HSD11B2) buffers the impact of maternal glucocorticoid exposure by converting cortisol/corticosterone into inactive metabolites. However, previous studies indicate that maternal adversity during the prenatal period can lead to a down-regulation of this enzyme. In the current study, we examined the impact of prenatal stress  in Long Evans rats on HSD11B2 mRNA in the placenta and fetal brain (E20) and assessed the role of epigenetic mechanisms in these stress-induced effects. In the placenta, prenatal stress was associated with a significant decrease in HSD11B2 mRNA, increased mRNA levels of the DNA methyltransferase DNMT3a, and increased DNA methylation at specific CpG sites within the HSD11B2 gene promoter. Within the fetal hypothalamus, though we find no stress-induced effects on HSD11B2 mRNA levels, prenatal stress induced decreased CpG methylation within the HSD11B2 promoter and increased methylation at sites within exon 1. Within the fetal cortex, HSD11B2 mRNA and DNA methylation levels were not altered by prenatal stress, though we did find stress-induced elevations in DNMT1 mRNA in this brain region. Within individuals, we identified CpG sites within the HSD11B2 gene promoter and exon 1 at which DNA methylation levels were highly correlated between the placenta and fetal cortex. Overall, our findings implicate DNA methylation as a mechanism by which prenatal stress alters HSD11B2 gene expression. These findings highlight the tissue specificity of epigenetic effects, but also raise the intriguing possibility of using the epigenetic status of placenta to predict corresponding changes in the brain.Maternal exposure to stress during pregnancy is associated with significant alterations in offspring neurodevelopment and elevated maternal glucocorticoids likely play a central role in mediating these effects. Placental 11β-hydroxysteroid dehydrogenase type 2 ( In humans, the experience of stress during pregnancy is associated with increased risk of preterm birth, reduced birth weight, and smaller head circumference HSD11B2 gene leads to hypertension, excess mineralocorticoid activity, and increased anxiety-like behavior in adulthood HSD11B1 mutation leads to attenuated negative-feedback of the HPA response to stress and improved cognitive performance in aging In mammals, the placenta serves as a critical interface between maternal and fetal physiology and forms a barrier to maternal glucocorticoids nd–3rd trimester of human pregnancy) was found to decrease placental 11ß–HSD2 enzymatic activity and decrease mRNA levels of this gene HSD11B2 mRNA levels HSD11B2 mRNA levels have also been found associated with intrauterine growth retardation and pre-term birth There is increasing evidence that maternal adversity during pregnancy may lead to a down-regulation of 11ß–HSD2. In rats, chronic restraint stress during gestational days 11–20 . All procedures were performed in accordance with guidelines of the NIH regarding the Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee (IACUC) at Columbia University.16 female and 8 male Long Evans rats (purchased from Charles River) were maintained on a 12:12 hour light-dark schedule with white lights on at 0800 h and off at 2000 h and housed 2 per cage (same-sex) in 26×50×22 cm polycarbonate cages in the animal facility at the Department of Psychology, Columbia University. Food and water were available Within the sample of 16 mated females, 12 females became pregnant following the 1-week mating period. At gestational day 14, all pregnant females were singly-housed and assigned to either non-stress  or stress (n = 6) treatment conditions. Control females were left undisturbed throughout gestational days 14–20. Stress females were exposed to restraint stress, through placement in a 19×29×12 cm cage  for 1-hour/day from gestational days 14–20. The timing of stress exposure was randomized to prevent habituation. At gestational day 20, pregnant dams were sacrificed 1 hour after restraint stress through rapid decapitation and trunk blood was collected for assay of corticosterone levels. Plasma corticosterone was assayed using an RIA kit  and this assay confirmed elevated levels of corticosterone in stressed compared to control dams .via caesarean section at gestational Day 20. Pups were decapitated and whole brains extracted. Placenta and fetal brains were snap-frozen, and stored at −80C until further processing. Cortex and hypothalamus of E20 fetal brains were dissected in a cryostat cooled to −20C. Placenta samples were dissected such that a pie slice including both the basal zone and inner labyrinth zone was used. Samples were weighted and homogenized in 700 µl lysis buffer RLT-Plus (Qiagen) with 0.1% β-mercaptoethanol using a tissue homogenizer (Omni) for 15–20 seconds. To analyze both mRNA and DNA methylation from the same sample, RNA and DNA from fetal cortex, hypothalamus, and placenta samples were isolated simultaneously using a dual RNA/DNA extraction kit (Qiagen). cDNA was synthesized using a reverse transcription kit (Applied Biosystems) according to manufacturer’s protocol. Samples were stored at −20C until further processing. Subsequent analysis of gene expression and CpG methylation was conducted using tissue from 1–2 offspring each of 4 control and 4 stress dams. Selection was based on average pup weight at the time of sacrifice. Only litters in which average pup weight was greater than 2.5 g were included in this sample and average pup weight did not differ significantly between treatment groups.Feti and placenta were extracted HSD11B2), 11β-hydroxysteroid dehydrogenase -1 (HSD11B1), DNA methyltransferase-1 (DNMT1), and DNA methyltransferase-3a (DNMT3a) were conducted with the 2ddCT method Tissue  from 2 pups per litter from 4 control and 4 stress dams were included in the gene expression analyses. Relative gene expression was measured by real-time quantitative PCR on a 24-well 7500Fast qPCR thermocycler using SybrFast (Applied Biosystems) with standard amplification and Ct calculation protocols by Applied Biosystems. All primers were designed to span exons and were tested for specificity (single melt curve peak) and efficiency (87–105%). Primer pairs are included in Rattus norvegicus HSD11B2 gene  samples from 1 pup per litter from control and stress dams were run in duplicate and included in the DNA methylation analyses. Purified DNA was analyzed (EpigenDX) for CpG methylation by bisulfite pyrosequencing. Samples were bisulfite converted and PCR amplified . 50 ng of DNA was amplified using a PCR amplification kit (Qiagen) according to standardized protocol  with Sybr DNA loading dye. Immediately after amplification samples were run on a 2% gel and imaged under UV light. Lanes expressing a bright band were identified as male. Multiple SRY loci have been identified in the rat genome In order to determine the sex of pups, DNA purified from placenta of each pup was amplified using primers for All statistics were performed using SPSS . Analysis of average ddCT levels to determine differences in gene expression was conducted with a 2-way ANOVA with tissue type and stress treatment as factors. Tukey’s HSD post-hoc tests were used to compare tissue-specific gene expression levels and two-tailed Student’s t-tests were used to determine significant differences associated with maternal stress. Correlation between DNA methylation levels at specific CpG sites was conducted with two-tailed Pearson correlation coefficients. One-way ANOVA was used to compare DNA methylation between tissue types. Repeated measures ANOVA was used to determine the impact of maternal stress on DNA methylation and Tukey’s HSD was used to compare stress effects at specific CpG sites. Analysis of stress effects on overall levels of DNA methylation averaged across sites was conducted using two-tailed Student’s t-tests. Though sex-specific effects on gene expression and DNA methylation were not observed in this sample, sex was used as a covariate in the analyses. Significance was set at the p<0.05 level.HSD11B2 gene expression indicated a main effect of tissue  = 15.37, p<.001), a main effect of stress  = 5.46, p<.05), and a significant tissue by stress interaction  = 6.66, p<.01). Overall, there were significantly higher levels of HSD11B2 mRNA in the placenta (p<.01) compared to fetal hypothalamus and cortex . Prenatal stress was associated with a significant decrease in HSD11B2 mRNA in the placenta . Overall, levels of DNMT1 mRNA were found to be significantly higher in the placenta compared the fetal hypothalamus and cortex (p<.001) and levels of DNMT3a mRNA were significantly elevated in the hypothalamus compared to the cortex and placenta  = −1.79, p = .09). Within the fetal cortex, DNMT1 expression was significantly increased in stressed offspring (t(14) = −2.24, p<.05; DNMT1 expression in the placenta. Analysis of the de novo methyltransferase DNMT3a, indicated a placenta-specific effect of stress on the expression of this enzyme. Prenatal stress was found to be associated with increased placental DNMT3a mRNA levels (t(14) = −3.71, p<.01; Analysis of HSD11B2 promoter and region from −378 to +56 is rich in CpG sites and contains several transcription factor binding sites for Sp1 and NF-κB, suggesting that this region would be a likely candidate for regulation of gene transcription by DNA methylation. Moreover, previous studies in rats report environmentally-induced differences in methylation within this region associated with variation in gene transcription HSD11B2 promoter  and increased methylation within the placenta .Analysis indicated main effects of tissue , stress , and a significant tissue by stress interaction  on CpG methylation within the HSD11B2. Stress, anxiety, and depression during pregnancy can have a long-lasting impact on the psychological health of children HSD11B2 within the placenta in late gestation DNMT1 and DNMT3a. Within the placenta, this stress effect was specific to the de novo methyltransferase DNMT3a, whereas in fetal hypothalamus and cortex, prenatal stress induced increased mRNA levels of DNMT1. Consistent with the reduced HSD11B2 mRNA in placenta, we find increased placental CpG methylation within several sites of the HSD11B2 gene promoter associated with maternal stress. In contrast, our analysis indicates decreased DNA methylation at several sites of the HSD11B2 gene promoter in fetal hypothalamic tissue associated with stress and no effect of this prenatal exposure on DNA methylation in the fetal cortex. These findings highlight the tissue specificity of epigenetic effects. However, we do identify several CpG sites within the placenta at which an individual’s DNA methylation levels do significantly predict those observed in the fetal cortex, raising the intriguing possibility of using the epigenetic status of placenta to predict corresponding changes in the brain. Overall, these findings provide novel evidence for the epigenetic regulation of HSD11B2 as a potential mechanism linking maternal stress during gestation, dysregulation of placental gene expression, and neurodevelopmental outcomes in offspring.In the current study we report a robust and tissue-specific effect of maternal stress during pregnancy in rats on mRNA levels of the enzyme HSD11B2 mRNA levels, the