er model . Recentler cells .  Another tier of Src regulation by RTKs was demonstrated by Jiang et al. who showed that EGFR, PDGFR, and fibroblast growth factor receptor (FGFR) phosphorylate Cbp upon ligand stimulation . The EGFIn a manner similar to many other signaling molecules, Src exerts its biologic action not only through its enzymatic activity and multidomain structure but also through its ability to interact with other signaling molecules in different cellular compartments . Due to The primary role of tyrosine phosphorylation is to generate docking sites for proteins containing SH2 or phosphotyrosine binding (PTB) domains, thereby promoting protein-protein interaction and the formation of the macromolecular complexes responsible for signal transduction . Many prTensins are the members of focal adhesion proteins that can serve as Src substrates. There are four members of the tensin family in mammals . Tensinsin vitro  [Previously, Davis et al. showed that Tensin-1 is Tyr phosphorylated in Src-transformed chicken embryo fibroblasts . Qian etn vitro  . They alStudies on the subcellular localization of Src reveal that it has been associated with the plasma, perinuclear, and endosomal membranes –109. AltThese data contrast with and extend previous reports of Src localization at the plasma membrane. One explanation for this discrepancy was that the biochemical fractionation techniques used in some prior studies did not differentiate between the plasma and endosomal membranes, which have similar densities and are thus likely to cofractionate . The preSrc exhibits a predominantly perinuclear pattern of expression in malignant cells in contrast to a more evenly cytoplasmic distribution in normal breast epithelial cells . The locSrc contribution to cell regulation and cancer development has been widely discussed in several review articles , 97, 138There is a large body of evidence that has demonstrated that Src kinase activity and protein levels are elevated in several cancers, including those of the colon and breast. A correlation has often been observed between increases in Src kinase activity and the progression of malignancy , 139–141Recently, Zhang et al. provided both clinical and experimental evidence that Src plays a critical role in the establishment of latent bone metastasis in breast cancer . Using ain vitro and tumor growth in vivo via its regulation of PTEN protein stability and function. Thus Rak may function as a tumor suppressor gene. Further understanding of its function may contribute to effective therapeutic approaches for both Rak- and PTEN-defective cancers [In 2009, Yim et al. showed that the ectopic expression of Rak  effectively suppressed breast cancer cell proliferation, invasion, and colony formation  cancers . G12D/Lkb1 murine model of lung tumor [G12D/Lkb1 lung tumors by inhibiting Src, with concomitant increase in the sensitivity of tumor towards PI3K-MEK inhibition. Using integrated genomic and phosphoproteomic analysis of mouse lung primary and metastatic tumors, Carretero et al. demonstrated that loss of tumor suppressor LKB1 led to the activation of Src and FAK in a Krasng tumor . Src andA large body of evidence, including that discussed above, has identified Src as a key molecule in tumor progression that can provide oncogenic signals for cell survival, EMT, mitogenesis, and invasion and angiogenesis and metastasis , 147. Duin vitro [Dasatinib suppressed invasion and induced cell cycle arrest in HNSCC cells in vitro , affectein vitro , and grein vitro  and coloin vitro  showed iin vitro , 163, anin vitro . Interesin vitro .Bosutinib showed activity against colon cancer in a murine model and was well tolerated. In cellular assays, bosutinib treatment resulted in a dose-dependent reduction in proliferation, invasion, and migration of breast cancer cells , 167. FuSaracatinib  is another ATP-competitive inhibitor of SFKs, with activity against ABL and activated mutant forms of EGFR (L858R and L861Q) . In a paIn a recent phase II trial with dasatinib as a first line of treatment for metastatic NSCLC several patients had prolonged stable disease and one patient had a near complete response that persisted 2 years after the start of therapy, suggesting that there was a subset of patients with NSCLC who benefited from Src inhibition . AnotherIn a phase II trial in 2008, Yu et al. demonstrated that dasatinib improves the overall survival in castration resistant prostate cancer . Based ohttp://www.clinicaltrial.gov/). All these therapeutic agents appear to be well tolerated and we eagerly await their detailed clinical results. M475271 is an oral inhibitor of Src and vascular endothelial growth factor receptor (VEGFR) that has shown preclinical activity in lung adenocarcinoma cell lines . AnotherOur understanding of Src structure and function, regulation, and localization has increased dramatically since its discovery. One-hundred years after the original description of Src, this protein continues to attract keen interest because of its multiplicity of actions in the molecular signaling pathways underlying developmental as well as oncogenic events. Many studies have addressed the molecular mechanisms of Src regulation in cells and tumor tissues. In order to clarify and fully elucidate the normal physiologic function of Src and other SFKs and to fully comprehend Src signaling networks in various cancers, Src interactions with specific targets or binding partners in different subcellular localization studies should be characterized in as much detail as possible. Special focus should be placed on the role of Src in bone metastasis because of the protein's role in osteoclast and osteoblast function. Moreover, preclinical reports of combination treatments involving chemotherapy, radiation therapy, and targeted therapies with a Src inhibitor warrant further investigation , 182.


