We sought to validate 19 of these in the replication study and find that five of these significantly associate with the corresponding meQTL SNPs from the primary study. We find that none of the 149 meQTL top SNPs is a significant expression quantitative trait locus in our expression data, but we observed association between expression levels of two mRNA transcripts and cis-methylation status. Our results indicate that DNA CpG methylation in abdominal SAT is partly under genetic control. This study provides a starting point for future investigations of DNA methylation in adipose tissue.Genetic variants that associate with DNA methylation at CpG sites  offer a potential biological mechanism of action for disease associated SNPs. We investigated whether meQTLs exist in abdominal subcutaneous adipose tissue (SAT) and if CpG methylation associates with metabolic syndrome (MetSyn) phenotypes. We profiled 27,718 genomic regions in abdominal SAT samples of 38 unrelated individuals using differential methylation hybridization (DMH) together with genotypes at 5,227,243 SNPs and expression of 17,209 mRNA transcripts. Validation and replication of significant meQTLs was pursued in an independent cohort of 181 female twins. We find that, at 5% false discovery rate, methylation levels of 149 DMH regions associate with at least one SNP in a ±500 kilobase  There is growing interest in the role of epigenetic factors in predisposition to common, and especially metabolic, diseases. Epigenetic effects are mitotically heritable alterations in gene expression that occur without alterations in the DNA sequence These epigenetic marks are generally not inherited across generations Many studies on CpG methylation have been carried out in blood We collected abdominal fat biopsies from the Oxford Biobank We extracted DNA from abdominal SAT samples and determined the methylation status of 51,195 genome-wide probe set regions using Differential Methylation Hybridisation . This method measures the quantity of methylated DNA after a methylation sensitive restriction digest by hybridisation onto a custom microarray 2-transformed probe intensities for each probe set and the median log2-transformed chip-internal signal control probe intensities Each biological sample was analysed in duplicate by hybridisation onto two DMH microarrays, which were randomized across three batches. Intensity values of each probe were averaged over the two duplicate chips per sample. A methylation score for each DMH region was calculated as the difference between the median logThe replication cohort consists of subcutaneous adipose samples from 181 female individuals Table 2 −7 and (ii) minor allele frequency (MAF) >1% and genotype rate >99%, or >95% if MAF >5%. We imputed 11,116,176 SNPs using IMPUTE (v2.1.2) 302,765 genetic markers from the 38 individuals were genotyped using the Illumina 317k BeadChip platform  and filtered for (i) Hardy-Weinberg p-value ≤5.7x10Genotyping of the 181 replication samples was performed as part of the TwinsUK dataset (N ∼ 6000) with a combination of Illumina arrays . Intensity data for each of the three arrays were pooled separately (with 1M-Duo and 1.2MDuo 1M pooled together) and the Illumina calling algorithm assigned genotypes. No calls were assigned if the maximum posterior probability was less than 0.95.After quality control 2-transformed probe intensities We profiled mRNA expression on the Affymetrix human GeneChip HGU133 Plus 2.0 array , covering the transcription level of 17,209 Ensembl-annotated genes in 31 of the 38 abdominal SAT samples with data for methylation and genotypes. Using a custom chip definition file as described by Dai et al. limma R package To test for association of DNA methylation with MetSyn, we applied linear models with the cis-meQTL association analysis using 29,441 filtered DMH regions, where we tested SNPs in a ±500 kb region around the probe set for association in a linear additive model using PLINK qvalue package in R We carried out a cis-meQTL findings from the DMH primary cohort, we first assessed how many probes on the Illumina 27 k array were located within a 1 kb distance of DMH probe sets with significant meQTLs in the primary DMH cohort. This distance was chosen because of the high correlation of CpG methylation within 1 kb observed in previous studies cis-SNPs  in a ±500 kb region we applied a linear mixed effects model in R To replicate any Subsequently, to assess the effect of CpG methylation on mRNA expression, we matched each DMH probe set that was significantly associated with a meQTL SNP at the 5% FDR level with the mRNA transcript of which the transcription start site (TSS) was closest in distance. The rationale for this approach is that CpG methylation represses transcription at gene promoters cis-eQTL association with transcripts previously matched to the DMH regions under genetic control. Analogous to the meQTL analysis, we used a linear additive model with allelic dosage as a predictor, while adjusting for age, gender and case/control status for MetSyn. We applied an FDR threshold of 5% to all analyses.We tested the meQTL SNPs for First, we used DAPPLE Second, for the gene pathway analysis, we assigned each meQTL SNP to an Ensembl-annotated gene with closest TSS. This was used as input for a gene set enrichment analysis (GSEA) using MAGENTA Third, to detect whether the meQTL hits in SAT are enriched for associations previously found by GWAS, we carried out a permutation test  using a rank-sum statistic, where 149 random SNPs matched for MAF ±0.05, distance to genes ±500 kb and local recombination rate ±1 cM/Mb were chosen in each permutation.cis-meQTL probe sets were statistically over-represented at each term in the GO database Fourth, we tested whether those genes that overlapped We first set out to investigate whether the data measured by DMH fit with previously reported patterns of methylation .We then investigated whether the DMH methylation score at each probe set associated with MetSyn case/control status or BMI. At the 5% FDR level, we found no DMH probe sets that were significantly associated with MetSyn or BMI and the resulting p-value distribution remained within a 95% confidence interval generated by 1000 permutations of the methylation scores Figure S1C) and four autosomal DMH probe sets significantly associated with gender at the 5% FDR level .Additionally, we tested for association of methylation score with age and gender, excluding sex chromosomes in the latter case. We observed an association signal of gender with methylation score . These top meQTLs show a median distance of 80.7 kb between the DMH probe set and the lead SNP , available . We then tested the 19 Illumina 27 k probes for associations with all SNPs within a ±500 kb cis-region. We found that only four probes for which the primary SNP replicated associated at the 5% FDR level with other nearby SNPs in the replication data (Table S4). In one case, the meQTL SNP was the same in the primary and replication study. A conditional analysis revealed that the weaker association signal of the primary and the replication study disappeared when adjusting for the SNP more strongly associated in either study (Table S5). This means that in some cases, there was an independent stronger cis-meQTL in the replication than the one detected in the primary study, where the difference was potentially caused by heterogeneity between the two sample populations or stochastic effects. In all cases the top SNP from the replication study was also significantly associated with methylation in the primary data (Table S5), which supports the replication.Of the 149 significant DMH probe sets with meQTL SNPs, 19 have a corresponding Illumina 27 k CpG probe  that associate with the same SNP in our data (FDR <5%). One additional probe (cg04007936 [CARHSP1]) was also under significant genetic regulation although the SNP associated is independent (r2<0.1) of the lead SNP found in our study. In the latter case, this could indicate a general tendency to genetic control of methylation in this region or tissue specific differences.Further, we investigated whether the meQTLs found in our study are also meQTLs reported in other tissues (i.e. LCLs or brain tissue) cis-mRNA expression of the transcript with the closest TSS. Firstly, when taking all 27,718 DMH probe sets into account no associations remained significant at the 5% FDR level . In general, highly correlated associations between DMH probe sets and nearby transcripts tended to occur when the distance between the methylation site and TSS was short . Results indicated that expression of two out of 149 mRNA transcripts were significantly associated with DMH methylation score at the 5% FDR level: EpiGII2424610Q , and EpiGII8555714Q . Distances between TSS and the correlating DMH probe set were 324 kb and 13 kb, respectively. In both cases, the correlation between DMH methylation and mRNA expression levels was negative.We then investigated the association of DMH methylation scores with Figure S2D). No associations were significant at the 5% FDR level. Thus, we did not observe control of gene expression by the meQTL SNPs.To investigate whether the 149 meQTL SNPs are also eQTL SNPs, we tested association of 149 lead meQTL SNP genotypes with mRNA expression  had significant enrichment in the 149 genes tested (p = 7.40×10−3).We then investigated whether the meQTLs are enriched in genes associated with