stress-induced DNA hypomethylation of this gene that we observe in hypothalamic tissue may be an epigenetic precursor to these buffering effects. Time course analysis of HSD11B2 gene expression and DNA methylation throughout the prenatal stress exposure and into the postnatal period may thus be a key strategy for determining the dynamics of glucocorticoid programming mechanisms.Though placental 11ß-HSD2 can function as an enzymatic buffer against the deleterious effects of exposure to maternal glucocorticoids, it is clear that this enzyme can be down-regulated by adverse prenatal experiences HSD11B2 has previously been explored outside the context of studies on maternal stress. In humans and in rats, the promoter and first exon of the HSD11B2 gene  are rich in CpG sites and DNA methylation levels at CpG sites within this region are related to the expression of this gene HSD11B2 through in vitro or in vivo treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine has been found to increase HSD11B2 expression HSD11B2, CpG methylation at Sp1 and NF1 recognition sequences prevents binding of these transcription factors and diminishes the transcriptional activity of HSD11B2HSD11B2 promoter methylation and decreased HSD11B2 mRNA has also been observed in neonatal offspring exposed to in utero magnesium deficiency HSD11B2 expression and increased HSD11B2 promoter methylation in kidneys at birth and these epigenetic effects lead to altered transcription factor binding of Sp1 and NF-κB HSD11B2 dysregulation. The sex-specificity of environmentally induced changes in DNA methylation is increasingly evident and indeed epigenetic modifications may be involved in the normal process of sexual differentiation HSD11B2 expression or DNA methylation in the current study, it may be that large samples are needed to detect sex-differences in the epigenetic regulation of this gene  or that sex-differences are more likely to emerge in later development.Investigation of the epigenetic regulation of HSD11B2 levels we have found in response to maternal stress during pregnancy may involve the up-regulation of DNA methyltransferase levels in the placenta of stressed offspring. Our data indicate a placenta-specific increase in DNMT3a mRNA in response to maternal stress. This stress-induced effect has implications for genome-wide epigenetic changes, and may account for the diverse phenotypic outcomes associated with maternal adversity during pregnancy. DNMT1 and DNMT3a are enzymes active throughout the lifespan and thus could potentially serve as a mechanism for long-term epigenetic regulation, though it is possible that DNMT3b (only active early in development) is similarly altered by prenatal stress to mediate HSD11B2 promoter methylation. Our data are consistent with previous findings indicating an up-regulation of DNMTs in the placenta of mice exposed to 1st trimester maternal stress DNMT1 or DNMT3a in mice has been found to produce embryonic and postnatal lethality and widespread epigenetic changes – particularly amongst imprinted genes – in embryonic tissues DNMTs and DNMT3L may account for the changing epigenetic and transcriptional profiles in this tissue during the course of pregnancy A candidate mechanism for the increased placental DNA methylation and increased HSD11B2 mRNA, DNMT expression, and CpG methylation within the HSD11B2 gene promoter. Despite stress induced decreases in HSD11B2 CpG methylation in the fetal hypothalamus, we do not find differential expression of hypothalamic HSD11B2 and within the fetal cortex we find stress-induced increases in DNMT1 without corresponding changes in the DNA methylation or expression of HSD11B2. The regulation of HSD11B2 expression is complex and these paradoxical findings suggest that gene regulation in response to prenatal stress is also accomplished by other epigenetic mechanisms, such as chromatin remodeling, as well as regulation by transcription factors known to influence HSD11B2 transcription  promoter in fetal cord blood samples and the degree of CpG methylation detected in these cells predicts salivary cortisol levels of infants at 3 three months of age NR3C1 DNA methylation in blood samples from children/adolescents that were in utero during the stressor e.g. blood, placenta) and target tissues  has yet to be established and will likely vary dependent on the target gene, the nature of the environmental exposure, and the timing of sampling. Our data indicate that though at most CpG sites within the HSD11B2 gene promoter, DNA methylation levels are equivalent across placenta, hypothalamus, and cortex, within-individual correspondence in the methylation patterns between these tissues is limited. Though it may be possible to predict brain DNA methylation from the degree of CpG methylation observed in placenta – the biological relevance of the epigenetic markers identified in the current study has yet to be established. Future studies are needed both in humans and animals to address the issue of tissue specificity in environmentally-induced epigenetic variation and to elucidate the mechanisms contributing to the concordance and discordance in tissue-specific epigenetic profiles.The translation of


SNAP25 gene and has a hyperactive phenotype similar to that of ADHD. Such mice are 3 fold more active compared to their control littermates. Genetic association studies support a role for allelic variants of the human SNAP25 gene in predisposing to ADHD.The Coloboma mouse carries a ∼2 cM deletion encompassing the SNAP25 gene in 1,107 individuals (339 ADHD trios). To assess the functional relevance of the SNAP25-ADHD associated allele, we performed quantitative PCR on post-mortem tissue derived from the inferior frontal gyrus of 89 unaffected adults. Significant associations with the A allele of SNP rs362990  and three marker haplotypes  were observed. Furthermore, a significant additive decrease in the expression of the SNAP25 transcript as a function of the risk allele was also observed. This effect was detected at the haplotype level, where increasing copies of the ADHD-associated haplotype reduced the expression of the transcript.We performed association analysis across the SNAP25 confers risk to ADHD and reduces the expression of the transcript in a region of the brain that is critical for the regulation of attention and inhibition.Our data show that DNA variation at  Attention deficit hyperactivity disorder (ADHD) is a highly heritable disorder of childhood with significant functional impairment and negative lifetime outcomes across all developmental stages. The disorder is marked by disruption of catecholamine signaling, with mainstay treatments for the disorder targeting the dopamine and noradrenaline transporters and the alpha 2a adrenoreceptor 2+-triggered neurotransmitter exocytosis Synaptosomal-associated protein (SNAP-25) is a presynaptic plasma membrane protein that is specifically and highly expressed in nerve cells. The gene encodes a protein component, which interacts with the membrane associated protein syntaxin, and vesicle-associated membrane protein (VAMP) to form the SNARE complex. This complex interacts with membrane proteins known as synaptotagmin to make up a core complex essential for docking and holding synaptic vesicles at the presynaptic membrane in preparation for CaSNAP25 and the gene encoding phospholipase C beta (PLCB-1) SNAP25 copy) may contribute to the hyperactive behavior of the coloboma. Similarly, NA depletion using DSP-4(N-(2-chloroethyl)-N-ethyl-2 bromobenzylamine hydrochloride) significantly reduced hyperactivity in the Coloboma mouse, supporting the noradrenaline hypothesis The Coloboma mouse bears a semi-dominant mutation (cm/+) in which the heterozygous form results in the mutant type while the homozygous is lethal. The mutation is a ∼2 Cm deletion encompassing genes including SNAP25 gene might confer susceptibility to ADHD. An initial analysis SNAP25 gene Genetic association studies suggest that allelic variations in the SNAP25 influence expression within the IFG, with decreased SNAP-25 expression (as expected from the Coloboma mouse) associated with the ADHD risk variants.Here we sought to provide additional evidence for a role of SNAP-25 in ADHD by performing dense SNP mapping across the gene in nuclear families with ADHD. To assess the functional relevance of ADHD-associated variants, we performed quantitative PCR (qPCR) of the SNAP-25 transcript in a large sample (N = 89) of post-mortem brains from non-clinical individuals. Our qPCR work focused on expression of the transcript in human inferior frontal gyrus (IFG), an area of the cortex that has consistently been implicated in ADHD from both structural and functional imaging analyses SD = 3.67) and were predominantly male (87%). All probands fulfilled DSM-IV diagnostic criteria for ADHD. Of these, N = 44 (13%) had the inattentive subtype, and N = 19 (5.6%) had the hyperactive impulsive subtype; the remainder had the combined subtype (81.4%). One hundred and seventy-nine probands (52.8%) had a comorbid diagnosis of oppositional defiant disorder (ODD) and 34 (10%) fulfilled criteria for comorbid conduct disorder (CD). Frequencies of ADHD subtypes and comorbidities were similar across the recruitment sites. We obtained a written consent from the next of kin, caretakers, or guardians on the behalf of the minors/children participants involved in our study. The consent was in accordance with the declaration of Helsinki and the ethical approvals from the University of Queensland, Australia, Trinity College Dublin, Ireland, and the University of Birmingham, UK”A total sample of 1,017 individuals, comprising 339 Caucasian ADHD probands and their parents (full trios) across three similar sub-samples was investigated. The first cohort comprised 185 full trios recruited throughout Ireland from child psychiatric clinics and schools in West County Dublin and from the Hyperactive and Attention Deficit Children's Support Group of Ireland. Consensus diagnoses were made according to DSM-IV ADHD either with or without comorbidity. These diagnoses were based on all available clinical information and the Child Behaviour Checklist (CBCL), the Conners' Parents and Teachers Rating Scales, and the Comprehensive Teachers Rating Scale (ACTeRS) In addition to the ADHD samples, 89 unaffected Caucasian brain samples  were obtained from the Australian Brain Bank. Of these, 63 (71%) were from male subjects. None of these individuals were diagnosed with ADHD or other psychiatric condition. The mean age of the sample was 51.6 years (SD = 12.2), PH ranged from 5.8–7 and the post-mortem interval (PMI) was 28.2 hours (SD = 14).CTTGAAGCATCCCAGGAAGA and the reverse 5′ GAAGGAAAAATGTTGGGGTTT 3′. The PCR product (214 bp) was then restricted with the enzyme Cac81 and the DNA fragments were fractionated on a 3% agarose gel. The G allele was characterized by 164 bp and 51 bp fragments while the A allele was characterized by 215 bp fragment.Fifteen SNPs were included in the current investigation . SNPs weApproximately 100–200 mg of inferior frontal gyrus (IFG) tissue of each sample was used to extract RNA and DNA using TRIZOL reagent as recommended by the manufacturers (Invitrogen). As PH is considered a good indicator of RNA