In this review, we will summarize the current literature on BET family proteins, their potential as therapeutic targets in brain tumors as well as other malignancies, and the preclinical and clinical investigations that have been undertaken to date.The bromodomain and extraterminal (BET) family proteins associate with transcriptional activation through interaction with acetylated chromatin, therefore playing a key role as epigenetic regulators. BET proteins serve to regulate the expression of importance oncogenes, including those involved in apoptosis as well as cell cycle progression. Due to this potential as an epigenetic target, small molecule inhibition of BET proteins have been investigated and demonstrate promising activity in both solid and hematologic malignancies, including brain tumors. Glioblastoma multiforme (GBM), subsets of medulloblastoma, and diffuse intrinsic pontine glioma (DIPG) are types of brain tumors with dismal prognoses, and as such have been the subjects of preclinical studies using BET inhibitors both  The lysine acetylation on the N-terminals tails of histones is associated with activation of transcription through opening of chromatin architecture . This hiIn the past 10 years, research has established a compelling rationale for targeting BET proteins for their oncogenic potential. BRD4 influences mitotic progression by binding to transcriptional start sites of genes and directing post-mitotic transcription . BRD4 alEpigenetic proteins such as BET have long been considered potential therapeutic targets and as such investigators had pursed them in ligand discovery. The oncogenic potential of BET proteins was first identified in nuclear protein in testis (NUT) midline carcinoma, a highly malignant solid tumor in which oncogenesis is driven by fusion of BRD3 or BRD4 with the NUT protein.This fusion results in blockade of epithelial differentiation and promotion of tumor growth . This diFilippakopoulos et al. were the first to describe a histone binding module inhibitor. They reported JQ1, a cell-permeable small molecule that competitively binds to bromodomains with high potency and specificity. When JQ1 binds, it displaces the BRD4 fusion oncoprotein from the chromatin, thus inducing cell-cycle arrest and initiating apoptosis. The discovery of the first BET inhibitor by this group led to the development of other BET inhibitors that probe throughout the BET family . Other BET inhibitors include I-BET151/762, PF-1, and RVX-208. Long et al. used a Hedgehog-dependent report cell line (Light2 cells) to target different epigenetic modulators to find that I-BET151, a BET protein inhibitor attenuates Hedgehog activity. As therapy with I-BET151 mimicked BRD4 knockdown, their data suggests that BRD4 regulates the Hedgehog signaling pathway . I-BET76in vivo and in vitro and shown to have potent antiproliferative effects not only of NUT (nuclear protein in testis)-midline carcinoma, but also hematologic malignancies, including leukemia and multiple myeloma, and other solid tumors, including lung, thyroid, liver, colorectal, prostate, and skin cancers, neuroblastoma, sarcoma, and brain tumors [Over the past few years, these newly described small molecule BET inhibitors have been tested n tumors -34.in vitro cytotoxic response to JQ1 in a panel of primary ALL cells. This response was independent of prognostic features but did depend on high expression of MYC and coupled with transcriptional downregulation of various pro-survival pathways. JQ1 decreased c-MYC protein stability and also reduced progression of DNA replication forks. JQ1 sensitized the ALL cells to dexamethasone therapy [Pediatric malignancies for which BET inhibition has been investigated for therapeutic potential include acute lymphoblastic leukemia (ALL) and neuroblastoma. Pediatric B-precursor ALL is the most common childhood cancer and in most cases a highly curable disease. However, the treatment resistance and long-term toxic side effects of current therapies in a subset of patients pose the need for more targeted therapies, prompting the consideration of BET inhibition as a potential therapeutic approach . Ott et  therapy .in vivo models of neuroblastoma [in vitro and subcutaneous neuroblastoma xenografts in vitro. They also looked at neurofilament expression to assess whether JQ1 induced neural differentiation. They found BET inhibition to attenuate progression and promote neuronal differentiation of neuroblastoma in vitro and in vivo in mice [Neuroblastoma is the most common extracranial solid tumor in childhood. While children diagnosed at younger ages and earlier stages tend to have favorable prognosis, this diagnosis continues to carry a dismal prognosis for those diagnosed with advanced stage or relapsed disease. Many of the high-risk neuroblastoma cells are MYCN-amplified; therefore novel therapeutic strategies directed toward this target are continually being studied. Puissant et al. conducted a cell-based screen of genetically defined cancer cell lines using a prototypical BET bromodomain inhibitor to reveal a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. Neuroblastoma is frequently associated with amplification of MYCN, and bromodomain-mediated inhibition of MYCN attenuated growth and induced apoptosis, conferring a survival advantage in three blastoma . Lee et  in mice . Extensive preclinical work has been performed to determine the potential efficacy of BET in brain tumors. Glioblastoma multiforme (GBM), the most common and aggressive primary malignant brain tumor, bears a dismal prognosis and therefore presents a challenge for development of novel therapeutic strategy. In considering epigenetic proteins and their recent emergence as novel anticancer targets, several studies have looked at BET proteins as potential targets for therapy. in vivo efficacy. They found that JQ1 induced marked G1 cell-cycle arrest and apoptosis, resulted in significant changes in the expression of important GBM genes, including c-MYC, p21CIP1/WAF1, hTERT, Bcl- 2, and Bcl-xL. They also found that the efficacy of JQ1 was not compromised by Akt hyperactivation or p53/Rb inactivation, indicated that these often-mutated signaling pathways may not confer resistance to JQ1. The orthotopic GBM tumors also showed significant growth repression with JQ1. The results of these studies support the potential broad therapeutic use of BET bromodomain inhibitors in the treatment of GBM tumors [in vitro and in vivo. They found that EGFRvIII, the most common EGFR mutation, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in this SOX9, FOXG1-dependent manner, thereby identifying the role of epigenetic and transcriptional remodeling in EGFR-dependent tumorigenesis and suggesting a plausible basis for epigenetic therapy [One BET inhibitor that has been studied with GBM is JQ1. Cheng et al. assessed JQ1 in a panel of genetically heterogeneous GBM samples. They used ex vivo cultures derived from primary GBM xenograft lines and orthotopic GBM tumors to test M tumors . Liu et  therapy . in vitro and in vivo [Another inhibitor that has been evaluated in terms of efficacy in GBM tumors is I-BET 151. Pastori et al. (2014) perfomed NanoString analysis of GBM tumor samples to demonstrate the significant overexpression of BRD2 and BRD4 RNA in GBM as well as the reduced cell cycle progression with disruption of BRD4 expression. They also used cell proliferation assays of GBM cell lines and stem cells and performed xenograft experiments to determine the efficacy of I-BET151, a BET protein inhibitor. Their studies showed that treatment with I-BET151 arrests cell cycle progression, reducing GBM cell proliferation  in vivo . To unde in vivo . The resin vivo [. The preclinical data in these studies supports the pursuit of testing BET inhibitors as molecular targeted therapeutic options for patients with MYC-driven medulloblastoma.Medulloblastoma is the most common malignant brain tumor in children. The current consensus is of at least four distinct subtypes, including Wingless (WNT), Sonic Hedgehog (SHH), and groups 3 and 4 , 42. Ampin vivo . The prein vitro and in vivo [Medulloblastoma has other potential therapeutic targets for non-MYC-driven tumors. Tang et al. first described the