V. anguillarum is a commonly encountered disease in Atlantic cod farms and several studies indicate that the initiation of infection occurs after the attachment of the pathogen to the mucosal surfaces  of fish. Therefore it is necessary to investigate the role of different mucosal components in fish upon V. anguillarum infection. The present study has two parts; in the first part we analyzed the differential expression of skin mucus proteins from Atlantic cod naturally infected with V. anguillarum using two dimensional gel electrophoresis coupled with mass spectrometry. In the second part, a separate bath challenge experiment with V. anguillarum was conducted to assess the mRNA levels of the genes in skin tissue, corresponding to the selected proteins identified in the first part.Vibriosis caused by V. anguillarum revealed key immune relevant proteins like calpain small subunit 1, glutathione-S-transferase omega 1, proteasome 26S subunit, 14-kDa apolipoprotein, beta 2-tubulin, cold inducible RNA binding protein, malate dehydrogenase 2  and type II keratin that exhibited significant differential expression. Additionally a number of protein spots which showed large variability amongst individual fish were also identified. Some of the proteins identified were mapped to the immunologically relevant JNK  signalling pathway that is connected to cellular events associated with pathogenesis. A bath challenge experiment with V. anguillarum showed differential expression of beta 2-tubulin, calpain small subunit 1, cold inducible RNA binding protein, flotillin1, and glutathione S-transferase omega 1 transcripts in the skin tissue of cod during early stages of infection.Comparative proteome analysis of skin mucus of cod upon natural infection with V. anguillarum pathogenesis. The role of some of these proteins in vibriosis in cod described in this paper can be considered unconventional with respect to their established functions in higher vertebrates. Based on the differential expression of these proteins they are possibly important components of fish defence against bacteria and innate immunity at large. The feasibility of utilizing these proteins/genes as markers of bacterial infection or stress in cod needs to be explored further.Differentially expressed proteins identified in the cod skin mucus point towards their possible involvement in  Fish like Atlantic cod, of the gadoid lineage, are thought to rely extensively on innate immune components ,2 to warVibrio anguillarum strains O2α and O2β are commonly associated with vibriosis in cod  search engine and searched against NCBInr with the following parameters: maximum one missed cleavage by trypsin, peptide mass tolerance 100 ppm, MS/MS ion tolerance set to 0.1 Da, fixed carbamidomethyl modification and variable oxidation modification. Protein hits not satisfying a minimum score of 40 and with a low sequence coverage were further searched against vertebrate EST database, taxonomy Actinopterygii. An interaction network of selected proteins was generated based on their mammalian orthologues (generated through BLASTp searches in NCBI database) using Ingenuity pathway analysis [http://www.ingenuity.com].The peak list files obtained from LC-MSMS analyses were submitted to MASCOT , after retrieving their full length or partial sequences from Atlantic cod genome database [http://www.ensembl.org] or NCBI dbEST and they were then evaluated using Netprimer . The oligonucleotide primers and their attributes are shown in Table A tool box of genes identified from the proteome experiment were employed for quantitative real-time PCR. Primers were designed using Primer3 software available online [(-1/slope)-1*100 . The Ct values obtained for each gene was transformed into relative quantities by setting the highest value to 1 according to the requirement of geNORM software [http://medgen.ugent.be/~jvdesomp/genorm/] to obtain the normalization values for each sample calculated based on the geometric mean of reference genes ubiquitin (Ubi) and acidic ribosomal protein (Arp). The normalized values of individual samples were used for statistical analysis.qPCR was performed with StepOne thermocycler (Applied Biosystems (AB), CA, USA). A 10 μL PCR mix per reaction was prepared with 1 μL of primers (5 pM each), 5 μL of Fast SYBR Green Master Mix (AB) and 4 μL of 25x diluted cDNA template. The reaction mix was dispensed into 96 well plates and covered with adhesive optical film (AB) and amplification was performed according to the following conditions: holding stage for 20 sec at 95°C for activation of fast Taq DNA polymerase followed by 40 cycles of denaturation at 95°C for 3 sec and annealing/extension for 30 sec at 60°C. Fluorescence data was acquired during the annealing/extension step. The samples were run in duplicates and pooled DNase treated RNA and no template negative controls were also included in the scheme. A 3-fold dilution series from pooled cDNA were included in each plate for each gene and efficiency (E) of PCR reaction for each set of primers was calculated based on standard curve method according to the formula E = 10The statistical analyses of qPCR data were carried out using GraphPad Prism software . The sample sets were tested for normality using Kolmogorov-Smirnov test and data was log transformed where necessary. Transcript profiles for each gene from bacterial challenge group were compared with the respective values from mock challenge control groups, at each time point, using unpaired student’s t-test. Zero hour samples that are included in the bar charts as a basal reference point were not included for statistical comparisons.V. anguillarum infected cod. The skin mucus 2D gels showed good resolution and reproducibility  of protease activity in the presence of gastrointestinal