purity and integrity, this was measured and found to range from 5.8–7. The optical density (OD) at 260/280 of RNA preparations ranged from 2–2.1 indicating good quality of the RNA preparation.SNAP25) and the reference genes β2micoglobulin (β2M) and beta actin (ACTB) gene primers were designed to amplify DNA regions involving exon 7 and 8 of SNAP25, exons 1 and 2 in β2M and exon 3 and 4 of ACTB. The qPCR primers were designed using the INTEGRATED DNA TECHNOLOGIES Oligo Design Tool, which is freely available at http://www.idtdna.com/scitools/scitools.aspx. The SNAP-25-qPCR forward primer sequence was 5′ATGGATGAAAACCTAGAGCAGG 3′ and the reverse was 5′ACACTTAAC CACTTCCCAGC 3′. The β2M forward primer sequence was 5′ GGCATTCCTG AAGCTGACAG 3′ whereas the reverse was 5′TGGATGAAACCCAGACACATAG 3′. The ACTB qPCR forward primer was 5′ACCACACCTTCTACAATGAGC 3′ and the reverse was 5′ GCGTACAGGGATAGCACAG3′. In this context, β2M and ACTB were used as reference genes as they have stable gene expression in various tissues including brain. A standard Invitrogen procedure was used to synthesize first cDNA strands of the samples. Relative quantification was performed using SNAP25 (target gene) compared to β2M and ACTB (reference genes). PCR cycling was performed on a Roche LightCycler-480. Cycling conditions were 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 60°C for 10 sec and 72°C for 20 sec. This was followed by a melting curve cycle at 95°C for 5 sec and 1 min at 65°C. mRNA relative was expression quantified using normalized threshold cycles (Ct) of target relative to reference genes.To minimize RNA contamination with the DNA, samples were treated with DNASE-I and cleaned using RNeasy Mini Kit as recommended by manufacturers . Furthermore, to prevent any possible interference by DNA contamination when conducting qPCR, the target (http://www.broad.mit.edu/mpg/haploview) using the method of Gabriel et al. Assessment of Hardy Weinberg Equilibrium for all SNPs was undertaken using parental DNA of the ADHD participants. All genotypes were in Hardy Weinberg equilibrium. Genetic association across a 15 SNP array was performed using the transmission disequilibrium test (TDT). Haplotype analysis to test for the transmission of multi locus haplotypes was performed by applying the default block definition in HAPLOVIEW . Permutation testing  revealed that only rs362990  was associated with ADHD, with a trend towards association for rs6108461 (p-corrected = 0.059).Fifteen SNPs were examined at the 2 = 8.37, p-corrected  = 0.019, OR = 1.4). An exploratory haplotype analysis using sliding window of 8 SNPs (∼20 kbp) extending from intron 3 (rs6108461) to the 3UTR (rs8636) enhanced the risk association signal . Finally, a haplotype comprised of the alleles GTC in the second haplotype block . This implies that this haplotype may confer protection against the development of ADHD.Although strong LD is apparent across the gene , three dlock see  was less2 change  = 5.6.2%]. Similar results were found for rs6108461 . The strong LD between these SNPs meant that controlling for one removed the effect of the other in the above analyses. Possession of the ADHD risk haplotype (AAC) was also significantly associated with expression of SNAP-25 in the IFG tissue [2 change  = 6.3%]. As expected, increasing possession (0 vs. 1 vs. 2 copies) of the risk haplotype was associated with decreased expression. Conversely, increasing possession of the protective haplotype was associated with increased SNAP-25 expression [2 change = 7.7%].A significant association between rs362990 and relative expression of the SNAP-25 transcript was observed . SpecifiSNAP25 gene that associate with ADHD are also associated with functional changes in the expression level of the transcript in a region of the brain that is an established pathological locus for ADHD. The current investigation provides further support for the notion that SNAP25 is a genetic risk factor for ADHD. Although nominal associations with two SNAP25 SNPs (rs6108461and rs362990) were observed, only the association with rs362990 survived permutation testing. Haplotype analysis enhanced the association (OR = 1.62), suggesting that the region between intron 3 and the 3′UTR of SNAP25 may harbor an important susceptibility variant for ADHD.The results of the current study show that DNA variants of the SNAP25 an important candidate gene for ADHD.SNAP-25 is important for axonal growth and synaptic plasticity, which are essential steps for wiring the nervous system. Selective inhibition of SNAP-25 expression prevents neurite elongation of cortical neurons SNAP25 gene variants and ADHD. All these studies have examined multiple SNPs and tested for association of single SNPs and haplotypes. The associated SNPs and their position within the SNAP25 gene are presented in SNAP25; see 2 = 12.7, p-corrected = 0.003, OR = 1.62) indicating that this region may harbor a risk/functional variant for ADHD.Our results are consistent with some but not all previous association/linkage studies. Apart from the current investigation, a number of studies have tested for association between SNAP25, several SNPs showed evidence of association with the symptoms of ADHD in a quantitative GWAS SNAP25 markers including rs362562 SNAP25 variations in ADHD.Furthermore, it is important to emphasize that the quartile–quartile (QQ) plot for ADHD genome wide association (GWAS) for SNPs in or near candidate genes SNAP25 variants may increase risk to ADHD. Specifically, the lowered expression of SNAP-25 in regions of the cortex that are critical for attention and inhibition, such as the IFG, may ultimately decrease the efficiency of neurotransmitter release and synaptic function, impairing behavior and cognition and conferring risk to ADHD. It should be noted however that the above hypothesis must be viewed with some caution since the current study did not test the expression of SNAP25 in post-mortem samples derived from individuals with ADHD.Computer simulation SNAP25 mutations introduced to the C terminal of the protein at AA positions 78, 81 and 202 resulted in a near elimination of exocytosis Recent biochemical analyses have shown that SNAP-25 amino acids (AA) 7–83 and 141–204 are essential motifs that are spontaneously assembled into helical SNARE complexes with Syntaxin1 and synaptobrevin 2 motifs SNAP25 as a susceptibility locus for ADHD. We hypothesize that the region between intron 3 and the 3UTR of SNAP25 may harbor functional variants that confer risk to ADHD. Finally, we stress the importance of independent replication of our findings preferably in different ethnic samples.In summary, this study provides support for the involvement of


The five year survival of juvenile systemic sclerosis (jSSc) is around 95%. Patients, who died in the two retrospective cohorts, died mostly in the first 24 months of disease course. Autologous bone marrow transplantation (ABMT) seems to be a promising therapeutic approach for adult patients ( ASTIS (EU) and SCOT (USA)Trial) with severe disease course. Around 8 patients with jSSc are transplanted according the EBMT registry. Currently no consent based inclusion or exclusion criteria for ABMT in jSSc exists.Aim of the survey was to get a feeling from paediatric rheumatologists, when they would apply autologous bone marrow transplantation as a treatment option.Paediatric Rheumatologist - members of the PRES Juvenile Scleroderma Working Group and participants of the Paediatric Rheumatology E-mail Board were asked via Internet to fill out the survey22 centres responded, all of them were academic centres. BMT would be considered for 14 of the 22 colleagues after nonresponse to cyclophosphamide, 10 of 22 after non response to two DMARDs and 12 of 22 after nonresponse to Rituximab. 19 of 22 would consider transplantation if the CHAQ score ≤ 2 , 20 of 22 if the CHQ is less than 40%. 21 of 22 would think about transplantation if the modified Rodnan skin score is more than 30 and 15 of 22 if the DLCO is less than 50%, 18 of 22 if the WHO functional class is 3, 14 of 22 if the FVC less than 60%, 15 of 22 if the pulmonary arterial pressure more than 40 mm/hg and 11 of 22 if left ventricular ejection fraction is less than 40%.This survey represents an impression, when pediatric rheumatologist would consider ABMT. It is a starting point for a possible evolving ABMT program for this orphan disease


To compare the hemostatic effect of sulfur hexafluoride 20% (SF6 20%) with lactated Ringer’s solution for prevention of early postoperative vitreous hemorrhage following diabetic vitrectomy.In a prospective randomized clinical trial, 50 eyes undergoing diabetic vitrectomy were divided into two groups. At the conclusion of surgery, in one group the vitreous cavity was filled with SF6 20% while in the other group lactated Ringer’s solution was retained in the vitreous cavity. The two groups were compared for the rate of early postoperative vitreous hemorrhage.The incidence of vitreous hemorrhage was lower in the SF6 group than the Ringer’s group 4 days , 7 days  and 4 weeks  after vitrectomy.In comparison with lactated Ringer’s solution, SF6 20% had a significant hemostatic effect especially in the early postoperative period after diabetic vitrectomy and reduced the incidence of vitreous hemorrhage. Vitreous hemorrhage is one of the most serious complications of vitrectomy for proliferative diabetic retinopathy. The incidence of this complication ranges from 8% to 75%.7Postvitrectomy vitreous hemorrhage decreases visual acuity and impairs fundus visualization, it interferes with laser photocoagulation and detection of other retinal complications, furthermore it can predispose to cellular proliferation and may also increase intraocular pressure (IOP).Thompson et alRegarding the lack of data on the incidence of vitreous hemorrhage following diabetic vitrectomy using lactated Ringer’s solution, this pilot study was performed to evaluate the hemostatic effect of SF6 20% as compared to lactated Ringer’s solution in the early postoperative period following diabetic vitrectomy.In a prospective randomized clinical trial, 67 eyes of 67 patients undergoing diabetic vitrectomy who met the inclusion criteria were randomly divided into two groups using a random number table. Informed consent was obtained from all patients preoperatively. Indications for vitrectomy were nonclearing vitreous hemorrhage and progressive fibrovascular proliferation unresponsive to panretinal laser photocoagulation. Patients with orthopedic or systemic problems who were unable to maintain prone position, monocular patients, subjects who were obliged to travel by air immediately after the operation, those who required concomitant cataract surgery, as well as patients with rubeosis iridis, extensive tractional retinal detachment with or without rhegmatogenous retinal detachment were excluded. Patients who required silicone oil injection intraoperatively were also excluded from the study. Seventeen eyes were excluded due to intraoperative use of silicone oil. Eventually 50 eyes of 50 patients fulfilled the inclusion and exclusion criteria of the study and were analyzed.All eyes underwent standard three port pars plana deep vitrectomy by two surgeons; complete