use of BET inhibitors in Hedgehog-driven cancers, including medulloblastoma. They found medulloblastoma as well as atypical teratoid/rhabdoid tumors (ATRT), another pediatric brain tumor with non-canonical Hedgehog signaling, to respond to treatment with BET inhibitor JQ1 . As desc in vivo .Diffuse intrinsic pontine glioma (DIPG), the most common brainstem tumor of childhood, is almost uniformly fatal, and current treatment options provide very little survival advantage. The high level of MYCN in diffuse intrinsic pontine glioma (DIPG) led investigators to test sequential therapy with the bromodomain inhibitor JQ1, which targets MYCN, and the gamma-secretase inhibitor MRK003, which targets NOTCH. Taylor, et al., found that this dual targeting with JQ1 and MRK003 inhibited DIPG growth and induced apoptosis, suggesting this as a potentially effective therapeutic regimen .With the promising results of preclinical trials, there are currently several open trials with different BET inhibitors that are actively recruiting participants with various hematologic and solid malignancies. Several of these trials are open or have recruited solid tumors of all types, including brain tumors, and each of them tests a different BET inhibitor with slight variation in mechanism. JQ1 is not being tested in clinical trials due to its short half life. Open trials include a GlaxoSmithKline-sponsored phase I/II study of GSK525762 in subjects with NUT midline carcinoma and other cancers, an Incyte Corporation-sponsored phase I/II of INCB054329 in subjects with advanced malignancies, and Tensha Therapeutics phase I, multicenter, open-label study of TEN-010 for patients with advanced solid tumors. While neither of the following studies is actively recruiting, GlaxoSmithKline has a dose escalation study of GSK2820151 in subjects with advanced or recurrent solid tumors, and Bayer has a phase I study of BAY 1238097 for subjects with advanced malignancies. The only trial in which a BET inhibitor has specifically been tested in brain tumors is a phase IIa trial of OTX015, which tested dose optimization in recurrent GBM patients. This was a non-randomized, multicenter study of single agent use of the inhibitor following failure of front-line therapy; the trial was terminated after one year. There are no current open trials with BET inhibitors in pediatric brain tumors.in vivo and in vitro. There are several open early phase clinical trials that are currently testing the efficacy of these inhibitors in humans. Brain tumors, including GBM, DIPG, and subsets of medulloblastoma, bear dismal prognoses with current therapies, and while BET protein inhibitors have been positively tested in preclinical trials with these pathologies, clinical trials currently are in early stages. Future directions include more extensive preclinical trials of brain tumors with various BET protein inhibitors as they continue to be developed, as well as translation of promising results to phase I/II clinical trials.It is now apparent that BET family proteins play a critical role in transcriptional activation and have oncogenic potential. This discovery that BET proteins can act as potential therapeutic targets in cancer has led to the development of several small molecule BET protein inhibitors. Preclinical studies have shown promise in a number of hematologic and solid malignancies both


BTLA, B-and-T lymphocyte attenuator, is known as an inhibitory molecule in different immune cells. Its cytoplasmic region contains three motifs . ITIM and ITSM are inhibitory motifs that suppress T cell function upon ligation of the BTLA ligand, HVEM (Herpes virus entry mediator). However, the physiological function of Grb2 motif, known to interact with p85 PI3K, remains unclear. The aim of this study was to investigate the role of the Grb2 motif of BTLA in CD8+ TIL and murine CD8+ T cells.Our recent data have shown that a subset of CD8+BTLA-human TIL or BTLA-/- mouse T cells to uncover BTLA downstream signaling pathways and in vivo anti-tumor effect following TIL transfer.To dissect the signaling pathway of BTLA's motifs, we generated retroviral vectors containing BTLA and its mutants by substitution of tyrosine (Y) for phenylalanine (F) in either the Grb2 motif (∆Grb2) or ITIM and ITSM (∆ ITIM and ITSM). BTLA and its mutants were overexpressed in either CD8AKT and MAPK pathway was significantly suppressed in the Grb2 mutant upon HVEM ligation. Greater proliferation was pronounced in ITIM and ITSM mutant mouse T cells when stimulated with DCs pulsed with the cognate peptide, while less proliferation was observed when Grb2 was inactivated. NOD scid gamma (NSG) mice were engrafted human derived melanoma and TIL overexpressing BTLA mutants for the investigation of an anti-tumor effect. ITIM and ITSM mutant TIL exhibited better tumor burden control and were present at higher frequency in peripheral blood following adoptive transfer.Our study revealed that the Grb2 motif provided a positive signal that favors anti-tumor responses. Therefore, the strategy to inactivate ITSM and ITSM may enhance persistence following infusion and resulting in mediating tumor control.


Water diffusion within smectite clay interlayers is reduced by confinement and hence is highly determined by the interlayer spacings that are adopted during swelling. However, a molecular understanding of the short- and long-range forces governing interlayer water structure and dynamics is lacking. Using molecular dynamics simulations of water intercalated between pyrophyllite (smectite prototype) layers we provide a detailed picture of the variation of interlayered water mobility accompanying smectite expansion. Subtle changes in hydrogen bond network structure cause significant changes in water mobility that is greater for stable hydration states and reduced for intermediate separations. By studying pyrophyllite with and without external water we reveal that long-range electrostatic forces apply a restraining effect upon interlayer water mobility. Our findings are relevant for broad range of confining nanostructures with walls thin enough to permit long-range interactions that could affect the mobility of confined solvent molecules and solute species. They influence soil permeability and chemistry and have important technological roles such as substrates for catalysisi.e., 1W, 2W, 3W, …) are observed experimentally12457e.g., 1–2W, 2–3W, 3–4W, …) is a first-order phase transition243The sorption and transport properties of water in smectite system are highly correlated with its hydration and swelling behavior12The dynamics of water molecules confined between smectite layers differ from bulk water. Both simulations and experiments6911121416We used molecular dynamics (MD) simulation to predict the mobility of water molecules within two uncharged smectite layers  as a function of layer separation. The ideal pyrophyllite structure does not contain Si or Al vacancies or metal substitutions that confer a permanent charge to the layer and thus do not require the presence of interlayer cations for charge compensation. As shown by the plots of interlayer water density profiles , well-de691112Because prior work has suggested that transient states are characterized by disruption of the interlayer hydrogen bond (HB) network, we analysed the water HB pattern using a simple geometric criterion14e.g., 1W, 1–2W; The profiles of HB acceptors and donors demonstrate that the water molecules are oriented differently inside the interlayer as expansion progresses. At small separations, the HB-acceptors (water O atoms) are located closer to the clay-plates than HB-donors (water H atoms), which in turn populate the center of the interlayer . The cutoffs for the Lennard-Jones interactions and real space in Ewald summation were set to 20 Å. The structure of pyrophylliteMolecular dynamics simulations were carried out initially in the NPT ensemble at the ambient temperature and pressure for 5 ns 2121Diffusion coefficient was calculated from the mean square displacement via the Einstein relation2114How to cite this article: Zarzycki, P. and Gilbert, B. Long-Range Interactions Restrict Water Transport in Pyrophyllite Interlayers. Sci. Rep.6, 25278; doi: 10.1038/srep25278 (2016).