mucus [The rather low number of significantly different protein spots identified in this study could be linked to the possible variation in the progression of al mucus . This coV. anguillarum are limited. To gain a better understanding of the possible role of identified proteins in disease progression and inflammation, a protein-protein interaction network generated by Ingenuity Pathway Analysis (IPA) is shown in Figure V. anguillarum infection.Proteomic studies to elucidate host responses in fish infected with V. anguillarum[V. anguillarum[V. anguillarum infection in cod might have resulted in low levels of CAPNS1 observed in the present study  was found to be downregulated in the present study  are stress proteins with RNA binding domain that can regulate cellular activity under stressful conditions such as hypoxia  or durinV. anguillarum lipopolysacharide (LPS) as shown in a comparative proteome study of Moraxella sp. infected kidney in sea bream (Sparus aurata) [Malate dehydrogenase 2  (MDH2) is a mitochondrial isozyme of malate dehydrogenase that is primarily involved in citric acid cycle . Conditi aurata) .Tubb2 downregulation has been reported in Chinese white shrimp (Fenneropenaeus chinensis) challenged with V. anguillarum[Tubb2 in the mucosal cells associated with phagocytic processes.Keratin is a cytoskeletal protein whose primary function is to protect cells from mechanical and non mechanical injuries . Recent guillarum. Being aCyprinus carpio) it has been shown to have antibacterial effects in vitro[V. anguillarum infected European seabass (Dicentrarchus labrax) [14-kDa apolipoprotein (apo-14kDa) is a fish specific high density lipoprotein (HDL) orthologous to mammalian apolipoproteinA-II (apoA-II) , the exp in vitro. Further labrax) .Gsto1 transcripts were found in different tissues including skin [Venerupis philippinarum) infected with V. anguillarum[Eriocheir sinensis) injected with Aeromonas hydrophila, an upregulation of novel gst transcripts was noted as early as 6 h post infection [gst reported in the aforementioned studies could be due to reactive oxygen metabolites induced by the V. anguillarum infection as shown in a study where the measurement on enzyme activity complemented the gene expression data [Glutathione-S-transferase omega-1 (GSTO1) is an enzyme involved in biotransformation of compounds including toxic substances and oxidative stress products, transport of ligands, and regulation of signaling pathways . In cod,ing skin . In the guillarum and Chinnfection . The uprion data .V. anguillarum infection [A. salmonicida[Proteasome 26 subunit is involved in ATP dependent degradation of ubiquitinated proteins, which are instrumental in oxidative stress response and partakes in generating peptide fragments of invading pathogen for antigen presentation . Proteasnfection  and in Almonicida. In the V. anguillarum infection.Other important proteins that exhibited a differential expression trend in this study, including profilin-2, flotilin-1, peroxiredoxin-6, apolipoprotein AI are also important from an immune perspective. The genes corresponding to these proteins could also be considered as potential biomarkers in studies involving Trichodina spp [In addition to bacterial infection, Atlantic cod may be susceptible to concurrent or secondary infections with parasites like dina spp . AttemptV. anguillarum H610 challenge protocol at Institute of Marine Research (IMR), Bergen is robust and well established as reported in different studies [In order to understand if the changes in the mucus proteins, arising from natural infection, correspond to the alterations of the respective genes in skin, a controlled bath challenge experiment was performed. The observations were made only during early stages of infection. The  studies ,54.Capns1 and Gsto1 were upregulated at 4 h post challenge whereas Flot1 was significantly downregulated compared to the mock challenged controls at 4 h and 48 h post infection  transcripts were quantified in the skin and gill tissue of the infected fish. The il1β mRNA levels were found to be significantly upregulated in gills at 48 h post bath challenge compared to the control [see Additional file il1β transcripts of skin showed no changes at the two time points (data not shown).Since the proteins were identified through ESTs, the genes had to be cloned. The qPCR expression patterns of selected genes are shown in Figure V. anguillarum infection and it can be surmised that they may play important role in the progression of vibriosis in cod. Fish skin has been seldom targeted for gene expression studies, in challenge experiments and infection scenarios, as most of them focused on systemic immune-relevant organs like spleen and head kidney. The expression of key immune genes in skin has been explored primarily in the case of ectoparasite infections [The gene expression data and proteomic data need not always show a one to one correspondence . Neverthfections . A recenfections  taken toThus, through the proteomic approach we were able to identify differentially regulated proteins in skin mucus that might be relevant targets for gene expression studies as evident from this study. Going further, the ultimate objective of this study is to utilize the mucosal proteins as biomarkers for early assessment of fish health status, aimed at controlling bacterial diseases including vibriosis.V. anguillarum infection of cod. More importantly, these proteins are shown to be closely associated with key pathways that mount an immune response in the host. The early induction of mRNA transcripts of some of the selected genes also points towards their involvement along with other acute phase proteins as primary orchestrators in the fish mucosal immunity against bacterial infection.We identified a group