posterior vitreous detachment was induced and delamination and segmentation of preretinal fibrovascular tissue was performed. We decided to use lactated Ringer’s solution instead of BSS during diabetic vitrectomy in both groups because BSS contains sodium citrate which has anticoagulant properties and may promote vitreous hemorrhage.t-test for mean values; P values <0.05 were considered significant.The extent of vitreous hemorrhage was graded using indirect ophthalmoscopy according to the Diabetic Retinopathy Vitrectomy Study (DRVS) as follows:Fifty eyes of 50 patients were enrolled and randomized to SF6 20% (25 eyes) versus lactated Ringer’s solution (25 eyes). Mean age was 53.8±11.3  years in the SF6 group and 51.4±12.5  years in the Ringer’s group (P=0.417). Seventeen (68%) patients in the Ringer’s group and 13 (52%) patients in the SF6 group were female (P=0.248). The incidence of vitreous hemorrhage was higher in the Ringer’s group at all postoperative visits .Eleven (22%) patients had systemic hypertension preoperatively and were receiving antihypertensive agents. The incidence of vitreous hemorrhage during the follow-up period was 54.5% (6 cases) among hypertensive patients and 43.5% (16 cases) among normotensive patients (P=0.425). Forty-two eyes were phakic of which 3 eyes (6%), including two eyes in the SF6 group and one eye in the Ringer’s group, developed cataracts postoperatively. Mean IOP was 12.13±1.24 mmHg in the SF6 group and 12.16±0.34 mmHg in the Ringer’s group (P=0.951) 4 weeks postoperatively. Forty-one (82%) patients had type II and 9 (18%) patients had type I diabetes mellitus. The incidence of vitreous hemorrhage during the follow-up period was 46.3% (19 cases) in type II diabetic patients and 44.4% (4 cases) in type I patients (P=0.87).Visual acuity was hand motions or worse in 26 (52%) patients and better than hand motions but less than 6/120 in 24 (48%) patients preoperatively with no significant difference between the two groups (data not presented). At final visit, 11 (22%) eyes had visual acuity worse than 6/120, including 7 (28%) eyes in the Ringer’s group and 4 (16%) eyes in the SF6 group; 13 (26%) eyes had visual acuity between 6/120 and 6/60, including 8 (32%) eyes in the Ringer’s group and 5 (20%) eyes in the SF6 group; and 26 (52%) eyes had visual acuity better than 6/60, including 10 (40%) eyes in the Ringer’s group and 16 (64%) eyes in the SF6 group. The difference in postoperative visual acuity between the study groups was not statistically significant (P=0.235).Despite laser treatment for proliferative diabetic retinopathy, the ocular sequelae of diabetes mellitus remain one of the most common indications for deep vitrectomy. Postoperative vitreous hemorrhage is a common complication of diabetic vitrectomy with a reported incidence of up to 75%. Advances in surgical technique and instrumentation including endodiathermy and endolaser photocoagulation have reduced the incidence of this complication to 25%.Various substances have been used to prevent vitreous hemorrhage after diabetic vitrectomy. De Bustros et alKoutsandrea et alYang et al In our study, we employed lactated Ringer’s solution instead of BSS, because the latter contains citrate which can promote bleeding during and after surgery. Contrary to Koutsandrea et al,It summary we may conclude that SF6 20% in the vitreous cavity provides temporary tamponade and reduces the incidence of postoperative diabetic vitreous hemorrhage; disadvantages include the potential for inducing cataracts and prone positioning.


The Meta-Analysis of Glucose and Insulin related traits Consortium (MAGIC) recently identified 16 loci robustly associated with fasting glucose, some of which were also associated with type 2 diabetes. The purpose of our study was to explore the role of these variants in South Asian populations of Punjabi ancestry, originating predominantly from the District of Mirpur, Pakistan.SLC30A8 rs11558471 SNP was nominally associated with fasting glucose . Of interest, the MTNR1B rs10830963 SNP displayed a negative β value for fasting glucose in our study; this effect size was significantly lower than that seen in Europeans (p = 1.29×10−4). In addition to previously reported type 2 diabetes risk variants in TCF7L2 and SLC30A8, SNPs in ADCY5 (rs11708067) and GLIS3 (rs7034200) displayed evidence for association with type 2 diabetes, with odds ratios of 1.23  and 1.16  respectively.Sixteen single nucleotide polymorphisms (SNPs) were genotyped in 1678 subjects with type 2 diabetes and 1584 normoglycaemic controls from two Punjabi populations; one resident in the UK and one indigenous to the District of Mirpur. In the normoglycaemic controls investigated for fasting glucose associations, 12 of 16 SNPs displayed β values with the same direction of effect as that seen in European studies, although only the SLC30A8 rs11558471 SNP was nominally associated with fasting glucose in our study, the finding that 12 out of 16 SNPs displayed a direction of effect consistent with European studies suggests that a number of these variants may contribute to fasting glucose variation in individuals of South Asian ancestry. We also provide evidence for the first time in South Asians that alleles of SNPs in GLIS3 and ADCY5 may confer risk of type 2 diabetes.Although only the  Type 2 diabetes, a disease that is 4- to 6-fold more common in South Asian individuals than Europeans, is characterised by impaired glucose homeostasis resulting from a combination of beta cell dysfunction and insulin resistance. This inability to adequately regulate blood glucose levels is also linked to both micro- and macro-vascular complications. Recently, the Meta-Analysis of Glucose and Insulin related traits Consortium (MAGIC) identified 16 genetic variants robustly associated with fasting glucose in non-diabetic populations of European origin Informed written consent was obtained from all study participants and the studies were approved by the Birmingham East, North and Solihull Research Ethics Committee (UKADS participants) and the Baqai Institute of Diabetology and Endocrinology Institutional Review Board (DGP participants). UKADS registered clinical trial number; ISRCTN38297969.Participants in the investigation  were froAll subjects  were genotyped for the 16 SNPs using the KASPar method . Genotyping success rates were >96% for each SNP. Approximately 10% of samples were genotyped as blind duplicates resulting in an error rate of <1% for each SNP. Genotype counts in the two study populations are shown in p<0.05) and study-wide significance . Genetic risk scores (GRSs) were constructed to investigate the additive effect of multiple SNPs on fasting glucose levels and type 2 diabetes. For all GRS analyses, only those individuals successfully genotyped at ≥12 SNPs (nmax = 3231) were included. Firstly an allele count GRS (acGRS) was constructed. For each individual, the average number of risk alleles per successfully genotyped SNP  was multiplied by the total number of SNPs included in the GRS. This produced a GRS approximating a simple risk allele count where all SNPs were successfully genotyped. Two weighted GRSs (wGRS) were also produced, one for the fasting glucose analyses and one for the type 2 diabetes analyses. Risk alleles of each SNP were weighted by a SNP-specific β-value, obtained from the MAGIC study TCF7L2 and SLC30A8 variants, as these SNPs have previously displayed association with the disease in the UKADS and DGP study populations http://ibgwww.colorado.edu/~pshaun/gpc/) http://hydra.usc.edu/gxe) Deviation from Hardy-Weinberg equilibrium (HWE) in the non-diabetic groups was tested using an exact test implemented in Haploview SLC30A8 rs11558471 variant displayed a nominally significant association with fasting glucose levels  (MTNR1B rs10830963 effect size was markedly lower in the current study compared with the Europeans from the MAGIC study p = 1.29×10−4). The acGRS showed a stronger association with fasting glucose than did the wGRS   . No evidctively) .TCF7L2 and SLC30A8 variants, which have previously demonstrated association with type 2 diabetes in the UKADS/DGP study populations ADCY5 rs11708067  and GLIS3 rs7034200  SNPs conferred risk of the disease in this study  than the acGRS  .None of the 16 studied variants displayed strong evidence for heterogeneity of effect on type 2 diabetes risk between the current study and the MAGIC study. In addition to the is study . The str= 0.001) . The strSLC30A8 rs11558471 variant was nominally associated with fasting glucose levels. Twelve of the 16 SNPs displayed positive β-values, however, suggesting that a number of these variants may be true determinants of fasting glucose levels in our study populations  have negative β-values in the current study  was not.In this study we investigated the effects of 16 SNPs on fasting glucose levels and type 2 diabetes risk in two South Asian populations of Punjabi ancestry. Only the ulations , even thnt study , and thiGCK, GCKR, G6PC2 and MTNR1B have been shown to be associated with fasting glucose levels in Indian Asians, with similar effect sizes to those seen in Europeans GCK and GCKR SNPs studied in Indian Asians (rs4607519 and rs1260326 respectively) are in strong linkage disequilibrium (LD) with the SNPs genotyped in this study  with the variant (rs10830963) genotyped in our South Asian cohort and reported as the sentinel MTNR1B SNP in the MAGIC study MTNR1B SNPs and their association with glucose levels in South Asians may be useful in fine-mapping the aetiological variant.It is of interest to note that variants within or near t is low , it is nTCF7L2 and SLC30A8 have previously been associated with type 2 diabetes in our Punjabi populations ADCY5 and GLIS3 were associated with the disease in the current study. To our knowledge, this is the first time that either of these SNPs has been implicated in type 2 diabetes development in a South Asian population. The ADCY5 gene encodes adenylate cyclase 5, an enzyme that catalyses the generation of cAMP, a second messenger vital in a number of biological processes. It has been demonstrated in a large meta-analysis of Europeans that the rs11708067 SNP within ADCY5 is strongly associated (p≤3.6×10−8) with type 2 diabetes, fasting glucose levels and HOMA-B (a measure of β-cell function) but is not associated with HOMA-IR (a measure of insulin resistance) ADCY5 gene (rs9883204), in LD with the variant investigated in this study, is associated with foetal growth and birth weight in utero, potentially providing a common mechanism linking reduced foetal growth with increased risk of type 2 diabetes. The GLIS3 gene encodes the transcription factor GLIS family zinc finger 3 isoform, a protein that regulates target gene transcription and has been shown to play a key role in β-cell generation in mice GLIS3 gene lead to a syndrome of neonatal diabetes and congenital hyperthyroidism GLIS3 rs7034200 SNP only displayed a weak association with type 2 diabetes in the MAGIC study TCF7L2 SNP, three of the four variants most strongly associated with type 2 diabetes in the MAGIC study MTNR1B rs10830963, PROX1 rs340874 and GCKR rs780094) have odds ratios of less than one in our South Asian populations Click here for additional data file.