the six phenotypes (i)-(vi) above. Using MAGENTA, we carried out a gene pathway enrichment analysis, assigning each DMH region to a gene, similar to the mRNA transcript assignment. For the six traits examined , MolOBB genotype data is available at the European Genome-phenome Archive (EGAS00000000102) and MolOBB methylation data is available at Epigenetic marks, especially DNA methylation, play an important role in cellular differentiation and gene regulation in adipose tissue Figure S1D), which is in line with a previous report We first investigated whether DMH methylation associated with MetSyn status, BMI, age or gender. We find no associations of DMH methylation with MetSyn case control status or BMI. Changes in DNA methylation associated with BMI have been previously reported in a longitudinal study cis-meQTLs in abdominal SAT. Moreover, we observe that most cis-meQTLs act over a much shorter distance than the arbitrarily defined cis window of ±500 kb. In our replication study we attempted to replicate 19 out of 149 significant meQTL signals and find that five out of 19 DMH probe sets that reached significance in the primary study are also found to be significantly associated and directionally consistent in the replication study. The associations of methylation with SNPs in these five regions are consistent throughout the datasets, as each top cis-meQTL SNP within a ±500 kb region also is an meQTL in the other data set (Table S5). As methylation was measured on a different platform, the replication study also provided validation of the DMH method used in the primary study. There are three main aspects between the two platforms that may have an effect on the replication rate: (i) targeting of single vs. multiple CpGs, (ii) imperfect correlation between the DMH probe sets and Illumina 27k probes (within 1 kb) used as methylation proxies and (iii) methodological differences between the two assay techniques. Different genotyping scaffolds have been used and we use very stringent quality control measures pre-imputation, which means that even though different chips are used, the filtering on the minor allele count we do only allow high quality data, and variants with low allele frequency will be filtered out The results of our meQTL study indicate that there are a number of Importantly, in a recent report investing imputation accuracies using different methods and sizes of European reference panels, it was shown that for common variants with a minor allele frequency >5% the imputation accuracy performs similarly well cis-mRNA transcripts are found, but association cannot be ruled out due to the relatively low statistical power in this study. Using similar assumption as above, and adjusting for 149 tests, we have 80% power to detect SNP-mRNA associations explaining 41% of variation in expression No significant associations of the 149 meQTL SNPs with expression of et al.et al.Despite the current understanding that promoter methylation acts as a suppressor of mRNA translation, the observation that many meQTLs do not influence mRNA expression is consistent with the study in brain tissues by Gibbs cis-mRNA’s significantly associated with meQTL DMH probe sets, encode for protein products that have previously been implicated in type 2 diabetes and MetSyn. TNFRSF11B, also known as RANKL, was previously characterized as an extracellular negative regulator of osteoprotegrin, which acts as a decoy receptor when secreted TNFRSF11B is normally secreted by osteoblasts GOT1, also known as AST1, is a liver transaminase that plays a role in amino acid metabolism, the urea cycle and the Krebs cycle GOT1 gene promoter has been shown to be regulated by glucocorticoids, cAMP and insulin We find that two Overall, we show for the first time that meQTLs are present in adipose tissue. This indicates a direct genetic influence on DNA methylation and also an indirect influence on the general molecular phenotype of adipose tissue. Defining the genetic influence on both gene expression and CpG methylation in abdominal adipose tissue can help towards characterising this type of tissue and understanding molecular pathways associated with obesity.Figure S1Association of CpG methylation with metabolic syndrome and other phenotypes. A: Association of methylation score with metabolic syndrome case/control status (linear regression). B: Association of methylation score with BMI. C: Association of methylation score with gender, chromosomes 1–22 only D: Association of methylation score with age. Grey bands correspond to 95% confidence intervals calculated by 1000 permutations of sample labels.(TIF)Click here for additional data file.Figure S2Association of mRNA expression with CpG methylation. A: Analysis of 27,718 DMH probes for association with downstream transcripts expression levels B: Analysis of 149 DMH probes with meQTLs for association with downstream transcripts expression levels. C: -log10(p) values plotted against DMH probe set-TSS distance. D: Analysis 149 meQTL SNPs (top hit for each probe) for association with mRNA transcript expression levels. Grey bands correspond to 95% confidence intervals.(TIF)Click here for additional data file.Table S1Full list of significant SNP-DMH probe set pairs.(XLS)Click here for additional data file.Table S2All primary meQTL hits tested for replication.(XLS)Click here for additional data file.Table S3Gene set enrichment analysis results.(XLS)Click here for additional data file.Table S4500 kb cis-meQTL analysis of 19 probes in the replication study.(XLS)Click here for additional data file.Table S5Conditional analysis between top meQTL SNPs found in the primary and replication study.(XLS)Click here for additional data file.Supplementary Information S1Supplementary methods and power calculations.(PDF)Click here for additional data file.


MED10, MSI2 and three long intergenic non-coding (LINC) RNAs. Both MED10 and MSI2 have been previously linked with WNT/GSK3β/β-Catenin pathway function suggesting that this is an important mediator of lithium action in this screen. The methodology applied here provides a rapid, objective and economic approach to define the genetic contribution to drug action, but could also be readily adapted to any desired in vitro functional selection/screening paradigm.Identifying the biological pathways mediating the action of a therapeutic compound may help the development of more specific treatments while also increasing our understanding of the underlying disease pathology. Salts of the metal lithium are commonly used as a front-line mood stabilizing treatment for bipolar disorder. Lithium's action has been variously linked to inositol phosphate metabolism and the WNT/Glycogen Synthase Kinase 3β (GSK3β)/β-Catenin signalling cascade, but, to date, little is known about which of these provides the principal therapeutic benefit for patients and, more specifically, which constituent genes, through presumed sequence variation, determine differences in patient response to treatment. Here, we describe a functional screen in which SH-SY5Y neuroblastoma cells were randomly mutated through genomic integration of the pMS1 poly A ‘gene trap’ plasmid vector. Lithium normally induces differentiation of neuroblastoma cells, but a small proportion of mutated cells continued to proliferate and formed colonies. Rapid amplification of cDNA ends (RACE)-PCR was used to identify the ‘trapped’ gene in each of these lithium-resistant colonies. Heterozygous, gene trap integrations were identified within ten genes, eight of which are likely to produce loss-of-function mutations including  Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. The unicellular approach is quick, economically attractive and permits a wide range of selection criteria. In contrast, screens of higher organisms offer a more accurate model of human development, physiology and disease—but with increasing costs, time, use of animals and a more restricted repertoire of screening criteria.Mutation screens in unicellular organisms such as bacteria and yeast have proved a valuable approach to dissect biological processes. They have also been applied to metazoans such as e.g. NeoR: Neomycin/G418 resistance) to permit selection of productive gene trap events. Thirdly, these hybrid mRNAs comprising endogenous and gene trap-derived sequences can be readily amplified by rapid amplification of cDNA ends (RACE)-PCR protocols to identify the gene that has been trapped. To date, mammalian gene traps have been most commonly used to mutate mouse embryonic stem cells which are then used to generate mouse strains for large-scale phenotypic screens Human laboratory cell lines are frequently used as tools to measure specific responses to drug treatments or to characterize specific gene mutations. However, they may also offer a useful ‘middle ground’ for primary mutation screens. One cell-based mutation approach has used RNAi libraries to knock down gene expression to expose specific cellular phenotypes. Some issues with this approach have been recently described, mostly concerning the specificity and efficiency of gene inhibition in vivo biological question. In this instance we were interested in uncovering the biological processes affected by the mood stabilizer lithium in the context of its use for the treatment of the psychiatric illness bipolar disorder. Lithium is currently thought to offer neurotrophic or neuroprotective properties through the inhibition