of proteins, of which the involvement of some can be considered novel in The authors declare that they have no competing interests.Conceived and designed the experiments: BR, VK and MFB. Performed the experiments: BR and LJ. Analyzed the data: BR and MFB. Wrote the paper: BR, VK and MFB. All authors read and approved the final manuscript.Relative expression of interleukin-1β (il1β) in gill tissue of Atlantic cod bath challenged with Vibrio anguillarum. Quantification of il1β mRNA level by real-time PCR at 4 h and 48 h post bath challenge. Values are indicated as means ± SEM (N = 5). Transcript levels normalized to reference genes: Ubiquitin (Ubi) and acidic ribosomal protein (Arp). Asterisk (*p < 0.05) above the error bars indicate statistically significant difference (Student’s t-test) between infected and control fish, at each time point.Click here for file


As such, there is a need to characterize this natural variability of seawater carbonate chemistry, especially within coastal ecosystems. Since 2009, carbonate chemistry data have been collected on the Florida Reef Tract (FRT). During periods of heightened productivity, there is a net uptake of total CO2 (TCO2) which increases aragonite saturation state (Ωarag) values on inshore patch reefs of the upper FRT. These waters can exhibit greater Ωarag than what has been modeled for the tropical surface ocean during preindustrial times, with mean (± std. error) Ωarag-values in spring = 4.69 (±0.101). Conversely, Ωarag-values on offshore reefs generally represent oceanic carbonate chemistries consistent with present day tropical surface ocean conditions. This gradient is opposite from what has been reported for other reef environments. We hypothesize this pattern is caused by the photosynthetic uptake of TCO2 mainly by seagrasses and, to a lesser extent, macroalgae in the inshore waters of the FRT. These inshore reef habitats are therefore potential acidification refugia that are defined not only in a spatial sense, but also in time; coinciding with seasonal productivity dynamics. Coral reefs located within or immediately downstream of seagrass beds may find refuge from OA.Ocean acidification (OA) is expected to reduce the calcification rates of marine organisms, yet we have little understanding of how OA will manifest within dynamic, real-world systems. Natural CO The saturation state of a given carbonate mineral is a key driver of inorganic carbonate mineral kinetics 3 exoskeleton by scleractinian corals Ocean acidification (OA) is the global decline in seawater pH due to the uptake of carbon dioxide (CO2 and have low aragonite saturation states (Ωarag), such as upwelling areas in the eastern tropical Pacific, are OA “hotspots” in that rising CO2 will force the already low Ωarag to potentially critical levels arag found in upwelling areas could also make organisms there more tolerant to future OA 2 sinks may serve as OA refugia because calcareous organisms will experience higher Ωarag relative to the open ocean.Despite these concerns, we still have only a rudimentary understanding of the spatial and temporal variability of carbonate chemistry within reef environments. There is a pressing need to ascertain which locations, habitats or regions may be relatively susceptible or even resilient to OA. This is a challenging undertaking which will take years, if not decades to unravel, as a given areas risk to OA will be a function of localized biogeochemical feedbacks that may locally alter the rates of OA 2 uptake of seagrasses 2 uptake associated with seagrass beds has the potential to create OA refugia, carbonate chemistry was repeatedly sampled across an inshore-to-offshore gradient in the upper, middle, and lower FRT over two years. During periods of heightened productivity, there is a net uptake of total CO2 (TCO2) which significantly increases aragonite saturation state (Ωarag) values on inshore patch reefs of the upper FRT.Seagrass beds are often located near coral reefs and the Florida Reef Tract (FRT) is no exception 2 (TCO2) was measured coulometrically and total alkalinity (TA) was determined using gran titration. The remaining carbonate parameters were calculated from these values and in situ temperature with CO2SYS Discrete seawater sampling was started in April 2009 from paired inshore and offshore sites in the upper Keys (UK) . No specU-tests were used when they were not. One-way ANOVAs  were used to ascertain significant effects of region  and season within the inshore and offshore grouping. When significant differences were indicated, post-hoc t-tests were used to determine the relationship of regions/seasons.Statistical comparisons were made between inshore and offshore sites in the UK, middle (MK), and lower (LK) Florida Reef Tract (FRT) after data were divided into seasons. Student's t-tests (α = 0.05 significance level) were used when data were normally distributed, whereas Mann-Whitney 2, TA, pCO2, and Ωarag were subtracted from their paired offshore values to determine the magnitude (Δ) of the gradient between inshore and offshore sites. A positive Δ indicates that inshore values are higher than offshore, whereas a negative Δ shows the opposite. TCO2 and TA values were normalized to salinity (nTCO2 = TCO2×35/S) prior to calculating Δ. Mean [± std. error of the mean (SE)] values by season, as well as the sum of the mean values across all seasons (Σ) are presented. Means represent the average gradient encountered by season, whereas the summed values indicate the annual net direction and magnitude of the gradient. TA-TCO2 plots were used to infer the dominant forcing mechanisms of the carbon cycle eliciting the inshore-to-offshore gradient in carbonate chemistry Inshore values of TCO2 and TA relative to offshore during spring and summer , and 2) nTA