Several novel susceptibility loci for type 2 diabetes have been identified through genome-wide association studies (GWAS) for type 2 diabetes or quantitative traits related to glucose metabolism in European populations. To investigate the association of the 13 new European GWAS-derived susceptibility loci with type 2 diabetes in the Japanese population, we conducted a replication study using 3 independent Japanese case-control studies.MTNR1B, GCK, IRS1, PROX1, BCL11A, ZBED3, KLF14, TP53INP1, KCNQ1, CENTD2, HMGA2, ZFAND6 and PRC1) with type 2 diabetes using 4,964 participants  from 3 independent Japanese samples. The association of each SNP with type 2 diabetes was analyzed by logistic regression analysis. Further, we performed combined meta-analyses for the 3 studies and previously performed Japanese GWAS data . The meta-analysis revealed that rs2943641 in the IRS1 locus was significantly associated with type 2 diabetes,  and 3 SNPs, rs10930963 in the MTNR1B locus, rs972283 in the KLF14 locus, and rs231362 in the KCNQ1 locus, had nominal association with type 2 diabetes in the present Japanese samples (P<0.05).We examined the association of single nucleotide polymorphisms (SNPs) within 13 loci (IRS1 locus may be common locus for type 2 diabetes across different ethnicities.These results indicate that  Type 2 diabetes is a chronic metabolic disorder characterized by hyperglycemia, variable degrees of insulin resistance, and impaired insulin secretion. The total number of individuals with diabetes mellitus is estimated to be nearly 300 million worldwide, and its prevalence continues to increase in many countries, including Japan. Although the precise mechanisms underlying the development and progression of type 2 diabetes have not been elucidated, it is considered that genetic factors play an important role in the pathogenesis of the disease KCNQ1) locus, ubiquitin-conjugating enzyme E2E 2 (UBE2E2) locus and C2 calcium-dependent domain containing 4A (C2CD4A)-C2CD4B locus with type 2 diabetes Currently, approximately 40 susceptibility loci for type 2 diabetes, mostly discovered through genome-wide association studies (GWAS), have been confirmed in populations of European descent  IRS1) was identified by GWAS on French patients Because many of these loci have also been shown to be associated with type 2 diabetes in other ethnic populations, including Japanese, these loci may be considered convincing susceptibility loci for type 2 diabetes across different ethnicities In this study, we aim to evaluate the contribution of these new susceptibility loci identified in European GWAS to conferring susceptibility to type 2 diabetes in the Japanese.We selected 4,964 individuals, 2,839 cases and 2,125 controls, from 3 independent Japanese samples.st study): DNA samples were obtained from peripheral blood samples of type 2 diabetes patients recruited from the outpatient clinics of the Shiga University of Medical Science and the Kawasaki Medical School , We also examined 716 controls who were enrolled in an annual health check conducted either at the Juntendo University or the Keio University .RIKEN case-control study (1nd study): We selected 724 individuals with type 2 diabetes from the outpatient clinic of the Toyama University Hospital . We also examined 763 controls with HbA1c<6.0%, age≥50, no family history of diabetes mellitus for the first and second degree relatives .Toyama University study (2rd study): We recruited 485 individuals with type 2 diabetes from the outpatient clinic of the St. Marianna University School of Medicine . We also examined 646 controls, who were enrolled in an annual health check conducted at the St. Marianna University School of Medicine St. Marianna University study  GCKR locus and rs2191349 in the DGKB- TMEM195 locus were shown to be associated with type 2 diabetes in the Japanese We first selected 9 single nucleotide polymorphisms (SNPs) from previous reports, rs2943641 within the locus near the SNP genotyping was performed by the multiplex-polymerase chain reaction (PCR)-invader assay, as described previously http://www.sph.umich.edu/csg/abecasis/CaTS/).We performed the Hardy-Weinberg Equilibrium (HWE) test according to the method described by Nielsen et al ADCY5 locus, and rs7957197 in OASL locus) are monoallelic in the Japanese populations. The genotype distributions of rs13292136 in CHCHD9 showed significant deviation from the Hardy-Weinberg equilibrium proportion in the control group  was consistent with that of a previous study in European populations 2<80), whereas no heterogeneity in effect size (odds ratio) was observed on the remaining loci (data not shown). We could not observe remarkable differences in LD structures around each locus between Japanese and European populations — may also have some effects. Further studies are required to elucidate the association of these as well as other loci with susceptibility to type 2 diabetes, and to understand the biological significance of these genes and their polymorphisms.In conclusion, these results indicate that the Figure S1Linkage disequilibrium structures for 500 kb region around each SNP locus in JPT and in CEU. Pairwise correlation structure analyzed by Haploview (http://www.broadinstitute.org/haploview/haploview). The plot includes pairwise D′ values from the HapMap release 27.(PDF)Click here for additional data file.Table S1Genotype data for 15 SNPs in the 3 independent Japanese samples.(DOC)Click here for additional data file.Table S2The associations of the 14 SNPs with type 2 diabetes in 3 independent Japanese samples.(DOC)Click here for additional data file.Table S3Power estimation for each SNP locus in the present study.(DOC)Click here for additional data file.


FYVE  and coiled coil containing 1 (fyco1) causes human cataract suggesting a role for autophagy in lens function. Here, we analyzed the range and spatial expression patterns of lens autophagy genes and we evaluated whether autophagy could be induced in lens cells exposed to stress.Mutation of the autophagy gene Autophagy gene expression levels and their spatial distribution patterns were evaluated between microdissected human lens epithelium and fibers at the mRNA and protein levels by microarray data analysis, real-time PCR and western blot analysis. Selected autophagy protein spatial expression patterns were also examined in newborn mouse lenses by immunohistochemistry. The autophagosomal content of cultured human lens epithelial cells was determined by counting the number of microtubule-associated protein 1 light chain 3B (LC3B)-positive puncta in cells cultured in the presence or absence of serum.A total of 42 autophagy genes were detected as being expressed by human lens epithelium and fibers. The autophagosomal markers LC3B and FYCO1 were detected throughout the newborn mouse lens. Consistently, the autophagy active form of LC3B (LC3B II) was detected in microdissected human lens fibers. An increased number of LC3B-positive puncta was detected in cultured lens cells upon serum starvation suggesting induction of autophagy in lens cells under stress conditions.The data provide evidence that autophagy is an important component for the function of lens epithelial and fiber cells. The data are consistent with the notion that disruption of lens autophagy through mutation or inactivation of specific autophagy proteins could lead to loss of lens resistance to stress and/or loss of lens differentiation resulting in cataract formation. The eye lens is an avascular encapsulated organ whose function is to focus light on to the retina where visual information is translated into nerve signals and ultimately perception of images by the visual cortex of the brain. Disruption of lens transparency as a consequence of developmental defect or damage to lens cells and their components causes cataract formation –3 which One intriguing system that may be important for lens differentiation and resistance to environmental damage is macroautophagy (hereafter termed autophagy) which operates in the degradation and recycling of damaged organelles and proteins in many other tissues . AutophaATG5) did not disrupt lens fiber cell differentiation despite the occurrence of autophagy in lens cells , tuberous sclerosis complex 1 [tsc 1], UV irradiation resistance-associated gene [uvrag], astrocyte-elevated gene-1 [aeg-1], high temperature requirement  factor A2 [omi/htrA2], phosphatase and tensin homolog [pten], autophagy related 14 [atg14], Bax-interacting factor 1 [bif1], high mobility group box 1 [hmgb1], v-ral simian leukemia viral oncogene homolog B [ralB], retinoblastoma 1 inducible coiled coil-1/focal adhesion kinase (FAK) family interacting protein of 200 kDa [rb1cc1/fip200], forkhead box O1 [foxO1], forkhead box O3 [foxO3], and PKR-like ER kinase/eukaryotic translation initiation factor 2-alpha kinase 3  [perk/eif2alpha3k]), eight genes were involved in expansion of autophagic vesicles , six were genes involved in autophagosome fusion to lysosomes  and eight genes were involved in specific autophagy sub- pathways including mitophagy  and chaperone mediated autophagy , which was present on the microarray but not definitively identified to be expressed, was also analyzed. These genes included the autophagy induction genes beclin 1, atg14, fip200, ralb, the autophagy expansion/closure genes atg4a, atg12, map1lc3b/atg8, the autophagy fusion/degradation genes rab7, fyco1, the mitophagy genes nix/bnip3L, pink1 and the adaptor gene sequestosome 1 (p62). The data confirmed that all of the analyzed genes were expressed in human lens epithelium and fiber cells  in lens fibers suggests that autophagy is a functional process throughout the lens. Since autophagy is a response to stress in many tissues  we examifyco1 causes human cataract [Autophagy has been shown to be important for the development, differentiation and protection against oxidative stress in multiple tissues ,88, howecataract , suggestcataract .As a first step toward examining the potential function of autophagy in the lens, we analyzed the expression levels of autophagy genes in microdissected human lens epithelium and fiber cells. Our analysis revealed the lens epithelium and fiber cell expression of 14 genes involved in autophagy induction, eight genes involved in expansion of autophagy vesicles, six genes involved in autophagosome fusion to lysosomes and eight genes involved in specific autophagy sub-pathways including mitophagy and chaperone mediated autophagy . The datTo date, few reports on the role of autophagy in lens function have been published. Matsui et al.  reportedmap1lc3b [bnip3l/nix [beclin1 [rb1cc1/fip200 [bif-1 [lamp2 [beclin 1 and fip200 were embryonic lethal and atg3 was neonatal lethal. Lamp2 knockout mice were viable but exhibited increased mortality and bif-1, map1LC3b and bnip3l/nix were viable. Only two genes (apart from fyco1) that were identified in the present report have a previously reported lens function; these are fox01 and fox03 [atm), has also been reported to be important for lens resistance to cataract [In addition to the ATG5 mouse knockout, other knockout mice have been made for some of the genes we detected in the lens but unfortunately no lens or eye phenotype has been described for any of the mice. These include map1lc3b , bnip3l/ip3l/nix ,91, becl[beclin1 , rb1cc1/1/fip200 , bif-1 [0 [bif-1 , and lam1 [lamp2 . Of thesnd fox03  which wecataract . It is iIn summary, the present data provide evidence for a significant role for autophagy in lens function. Autophagy and mitophagy are likely important for lens cell removal of damaged proteins that could cause cataract upon their accumulation and the degradation of lens organelles during epithelial cell differentiation into fiber cells. Further studies on the role of autophagy in lens resistance to stress and lens cell differentiation are likely to provide additional insight into our understanding of lens development, maintenance and cataract formation.