Insulin producing beta cell and glucagon producing alpha cells are colocalized in pancreatic islets in an arrangement that facilitates the coordinated release of the two principal hormones that regulate glucose homeostasis and prevent both hypoglycemia and diabetes. However, this intricate organization has also complicated the determination of the cellular source(s) of the expression of genes that are detected in the islet. This reflects a significant gap in our understanding of mouse islet physiology, which reduces the effectiveness by which mice model human islet disease.To overcome this challenge, we generated a bitransgenic reporter mouse that faithfully labels all beta and alpha cells in mouse islets to enable FACS-based purification and the generation of comprehensive transcriptomes of both populations. This facilitates systematic comparison across thousands of genes between the two major endocrine cell types of the islets of Langerhans whose principal hormones are of cardinal importance for glucose homeostasis. Our data leveraged against similar data for human beta cells reveal a core common beta cell transcriptome of 9900+ genes. Against the backdrop of overall similar beta cell transcriptomes, we describe marked differences in the repertoire of receptors and long non-coding RNAs between mouse and human beta cells.The comprehensive mouse alpha and beta cell transcriptomes complemented by the comparison of the global (dis)similarities between mouse and human beta cells represent invaluable resources to boost the accuracy by which rodent models offer guidance in finding cures for human diabetes.The online version of this article (doi:10.1186/1471-2164-15-620) contains supplementary material, which is available to authorized users. Neurog3) [Neurog3+ early endocrine progenitors undergo stepwise differentiation along parallel lineages to develop into mature alpha and beta cells [Pancreatic beta and alpha cells are clustered together in pancreatic islets to ensure tight coordination of the secretion of insulin and glucagon, whose opposing actions on hepatic glucose metabolism are essential for glucose homeostasis. Yet, despite the functional juxtaposition of insulin and glucagon, pancreatic beta and alpha cells derive from a common progenitor population that during embryonic development is first uniquely defined by the expression of the basic helix-loop-helix transcription factor neurogenin 3 (Neurog3) . Under tta cells –5. It fota cells , 7. Furtta cells . In contta cells . The serta cells . It follIns1) promoter and crossed it to a GFP reporter line that labels alpha cells. Bitransgenic offspring of a cross between both lines then enabled the collection of purified populations of alpha and beta cells from the same islets. These tools enabled the first transcriptome-wide comparison of murine alpha and beta cells by RNA-seq, which revealed over 2500 genes that are differentially expressed  < 0.1%) between both populations, with insulin the second-most significantly enriched in the beta cell fraction. Next generation sequencing combines the tremendous benefits in sensitivity and dynamic range of techniques traditionally used to assess expression of targeted sets of genes, such as quantitative PCR, with the scale of microarray and the distinct benefit that no a priori sequence information is required, which enables novel transcript discovery. We take advantage of our data to conduct the first transcriptome-wide assessment of the similarities and differences that exist between rodent and human beta cells. These data provide an unprecedented global view of the core beta cell transcriptome that is conserved across species, while simultaneously highlighting marked differences in the expression of receptors and associated long non-coding RNAs (lncRNAs) between beta cells of both species. An unbiased and comprehensive comparison between mouse and human beta cells is an invaluable resource that can facilitate the translation of preclinical findings in rodent models towards therapeutic strategies aimed at alleviating or curing diabetes.The intimate clustering of beta and alpha cells in pancreatic islets subserves the tight coordination of the secretion of insulin and glucagon, but has long complicated the process of obtaining highly pure populations of alpha and beta cells from isolated islets. To overcome this challenge, we have developed a reporter strain that fluorescently marks all beta cells by the nuclear expression of mCherry under control of the mouse insulin 1  enriched in beta cells. The Insulin 2 (Ins2) and Ins1 genes are the second- and third most significantly enriched genes in beta cells compared to alpha cells, after the beta cell-specific transcription factor Mafa  and the peptide hormone Urocortin3 (Ucn3)  enriched in the alpha cell fraction, including the mouse alpha cell-specific transcription factors Arx, Irx1, Mafb, as well as glucagon (Gcg) , somatostatin (Sst), peptide YY (Pyy) and islet amyloid polypeptide (Iapp), is between 6.5 and 33-fold lower than the expression of Gcg, which confirms the successful enrichment of alpha cells , glucose-6-phosphate convertase 2 (G6pc2), proprotein convertase subtilisin/kexin type 1 (Pcsk1), Ero 1-like beta (Ero1lb), and synaptotagmin-like 4  were highly significantly enriched in beta cells, while genes encoding the ATP-sensitive potassium channel and voltage-gated calcium channels were equally abundantly expressed in alpha and beta cells , best known as the principal hypothalamic initiator of the stress response, is not detectable in mouse islets  and over 10-fold difference in beta cells of one species over the other. This approach identified 725 genes that were either uniquely expressed  or significantly enriched  in mouse beta cells  and robustly (>10-fold) enriched in mouse beta cells, but still detectable in human beta cells, constitutes less than 1.5% of the common beta cell transcriptome    Figure J and cil) Figure K are robIl1r1  locus aptly recapitulates all aspects of our study. Il1r1 is the most abundantly expressed cell surface receptor on mouse beta cells by a considerable margin with an RPKM value > 200. Expression of Il1r1 is significantly enriched in mouse beta over alpha cells (p < 1.9 × 10-15) and the mouse Il1r1 promoter is associated with strong transcription factor binding by the beta cell transcription factors PDX1, NKX6.1 and NEUROD1  and significantly differentially expressed  genes. Further validation of transcriptome differences between mouse and human beta cell transcriptomes was obtained by demonstrating that species-specific enrichment of gene expression correlates well with species-specific enrichment of ChIP signatures for the beta cell specific transcription factors Pdx1 [Prlr, Ghr and Cntfr, three related receptors that respond to structurally similar ligands [in vitro have been largely unsuccessful [GHR and relative absence of PRLR are not conducive to robust responses to their potential mitogenic ligands.Importantly, our transcriptome data now offer the opportunity for the systematic comparison of gene expression by mouse and human beta cells. We identified a core set of 9906 genes that are shared between mouse and human beta cells. It is no surprise that this set contains many of the key genes required for the stimulus-appropriate release of insulin such as ors Pdx1  and Nkx6ors Pdx1 , 49. Amo ligands , that ar ligands  and GH a ligands  and impl ligands , 44. Howccessful , 53 and ccessful . While tccessful , the lacIl1r1, which responds to the pro-inflammatory cytokines IL-1α and IL-1β. Importantly, the human islet is not devoid of IL1R1 transcript, in line with a body of literature that established that mouse and human beta cells respond to sustained IL-1β stimulation with a reduction in function and an increase in apoptosis [Il1r1 expression in mouse islets could betray a more prominent role for locally produced IL-1β in beta cell glucotoxicity in mouse islets and potentially render mouse beta cells more sensitive to IL-1β-mediated pro-inflammatory insults. This observation is important, as local inflammation in the islet precipitated by obese conditions is thought to contribute to beta cell failure and exacerbate diabetes [The most abundantly expressed cell surface receptor in mouse beta cells, measured by gene expression, is poptosis –58. The diabetes .Il1r1 and Pparg, were flanked by mouse-specific novel lncRNAs. We therefore leveraged our transcriptome data to identify 145 novel lncRNAs that are enriched for significant transcription factor binding of key beta cell transcription factors and are co-regulated with the nearest protein-coding gene in line with previous observations [Several of the genes that displayed markedly higher expression in mouse over human beta cells, such as rvations , 60, 61.rvations . Indeed,rvations  revealedrvations , 64. Thervations , 20 is nRodents are commonly used to study the islets of Langerhans, with the ultimate goal of improving the outlook of diabetic patients and despite considerable differences in islet architecture and innervation between rodent and primate islets. Our comprehensive beta and alpha cell transcriptomes will greatly enhance our understanding of normal islet physiology and yield significant new leads to direct the behavior of its two primary endocrine constituents. Importantly, our data leveraged against recently published transcriptomes for FACS-purified human islet populations will now facilitate routine cross comparison between mouse and human alpha and beta cell transcriptomes. This is an invaluable resource for all with an interest in islet physiology and disease that stands to improve the translatability of rodent studies  by ensurAnimals were maintained on a 12-h light/12-h dark cycle with free access to water and standard rodent how. DNA encoding a fusion between human histone-2b and monomeric Cherry (H2b-mCherry) was ligated in the Spe I site of the mouse insulin 1 promoter (pBS.MIP1(-SpeI); generously gifted by Dr. Mark Magnuson). The resulting mIns1-H2b-mCherry reporter construct  per the manufacturer's instructions, taking great care to avoid carry over of ethanol following the column washes. Following elution in 30 microliter elution buffer, RNA quality was verified by BioAnalyzer . Indexed sequencing libraries were constructed using the TruSeq RNA sample Prep Kit v2  and sequenced at 50 cycles, single read on an Illumina HiSeq 2000 platform.http://homer.salk.edu) [Ets1, Pax6, Pdx1, Myc). The full mouse transcriptome was scanned using default parameters. The consensus site motifs used were taken from the JASPAR Database [Sequencing reads from two beta cell populations and two alpha cell populations were mapped using STAR  to the malk.edu)  and uploalk.edu)  was usedDatabase .To identify novel lncRNA, RNA-Seq libraries from all replicates of alpha and beta cells were combined into a single meta-experiment to maximize sensitivity. Two approaches where used to identify transcripts. First, Cufflinks  was usedIslets were sonicated in 300 μl of KREBS ringer buffer, then centrifuged 10 min at 8000 K. The supernatant was transferred to a fresh tube. Stock samples were stored at -20 C. For each assay the samples were diluted in the assay buffer supplied with each individual kit. Human and mouse insulin, glucagon and IAPP islet peptide content were measured with commercially available RIA or ELISA kits  per the manufacturer's instructions. As cross-reactivity for mouse IAPP had not been determined by EMD Millipore, a separate rat/mouse IAPP  standard curve was run in equivalent doses and shown to be superimposable with the human IAPP curve supplied with the ELISA, confirming 100% cross-reactivity with mouse IAPP  and declared exempt from approval by the Institutional Review Board of the Salk Institute for Biological Studies.CB is the director of the Razzavi Newman Integrated Genomics and Bioinformatics Core at the Salk Institute for Biological Studies and is an expert bio-informatician, who wrote Homer (Hypergeometric Optimization of Motif Enrichment), a suite of tools for Motif Discovery and next-generation sequence analysis. MOH is a Staff Scientist at the Salk Institute's Clayton Foundation Laboratories for Peptide Biology and will continue to grow his research program at the University of California, Davis in the fall of 2014. His group studies the role of Corticotropin Releasing Hormone (CRH) and the related peptide hormone Urocortin3 in the pancreas. The direct actions of these and other peptide hormones on the pancreas add a novel layer of complexity to the intricate network of signaling molecules that in concert governs beta cell mass and endocrine output of the pancreas.Is a figure validating the expression of eGFP in alpha cells of the S100b-eGFP reporter mouse.(PDF 888 KB)Additional file 1:Is a table listing the 100 most significantly enriched genes in mouse beta cells.(XLSX 48 KB)Additional file 2:Is a table listing the 100 most significantly enriched genes in mouse alpha cells.(XLSX 46 KB)Additional file 3:Is a side-by-side comparison of our alpha and beta cell transcriptomes with the beta cell transcriptomes derived from MIP-GFP + beta cells [.(PDF 277 KB)Additional file 4:ta cells .Additional file 5:Is a figure highlighting the differential expression of genes important for glucose uptake and glycolysis, stimulus secretion coupling and insulin exocytosis in mouse beta and alpha cells.(PDF 536 KB)Additional file 6:Is a figure containing single channel images for the immuno-fluorescent panels of Figure .(PDF 2 MB)Additional file 7:Is a table listing the genes comprising the common beta cell transcriptome as defined by expression > 1 RPKM in beta cells of both species.(XLSX 815 KB)Additional file 8:Is a table listing genes that are significantly enriched or uniquely expression in mouse or human beta cells.(XLSX 214 KB)Additional file 9:Is a side-by-side comparison of a standard curve of mouse and human IAPP as measured by ELISA, demonstrating 100% cross-reactivity.(PDF 121 KB)Additional file 10:Is a table listing novel transcripts discovered in purified mouse beta and alpha cells and the regulation of their expression by glucose.(XLSX 90 KB)Additional file 11:Is a count table with all genes in our alpha and beta transcriptomes.(XLSX 4 MB)Additional file 12:


Genetic heterogeneity between pGBM and sGBM has been documented as have differences in the methylation of individual genes. A hypermethylator phenotype in grade IV GBMs is now well documented however there has been little comparison between global methylation profiles of pGBM and sGBM samples or of methylation profiles between paired early and late sGBM samples.Grade IV glioblastomas exist in two forms, primary (We performed genome-wide methylation profiling of 20 matched pairs of early and late gliomas using the Infinium HumanMethylation450 BeadChips to assess methylation at >485,000 cytosine positions within the human genome.IDH1 mutation in sGBM tumors. In 80% of cases, the hypermethylator status was retained in both the early and late tumor of the same patient, indicating limited alterations to genome-wide methylation during progression and that the CIMP phenotype is an early event. Analysis of hypermethylated loci identified 218 genes frequently methylated across grade II, III and IV tumors indicating a possible role in sGBM tumorigenesis. Comparison of our sGBM data with TCGA pGBM data indicate that IDH1 mutated GBM samples have very similar hypermethylator phenotypes, however the methylation profiles of the majority of samples with WT IDH1 that do not demonstrate a hypermethylator phenotype cluster separately from sGBM samples, indicating underlying differences in methylation profiles. We also identified 180 genes that were methylated only in sGBM. Further analysis of these genes may lead to a better understanding of the pathology of sGBM vs pGBM.Clustering of our data demonstrated a frequent hypermethylator phenotype that associated with This is the first study to have documented genome-wide methylation changes within paired early/late astrocytic gliomas on such a large CpG probe set, revealing a number of genes that maybe relevant to secondary gliomagenesis. While secondary glioblastomas (sGBM) develop slowly through progression from lower grade diffuse or anaplastic astrocytomas and more commonly occur in younger patients. pGBM and sGBM represent not only clinically distinct entities but also demonstrate distinct genetic heterogeneity. For example, pGBM demonstrate mutation of the PTEN gene and frequent loss of heterozygosity on chromosome 10q (inclusive of the PTEN gene locus), amplification of EGFR, deletions of CDKN2A (p16), while sGBM and their lower grade precursor lesions have frequent mutations of the TP53 gene and the IDH1 gene [Gliomas are classified into 4 grades according to the WHO classification system. These range from curable World Health Organization (WHO) grade I tumors (pilocytic astrocytomas) to the highly malignant WHO grade IV glioblastoma (GBM) with mean survival < 1year. In between these two grades are WHO grade III malignant tumors (anaplastic astrocytomas) with median survival rates of 2–3 years after diagnosis and WHO grade II (diffuse astrocytomas) considered as low grade gliomas with median survival rates of 6–8 years after diagnosis [DH1 gene . Recent MGMT promoter that has provided an exciting and clinically relevant epigenetic marker in gliomas. The MGMT gene encodes for an O-6-methylguanine methyltransferase that removes alkyl groups from the O-6 position of guanine. Thus loss of its activity greatly impairs a cells ability to tolerate alkylating agents and studies have shown that MGMT-promoter methylation is associated with longer survival of patients treated with alkylating agents such as temozolomide [IDH1) are thought to be largely responsible for the glioma hypermethylator phenotype due to the massively increased production of the 2-hydroxyglutarate oncometabolite and have recently been shown to be sufficient to result in a hypermethylator phenotype in glioma cell lines [In recent years large scale genome-wide epigenetic studies have been performed with the aim of developing clinically relevant biomarkers for glioblastoma -11. A gozolomide ,13. Recezolomide . The gaill lines ,15. At lll lines  and althRecent technological advances have made it possible to quantitatively assess genome-wide methylation at the individual CpG loci level using the Illumina Infinium BeadChips. The most recent version of this BeadChip (Infinium HumanMethylation450 BeadChip) is able to quantitatively assess the levels of methylation at specific CpG loci throughout the genome, including CpG islands and regions of much lower CpG dinucleotide density. In this report we utilized these comprehensive Infinium HumanMethylation450 BeadChip arrays to define genome-wide methylation in paired samples of early/late astrocytic gliomas and to demonstrate any alterations induced by progression.Forty DNA samples from 20 astrocytoma/glioma patients were used in this study. These patient samples consisted of; 10 WHO grade II astrocytomas, 15 WHO grade III astrocytomas and 15 WHO grade IV glioblastomas. The 40 DNA samples represent 20 cases of paired early and late lesions from the same patient. The DNA was extracted from tissue samples consisting of a minimum of 80% tumor. The DNA from four non-disease brain samples was used to provide the normal, expected levels of methylation. Ethical guidelines were followed for patient sample collection and all samples have been anonymised. Research was conducted according to the principles expressed in the Declaration of Helsinki. Patients gave written informed consent for analysis of tumor samples. The study was approved by the Institutional Ethics Committees of University of Technology Dresden and University of Birmingham.The Illumina Infinium HumanMethylation450 array  was performed on 0.5 μg bisulfite modified patient DNA according to manufacturers’ instructions. Bisulfite modification of DNA and array hybridization was carried out by Cambridge Genomics Services. Raw data was obtained using Genome Studio software from Illumina. The raw data were processed using the lumi R  package Probes demonstrating detection p-values greater than 0.01 in any sample were removed along with probes located on the X and Y chromosomes. To ensure tumor specific hypermethylation, probes showing a beta value ≥0.25 in any of the four normal samples were also removed. Hypermethylation was subsequently determined as a beta value ≥0.5. This was considered relevant if present in >30% tumor samples. Additional filtering was achieved limiting selection to genes for which the hypermethylation criteria were met in ≥3 probes associated to that gene.The top 2000 most variable loci for each clustering event were determined by selecting the 2000 probes with the greatest standard deviation across all the given samples. Clustering was performed using the Cluster3 program (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm#ctv) and visualized using the Java TreeView program (http://jtreeview.sourceforge.net/). Unsupervised hierarchical clustering was performed using the Euclidean based algorithm.Clone sequencing was used for array validation. 0.5 μg of DNA for each sample was bisulfite modified using the Qiagen EpiTect kit  according to manufacturers’ instructions. PCR reactions were performed using FastStart Taq DNA polymerase  on a semi-nested basis for all genes using the primers listed in Additional file IDH1 and IDH2 genes [IDH1 forward primer 5′-CTCCTGATGAGAAGAGGGTTG-3′ and IDH1 reverse primer 5′-TGGAAATTTCTGGGCCATG-3′ were used to sequence codon 132 and the IDH2 forward primer 5′-TGGAACTATCCGGAACATCC-3′ and IDH2 reverse primer 5′-AGTCTGTGGCCTTGTACTGC-3 were used to sequence codon 172 of IDH2. Twenty nanograms of genomic DNA were used as starting material for a 25 μl total volume PCR reaction using Go Taq polymerase. An annealing temperature of 58°C was used for 35 cycles. PCR products were bi-directionally sequenced using cycle sequencing on an ABI 3730x .Previously described primers were used to amplify 129 bp and 150 bp fragments of the H2 genes . The IDHIDH1 and IDH2 mutation status for these tumors was identified using the cBioPortal for Cancer Genomics .Illumina Infinium HumanMethylation450 BeadChip array data was used for the following 19 TCGA primary glioblastomas: TCGA-06-5416, TCGA-06-0171, TCGA-26-5136, TCGA-06-0190, TCGA-06-5418, TCGA-06-0210, TCGA-26-5135, TCGA-26-5134, TCGA-26-5132, TCGA-12-5295, TCGA-06-5414, TCGA-06-0211, TCGA-26-5133, TCGA-06-5417, TCGA-06-0221, TCGA-26-1442, TCGA-06-6389, TCGA-06-6701, TCGA-15-1444. All array data was downloaded from the TCGA Data Portal (https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp). IDH1 and IDH2 mutation status, 14 out of 20 (70%) samples demonstrated mutation in the IDH1 R132 codon. No IDH2 mutations were detected  demonstrating an IDH1 mutation  demonstrated very similar methylation patterns between early and late grades  and those that demonstrate a partially remaining CIMP+ve between early and late lesions or greatly reduced (becoming CIMP-ve) degree of methylation after progression   and major cluster 2    were chosen from the list of 218 genes to confirm array values with regard to methylation. We chose two genes that had not previously been shown to be methylated in GBM (ALS2CL and GNMT) and one gene that has (WNK2) [To try and identify genes important throughout secondary gliomagenesis it was assumed that genes hypermethylated in all glioma grades would be the most relevant. This analysis identified 939 hypermethylated CpG loci across all grades for further analysis. This list represents 232 genes and was, as before, reduced to 218 genes (represented by 914 CpG loci) by selecting genes that were represented in the list by ≥3 CpG loci probes. The gene list and beta values for these probes are provided in Additional file +ve group, one of which had the IDH1 p.R132H change. To further expand this analysis we then downloaded all pGBM IDH1 p.R132H mutated samples that had available Infinium HumanMethylation450 BeadChip methylation data . Clustering of the total 19 pGBM samples with our 15 grade IV sGBM samples showed the CIMP phenotype within all five pGBM IDH1 mutated samples, which clustered together with our sGBM CIMP+veIDH1 mutation positive samples, indicating the CIMP+ve phenotype induced by IDH1 mutation in pGBM is similar in sGBM. Two additional pGBM samples also clustered within this group, completing the smaller of the two major cluster groups  identified 180 genes that were only methylated in sGBM samples according to our criteria , we chose to use our sGBM data prior to adjustment for this particular analysis. Using 15 of TCGA grade IV pGBM and our 15 grade IV sGBM data, we clustered the most variable 2000 loci and observed that the pGBM samples largely clustered together, while the sGBM samples clustered into two separate groups dependent upon their CIMP phenotype (data not shown). This suggests there is also epigenetic heterogeneity between pGBM and sGBM, at least in terms of DNA methylation, but interestingly there were three of the pGBM samples that clustered within the sGBM CIMPIDH1 gene that has been shown to result in a CpG island methylator phenotype (CIMP). In this report we have used the latest Illumina Infinium HumanMethylation450 BeadChips to assess the genome-wide methylation of 20 secondary glioblastomas and their matching lower grade precursors. Sandoval et al. [Secondary GBM represents a smaller subset (5%) of GBM tumors which develop from preexisting lower grade tumors (grade II/III), are more often seen in younger patients and patients with sGBM have longer survival times . These tl et al.  recentlyl et al. . While wIDH1 mutation and that in the lower grade precursors this nearly uniformly resulted in a CIMP phenotype. We saw one case  where there was evidence of an IDH1 mutation but no CIMP phenotype. We also saw one case (P19.E) where there was no evidence of IDH1 or IDH2 mutation but was CIMP positive. However, it has previously been suggested that even when negative for the known IDH1 p.R132H mutation, it is possible that other IDH1 mutations could be present in some cases that might therefore potentially affect CIMP status [IDH1 mutation and CIMP that we have seen in our study suggests this is an early and important event in gliomagenesis and that if not acquired at an early stage is not gained during progression as no later stage glioblastoma presented with CIMP where the precursor did not. Although the total number of samples is small the large degree of IDH1 mutation and CIMP argues strongly that this is true. In addition to increased overall survival, IDH1 mutation status has been shown to correlate with genetic features including the presence of MGMT methylation and codeletion of 1p and 19q, as well as inversely correlating with EGFR amplification, chromosome 10 loss and chromosome 7 polysomy [We demonstrated that these samples had the expected high levels of P status . The earpolysomy ,24 and tIDH1 mutation, split approximately in half to follow two paths after progression. Some samples appeared to fully retain and maintain CIMP in their higher grade lesions whatever level CIMP hypermethylation was observed within the lower grade precursor lesions, presumably due to the importance of this high level of general hypermethylation to the tumors survival. Interestingly, some samples notably reduced their levels of general hypermethylation, some retaining what we defined as CIMP and some losing it. This could potentially be due the initial lower grade lesion demonstrating epigenetic heterogeneity with different cells having differing hypermethylation patterns that together present as CIMP positive. If a subset of these cells contained hypermethylation of a particular tumor suppressor that resulted in a considerable growth advantage then these cells could grow out and progress to be the higher grade lesion. This lesion would still have the evolutionary pressure to maintain the hypermethylation of this specific tumor suppressor but not necessarily the need to maintain a global methylation phenotype, although in general you would expect some degree of maintenance by the IDH1 mutation, it is plausible that due to changing tumor heterogeneity this would be visualized at a lesser extent. Unfortunately we were unable to assess different regions from within the same tumor to investigate this hypothesis. Furthermore, we observed that these differences were not simply due to pairs progressing from grade II to grade III compared to grade III to grade IV or grade II to grade IV. Due to the relatively small size of our cohort we were unable to identify the specific genetic differences that may support this hypothesis as we would assume them to be tumor specific. Nonetheless this is an interesting observation that could possibly affect the effectiveness of therapies based on demethylating agents on these tumors. Naturally, we would assume they would be more effective in samples that at some stage demonstrated CIMP but they may still be effective in samples that do not demonstrate CIMP in the later grades if CIMP was present in the precursor lesion. It is hard to estimate whether a demethylating agent would be more effective on tumors dependent on global hypermethylation or are reliant on the hypermethylation of only a small number of targets. Promisingly, 5-azacitidine has recently been shown to be effective in reducing selected promoter methylation, tumor growth, cell proliferation and inducing differentiation in an in vivo primary xenograft IDH1 mutant glioma [The effects of tumor grade progression on the genome-wide methylation of these paired samples of sGBM tumors and their earlier lower grade lesions could be assessed in the most comprehensive manner to date due to the large amount of data provided by the Illumina Infinium HumanMethylation450 