at the LK offshore site, where there was a significant depletion in TA during summer . Spring and summer TCO2 and TA values were significantly lower than the winter values at all inshore sites (p<0.05).Seasonal effects were pronounced at the inshore sites, while the offshore sites were relatively stable  and S2. g summer . The onlore site . Ωarag v2, indicating that net primary productivity is the dominant mechanism forcing this gradient. The magnitude of the TCO2 depletion in the spring and summer is sufficiently large to elevate Ωarag-values despite a coincident decline in TA. The standing crop, abundance, and productivity of the dominant species of seagrass in the Florida Keys, Thalassia testudinum, follow a sinusoidal pattern, peaking from June to July, and reaching minimum values in January 2 at the inshore sites.The inshore-to-offshore variability in carbonate chemistry on the FRT is primarily driven by large changes in TCO2 in the Florida Keys during a recent mapping study that included terrestrial and marine habitats 2 reported herein. Seagrasses are usually net autotrophic ecosystems 2 at all inshore sites  in the LK, versus the biogenic coral reef deposits of the UK and MK, may be similar to what is occurring today on the FRT.Inshore patch reefs of the upper FRT may be OA refugia. The thermal extremes of the MK eliminate this area as a potential refuge. The ΣΔΩLK sites  and S2. offshore . It is noffshore  may be r2 production 22, indicative of net calcification  nTCO2, (B) nTA, (C) pCO2, and (D) Ωarag plotted by season for inshore sites from upper, middle, and lower keys. Error bars represent standard error of the mean. Means represent the average of mean values for each sampling excursion grouped by season. Non-matching letters indicate significant differences .(TIF)Click here for additional data file.Figure S2Carbonate Chemistry at Offshore Sites by Season. Mean values for (A) nTCO2, (B) nTA, (C) pCO2, and (D) Ωarag plotted by season for offshore sites from upper, middle, and lower keys. Error bars represent standard error of the mean. Means represent the average of mean values for each sampling excursion grouped by season.(TIF)Click here for additional data file.Figure S3Seawater temperature at paired inshore and offshore sites.In situ temperature data, collected every 30 min from paired inshore (green lines) and offshore (blue lines) sites for (A) upper, (B) middle, and (C) lower Keys. Depths of temperature values are 5 m.(TIF)Click here for additional data file.Table S1Timing of discrete sampling at paired inshore and offshore sites in the upper (UK), middle (MK), and lower (LK) Florida Keys. WS indicates sample taken aboard the R/V Walton Smith in coordination with the South Florida Program's repeat biophysical oceanographic cruises rather than small boat sampling (X).(DOC)Click here for additional data file.Table S22 trendline equations and R2 by site and season.nTA-nTCO(DOC)Click here for additional data file.Table S3Kruskal-Wallis results to identify seasonal differences within each of the six sites.(DOCX)Click here for additional data file.


Delivery in von Willebrand disease (VWD) represents a significant hemostatic challenge because of the variable pattern of changes observed during pregnancy of von Willebrand factor (VWF) and factor VIII (FVIII), the protein carried by VWF. Since a wide heterogeneity of phenotypes and of the underlying pathophysiological mechanisms is associated with this disorder, a prompt and careful evaluation of pregnant women with VWD is requested in order to plan the most appropriate treatment at time of parturition. VWF and FVIII increase significantly during pregnancy in normal women, already within the first trimester, reaching levels by far >100 U/dL by the time of parturition. Women with VWD, levels at baseline of VWF and FVIII >30 U/dL have us a high likelihood to achieve normal levels at the end of pregnancy; thus specific anti-hemorrhagic prophylaxis is seldom required. Women with basal level <20 U/dL usually have a poor increase since most of these women carry mutations associated with increased VWF clearance or are compound heterozygous for different VWF mutations; that prevent the achievement of satisfactory hemostatic levels. While women with mutations associated with increased clearance show a full, albeit transitory correction of their hemostatic deficiency after desmopressin administration, compound heterozygous need replacement therapy because they do not respond well to this agent. Patients with abnormal VWF:RCo/VWF:Ag ratio at baseline (e.g. <0.6), typically associated with type 2 VWD, maintain the abnormality throughout pregnancy and VWF:RCo usually does not attain safe levels ≥50 U/dL. These women require replacement therapy with VWF-FVIII concentrates. Delayed post-partum bleeding may occur when replacement therapy is not continued for some days. Tranexamic acid may be useful at discharge to avoid excessive lochia. Von Willebrand disease (VWD) is a common autosomal inherited bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (VWF), a multi-adhesive protein which binds platelets to exposed subendothelium and carries factor VIII (FVIII) in circulation.Women may undergo to naturally-occurring physiological events  that may cause undue bleeding complications even in absence of a specific bleeding disorder. At the time of delivery, several obstetric complications may cause bleeding with or without associated hematologic alterations.3Uterine atony is still the leading cause of hemorrhage at parturition accounting for approximately 75% of all cases with PPH and may be burdened with a high risk of being transfused or even death in otherwise healthy women.There are conflicting results in the literature about the correct estimation of the risk and the severity