Drosophila, the MSL  complex up regulates transcription of active genes on the single male X-chromosome to equalize gene expression between sexes. One model argues that the MSL complex acts upon the elongation step of transcription rather than initiation. In an unbiased forward genetic screen for new factors required for dosage compensation, we found that mutations in the universally conserved transcription elongation factor Spt5 lower MSL complex dependent expression from the miniwhite reporter gene in vivo. We show that SPT5 interacts directly with MSL1 in vitro and is required downstream of MSL complex recruitment, providing the first mechanistic data corroborating the elongation model of dosage compensation.In  Spt5 gene that encodes a universally conserved elongation factor. SPT5 closes the RNA polymerase II clamp around the DNA template to prevent pausing or premature termination. We find that the dosage compensation complex genetically and physically interacts with SPT5 on actively transcribed genes providing direct molecular support for the elongation model of dosage compensation.Drosophila males hypertranscribe most of the genes along their single X chromosome to match the output of females with two X chromosomes. It had been difficult to imagine how the MSL dosage compensation complex could impose a modest, but essential, ∼two-fold increase by interacting with hundreds of different factors that control transcription initiation for such a diverse collection of genes. An alternative model proposed that dosage compensation instead acted at some step of transcription elongation common to all genes. We performed a genetic screen for mutations that subtly reduce dosage compensation and recovered mutations in the  Drosophila dosage compensation is widely used as a model system to investigate how transcription is regulated by large scale chromatin modifications roX (RNA on X) RNAs A long-standing puzzle is the biochemical mechanism by which the MSL complex up regulates X-linked genes, each of which is controlled by different transcription factors. An elegant model that solves this problem posits that MSL complex does not act with diverse gene-specific transcription factors to alter initiation, but rather at the elongation step of transcription common to all genes Spt5, a universally conserved transcription elongation factor. We found that SPT5 is required for dosage compensation in males and extensively colocalizes with the MSL complex on the X-chromosome. Moreover, we found that SPT5 and MSL1 directly interact with each other. We propose that SPT5 is required downstream of MSL complex recruitment to stimulate transcription elongation. The identification of SPT5 is strong mechanistic evidence supporting the elongation model of dosage compensation.To search for new factors involved in dosage compensation we performed an unbiased forward genetic screen that relies on a sensitive eye pigmentation reporter of MSL complex activity. This approach was designed to recover heterozygous mutations in genes that are essential for general transcription in both sexes but play an additional role in male dosage compensation. We recovered multiple alleles of roX1 transgenic males is a sensitive reporter of MSL activity roX1 transgenes occasionally land in repressive chromatin, the miniwhite marker is epigenetically silenced so that females have solid white eyes. Males have spotted eyes because the MSL complex binds the autosomal roX1 transgene and locally modifies the chromatin allowing miniwhite expression in a fraction of cells. We have previously described a strategy for isolating mutations that increased local MSL activity in vivo, but may associate with the MSL complex too weakly or transiently to copurify with MSL proteins. Second, our genetic strategy retains a wild type allele of the relevant gene allowing us to capture factors that have additional essential functions. Homozygous mutations in such factors would be lethal to both sexes and thus would have been missed in earlier genetic screens based on the male specific lethal phenotype.The eye color of miniwhite reporter linked to roX1. Mutants that were also recessive lethal to both sexes were placed in complementation groups (roX1 transgenes inserted in distinct repressive locations reasoning that those were more likely to affect dosage compensation rather than the particular silencing factors acting on flanking chromatin (miniwhite gene in our roX1 reporters. We tested the effect of the new mutants on m4In(1)w which displays classic position effect variegation in both sexes. Most of the candidate modifiers of dosage compensation did not affect pigmentation in m4In(1)w wm4  arguing Spt5 and Elongin-C are located . However, when we tested the MGE-3Spt5 mutation Complementation group C was chosen for detailed analysis. Meiotic recombination placed the locus near the polytene bands 56C-F but none of the available chromosome deficiencies uncovered the mutation  located . AvailabroX1 lines just like Spt5 mutations had. We crossed six different roX1 mosaic lines that carry roX1 transgenes in diverse chromatin environments to 190 deficiencies. The idea was that any deficiency that affected the eye coloration of multiple roX1 reporter lines was more likely to affect some aspect of dosage compensation rather than the particular repressive environment surrounding the different inserts. We found that only 10 intervals reduced MSL complex reporter activity . This shows that a 50% reduction in SPT5 levels does not alter the phenotype from hypomorphic white alleles or miniwhite. We conclude that Spt5 mutations dramatically affected the probability that males overcome silencing not because of global reduction of transcription across the genome or the white promoter itself, but rather because SPT5 plays some role in dosage compensation to which our roX1 reporter is responsive.We were still concerned that the entation . We alsoSpt5 affected dosage compensation of the X chromosome in addition to the roX1 eye color reporter transgene. Because SPT5 is essential for most transcription, homozygous null animals die early in development. The viability of Spt5/+ males demonstrates that dosage compensation must be adequate even with reduced SPT5 levels. The same is true for any of the msl/+ heterozygotes. However, males with limiting MSL complex might be more sensitive to reduced levels of SPT5. Males missing either roX1 or roX2 are alive but males missing both roX RNAs have greatly reduced male viability roX1 roX2 double mutant males are completely lethal but can be rescued by an autosomal roX transgene msl1 or mle showed reduced viability when roX1 RNA is also limiting roX1 RNA was limiting consistent with a role in dosage compensation S192, a mutation in the elongation factor ELL did reduce male viability. Others have reported that ELL RNAi lines display male specific lethality consistent with a role in dosage compensation roX1 RNA levels.We used a sensitized genetic background to see if ensation . We assaiability . Howevermsl2 mRNA. Ectopic dosage compensation can be induced in females by artificial expression of MSL2 by the [H83M2] transgene that escapes SXL regulation Spt5 weakens dosage compensation then that might reduce the toxic effects of inappropriate dosage compensation in [H83M2] overexpression females. When [H83M2] females also carried a mutation in Spt5, the female-specific developmental delay was modestly rescued  of most Drosophila genes, with additional binding across the transcribed regions In order to place SPT5 in the dosage compensation pathway, we focused on two of its most intensively studied roles. First, unphosphorylated SPT5 binds to and pauses RNA polymerase over the transcription start site (TSS). Release from this 5′ pause requires phosphorylation by P-TEFb at multiple sites at the C-termini of both RNA polymerase large subunit and SPT5 Spt5 null tissue. However, we can approximate that condition by using elongation inhibitors DRB and flavopiridol that block P-TEFb phosphorylation of RNAPII CTD Ser2 and SPT5 that are necessary for pause-release and entry into elongation The Pause Release model is less appealing because it calls for MSL action at the TSS, when MSL complex is instead predominantly located farther downstream Modified histone H3K36me3 is found within active genes with a 3′ bias similar to the MSL complex. This modification may provide one component of MSL targeting specificity through the MSL3 chromodomain To examine MSL binding at a higher resolution than is possible with polytenes, we turned to ChIP analysis of male S2 cells. We measured MSL1 binding to the 5′ and 3′ ends of two highly validated target genes after treatment with flavopiridol. If the scarcity of MSL complex at the 5′ ends of X-linked genes was caused by released RNAPII/SPT5 complex quickly carrying it into the body of genes, we might be able to trap MSL complex over the TSS by treatment with P-TEFb kinase inhibitors. The Pause Release Model predicts that flavopiridol treatment should cause the MSL signal to accumulate at the 5′ end of genes with a corresponding loss at the 3′ end. However, just as was seen with the polytene experiments, flavopiridol treatment did not alter MSL complex distribution as measured by ChIP . These rroX RNAs with regions of SPT5 in vivo, the data show that SPT5 and MSL complex have the ability to interact via MSL1 PEHE.We tested whether the genetic interactions observed between SPT5 and dosage compensation might arise from direct physical contacts. Early attempts to purify intact MSL complex did not recover SPT5 as a partner suggesting that if such interactions occur, they are