BeadChips. Firstly, as mentioned above samples lacking CIMP in their precursor lesions never gained it via progression, presumably due to the early gain of some other genetic or environmental factor capable of driving gliomagenesis without the subsequent need for hypermethylation. While those samples presenting with CIMP in their precursor lesion, largely in association with t glioma .Further evidence for the loss of some hypermethylation due to tumor grade progression was observed when the levels of hypermethylated loci and genes were assessed simply by the grade of each tumor rather than looking for differences between paired samples. We noticed a trend towards decreasing levels of methylated targets with increasing tumor grade which has previously been documented ,20. ThisBy analyzing grade II, III and IV tumors separately, we were able to identify a list of genes where hypermethylation was retained in all 3 grades, likely representing the most generally important methylated genes within this cohort of sGBM tumors. This identified preferential hypermethylation of several genes associated with cell morphology, cellular movement, cellular development, cellular growth and proliferation, and cellular assembly and organization, with many of these select genes having been previously associated with cancer. Due to the relatively small number of tumors assessed, this analysis would greatly benefit from expansion into a larger cohort that could highlight which genes and pathways are most important to sGBM gliomagenesis and progression.IDH1 mutation, demonstrating a very similar hypermethylation profile to CIMP positive grade IV sGBM. This represented a specific epigenetic overlap between a subset of the pGBM and sGBM tumors. Included in this were two pGBM tumors exhibiting CIMP that lacked mutation in IDH1 or IDH2 that could possibly retain other mutations capable of resulting in CIMP such as could be present in our 19th pair. Overall, this small sGBM/pGBM analysis offers an insight into different tumorigenic processes giving rise to these different types of GBM tumors.By comparison of methylation profile of our grade IV lesions with a subset of the publically available methylation profiles of grade IV pGBM provided by the Cancer Genome Atlas (TCGA) network we demonstrated that in general the methylation profiles between these two tumor types differ in a similar manner to their respective genetic alterations. This was further observed when comparing the functions of genes commonly hypermethylated in grade IV sGBMs compared to grade IV pGBMs with sGBMs preferentially hypermethylating genes involved in cell death, survival and maintenance pathways and pGBMs preferentially hypermethylating genes that alter or control gene expression. Interestingly, a small number of the pGBM tumors demonstrated CIMP that was also largely associated with In summary, this data offers an insight into different epigenetic, methylation-related processes that give rise to these different types of GBM tumors and provides interesting rationales for further study of this kind on much larger cohorts. The increased use of genome-wide analysis of methylation using technologies such as the Illumina Infinium HumanMethylation450 BeadChips, that are relatively cheap and can be performed using both archival tissue DNA from FFPE blocks and small amounts of DNA acquired from biopsies, may well increase their usefulness as diagnostic or therapeutic markers. Thus, providing a greater understanding on these tumor specific methylation patterns may prove useful in a number of ways.The authors declare that they have no competing interests.FL and VKH designed the study and drafted the manuscript. ERM and GC helped design the study. VKH carried out the clone sequencing and in silico/bioinformatics analysis. TS carried out IDH1 mutation analysis. CJR carried out in silico/bioinformatics analysis. DK and GS provided the DNA samples and clinical information. JB developed the correction pipeline. WW helped with bioinformatic analysis. All authors’ read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/506/prepubPrimer sequences are provided for ALS2CL, GNMT and WNK2 beta value validation analysis.Click here for fileSample information is provided for the patient samples used in this study. (a) Pair numbers used in this study. (b) sample numbers used in this study. (c) WHO grade of each tumour given as either astrocytoma (astro) grade II or III; or GBM (glioblatoma multiforme); astrocytoma WHO grade IV. (d) KPS; Karnofsky Performance Score at the time of initial admission. (e) tumor location (hemisphere) (f) RTx; whether radiotherapy was received (g) CTx; whether chemotherapy was received (h) IDH1 mutation status; mutated (mut) or wild type (wt) for the recurrent p.R132H mutation.Click here for filest exon, 3′UTR, 5′UTR, gene body, within 1500 base pairs of the transcription start site (TSS1500) or within 200 bp of the transcription start site (TSS200). Genomic locations include: CpG islands (island), north CpG island shelves (N shelf), south CpG island shelves (S shelf), north CpG island shores (N shore) south CpG island shores (S shore) or unclassified regions (open sea). Hypermethylated CpG loci distributions are almost identical in each grade. We also show the distribution of CpG islands that were analyzed for hypermethylation events with respect to gene feature or genomic location, as determined by the array design. These include all probes on the array that associated with a gene, were not on either the X or Y chromosome and were not associated with a SNP.Pie charts for each grade illustrate the distribution of hypermethyaled CpG loci with respect to gene features or genomic location. Gene features include CpG loci within the following regions: 1Click here for fileTable S3. This table contains probe beta values for the genes listed in Table S2.This table contains the gene symbols for probes methylated in grade II, III and IV samples in 3 or more probes. Click here for fileClone sequencing results are shown for CpG island regions of three genes; ALS2CL, WNK2 and GNMT. Black and white circles represent methylated and unmethylated CpG dinucleotides respectively and each line represents a single clone. Methylation indexes are given for each sample as a percentage of methylated CpGs out of the total number of CpGs analysed. CpG dinucleotides analysed by the infinium assay are indicated by an arrow and beta values for these loci are shown next to each sample.Click here for fileIngenuity pathway analysis results for the 218 genes hypermethylated across grade II, III and IV tumor samples. Table S4. shows the top 5 molecular and cellular functions alongside the number of genes falling within each category and the p-value range. Gene symbols are given for genes that fall within any of these 5 categories. Gene symbols in bold indicate genes that have previously been associated with cancer. Table S5. illustrates the top 3 gene networks of the 218 hypermethylated genes and the genes present within each of these networks.Click here for fileGene lists for hypermethylated genes exclusively in sGBM or pGBM samples, following comparison between the lists for the two tumor types.Click here for fileVenn diagram to illustrate the cross-over between hypermethylated genes within the grade IV pGBM and grade IV sGBM specific lists in addition to the original list of universally methylated genes across grade II, III and IV secondary glioma samples.Click here for file