of bleeding at parturition in women with VWD. Historical series reported percentages of VWD women with post-partum bleeding ranging from 15 to 60%.13Only a single study has tackled the estimation of the risk of bleeding occurring in women before the diagnosis of VWD was made.Pregnancy is considered as a hypercoagulable state because the increase of several hemostatic factors is observed. Factor VII, X, fibrinogen and plasminogen activator inhibitor type 1 do increase, but free protein S decrease.A progressive increase of these moieties is also evident in women with type 1 VWD, the partial quantitative deficiency of the disorder, with levels >50 U/dL in the third trimester.Type 2A VWD is characterized by the lack of high molecular weight multimers and an abnormal VWF:RCo/VWF:Ag (<0.6).Worsening of an already existing thrombocytopenia is the most important change observed in type 2B VWD women during pregnancy because an increased release of abnormal multimers with enhanced affinity for glycoprotein Ib on platelet surface occurs.Type 2M women usually show a significant correction of FVIII and VWF:Ag, while VWF:RCo does not attain levels around 50 U/dL, as is usually observed after desmopressin.26In type 2N, normalization of FVIII, which is more reduced compared to VWF in this type, usually occurs in women heterozygous or homozygous for the most frequent mutation R854Q responsible for this type,Women with typical type 3 VWD do not show any change during VWF because their endothelial VWF stores are lacking.Since pregnant women with VWD are at increased risk of postpartum hemorrhage if untreated,30In type 1 VWD pregnant women with FVIII:C and/or VWF levels lower than 30 U/dL, the administration of desmopressin usually after umbilical clamping and for 3–4 days thereafter is required,32In type 3 VWD women, VWF and FVIII do not increase during pregnancy and thus VWF/FVIII concentrates may be required during pregnancy to control intermittent vaginal bleeding and at delivery or for Cesarean section.30In patients with type 2A, 2B and 2M usually VWF:RCo does not attain normal levels and thus replacement therapy is similarly advised.33FVIII and VWF fall to baseline levels soon after delivery


Preclinical and clinical studies have shown for decades that tumor cells demonstrate significantly enhanced sensitivity to “fever range” hyperthermia  than normal cells, although it is unknown why cancer cells exhibit this distinctive susceptibility.To address this issue, mammary epithelial cells and three malignant breast cancer lines were subjected to hyperthermic shock and microarray, bioinformatics, and network analysis of the global transcription changes was subsequently performed.Bioinformatics analysis differentiated the gene expression patterns that distinguish the heat shock response of normal cells from malignant breast cancer cells, revealing that the gene expression profiles of mammary epithelial cells are completely distinct from malignant breast cancer lines following this treatment. Using gene network analysis, we identified altered expression of transcripts involved in mitotic regulators, histones, and non-protein coding RNAs as the significant processes that differed between the hyperthermic response of mammary epithelial cells and breast cancer cells. We confirmed our data via qPCR and flow cytometric analysis to demonstrate that hyperthermia specifically disrupts the expression of key mitotic regulators and G2/M phase progression in the breast cancer cells.These data have identified molecular mechanisms by which breast cancer lines may exhibit enhanced susceptibility to hyperthermic shock. Although the effectiveness of standard therapies such as surgery, chemotherapy, and irradiation has steadily improved over the years, cancer remains one of the most challenging problems of modern medicine. Among the major issues that complicate cancer treatment is the fact that cancerous cells are very difficult to therapeutically target with any specificity as they are in many respects similar to normal cells and have an astonishing ability of “hiding” their peculiarities. It has been known for over three decades that tumor cells demonstrate significantly more sensitivity to mild hyperthermia in “fever-range” temperatures 41-45°C) than normal cells -45°C tha. Mild hyHyperthermia over a short period  has been shown to induce irreversible cell damage and subsequent death in tumor cells, yet normal cells are remarkably spared . These eTo address this issue, we analyzed the hyperthermia-induced global gene expression profiles of a panel of breast cancer and mammary epithelial cell lines and used bioinformatics analysis to identify the unique gene networks distinct between the normal and cancer lines following this treatment. Furthermore, we confirmed our identified gene expression changes using qPCR and utilized flow cytometry to verify that these transcriptional alterations indeed reflect breast cancer specific responses to hyperthermia.C and C’ for mammary epithelial and breast cancer cells, respectively) and 45°C hyperthermic treatment . The 37°C control was grown under standard culture conditions. For the hyperthermia treatment, 45°C prewarmed conditioned media was immediately added to each treatment group and continuously maintained at this temperature for 30 minutes. After this time, the 45°C media was completely removed and replaced with 37°C conditioned media. The cells were then grown under standard culture conditions and harvested at the time point indicated for each experiment.MCF10A (ATCC #CRL10318) mammary epithelial cells and MCF7 luminal breast cancer cells (ATCC #HTB-22), MDA-MB-231 Basal B breast cancer cells (ATCC # HTB-26), and MDA-MB-468 Basal A breast cancer cells (ATCC #HTB-132) were purchased from ATCC and grown in standard culture conditions as previously reported . For heaTotal RNA was collected from each cell line  4 hours after completion of the hyperthermia treatment. RNA was amplified and biotin-labeled using Illumina TotalPrep RNA Amplification Kit (Ambion). 