transient Spt5, we wondered if the other modifier mutations found in the screen might identify a new class of factors needed for dosage compensation. We were able to map a few modifiers to previously characterized genes. In the case of deficiencies, we tested whether point mutations of candidate genes could recapitulate the effect of deficiency. That approach showed that Chromator was the relevant gene that dominantly suppresses the MSL complex dependent reporter expression in Df(3L)BSC21. CHRO is a chromodomain protein that localizes specifically to the interband regions and is implicated in maintaining chromosome structure Df(2R)vg-C vg-C . This inroX RNAs, and the fact that active MSL complex is tightly associated with transcribed chromatin. Extraction methods strong enough to release soluble MSL complex from chromatin may destroy critical contacts with key partners. Genetic approaches also face limitations. If an important partner performs additional functions beyond dosage compensation, mutations would likely be lethal to both sexes masking its interaction with the MSL complex.It is possible that dosage compensation in Drosophila is entirely a consequence of the known histone modifications carried out by its subunits, H4K16ac (MOF) and H2BK34ub (MSL2). However, if additional factors are required, new approaches may be needed to identify them. Biochemical purification is challenging due to the very large size of the MSL complex, the presence of the noncoding Spt5, a universally conserved transcription processivity factor for RNAPs, in the MSL pathway msl1, mle and ChroWe developed an unbiased forward genetic screen able to detect subtle changes in MSL activity that are not large enough to prevent dosage compensation of the male X, but sufficient to alter a sensitive eye pigmentation reporter. This screen implicated Spt5 mutations on the white eye color reporter was entirely dependent upon the adjacent roX1 locus that can recruit soluble MSL complex to any location in the genome. Spt5 mutations had no effect on white or miniwhite gene expression when not linked to roX1. Spt5 mutations acted on all mosaic roX1 reporter transgenes regardless of the chromatin environment surrounding the inserts. Interactions of Spt5 mutants and gain of function msl1 alleles suggest that SPT5 acts between MSL complex and RNA polymerase. Mutations in Spt5 selectively reduced male viability under limiting roX RNA conditions in a manner comparable to the effect of mle mutations. Additionally mutations in Spt5 partially suppressed the toxic effects of ectopic dosage compensation in females. An independent screen of the Drosophila deficiency collection showed that the Spt5 phenotype is rare. Removing one allele of almost any transcription related factor had no effect on the eye pigmentation levels of mosaic roX1 reporters arguing that dosage compensation is particularly sensitive to SPT5 protein levels. Finally, we found that the most ancient and conserved segment of the MSL1 protein physically binds to two different regions of the SPT5 protein consistent with the largely overlapping patterns of chromatin occupancy across the body of X-linked genes.Multiple lines of evidence support a role for SPT5 in dosage compensation. The effect of In eukaryotes SPT5 along with SPT4 forms the DSIF  hsp70 gene from Drosophila While regulated release from pause was originally described using the highly inducible MSL complex mediated H4K16ac is enriched within the body of genes and drives decondensation of chromatin possibly facilitating easier passage of RNAPII Spt5 in this report, our genetic approach also yielded additional candidates. So far, we have mapped two of these to Chro and Sin3A. CHRO, a chromodomain protein copurifies with the MSL complex While we have focused on the analysis of in vivo genetic strategy.An independent RNAi screen using an MSL complex dependent luciferase expression as a reporter in S2 cells also identified a role for CHRO and SIN3A in dosage compensation in vivo support for the elongation model of dosage compensation by linking the SPT5 elongation factor to the MSL complex Drosophila males have ∼1.4 fold more transcriptionally engaged RNAPII at the distal ends of X-linked genes as compared to autosomes also supports the idea of increased elongation Our results provide direct Mammals also contain a version of the MSL complex composed of MSL1, 2, 3 and MOF, but apparently lacking a large noncoding RNA component and RNA helicase + GMroX1-58D], [w+ GMroX1-60F], [w+ GMroX1-69C], [w+ GMroX1-75C], [w+ GMroX1-99F][w and + GMroX1-102C]y w roX1Mutagenesis was performed as described Plasmids for bacterial expression of MBP fusion SPT5 protein fragments, SPT5-N (aa 112–393), M (aa 389–733) and C (aa 732–1054) were a kind gift from Dr. John Lis Polytene squashes were prepared as described in Detailed protocol can be found in MBP-SPT5N, MBP-SPT5M, MBP-SPT5C, the unrelated protein MBP-MCP (MS2 phage coat protein), GST-MSL1 C-terminal domain fusion protein and GST were expressed and isolated from bacteria. Equivalent molar concentrations of MBP proteins bound to amylose beads were incubated with GST proteins. After three washes with 150 mM NaCl, 0.1% NP40 and 20 mM Tris for 10 mins at 4°C, proteins were eluted by boiling in SDS-loading buffer and separated on 8% SDS-polyacrylamide gels. Westerns were performed as described Figure S1spt5.A forward genetic screen to identify modifiers of MSL complex activity. (A) Approximately 16,000 males were screened and 48 modifier lines established. (B) Mutations were placed into complementation groups based on recessive lethality. The mutations scored as single hits are recessive lethals, which could be due to the modifier allele or an EMS induced secondary mutation. The mutants scored as viable suppressors lower MSL complex dependent red pigmentation and are homozygous viable. We identified 5 alleles of (TIF)Click here for additional data file.Figure S2Spt5 consistently lower mosaic eye pigmentation independent of autosomal roX1 insertion site. Shown above are flies hemizygous for the [GMroX1]/+ transgene at different positions in the genome shown on the side. Flies on the left are wildtype whereas flies on the right are heterozygous for S14FSpt5. All flies shown are males.Mutations in (TIF)Click here for additional data file.Figure S3Spt5 do not affect the white promoter. Males and female eye pigmentation is shown for the hypomorphic wa and we alleles with and without Spt5 mutations. Although Spt5/+ heterozygous males show dramatic pigment reductions from the dosage compensation [w+GMroX1] mosaic transgenes .Mutations is ansgenes , the sam(TIF)Click here for additional data file.Figure S4Validation of anti SPT5 antibodies. (A) Polytene chromosomes stained with anti-SPT5 antibodies. SPT5 is widely distributed on many sites on the genome. (B) Immunodepletion of anti-SPT5 antibodies with SPT5 fragments- SPT5N, SPT5M and SPT5C results in loss of signal indicating that the antibodies predominantly recognize SPT5. (C) Polytene chromosome spreads prepared from males subjected to 30 min heat shock at 37°C were stained with anti-SPT5 antibodies. In agreement with previous reports (TIF)Click here for additional data file.Figure S5SPT5 and MSL1 colocalize extensively on the X-chromosome. Polytene chromosome spreads were stained with antibodies against (A) MSL1 (green) and (B) SPT5 (red). As expected of a general transcription elongation factor SPT5 binds all over the genome. (C) Colocalization of MSL1 and SPT5.  A closer look at two regions of the X-chromosome. Arrow heads indicates MSL1 only (no SPT5) binding and arrows indicate SPT5 only (no MSL1) binding.(TIF)Click here for additional data file.Figure S6Treatment with Flavopiridol does not affect H3K36me3. (A) Polytene chromosome spreads prepared from salivary glands treated with either DMSO (control panel) or 500 nM Flavopiridol for 30 minutes. Chromosomes were stained with antibodies against H3K36me3. (B) Ser5P RNAPII and SPT5 colocalize on polytenes. After treatment with Flavopiridol, SPT5 is greatly reduced but low levels of SPT5 are found at polytene bands that are positive for Ser5 Phosphorylated RNAPII. (C) A close-up of Ser5P RNAPII and SPT5 colocalization in control and flavopiridol treated samples.(TIF)Click here for additional data file.Table S1roX1 lines were assayed in the deficiency screen (Bloomington Deficiency collection) and the deficiencies that dominantly suppressed the red pigmentation across more than four different mosaic roX lines are indicated. Candidate genes that have been mapped within those deficiencies are also shown. Only ten intervals (14/190 deficiencies) assayed had an effect on the eye phenotype indicating that a general suppression of transcription does not lower the expression of the dosage compensation reporter. Spt5 would be an eleventh locus, but is not uncovered in the Df collection.Results of Deficiency screen. Six mosaic (DOC)Click here for additional data file.Table S2Spt5 decreases male viability. In the absence of both roX1 and roX2 males are dead. The male specific lethality can be rescued by an autosomal GMroX1 transgene. In this assay roX1-,roX2-; [GMroX1-75C] virgins were crossed to balanced males carrying the indicated mutations. The male progeny of this cross that are roX1-,roX2-/Y;mutation/+;[GMroX1-75C]/+ are compared to the nonbalanced female progeny. All the flies therefore carry only one copy of the indicated mutation. The statistical significance was determined by Fisher exact fit test. Lowering (DOC)Click here for additional data file.Text S1Supplementary materials and methods.(DOC)Click here for additional data file.