of glycogen synthase kinase 3β (GSK3β), a component of the WNT signalling pathway BCL-2. Lithium has also been shown to affect the inositol pathway, specifically preventing inositol recycling by its inhibitory action on both inositol-1,4 bisphosphate 1-phosphatase and inositol-1-monophosphatase via the WNT pathway, promotes the differentiation of cells including stem and neuroblastoma cell lines Here, we describe how pMS1, a gene trap vector of the ‘poly A’ type 2, 37°C incubator. TryplE Express (Life Technologies) was used to passage cells. During lithium selection, FCS was reduced to 2% to reduce additional, serum-derived proliferation signals.SH-SY5Y cells were grown using standard techniques and conditions. In short, cells were cultured with DMEM/F12/Glutamax media  supplemented with 10% Foetal Calf Serum  and 1% penicillin/streptomycin  in T-25  and maintained in a 5% COScaI restriction enzyme, purified and then introduced into SH-SY5Y cells by nucleofection with Lonza Nucleofector utilizing Reagent V and two different protocols: G04 and A23. In each case, 106 cells were transfected with 2.1 μg of linearized pMS1. Cells were given a 1-week selection period in media supplemented with G418 . Resistant (productive gene trap integration) cells were seeded into 6-well plates at a density of 150,000 cells per ml.Gene trap vector pMS1 was acquired from Prof. William Stanford from the Stanford laboratory is at Ottawa Hospital Research Institute, Ottawa, Canada. The plasmid was linearized using Lithium chloride (Sigma-Aldrich) was dissolved in water, filtered and added to the medium. Titration experiments (data not shown) demonstrated that a final concentration of 9.5 mM was the minimal dose required to completely halt wild-type SH-SY5Y proliferation in the presence of 2% FCS. The medium was changed regularly. After 4 weeks, non-proliferating/moderately differentiated cells had mostly died leaving colonies of dividing cells. These were removed from the wells as either individual colonies or as low-complexity mixes of colonies and allowed to expand in T-25 flasks in the absence of lithium and with 10% serum restored.R gene and the 3′ exons of the endogenous trapped gene. The protocol and the primers used in the two-step amplification procedure were all based on information available at the website of Prof. Stanford's laboratory (http://www.cmhd.ca/genetrap/vectors.html). Briefly, the primer used for reverse transcription was 3CDS: 5′ AAG CAG TGG TAA CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TVN 3′. RACE 1 primers consisted of SD5-P3 (forward) 5′ CGC ATC GCC TTC TAT CGC CTT CTT GAC G 3′ and UPM (reverse), note that UPM is a mixture of 0.4 μM UPL 5′ CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA ACA ACG CAG AGT 3′ and 2 μM UPS 5′ CTA ATA CGA CTC ACT ATA GGG C 3′. The PCR protocol used was a two-step PCR where a second reaction was conducted using primers nested to RACE 1 primers, thus improving specificity. RACE 2 primers composed of 3′RACE1 (forward) 5′ CAA GCG ACG CCC AAC CTG CCA TCA CGA G 3′ and Nest1 (reverse) 5′ AAG CAG TGG TAA CAA CGC AGA GT 3′.RNA was extracted from expanded colonies/colony mixes using a Biometra/Analytik Jena, innuPREP RNA mini kit. cDNA was produced using Roche first-strand cDNA Synthesis kit for RT-PCR (AMV). RACE-PCR was performed to amplify a region of cDNA that spans the splice junction between the endogenous NeoEscherichia coli cells transformed with these ligation products. Antibiotic-resistant bacterial colonies were grown overnight in LB broth and plasmid DNA purified using a kit . Plasmid DNA was sequenced by Source Bioscience  using primer SD5-P3 (above).RACE-PCR products were purified and ligated into pGEM1  and chemically competent MSI2, MED10 and two long intergenic non-coding (LINC) genes, where we identified such mutation events. In addition, we identified six non-canonical insertional events in which, although clearly intronically inserted, the cloned RACE-PCR products suggested that the NeoR gene had preferentially spliced to a presumed cryptic splice acceptor and polyadenylation sequence within the intron rather than the next endogenous exon's splice genuine acceptor. Two of these indicated that the gene trap sequence had integrated in an orientation against the gene direction. The interpretation of these ‘non-canonical’ events is discussed in detail below.After a period of 3–4 weeks in 9.5 mM lithium selection a number of growing cell colonies began to emerge from a background of dying cells A and B. The molecular actions of lithium have been previously studied by direct assessment of its effect on particular candidate signalling pathways in vitro actions would be relevant to its mode of therapeutic action in patients. However, it is quite possible that the list of trapped genes from a different selection strategy that focused on, for example, the neuroprotective action of lithium might differ and be more closely relevant to its mood stabilizer action. The choice of 9.5 mM lithium treatment was based on the minimum concentration that completely inhibited SH-SY5Y cell proliferation. This is in the order of 10-fold greater than therapeutic concentrations typically observed in patients increase susceptibility to lithium differentiation, the fact that a proportion of genes lack  introns and the variation in dosage sensitivity effects between genes. The last of these factors is important because a gene trap mutation is a heterozygous loss-of-function event . The study of human genomic copy number variation and the molecular basis of dominant and recessive traits suggests that not all genes that are mechanistically important in any biological system under examination will have detectable/selectable phenotypes through this type of mutation There are, however, several caveats that should be appreciated. We chose to assess the biological actions of lithium by making use of its previously described effects on the proliferation of neuroblastoma cells MED10 and MSI2, that can be directly linked to the WNT/GSK3β/β-Catenin pathway previously implicated in lithium action, whereas none of the identified genes showed straightforward links to inositol metabolism. MED10 (Mediator complex subunit 10) is a component of a multi-protein complex responsible for converting signalling pathways into the transcription of target genes via interaction with RNA Polymerase II at gene promoters MSI2 (Musashi 2) encodes an RNA-binding protein that is known to regulate the balance between cellular proliferation and differentiation—a function that has been particularly well explored in the context of stem cells via signalling through the TCF1 and p21 proteins Msi2 knockdown has been shown to deregulate Wnt pathway target genes Our choice to study lithium was partly because its biological action is moderately well understood and could thus provide a framework for assessing the performance of the technique. Canonical gene trap insertions disrupted two genes, Two canonical insertions were identified in LINC RNA genes on chromosomes 1 and 3. This class of non–protein-coding genes is the subject of much current interest as they are catalogued and their diverse functional properties increasingly understood R gene and intronic DNA. For example, the non-canonical NeoR splicing event within LOC401164 occurs at the cryptic consensus splice acceptor sequence TCAG/G present in the intronic genomic sequence  is an intriguing finding as there is considerable  genetic evidence associating variants in this gene with increased risk of schizophrenia and bipolar disorder FRRS1  is involved in the uptake, and oxidative state regulation, of iron ions and is known to be expressed in the brain AGBL4  removes glutamate side chains from cytoskeletal proteins—a post-translational modification that has been linked to neurodegeneration LOC401164, was identified as an aligned, non-canonical gene trap event.As detailed in LPHN2  and a chromosome 5 LINC RNA may represent false positives because they do not conform to splicing-based disruption models, but they were still identified in cells that continued to proliferate in the presence of lithium. It is possible that more complex and locus-specific endogenous gene deregulation mechanisms could be responsible for their identification in the screen.The two non-aligned gene trap insertions in It should be pointed out that the screen described in this study has generated a set of candidate genes that will require further study to strengthen their link to clinical lithium action. Specifically, the deleterious action of the gene trap insertions on gene expression should be verified and a process of independent validation carried out  to confirm functional involvement. It will be important to determine, in the appropriate cohort, if genes identified in this screen have DNA variants responsible for individual patient differences in response to medication. We have demonstrated that the gene trap approach can usefully address questions of drug action and our findings can be interpreted, in part, as consistent with lithium's effect on the WNT pathway—particularly, the translation of this pathway into nuclear gene expression changes. Gene trap screens targeting laboratory cell lines are still in their infancy compared with RNAi screens or gene trap use in the large-scale production of mutant mouse strains, and yet they have the potential to offer rapid insights into functional biology as well as providing permanently, specifically and consistently genetically modified cell lines for more specific experimentation or small molecule screening