750 ng of biotinylated aRNA was then briefly heat-denatured and loaded onto expression arrays to hybridize overnight . Following hybridization, arrays were labeled with Cy3-streptavidin and imaged on the Illumina ISCAN. Intensity values were transferred to GeneSpring GX microarray analysis software (Agilent) and data was filtered based on quality of each call. Statistical relevance was determined using ANOVA with a Benjamini Hochberg FDR multiple testing correction . Data were then limited by fold change analysis to statistically relevant data points demonstrating a 2-fold or more change in expression. The microarray data from this experiment is publically available on the Gene Expression Omnibus (GEO Accession #GSE48398). All heatmaps shown represent the combined average of all biological and technical replicates.http://string-db.org).Pathway analysis to identify gene networks and biological processes affected by the gene expression changes was performed using Metacore software (Thomson Reuters). Protein-protein interaction networks were determined using String 9.05 . All RT-PCR experiments were performed in at least triplicate.Cells were harvested 24 hours post treatment via trypsinization and stained with propidium iodide as previous reported . Cell cyC and C’ , while cells grown at 45°C will be referred to as H and H’ . Total RNA was isolated 4 hours following hyperthermic treatment. We then performed microarray analysis of the global transcription changes using Illumina high density BeadArrays which measure the expression levels of more than 47,000 transcripts and known splice variants across the human transcriptome. Data was filtered based on quality of each call and statistical relevance was determined using ANOVA with a Benjamini Hochberg FDR multiple testing correction . Data were then limited by fold change analysis to statistically relevant data points demonstrating a 2-fold or more change in expression. When comparing the expression changes based on the C vs H and C’ vs H’ analysis, we discovered that hyperthermia induced very dramatic changes in gene expression in all cell lines tested as reflected by 7252 two-fold or greater statistically significant gene expression changes (p < 0.05) occurring in at least one of the four cell lines  and three malignant breast cancer lines from each of the known subtypes  were subjected to 30 minutes of fever range hyperthermic shock (or maintained at 37°C as a control) as described in the Materials and Methods section. To streamline identification of these treatment groups, cells grown at 37°C will be referred to as 0A and thGAPVD1, RASA1, RABEP1, CALM1, GMFB, PTPN11)   Figure  and survC vs H and C’ vs H’). Though this analysis provides us with information on how each individual cell line responds to hyperthermia relative to its normal growth temperature, it does not provide absolute comparisons of the transcriptome response of breast cancer cells relative to mammary epithelial cells following the elevated temperature. To better understand what provides breast cancer cells the selective disadvantage over mammary epithelial cells in response to hyperthermia we must identify those genes that are differentially expressed in breast cancer cell lines following hyperthermia from those of the mammary epithelial cell line following hyperthermia. To perform this analysis, we directly compared the gene expression changes that occurred for H’ vs H and identified genes whose expression was truly distinct between the breast cancer and mammary epithelial cell lines following hyperthermia.Our previous analysis compared the hyperthermic response of each individual cell line to its transcriptional expression baseline at the normal growth temperature (H’ (MCF7) vs H we identified 2708 genes whose expression was distinct at statistically significant levels . H’ vs H comparisons of the MDA231 and MDA468 lines yielded 919 and 750 significant gene expression changes, respectively. Heatmap analysis indicated a strong trend in the gene expression profiles between each of the three breast cancer lines following hyperthermia  relative to the mammary epithelial line (H)  or normal control temperature (C and C’) and collected the cells after 24 hours for cell cycle analysis using flow cytometry. The cells were collected 24 hrs after treatment as this is sufficient time to see the phenotypic effects on the cell cycle that would be induced by altered RNA expression. Propidium iodide staining of cells from each condition clearly revealed that a G2/M phase accumulation as a common event across all three breast cancer lines following hyperthermia even after 24 hours following the treatment, but did not occur in the mammary epithelial lines  compare the hyperthermia-induced changes in global gene expression patterns of the breast cancer cell lines to the mammary epithelial cells using a H’ vs H analysis. As a result of these studies, we identified several gene networks that reflect the hyperthermic response of breast cancer and mammary epithelial cells  and that clearly differentiate the response of breast cancer cells from that of mammary epithelial cells (including mitotic regulation and expression of histone and non-protein coding RNAs).While hyperthermic treatment of tumors has been utilized since the time of the ancient Greeks and modern medicine has implemented hyperthermia as an adjuvant treatment in various settings, use of this technique has been marred with limitations including the inability to target heat to the tumor without collateral damage