We also describe a new morphogenetic cell movement in the ventral first arch, sweeping cells anterior in the arch to the region where the lower jaw forms. This movement is negatively regulated by hand2 in an apparently edn1-independent fashion. These findings point to complexity of regulation by edn1 and hand2 at the earliest stages of pharyngeal arch development, in which control of growth and morphogenesis can be genetically separated.Endothelin-1 (Edn1) signaling provides a critical input to development of the embryonic pharygneal arches and their skeletal derivatives, particularly the articulating joints and the ventral skeleton including the lower jaw. Previous work in zebrafish has mostly focused on the role of Edn1 in dorsal-ventral (DV) patterning, but Edn1 signaling must also regulate tissue size, for with severe loss of the pathway the ventral skeleton is not only mispatterned, but is also prominently hypoplastic – reduced in size. Here we use mutational analyses to show that in the early pharyngeal arches, ventral-specific  However, it is unknown whether the phenotype is due to change in growth versus morphogenesis.Other recent work in zebrafish has identified additional signaling pathways, Notch and BMP, that interact with Edn1, and these studies fill in gaps in our understanding of arch patterning coming from just Edn1 signaling in isolation edn1 and hand2 mutants to focus on pharyngeal arch size regulation and examine edn1;hand2 double mutants to learn the epistatic relationships between these two genes. We extend the previous results showing edn1-dependent DV expansion, and we find that the expansion is due to growth, i.e., arch increase in size, rather than morphogenetic shape change. Previous work in zebrafish had demonstrated that edn1 functions upstream to hand2 in a positive pathway regulating DV patterning hand2 functions as a negative regulator of edn1-dependent ventral outgrowth. We provide direct evidence that the control of this outgrowth is at least in part by regulation of cell proliferation. Finally, we show that hand2 negatively regulates an anterior morphogenetic cell movement in the ventral first arch. This regulation appears to be independent of edn1 function.In this study we use edn1 or hand2 in zebrafish, developmental patterning of the embryonic pharyngeal arches is disrupted along the DV axis edn1 and hand2 single mutants are essentially as previously described edn1 single mutant. In the hand2 single mutant, a recognizable interhyal is absent, but the symplectic and joint are present. Thus it is in this intermediate region where the phenotypes of the two mutants differ.Previous work has established that with severe loss of function of either edn1 mutant both features are absent. In the hand2 mutant the joint is absent, but in the territory of the retroarticular process we see a locally expanded wedge- or fan-shaped region of cartilage, previously interpreted as being dorsalized As we interpret the first arch phenotypes of both single mutants, we also reach the conclusion that the mutants differ in the intermediate region. The dorsal pterygoid process and upper palatoquadrate cartilages are present in both single mutants. Ventrally in both, Meckel’s cartilage is extremely reduced. The intermediate region in WT forms the joint between the palatoquadrate and Meckel’s elements and a prominent retroarticular region of Meckel’s cartilage adjacent to the joint. In the edn1;hand2 double mutant has not been previously described. The most striking aspect is its overall phenotypic resemblance to the edn1 single mutant. This finding is consistent with previous understanding that the two genes act positively with respect to ventral cartilage development, and that they function on the same genetic pathway edn1 mutant rather than the hand2 mutant. In the second arch, the symplectic and joint are absent. Strikingly, in the intermediate region of the first arch where the single mutants perturb the WT condition in opposite directions  we also see an −edn1 -like reduction, showing that for this phenotype edn1 is epistatic to hand2. This finding indicates that the relationship between the two genes is complex, seemingly in opposite directions for the positive interaction responsible for overall cartilage reduction and for the negative interaction in expansion in the first arch intermediate region (see Discussion).The zebrafish edn1– hand2 interaction, we examined expression of fli1a:EGFP, a marker of neural crest derived ectomesenchyme, during early pharyngula stages ). At these stages patterning genes under control of edn1, including hand2, are transcriptionally upregulated and likely functional dlx2a expression as a proxy for arch morphology, discovered an edn1 mutant arch phenotype that becomes apparent during this period – a ventral-specific reduction in the DV extent of the pharyngeal arches, which normally are rapidly extending along this axis edn1 and hand2 single mutants, and the edn1;hand2 double mutant  is similar to that of the edn1 single mutant, and note that the reduction in these mutants is opposite to the expansion in the hand2 single mutant , and high PC1 values (WT and hand2 mutants) are not due to convergence and extension. The data do not show that arch convergence and extension is absent at this stage, only that the four genotypic groups do not differ from one another in this respect. Indeed, following the time course of changes by PCA suggests that the early arches are indeed undergoing convergence and extension, and that the genotype-specific changes occur progressively with time  and the epistasis between the two genes is the same; edn1 is epistatic to hand2.We used phospho-histone H3 labeling to examine proliferation, including senchyme . Double-senchyme . Strikine mutant . Genotype mutant . These fhand2 single mutant and the edn1;hand2 double mutant . We also note that in invitro studies in human cancer cell lines EDN1 promotes cell proliferation as well as inhibiting cell death We find that one edn1 being epistatic to hand2 in the control of early proliferation would place hand2 genetically upstream of edn1, were their interaction within a linear genetic pathway edn1 is molecularly upstream of hand2edn1-target genes Dlx genes function in the intermediate domain under positive control of edn1 as demonstrated by their downregulation in edn1 mutants Dlx genes are negatively regulated by hand2, as demonstrated by their upregulation in hand2 mutants edn1-hand2-Dlx interaction thus provides precedent for our model in Figiure 8A, and indeed it is possible that Dlx genes themselves mediate the regulation of early proliferation that our model requires. Talbot et al. described the expansion of the ventral-anterior region of the first pharyngeal arch in the zebrafish hand2 mutant being accompanied by an expansion of the intermediate domain Dlx genes hand2– Dlx gene interaction is involved in ventral proliferation is likely possible with currently available methods, but would be difficult because of redundancy of function of Dlx genes  and not well understood. It could well be that some specific instances of the human disorder could involve partial failure of anterior extension.hand2, both in terms of signaling pathways that converge on hand2 and in its role as a focal point in the regulation of downstream effector genes mediating a variety of functions – patterning Our work not only identifies Edn1 as a growth factor in pharyngeal arch development, but particularly highlights the complexity of regulation involving tf216bedn1Df(Chr01:hand2)s6fli1a:EGFP)zdf1p53−edn1 was identified using forward primer: 5′ GGTGCTCCAGCATCTTTGGGTC3′ and reverse primer: 5′TGTCTGTTCTGACTTACTCTGGTG3′ resulting in a 153 bp product. Digestion with MseI cleaved the PCR product from the mutant allele into 84 and 69 bp fragments. −hand2 was identified by PCR using forward primer: 5′ GCGGACAGTGAAACGTAGACC 3′ and reverse primer: 5′ GCCTTTCTTCTTTGGCGTCTGTC 3′ resulting in a 257 bp product from the WT allele. −p53 was genotyped as described Fish were raised under standard conditions Larvae were fixed at 4 or 6 days postfertilization (dpf) and stained with Alcian Blue for cartilage and Alizarin Red for bone as described Skeletal preparations were imaged on a Zeiss Axiophot 2. Static confocal images were captured on a Zeiss LSM 5 Pascal as previously described fli1a:EGFP expressing ectomesenchyme of the first two pharyngeal arches (PAs) were digitized at the positions shown in fli1a:EGFP. To provide a counterstain showing total cells within the arches . For geometic morphometric analysis, twenty landmarks outlining the es as in , the embfli1a:EGFP were counted.Proliferating cells were detected using anti-phospho-histone H3 antibody. Embryos were manually dechorionated, anesthetized with tricaine, and fixed in 4% paraformaldehyde in PBS overnight at 4°C. Prior to staining, embryos were washed with PBSTx and blocked in 10% normal goat serum (NGS) in PBDTx  for at least 2 hours. Embryos were incubated with rabbit anti-phospho-histone H3 (Millipore) at 1∶1000 dilution in blocking solution overnight at 4°C, washed with PBSTx, then incubated with Alexafluor 546 goat anti-rabbit IgG (Invitrogen) at 1∶500 dilution in PBSTx overnight at 4°C. Samples were visualized by confocal microscopy. Phospho-histone H3 positive cells were detected with Volocity. Regions of interest were set around the PAs and only cells positive for both phospho-histone H3 and TUNEL assay was performed to detect cell death. Embryos were dehydrated in 100% MeOH for 1 hour at -20°C then rehydrated by sequential incubation with 75%, 50%, and 25% MeOH in PBSTx at −20°C and with PBSTx at RT for 10 minutes each. Embryos were permeabilized by incubation in Proteinase-K (1 µg/mL) then fixed in 4% PFA for 20 minutes. Embryos were further permeabilized in Permeabilization Solution  for 30 minutes then washed with PBSTx. DNA fragmentation was detected by incubation with terminal deoxynucleotidyl transferase and TMR dUTP for 1 hour at 37°C (Roche). Embryos were washed in PBSTx then imaged.Figure S1The distinctive pharyngeal arch shapes of WT, edn1 and hand2 mutants arise progressively from 24 hpf to 32 hpf. PCA as in edn1 mutants, and light to dark blue triangles represent hand2 mutants. B. The same plot but showing the means for each genotype-age group. At the 24 hpf time point the three genotypes completely overlap. DV extension, captured by PC1, then increases markedly with developmental age for all genotypes, with −edn1 lagging behind WT and −hand2. The hand2 mutant shows progressive expansion in the ventral-anterior arch 1 region captured by negative PC2, whereas WT and −edn1 show slight change in the opposite direction.(TIF)Click here for additional data file.Figure S2Loss of function of the programmed cell death gene p53 does not rescue the skeletal phenotype of the edn1 mutant. Flat-mount of cartilage and bone stained with Alcian Blue and Alizarin Red. The skeletal phenotypes of the edn1 single mutant and the edn1;p53 double mutant appear identical, whereas phenotypic rescue would be expected if programmed cell death of pharyngeal arch precursor cells accounted for the −edn1 hypoplastic skeleton. Hence the experiment argues against cell death as an explanation for the reduced size of the arches.(TIF)Click here for additional data file.Table S1TUNEL labeling to detect cell death in the pharyngeal arches.(DOCX)Click here for additional data file.Movie S1hand2 negatively regulates anterior flow of ventral arch 1 cells.fli1a:EGFP animals were imaged by time lapse microscopy from 24–32 hpf. Movies are confocal projections in which individual cells were manually tracked at each frame (arrow). Cells travel from near the midpoint of arch1 to a location posterior to the stomodeum and the eye in wild types and edn1 mutants . Cells originating at a similar location in hand2 and hand2;edn1 double mutants travel much further, to a position well under the stomodeum and the eye . Scale bar: 50 µm(MOV)Click here for additional data file.


Ultrafiltration during intermittent haemodialysis has been associated with reduction in microcirculatory perfusion, as observed with sidestream dark-field (SDF) imaging . This teIn this single-centre, prospective, observational study patients with acute renal failure on CVVH were included after hemodynamic stabilization and written informed consent A fixed dose of net ultrafiltration was calculated for each patient, aiming at a negative total fluid balance of 50 ml/hour. Microcirculatory perfusion was observed with sublingual SDF imaging after 1 hour of zero balance CVVH (T1) and additionally after 1 hour of negative fluid balance ultrafiltration (T2). The primary outcome was a change in microvascular flow index (MFI) between T1 and T2. Data are presented as median (IQR). Differences are calculated with a nonparametric test for paired data.Eleven patients were eligible for the study; one denied informed consent. One patient could not be evaluated due to the unavailability of the research team and in two patients we were unable to obtain images of proper quality. The median APACHE II score was 26 21 to 29); at baseline LOS ICU was 5 (3 to 6) days and fluid balance +7.9 (5.1 to 14.2) l. Hemodynamic and microcirculatory variables are depicted in Table  to 29; aA negative net fluid balance of 50 ml/hour during ultrafiltration in CVVH is not associated with microcirculatory perfusion alterations.