It is not known whether or not delivering acupuncture triggers mechanismscited as placebo and if acupuncture or sham reduces radiotherapy-inducedemesis more than standard care.Cancer patients receiving radiotherapy over abdominal/pelvic regions wererandomized to verum (penetrating) acupuncture  in the alleged antiemetic acupuncture point PC6 or shamacupuncture  performed witha telescopic non-penetrating needle at a sham point 2–3 times/weekduring the whole radiotherapy period. The acupuncture cohort was compared toa reference cohort receiving standard care . The occurrence of emesis in each group was compared after amean dose of 27 Gray. Nausea and vomiting were experienced during thepreceding week by 37 and 8% in the verum acupuncture group, 38 and7% in the sham acupuncture group and 63 and 15% in thestandard care group, respectively. The lower occurrence of nausea in theacupuncture cohort (verum and sham) compared to patients receiving standardcare  0.6, 95 %confidence interval (CI) 0.5–0.8) was also true after adjustment forpotential confounding factors for nausea . Nauseaintensity was lower in the acupuncture cohort  compared to thestandard care cohort  (p = 0.002). The acupuncturecohort expected antiemetic effects from their treatment (95%).Patients who expected nausea had increased risk for nausea compared topatients who expected low risk for nausea .Patients treated with verum or sham acupuncture experienced less nausea andvomiting compared to patients receiving standard care, possibly through ageneral care effect or due to the high level of patient expectancy.ClinicalTrials.gov NCT00621660 Many cancer patients express interest in acupuncture for nausea both groups considered the treatment to beeffective, and 89% were interested in receiving the treatments in the futureBetween two and 31% of patients undergoing cancer treatment use acupuncturefor various kinds of symptoms The aims of the study were to compare nausea and vomiting experienced by a cohorttreated with verum or sham acupuncture with that experienced by a cohort receivingstandard care during radiotherapy, and to evaluate if expectations of nausea and ofacupuncture effects were related to the actual occurrence of nausea.The protocol for this trial and supporting CONSORT checklist are available assupporting information; see 3 volume and 25 Gray dose, antiemetic treatment orpersistent nausea within 24 hours prior to the start of radiotherapy andacupuncture treatment during the past year for any indication or ever fornausea.Two cohorts of patients treated for cancer in three Swedish oncology departmentswere included: one a standard care and the other an acupuncture cohort, seeAll patients gave their informed written consent and the Regional Ethical ReviewBoard in Linköping, Sweden, approved the study. The informed consent formused in the acupuncture cohort contained the information: “You willreceive an ordinary acupuncture treatment with needles penetrating the skin oranother treatment with needles placed just against the skin”. Thestudy-evaluator and all health-care professionals, with the exception of theacupuncture-providing therapists, were blind to the acupuncture allocation. Thestandard care group knew, of course, that no acupuncture was given. They hadbeen informed that the aim of the data collection was to evaluate the prevalenceof nausea during radiotherapy.The acupuncture and the standard care cohort were, except for studyparticipation, treated according to clinical routines, including the use ofrescue antiemetics. The standard care cohort received no acupuncture therapy.One physiotherapist at each hospital (performing 1412 and 607 treatments)performed both verum and sham acupuncture and they had five deputyphysiotherapists . Treatmentsstarted on the first day of radiotherapy, continued 30 minutes per session threetimes/week for two weeks, and then twice/week, until the end of radiotherapyaccording to a standardised treatment protocol. The patients were in a hotel,ward unit or at the radiotherapy department during the treatments, receivedeither in a sitting or a supine position. The physiotherapists treated one tothree patients simultaneously and maintained an everyday conversation, butavoided the subject of nausea.Verum acupuncture was administered bilaterally to thetraditional antiemetic point pericardium six (PC6) Sham acupuncture was administered bilaterally to a sham pointlocated two body-inches proximal to PC6, outside traditional acupuncture points.“Park's sham devise” Clinical data, listed in Type/dose of antiemetics and emesis during the previous 24-hours weremeasured by written established emesis questions At the end of the first, the sixth and the last verum or sham treatment thephysiotherapists asked the patients: “Do you believe that thetreatment that you have just received is effective in preventing or reducingnausea?” The four answer categories were “No, I do not think thetreatment is effective” and “Yes, I believe alittle/moderately/much that the treatment is effective”. Beforetreatment was started, the verum and sham treated patients answered thewritten question: “In relation to others, how do you estimate your ownrisk for becoming nauseous during the radiotherapy period?” to beanswered on a five-grade category scale from “Much lower risk”to “Much higher risk”.The acupuncture cohort was compared with the standard care cohort usingStudent's t-test regarding continuous data, Mann Whitney U-test regardingordinal or continuous, not normally distributed, data and by Fisher's exact(two categories), or Chi2-test (three categories or more), regarding categorydata. Relative risk (RR) for nausea with 95% confidence intervals (CI)was calculated for each of the different subgroups shown in Compared to the acupuncture cohort the standard care cohort comprised more men(p = 0.02), more patients with a testicular tumour(p = 0.001) and fewer patients consuming potentantiemetics; serotonin-receptor antagonists (p = 0.09) orcorticosteroids (p<0.001) . AccordiThe patients in the acupuncture cohort the past week and the past 24 hoursexperienced significantly less occurrence of nausea and vomiting than those inthe standard care cohort. The lower occurrence of nausea in the acupuncturecohort (37%) compared to the standard care cohort (63%) the pastweek  was also true when patients takingserotonin-receptor antagonists and corticosteroids were excluded  and afteThe intensity of nausea was lower in the acupuncture cohort  than in the standard carecohort  (p = 0.002). Within the acupuncturecohort, no statistically significant differences between the verum and the shamgroup were seen regarding nausea occurrence or intensity, vomiting or antiemeticconsumption the past 24 hours or the past week.The 27 patients in the acupuncture cohort who estimated their own risk forbecoming nauseous during the radiotherapy as higher than other patients had anincreased risk for nausea compared to the 44 patients who estimated that theyhad a lower risk for nausea than others . No statWe found lower occurrence of nausea and vomiting in patients treated with penetrating“deqi”-creating acupuncture or sham acupuncture compared to patients whohad received standard care. Almost all patients in the acupuncture cohort highlyexpected antiemetic effects from the treatment. Patients who expected nausea hadincreased risk for nausea compared to patients who expected low risk for nausea.http://www.ncbi.nlm.nih.gov/ pubmed/accessed 10/11/10, using thecombined search terms acupuncture, radiotherapy, nausea and vomiting). A Cochranereview of acupuncture for chemotherapy-induced nausea includedeleven studies There are many, not sham-controlled studies, reporting emesis-reducing effects ofacupuncture compared to standard care in chemotherapy-induced nausea The verum and the sham group received extra care compared to the standard care group,which may have reduced emesis: patient-therapist communication, the knowledge thatcontinuous contact with one single therapist would continue during the wholeradiotherapy period, the tactile stimulation from the therapists' hands, theextra time for rest and relaxation and the extra attention to the patient'ssymptoms through the daily emesis questions all are important elements of this extracare. The verum and sham performing therapists in our study might have had asupportive attitude. Arving and co-workers Both verum acupuncture and sham-acupuncture like tactile stimulation have been seento activate the limbic system Since almost all patients in the acupuncture cohort expected positive antiemeticeffects of the treatment, the positive expectation may be another factor thatreduced emesis. Expectations are known to influence intervention outcomes in generalSince nausea was prevalent in the standard care group and nausea may be associatedwith a reduced quality of life Emesis was measured using a well-established method www.valuta.se, date 080404). Costsfor needles may be approximately $0.72 USD .* A public hospital employing a physiotherapist for three hours spends $68USD  during the mean value of36 radiotherapy days in treatment costs $98 USD .** Consuming one tablet at a cost of 16.25 SEK Click here for additional data file.Checklist S1CONSORT Checklist.(0.22 MB DOC)Click here for additional data file.


Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed  The spatial organization of subcellular components and proteins at the nanometer scale on the level of whole cells or tissues is challenging for modern light microscopy methods. Well-established light microscopy techniques are capable of imaging large volumes, yet limited in their spatial resolution to about 200 nm in the imaging plane and >500 nm along the optical axis. A superior axial resolution was demonstrated through ‘array tomography’, a technique which starts off with the preparation of ultrathin sections of tissue that are labeled via immunofluorescence and imaged using widefield or confocal microscopy In the current study, we investigated the calyx of Held, a sample that illustrates particularly well the requirements of imaging methods in terms of imaging volume and resolution. The calyx of Held is a glutamatergic presynaptic terminal located in the auditory pathway dSTORM) tomoSTORM. 2D dSTORM imaging provides a lateral spatial resolution of ∼28 nm at an image size of up to 50×50 µm2. Array tomography provides an axial resolution of 40 nm or even less by virtue of the section thickness. By imaging many consecutive sections, tomoSTORM can cover large 3D volumes spanning tens of micrometers in all directions.Here, we present an approach that combines array tomography and single-molecule localization-based super-resolution imaging with photoswitchable organic fluorophores following the protocol for direct stochastic optical reconstruction microscopy  in the calyx of Held led mGFP  and widedSTORM revealed consecutive sections showing continuous variations of a typical calyx structure , consistent with the disrupted ultrastructure described above. Hence, we conclude that the super-resolution images obtained from HM20-embedded tissue closely reflect the known tissue ultrastructure.A ribbon of 53 serial sections cut at a thickness of 40 nm was labeled with an anti-GFP primary antibody and a secondary antibody coupled to Alexa Fluor 647. Imaging with tructure . The instructure . This mamembrane . To demo segment . In totatomoSTORM demonstrates a confinement of structures such as mitochondria within membranes . All necessary steps were taken to ameliorate suffering of the rats, including systemic anesthesia (isofluorane) and local wound anesthesia (lidocaine), post-surgery pain treatment, professional handling, behavioural observation and housing in individually ventilated cages at 21°C and 55% humidity. The stereotaxic injections into the ventral cochlear nucleus (VCN) of two Sprague Dawley rats and tissue processing were done as reported previously Resin embedding with LRWhite was done as described by Micheva et al. For Lowicryl HM20, tissue sections were high pressure frozen as described Ultrathin sections (40 nm) were cut with an ultramicrotome  using the procedure described by Harris et al. Following mounting on coverslips, the ribbons were washed in PBS (5 min) and immunostained following established protocols Electron microscopy was done following procedures described previously dSTORM images were recorded on a custom-built microscope using experimental protocols that were described earlier 2) for activation and 647 nm (1–5 k W/cm2) for read-out. Image reconstruction was performed using the rapidSTORM software package dSTORM image.dSTORM imaging was performed by sequentially imaging Alexa Fluor 647. As a first target, cytochrome c oxidase was labeled, imaged and bleached. Successful bleaching was verified in control experiments to exclude that Alexa Fluor 647 was driven into long-lived dark states and recovered to fluorescence. In a next round, the ribbon was stained for anti GFP with Alexa 647 and imaged. For alignment of the dSTORM images, we recorded wide field images of mGFP distribution in each of these steps. To achieve this in the first step, mGFP was stained and visualized with ATTO520.Dual-color dSTORM) and ATTO520 (standard resolution image used as reference image for registering multiple channels). By way of reference images, registration between two distinct (cytochrome c and mGFP) super-resolution channels could be achieved. The aligned and surface rendered image stacks were visualized in 3D using Amira 4.1.2 software  Click here for additional data file.Figure S1Image registration procedure for one-color imaging. In a first step (i), the sum-TIRF images of individual sections were registered using MultiStackReg (TIF)Click here for additional data file.


Exercise provides clear beneficial effects for the prevention of numerous diseases. However, many of the molecular events responsible for the curative and protective role of exercise remain elusive. The recent discovery of FNDC5/irisin protein that is liberated by muscle tissue in response to exercise might be an important finding with regard to this unsolved mechanism. The most striking aspect of this myokine is its alleged capacity to drive brown-fat development of white fat and thermogenesis. However, the nature and secretion form of this new protein is controversial. The present study reveals that rat skeletal muscle secretes a 25 kDa form of FNDC5, while the 12 kDa/irisin theoretical peptide was not detected. More importantly, this study is the first to reveal that white adipose tissue (WAT) also secretes FNDC5; hence, it may also behave as an adipokine. Our data using rat adipose tissue explants secretomes proves that visceral adipose tissue (VAT), and especially subcutaneous adipose tissue (SAT), express and secrete FNDC5. We also show that short-term periods of endurance exercise training induced FNDC5 secretion by SAT and VAT. Moreover, we observed that WAT significantly reduced FNDC5 secretion in fasting animals. Interestingly, WAT of obese animals over-secreted this hormone, which might suggest a type of resistance. Because 72% of circulating FNDC5/irisin was previously attributed to muscle secretion, our findings suggest a muscle-adipose tissue crosstalk through a regulatory feedback mechanism. Last January 2012 Boström and colleagues identified a new muscle-tissue-secreted peptide termed Irisin The transcriptional co-activator PGC1α is involved in many biological programs related to energy metabolism. PGC1α was initially implicated in the expression of UCP-1 and thermogenesis in brown fat through PPAR-γ Given the relevance of the FNDC5/irisin discovery as well as its controversy and potential functions, a need to obtain additional information regarding the nature of this hormone and its precise role in energy homeostasis, including its participation in food-related disorders, exists. We believe that the present study sheds light about FNDC5 secretion from skeletal muscle and more relevantly, we set a new paradigm by demonstrating that FNDC5/irisin is not just a myokine liberated by muscle tissue but also an adipokine released by white and to a lesser extent, by brown adipose tissue. Interestingly, we show that adipose-FNDC5/irisin expression and secretion varies according to the type of fat depot and that exercise and nutritional status influence its secretion. Moreover, we describe its deregulation in food-related disorders.The authors of this manuscript declare that the animal work in this study was performed according to the protocols approved by the Animal Care Committee of Santiago de Compostela University . Furthermore, the human adipose tissue specimen was obtained with written informed consent approved for this particular study by the Comité Ético de Investigación Clínica de Galicia – CEIC de Galicia (Spain) according to the Declaration of Helsinki.Male Sprague Dawley rats (160 g) were housed in 12-h light/12-h dark cycles with free access to a standard chow diet and water. After 5 days of acclimatization, weight-matched animals were assigned to one the following experimental groups (n = 5 per group): a) control ad libitum, b) 36 hours fasting, c) re-feed (36 hours fasting followed by 15 minutes of feeding), d) exercise (ad libitum with free access to the activity wheel for 1 or 3 weeks), e) control activity-based anorexia (ABA) and f) ABA as previously described and listed in The soleus and gastrocnemius muscles, the subcutaneous adipose tissue from the skin of the hips, the visceral fat located inside the peritoneal cavity around the internal organs and the brown (interscapular area) fat locations were processed for secretome collection based on a previously optimized protocol to minimize tissue damage Protein extracts from whole tissue samples and secretomes were processed as previously described Human adipose tissue was obtained from an obese patient (body mass index >35) who underwent laparoscopic gastrectomy surgery. The visceral fat was located in the hypogastric region around the internal organs, and the subcutaneous fat was located in the mesogastric region. The tissues were transported from the operating room to the laboratory in sterile KRH buffer with penicillin (100 U/ml) and streptomycin (100 μg/ml) and processed as described above for the rat adipose tissue.Five g of rat adipose tissue from the visceral and subcutaneous locations was washed three to four times with KRH buffer, cut in pieces less than 1 mg and resuspended in an equal volume of 0.2% pre-warmed (37°C) collagenase Type I . This tissue was placed in an orbital mixer at 37°C with continuous agitation for 90 minutes. Then, it was centrifuged for 5 minutes at 400 g at room temperature. The supernatant that contained mature adipocytes was recollected. The pellet was identified as the SVF.The cell line 3T3-L1 was grown in DMEM with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin until differentiation to adipocytes as previously described Total RNA was isolated from adipose tissue from five independent experiments  using Trizol  according to the manufactureŕs recommendations. Quantitative real-time PCR was performed using a Real Time PCR Systems Step One Plus (Applied Biosystems) with specific Taqman qRT-PCR primers as described elsewhere While characterizing FNDC5/irisin myokine regulation under different nutritional and pathological situations, we discovered that rat adipose tissue is able to express and secrete this novel peptide. Whole tissue and secretome samples from rat skeletal muscle and white adipose tissues were collected as indicated in the methods section following protocols previously described