to the neighboring cells, homogenous heat dispersion throughout the entire tumor, and intrinsic problems with targeting undetectable micrometastases. In recent years, advances in nanoparticle-enabled thermal therapy hold the promise to overcome many of these issues, thus a strong interest in treatment of tumors with hyperthermia has been renewed. While it has been established for decades that normal tissues exhibit enhanced thermotolerance relative to cancer cells ,2, the mC vs H and C’ vs H’ comparative analysis of the gene expression profiles of each cell line revealed that the mammary epithelial cells responded to increased temperature distinctly from the breast cancer lines, with altered regulation of gene networks controlling DNA damage response, cell cycle progression, apoptosis, and heat shock characterizing the mammary epithelial cell response. In contrast, the three breast cancer lines commonly altered gene networks encoding Rab and Ran G-protein regulators in response to hyperthermia.C vs H), while the three breast cancer lines showed no significant 2-fold or greater alterations in the expression of these genes (C’ vs H’). Despite this finding, we provide evidence that suggests the ability of mammary epithelial cells to properly modulate their heat shock response does not contribute to the selective disadvantage of breast cancer cells to hyperthermia. For instance, comparison of heat shock protein expression in the H’ vs H analysis revealed no significant difference in the relative abundance of these heat shock protein genes regardless of the cell type. As elevated expression of heat shock proteins has been observed in various types of cancers [Arguably the most studied response of cells to hyperthermia is that of heat shock protein activation and expression and one might guess that heatshock-protein mediated responses are likely responsible for the selective disadvantage of solid tumors to fever range hyperthermia. Numerous cell stresses have been shown to induce heat shock proteins, which act as molecular chaperones inside cells to modulate thermotolerance and protect cells from stress-induced death -29. MCF1 cancers -32, hypeC vs H and C’ vs H’ revealed a number of genes involved in DNA damage response whose expression was altered in the MCF10A cells, and similar changes were not observed in the three breast cancer lines. Despite this, no statistically significant changes in gene expression for these genes were observed in our H’ vs H comparison, suggesting that (similar to the heat shock proteins) this pathway may not clearly distinguish the selective disadvantage of breast cancer cells to hyperthermia.Hyperthermia has long been known as an effective radio- and chemo-sensitizing agent and it would be an attractive hypothesis that hyperthermia may impart a selective disadvantage to breast cancer cells via upregulation of DNA damage or reduction in its repair. Indeed, hyperthermia has been shown to induce chromosomal damage during S-phase  and inhiH’ vs H treatments to identify the unique gene networks that clearly differentiate the gene expression changes unique to the breast cancer cells following heat treatment. This comparative analysis identified cell cycle networks preferentially involved in mitotic progression as well as large scale changes in the expression of histones and non-protein coding RNAs as the major distinctions between the hyperthermic responses between the breast cancer lines and the MCF10A cells. 80% of the top 60 genes commonly expressed at higher levels in the three breast cancer lines following heat shock relative to the mammary epithelial line following heat shock were histones and non-coding RNA. This effect was due primarily to decreased expression of these genes in the MCF10A cells with no change or only a small upregulation in expression in the breast cancer lines, suggesting that mammary epithelial cells are repressing many of their core processes  following hyperthermic shock, while the breast cancer cells may continue performing these processes as normal. Similar findings have been reported following other cellular stresses whereby oxidative damage significantly decreases the expression of histones and ribosomal proteins [As our initial analysis compared only the heat shock response of each individual cell line relative to its transcriptional expression at normal growth temperature, we extended our analysis by directly comparing the transcriptional response of the proteins . In addiproteins , and hisproteins , suggestOur data revealed that a number of small nucleolar RNAs, which play key roles in ribosomal biosynthesis, were differentially regulated between the mammary epithelial and breast cancer cells following hyperthermia. Several small nucleolar RNAs are reportedly critical mediators of oxidative stress and their overexpression has been associated with reduced resistance to oxidative stress ,42. SmalH’ vs H analysis of our data. The genes that were differentially expressed at statistically significant levels included those involved in spindle assembly and chromosome separation, chromosome condensation in prometaphase, metaphase checkpoint, sister chromatid cohesion, and initiation of mitosis. Our confirmatory experiments using flow cytometry further revealed that hyperthermia treated breast cancer cells stalled in the G2/M phase of the cell cycle within 24 hours post-treatment, while the cell cycle profiles of heat-shocked mammary epithelial cells were similar to those grown at normal temperatures. Interestingly, it has been shown that cells vary in their susceptibility to heat in accordance to their phase in the cell cycle, with the highest heat sensitivity observed during mitosis due to damage to the mitotic apparatus, leading to inefficient mitosis and polyploidy. M- and