by our group Once the secretomes were characterized, we analyzed FNDC5/irisin secretion levels by immunodetection among the same secretome samples . BecauseGiven the interesting finding linking adipose tissue with FNDC5/irisin, we decided to better characterize the expression and secretion of this protein in adipose tissue under different nutritional and pathological situations. When comparing adipose tissues from different anatomical localizations, we observed that SAT expressed and secreted 40% more FNDC5/irisin than VAT; nevertheless, BAT barely expressed and secreted FNDC5/irisin compared with SAT and VAT under the ad libitum condition  and 3A. Because knowing whether adipose FNDC5/irisin plays a role in pathological situations is of interest, we examined FNDC5/irisin secretion and expression in obese animals. Rats with either diet-induced obesity (DIO) or genetic obesity (Zucker rats) showed a significant increase of FNDC5/irisin secretion in both SAT and VAT tissues compared with their lean counterparts . FurtherFNDC5/Irisin circulating levels were assessed in plasma samples from all the animals studied above . This anConsidering the described potent effect of FNDC5/irisin stimulating white adipose browning and thermogenesis In the case of pathological animal models, detection of Ucp1 expression in SAT and VAT adipose tissues from either obese or lean animals was not possible. The same lack of expression in SAT was also observed among ABA rats and their control counterparts ; howeverGiven the implication of PGC1α in energy metabolism, and its role in activating FNDC5 in exercising muscle, we analyzed its expression at protein level in all the experimental situations established in our study . GeneralIn an attempt to better characterize the nature of cells that secrete irisin within adipose tissue, we assessed irisin secretion in fractionated SAT and VAT rat primary cell cultures. The immunoblots of the resulting secretomes confirmed that mature adipocytes are responsible for adipose tissue irisin secretion in both cases . The difThe recent discovery of the PGC1α-dependent myokine FNDC5/irisin, which is able to stimulate brown-fat-like development and thermogenesis in white fat both in vitro and in vivo, has created a new field of research and many expectations in recent months Although Boström and colleagues described the discovery of FNDC5/irisin and its potential role, certain aspects regarding this protein were not fully clarified in their manuscript or elsewhere Regarding the experimental design chosen to analyze the role of FNDC5/irisin in the present work, it could be argued that contaminant or non-expected proteins might be present in the secretomes. The occurrence of unexpected proteins is related to unavoidable cell breakage and artifacts or to the ability of proteins to translocate between intra- and extracellular compartments in unconventional ways In the current study, we were unable to detect a significant increase of PGC1α expression in the exercised muscles of rats (after 3 weeks of voluntary wheel running followed by 12 hours rest before euthanasia), which parallels previous reports Knowing the importance of the role of adipose tissue on energy homeostasis and its participation in obesity The liver-forced expression of FNDC5 and the consequent increase of FNDC5/irisin circulating levels significantly improved glucose tolerance and reduced fasting insulin in obese and insulin-resistant mice according to Boström and colleagues Globally, FNDC5/irisin showed a secretion pattern similar to other well-known adipokines ; thus, our results also suggest a feedback pattern in which adipose tissue might be sensitive to FNDC5/irisin circulating levels. Because adipose tissue is determined to preserve its energy saving function to maintain energy balance, it stands to reason to find that elevated levels of circulating FNDC5/irisin correspond to decreased levels of irisin secretion by adipose tissue of the studied animals. Under this situation, irisin resistance might explain the high secretion pattern of WAT observed in the DIO animals; however, further research should be performed to confirm this hypothesis. In view of the fact that Zucker obese rats with no functional leptin receptor seem not to be affected by circulating FNDC5/irisin levels, other mechanisms related to leptin might be also implicated.Recently, Huh and collaborators published a report about the role of circulating irisin and FNCD5 in humans On the other hand, a recent study performed by Stengel and collaborators strongly supports our results Our data sets the basis for additional research since many questions remain to be answered concerning FNDC5/irisin such as the nature of the FNDC5/irisin receptor (which is yet to be discovered) and the precise role of the soluble long and short forms of FNDC5/irisin. In conclusion, this study reveals that rat skeletal muscle sheds the long form of FNDC5 whose secretion depends on the muscle fiber type. More interestingly, this work is the first to identify FNDC5/irisin as a potential adipokine secreted by WAT and especially by subcutaneous adipose tissue. Given that exercise, nutritional status and pathological conditions such as obesity influence WAT-secreted FNDC5/irisin, our findings suggest a regulatory mechanism influenced by circulating FNDC5/irisin in a feedback process.Table S1After 5 days of acclimatization, the weight-matched animals were assigned to one the following experimental groups (n = 5 per group). a) control ad libitum (CONT), b) 36 hours fasting (FAST 36 h), c) re-feed after 36 hours fasting followed by 15 minutes of feeding (RE-FEED), d) exercise ad libitum with free access to the activity wheel for 1 or 3 weeks (EXER), e) control activity-based anorexia (CABA), and f) ABA as previously described . Diet-induced obesity (DIO) animals (200 g) were fed with a 60% high fat diet D12492  over 9 weeks, and age-matched lean rats were used as a control. Male obese Zucker rats  and their control counterparts (CZ), lean Zucker rats , were purchased from Charles River Laboratories  at 10 weeks of age and fed chow ad libitum for 12 weeks.(DOCX)Click here for additional data file.


Globally, coral bleaching has been responsible for a significant decline in both coral cover and diversity over the past two decades. During the summer of 2010–11, anomalous large-scale ocean warming induced unprecedented levels of coral bleaching accompanied by substantial storminess across more than 12° of latitude and 1200 kilometers of coastline in Western Australia (WA).Extreme La-Niña conditions caused extensive warming of waters and drove considerable storminess and cyclonic activity across WA from October 2010 to May 2011. Satellite-derived sea surface temperature measurements recorded anomalies of up to 5°C above long-term averages. Benthic surveys quantified the extent of bleaching at 10 locations across four regions from tropical to temperate waters. Bleaching was recorded in all locations across regions and ranged between 17% (±5.5) in the temperate Perth region, to 95% (±3.5) in the Exmouth Gulf of the tropical Ningaloo region. Coincident with high levels of bleaching, three cyclones passed in close proximity to study locations around the time of peak temperatures. Follow-up surveys revealed spatial heterogeneity in coral cover change with four of ten locations recording significant loss of coral cover. Relative decreases ranged between 22%–83.9% of total coral cover, with the greatest losses in the Exmouth Gulf.The anomalous thermal stress of 2010–11 induced mass bleaching of corals along central and southern WA coral reefs. Significant coral bleaching was observed at multiple locations across the tropical-temperate divide spanning more than 1200 km of coastline. Resultant spatially patchy loss of coral cover under widespread and high levels of bleaching and cyclonic activity, suggests a degree of resilience for WA coral communities. However, the spatial extent of bleaching casts some doubt over hypotheses suggesting that future impacts to coral reefs under forecast warming regimes may in part be mitigated by southern thermal refugia. Coral bleaching associated with ocean-warming events have received much recent attention and are responsible for a significant global decline in both coral cover and diversity over the past two decades in situ methods. Remote sensing techniques using satellite-derived sea surface temperature (SST) observations are therefore used as an early warning system for predicting and monitoring mass bleaching events Accurate assessments of threats to coral reefs are difficult to gauge at regional scales (100 s–1000 s kms) through the use of The impacts of mass coral bleaching are varied and often difficult to assess, with regional scale patterns mediated through localised variability in physical and biological parameters Large-scale disturbances have previously had limited impact on the coral reefs of WA in situ temperature loggers (Sensus Reefnet Ultra) were deployed at 2–4 m at representative locations to corroborate satellite-derived temperature metrics. Tropical storm and cyclone track data were obtained from the Australian Bureau of Meteorology (BoM).Regional-scale temperature anomalies and heat stress data were obtained from the United States National Oceanic and Atmospheric Administration (NOAA) Coral Reef Watch (CRW) E50 product. Advanced Very High Resolution Radiometer (AVHRR) SST data from sensors onboard Polar-orbiting Observational Environmental Satellites (POES) at 0.5° (50 km ×50 km) resolution ReefTemp, a satellite mosaic product of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Marine and Atmospheric Research unit (http://www.cmar.csiro.au/remotesensing/reeftemp/web/ReefTemp.htm). We used this product over the NOAA CRW product as it provided us with a much finer-scale spatial resolution (ca. [1.8 km]2 versus ca. [50 km]2), thus increasing explanatory power and providing better model fit. Raw data for ReefTemp is administered through the BoM using data collected with AVHRR/3 sensors onboard NOAA-15 and NOAA-17 POES at 0.18° resolution (1.8×1.8 km) ReefTemp product are calculated by month against long-term averages from CSIRO’s 1993–2003 satellite SST climatology for Australian waters Satellite temperature metrics used for statistical models however were obtained from −2 (megajoules per metre squared) were obtained for the nearest BoM station to survey sites using data captured by MTSAT-1R, MTSAT-2 satellites and Geostationary Meteorological Satellite-5, Geostationary Operational Environmental Satellite-9 from the Japan Meteorological Agency and NOAA As the relative ratio between sun and shade, i.e. irradiance and cloud cover, can also influence the extent and intensity of coral bleaching 2), and coral cover change along 119 transects (4487 m2) across regions with each region divided into distinct geographic locations  in the tropical north (20° 27′S) and Perth (PER) in the temperate south (32° 1′S), . All surocations . All surThe data presented in this study were compiled from multiple research and monitoring programs across agencies, and collected with subtly differing methodologies. In most instances, sites were sampled three times: once at least one year prior to bleaching, once during or just after the temperature maxima, and once after the bleaching event when negligible paling of colonies was evident. The exceptions were a subset of transects in PER for which no coral cover data was available prior to bleaching and the Houtman Abrolhos Islands (HAI), where appreciable bleaching was evident during follow-up surveys. As surveys along some transects in PER occurred very near to the temperature maximum, it was assumed that no coral was yet lost and thus total cover of all coral observed (bleached and unbleached) during this survey was a reasonable proxy for total cover before the warming. At the HAI, bleaching data were averaged between March and May 2011 surveys as the bleaching episode was still evolving at the time of the March survey. Consequently, averaged data from the HAI were included in the bleaching assessment, however coral change data were excluded from analyses as it is possible that further change in cover occurred after our final survey. As resolution of imagery was not always sufficient to permit collection of quantitative information on assemblage composition due to turbidity and low light in some locations, we compiled a qualitative list of the dominant coral families and an indication of their relative abundance using visual assessments from researchers that conducted the in-water surveys.The timing of the bleaching surveys generally corresponded well with the peak in temperature between late January and early March 2011 . SpecifiH2,48 = 2.377, p = 0.305 ), bleaching  or the precision of those estimates .Benthic surveys were conducted along six to twenty transects of 20–100 m length per location using either digital photos or stereo video. Subsequent image analyses collected bleaching and coral cover data as percent of cover using coral point count methods from digital still images or by overlaying a grid over selected frames of video footage, both commonly accepted and widely used methods for assessing benthic cover and disturbance For the bleaching assessments, partial and complete bleaching were combined as any visible loss of pigment indicating a significant loss of zooxanthellae consequently represented a considerable stress to corals Coral bleaching and coral cover change were analysed using General Additive Mixed Models (GAMM) In situ water temperatures supported the regional patterns in SST anomalies. Temperature logger records confirmed that bleaching thresholds, defined as +1°C above the maximum long-term monthly mean, were exceeded by 0.5–3°C at all locations  formed within 1000 km of the WA coast , with cyCorals from the family Acroporidae were the dominant, or equal most dominant, taxa across three of the four surveyed regions . In the Anomalously high water temperatures caused widespread bleaching along much of the WA coastline south of MBI, although the extent of bleaching varied within and among regions. Highest bleaching was observed in the Exmouth Gulf in the NIN region, where 95% (±3.5) [mean (± se)] of all coral bleached. Conversely, at the southern extent of our study, 17% (±5.5) of coral bleached in PER. Regionally, MBI experienced high levels of bleaching across all locations, with ∼77% (±5.3) of corals bleaching on the Barrow Shoals and ∼34% (±5.3) on the Lowendal Shelf . SignifiF1.402 = 5.549, p<0.05) indicating that proportion of bleaching was a strong function of increasing temperature accumulation over the three month summer period , driven by higher incidence of bleaching on shallow reefs (<4 m) and lower than expected bleaching on deeper reefs (4–10 m) , with higher than expected bleaching in the MBI, less than expected at NIN and the HAI, with the exception of Gulf reefs, and expected levels for PER  . Similar for PER .Across regions, coral cover change was variable, with significant loss of coral at four of ten locations . Coral cF1.72 = 24.636, p<0.0001)  with the model generally explaining coral loss well across locations, with the exception of Ningaloo Gulf and South which lost a greater than expected amount of coral cover  . Summed <0.0001) . We alsoal cover . Althougal cover .Strong La-Niña conditions drove anomalously high water temperatures and storminess along the WA coast during the summer of 2010–11. Across more than 12° of latitude from tropical to temperate waters, significant and widespread coral bleaching followed by spatially patchy loss of coral were recorded. Whilst bleaching levels and post-disturbance loss of coral cover recorded here are comparable to similar large-scale events in the Caribbean Rising SSTs have previously been identified as the main threat to coral reefs along the NW coast of Australia, however, these threats are considered to be relatively low in comparison to other coral reef regions around the world Significantly, we report substantial mass bleaching and loss of corals from predominantly coastal locations across 12° of latitude from tropical to temperate waters. This occurred during one of the strongest recorded La-Niña years when the strength of the Leeuwin current was extremely high, driving exceptionally warm water southward into temperate waters Across regions, location, depth and maxDHD accounted for a large amount of overall variability in observed bleaching. Whilst coral bleaching often results from the interaction between various biological and physical parameters  were more likely to bleach than those in deeper water. The relationship between depth and coral bleaching may partially relate to depth-mediated flow regimes Taxonomic composition can also influence observed patterns of bleaching in-situ temperature logger records suggest that storms provided no local-scale relief from high temperatures, the weak relationship between bleaching and solar exposure (as a proxy for irradiance after The summer of 2010–11 was also very active cyclonically, with multiple storms tracking within close proximity to survey locations at or near the time of heating maxima. Storms and cyclones are likely to influence both bleaching and mortality in complex ways. Our results support other studies which show that tropical storms are generally documented to have a direct and detrimental effect on coral cover when passing within 50–70 km of coral reefs Symbiodinium communities in WA Impacts were observed across a range of taxa including mass fish kills, localised loss of abalone stock and high lobster mortality (reviewed in Across the world, coral reefs are increasingly imperiled This study is the first to document disturbance on a regional-scale for Western Australian coral reefs and as such provides a valuable baseline against which to measure the impacts of future disturbance events. Given the spatial extent of coral distribution along WA’s extensive coastline, institutional monitoring programs employing standardised methodologies are crucial in order to understand and document regional-scale disturbance events. This is critical as we continue to develop the ongoing capability to robustly assess the response of WA coral communities to disturbance under scenarios predicting increasing frequency of disturbance cycles


TP63, a member of TP53 tumor suppressor gene family, is expressed in bladder urothelium and skin over the external genitalia during mammalian development. It plays a role in bladder development. We have previously shown that −/−p63 mouse embryos developed a bladder exstrophy phenotype identical to human BEEC. We hypothesised that TP63 is involved in human BEEC pathogenesis. RNA was extracted from BEEC foreskin specimens and, as in mice, ΔNp63 was the predominant p63 isoform. ΔNp63 expression in the foreskin and bladder epithelium of BEEC patients was reduced. DNA was sequenced from 163 BEEC patients and 285 ethnicity-matched controls. No exon mutations were detected. Sequencing of the ΔNp63 promoter showed 7 single nucleotide polymorphisms and 4 insertion/deletion (indel) polymorphisms. Indel polymorphisms were associated with an increased risk of BEEC. Significantly the sites of indel polymorphisms differed between Caucasian and non-Caucasian populations. A 12-base-pair deletion was associated with an increased risk with only Caucasian patients (p = 0.0052 Odds Ratio (OR) = 18.33), whereas a 4-base-pair insertion was only associated with non-Caucasian patients (p = 0.0259 OR = 4.583). We found a consistent and statistically significant reduction in transcriptional efficiencies of the promoter sequences containing indel polymorphisms in luciferase assays. These findings suggest that indel polymorphisms of the ΔNp63 promoter lead to a reduction in p63 expression, which could lead to BEEC.Bladder exstrophy epispadias complex (BEEC) is a severe congenital anomaly; however, the genetic and molecular mechanisms underlying the formation of BEEC remain unclear.  TP63, plays a crucial role in the early bladder development. Two different genetic promoters of TP63 produce different forms of the protein with opposing properties. We have shown mice lacking p63 displayed a deformity complex identical to human BEEC. There are no genetic mutations in the p63 protein in BEEC, so genetic variants in the promoter could alter protein expression. Our hypothesis was that loss of p63 expression due to sequence polymorphisms in a promoter is a risk factor for BEEC. We found promoter sequence variants that were statistically associated with the disease and the sequence variant location varied between Caucasian and non-Caucasian patients. This is particularly important as Caucasian populations have a higher risk of BEEC. These findings provide an explanation of BECC and a base for further study of TP63 related genes in this disease.Bladder exstrophy epispadias complex is a severe congenital abnormality. The affected babies' bladders are born open, leaking urine constantly. Treatment involves multiple major reconstructive surgeries and the need for lifelong care for the complications of the disease. Although a number of studies have suggested a genetic cause of the disease, the genetic and molecular mechanism underlying the formation of BEEC remains unknown. One gene,  Bladder exstrophy epispadias complex  is a serious congenital anomaly present in 1 in 36,000 live births TP63 is regulated by two promoters, TAp63 and ΔNp63, located upstream to exon 1 and exon 3 respectively TAp63 protein isotypes are pro-apoptotic whereas ΔNp63 isotypes are anti-apoptotic and both isotypes may compete for the same set of target genes p63 have thin, non-stratified skin, a short tail, truncated limbs, and cleft palate and are perinatally fatal p63 is required for stratification of epithelia, including the bladder urothelium −/−p63 mouse embryos exhibit ventral midline defects identical to those of human BEEC, including ventral bladder and abdominal wall defects, separation of external genitalia, separation of pubic bones and rectus abdominis muscles, exomphalos, and ventrally translocated anus p63 is expressed in the bladder urothelium during development, TAp63 at an earlier stage (E9–11) and ΔNp63 later (E11–19). ΔNp63 is preferentially expressed along the ventral midline in the epithelium overlying the genital tubercle and ventral bladder. Moreover, the apoptotic activity of ventral urothelium of −/−p63 bladder is markedly increased whereas cell proliferation is much reduced. We believe that bladder epithelial apoptosis and failure of induction of the adjacent mesenchyme lead to the development of a BEEC-like phenotype in mice ΔNp63 is a risk factor for BEEC. In this study we show the expression of the two different TP63 promoters in BEEC patient tissue, and explore sequence of the ΔNp63 promoter in BEEC patients and controls. Our studies suggest that ΔNp63 is the dominant promoter in human tissue and its expression is significantly reduced in BEEC tissue in early bladder formation. We also find many sequence variants within the ΔNp63 promoter and three insertion/deletion polymorphisms were significantly associated with increased risk of BEEC. Furthermore the sites of these polymorphisms varied between Caucasian and non-Caucasian patients.A member of the p53 tumor suppressor family, the protein p63, is expressed in all stratified epithelia, including the bladder urothelium and the skin overlying the external genitalia during development TP63 plays a role in human bladder exstrophy, we first confirmed by real time-PCR that normal human foreskin (from circumcision) predominantly expresses ΔNp63 mRNA, whereas the TAp63 isoform was expressed at lower levels  was decreased and also decreased compared to the patient's own ventral foreskin (opposite side) (ΔNp63 appears to be mainly in the foreskin mucosa (non-keratinized epithelium) in an 8-year old BEEC patient  . Down-re patient . Strikincontrols . Tissue controls . Expresspression . The oldΔNp63 expression detected in their bladders ΔNp63 expression led us to examine if any mutations were present within the coding sequence of the TP63 gene in BEEC patients. We therefore sequenced all 15 exons of TP63 gene Our data corroborate a recently published study where three out of the five BEEC patients had no ΔNp63 expression in the absence of any exon mutation, the ΔNp63 promoter (2700 nucleotides upstream of exon 3) was sequenced in BEEC patients and normal controls. DNA was extracted from buccal swab samples from 163 BEEC patients and 285 ethnicity-matched controls from India, Bangladesh, China, Australia, Spain, Canada and USA. We found 7 single nucleotide polymorphisms  and 4 insertion/deletion  polymorphisms in the ΔNp63 promoter in both BEEC and control sequences  ΔNp63 promoter base pair (bp) indel polymorphisms was observed in patients compared to their ethnically matched controls. The 12 bp deletion (rs6148242) was only associated with increased risk of BEEC among Caucasians but not in non-Caucasian patients. Conversely, the novel homozygous 4 bp insertion (ss#541028600) was found to be only associated increased risk of BEEC amongst non-Caucasians. The homozygous 1 bp insertion polymorphism (rs5855273) was significant across both cohorts  were associated with increased risk of BEEC . The ind cohorts ; Table 5 cohorts .ΔNp63 promoters. We sub-cloned four variations of indel polymorphisms  TP63 is a candidate gene for human BEEC and dysregulation of p63 expression could be a contributing factor in BEEC pathogenesis.A number of studies suggest a genetic component in the etiology of BEEC. Precise regulation of patients . The onlΔNp63 is expressed ΔNp63 is the dominant isotype in human foreskin, that ΔNp63 expression was reduced in the urothelium of BEEC patients, and expression of the pro-apoptotic TAp63 was increased. No exon mutations have been discovered in TP63 suggesting that dysregulation may be caused by other regions of the gene such as the promoter regions. Sequence variants in promoters have been shown to be risk factors in numerous diseases including pneumoconiosis ΔNp63 promoter region revealed three indel polymorphisms in the 163 BEEC patients associated with a statistically significant increase in BEEC risk. The prevalence and the role of indel polymorphisms differed between Caucasian and non-Caucasian ethnic populations. While further studies are required to explain this, we speculate ethnicity-specific polymorphisms of up-stream or down-stream genes may further modify the final effect of the ΔNp63 promoter polymorphisms. One possible explanation may be that the polymorphisms may interfere with transcription binding sites which may differ in Caucasian and non-Caucasian populations. An example is the deletion of six-nucleotides in the CASP8 promoter destroys the Sp1 transcription factor binding site ΔNp63 promoter using MATCH software. Computational analysis predicted the 12 bp indel might affect binding of Hand1, GATA-1,2,3,6, Gfi1, LEF1, TCF1 and SOX10, whereas the 4 bp indel may affect binding of SREBP, EGR and CBF transcription factors. Further studies would be needed to verify these suggested interactions.Our murine model shows that the urogenital tubercle, the embryological origin of the foreskin, is one of few anatomical sites where ΔNp63 promoter are associated with increased risk of BEEC, most likely due to decreased transcriptional efficiency and therefore decreased expression of anti-apoptotic ΔNp63 isoforms during bladder development. The consequences of decreased expression of p63 isotypes could be complex and result in stimulation or negative regulation of a number of genes. One such gene, PERP, is a p63 regulated gene central to epithelial integrity and homeostasis PERP, SYNOP2, and the Wnt pathway as potentially contributing to BEEC etiology −/−p63 BEEC knockout model In this study, indel polymorphisms in ΔNp63 promoter are associated with increased risk of Bladder Exstrophy Epispadias Complex. We believe our results provide a base for further study of p63 related genes in this debilitating conditionWe conclude that insertion/deletion polymorphisms of The Research Ethics Committee of Royal Children's Hospital, Melbourne, Australia reviewed and approved the study. Informed consents were obtained from the patients, parents/guardians and the normal controls. Buccal swabs were used to collect DNA extracted with the BuccalAmp DNA Extraction Kit . All samples were codified and subject identities kept confidential. Collaborators from overseas centres  had the study approved by their institutional ethic committees prior to sample collection.ΔNp63 promoter by aligning and comparing the 5′ upstream regions of exon 3 of P63 gene sequences from humans, mouse and pig. The most conserved region 2785 bp (−2696 to +89) was selected as the putative region of ΔNp63. Sequencing was carried out in both directions however in a number of cases sequences from patients or controls were unreadable using a number of different combinations of primers. The PCR products were purified and then used as templates for direct DNA sequencing by the automated ABI Prism 3100 Genetic Analyzer and the Big Dye Terminator kit . Sequences were compared with reference sequences of p63 GenBank (Locus: NC_000003) using the BioEdit software (Ibis Biosciences). Data were analyzed with contingency tables, χ-square, odds ratio and 95% confidence intervals .Exon PCR amplification was performed using published protocols TAp63 and ΔNp63 primer sequences and published PCR conditions As previously described ΔNp63 promoter sequences with indel polymorphisms were cloned into pGL3 luciferase reporter vector . Human embryonic kidney (HEK-293)  was used for luciferase assays. Cells were cultured with DMEM , 10% fetal bovine serum at 37°C with 5% CO2. Cells were plated on a 96-well plate . The following day cells were co-transfected with 200 ng of pGL3-promoter constructs containing ΔNp63 promoters and TK Renilla plasmid DNA with 5 µl Lipofectamine Transfection Reagent . Cells were harvested 48 hours later and cell lysates assayed for luciferase activity with the Dual Luciferase Reporter Assay system . Luminescent signals from the firefly and Renilla luciferase reactions were measured in a FLUOstar Optima luminometer . Luciferase assay reagent was added, firefly luciferase luminescence measured, followed by Stop & Glo Reagent . The signal intensity from Renilla luciferase was used to normalize the signal from the firefly luciferase. One-way ANOVA with post-hoc Bonferroni's multiple comparisons test was applied to compare the relative expressions .The following submitter National Center for Biotechnology Information (NCBI) SNP (ss) accession numbers were assigned to the SNPs and indel observed in this study: promoter position −2657, ss#541026548; −2651, ss#541027004; −2431, ss#541027120; −2293-2282, ss#5411027737; −2260, ss#541027867; −1944, ss#541027977; −1287, ss#541028071; −1209, ss#541028198; −1059, ss#541028317; −71, ss#541028452 (2 bp); and −71, ss#5410228600 (4 bp).