S-phase arrested cells show increased susceptibility to heat-induced damage, while G1-phase cells are relatively heat resistance [Changes in cell cycle progression (particularly mitotic catastrophe) have been repeatedly shown to characterize the hyperthermic response of numerous cell types -49, thousistance -53.This study has been the first to shed light on the comparisons of transcriptome-level fever range hyperthermic responses of mammary epithelial cells to breast cancer cells. While this data points to a number of areas that potentially contribute to the selective advantage of normal breast epithelium over its malignant counterparts following hyperthermia, our studies were simplistic in that they utilized a cell culture monolayer system solely consisting of cells derived from normal or tumor breast tissue. We have gained solid insight into the responses of these particular cell types to fever range hyperthermia, however a tumor is a very complex entity. For instance, solid tumors are not only composed of the tumor cells, but also consist of endothelial, fibroblast, and immune cells which will each respond to hyperthermia in their own fashion and potentially affect the response of the tumor as a whole. Moreover, heterogenous heat distribution and dissipation due to a faulty tumor vascular system may induce uneven heating in the tumor itself, thus affecting some areas distinctly and differentially altering the tumor’s response to hyperthermia. Future studies should be undertaken to address these issues.Collectively, our data suggest that fever range hyperthermia affects breast cancer cells distinctly from mammary epithelial cells. These differences are largely attributed to alterations in the expression of genes involved in mitotic cell cycle progression, histones, and non-coding RNAs. Considering the hyperthermia induced G2/M cell cycle defects observed in the breast cancer cells but not the mammary epithelial cells, these data pose the question as to whether hyperthermia may function in a synergistic manner when combined with drugs that specifically target mitosis such as taxols and vinca alkaloid derivatives.C: 37°C treatment of mammary epithelial cells; C’: 37°C of breast cancer cells; H: 45°C of mammary epithelial cells; H’: 45°C of breast cancer cells; RNA: Ribonucleic acid; DNA: Deoxyribonucleic acid; aRNA: Amplified RNA; cDNA: Complementary DNA; mRNA: Messenger RNA; G1: Gap 1; S: Synthesis; G2: Gap 2; M: Mitosis; RT PCR: Real time polymerase chain reaction.The authors declare that they have no competing interests.CA contributed to experimental concept and design, cultured and treated cells, purified RNA, performed analysis and interpretation of microarray data, carried out network analysis, drafted and revised manuscript, gave final approval for version to be published. VK contributed to interpretation of network analysis, assisted with drafting and revising manuscript, gave final approval for version to be published. JS cultured and treated cells, purified RNA, drafting and revising manuscript, gave final approval for version to be published. AN performed flow cytometric cell cycle analysis and interpretation of data, gave final approval for version to be published. AA cultured cells, gave final approval for version to be published. RL contributed to experimental concept and design, drafted and revised manuscript, gave final approval for version to be published. CB contributed to concept and design, drafted and revised manuscript, gave final approval for version to be published. DM performed microarray experiments, performed bioinformatics analysis and interpretation of data, drafted and revised manuscript, gave final approval for version to be published. BB contributed to concept and design, acquisition of data, analysis and interpretation of data, drafted and revised manuscript, gave final approval for version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/81/prepub


VHL) mutation to cause clear cell renal cell carcinoma (ccRCC) remain poorly understood. We show that the TP53 tumour suppressor gene is mutated in approximately 9% of human ccRCCs. Combined deletion of Vhl and Trp53 in primary mouse embryo fibroblasts causes proliferative dysregulation and high rates of aneuploidy. Deletion of these genes in the epithelium of the kidney induces the formation of simple cysts, atypical cysts and neoplasms, and deletion in the epithelia of the genital urinary tract leads to dysplasia and tumour formation. Kidney cysts display a reduced frequency of primary cilia and atypical cysts and neoplasms exhibit a pro-proliferative signature including activation of mTORC1 and high expression of Myc, mimicking several cellular and molecular alterations seen in human ccRCC and its precursor lesions. As the majority of ccRCC is associated with functional inactivation of VHL, our findings suggest that for a subset of ccRCC, loss of p53 function represents a critical event in tumour development.The combinations of genetic alterations that cooperate with von Hippel–Lindau ( VHL) tumour suppressor gene is mutated, deleted or epigenetically silenced in up to 85% of all sporadic cases of ccRCC  accounts for approximately 80% of kidney tumours and thereby approximately 2.5% of all types of human malignancy. The von Hippel–Lindau , in the entire mouse  in ccRCC. Rather, ccRCC frequently (41%) harbour mutations in the chromatin remodelling gene PBRM1 . We demonstrate that combined mutation of Vhl and Trp53 causes dysregulation of cellular proliferation in primary mouse embryo fibroblasts (MEFs) and kidney epithelial cells and results in the formation of kidney cysts and neoplastic lesions in kidneys as well as tumours in genital tract organs.Genes that are mutated at high frequency in diverse human epithelial tumours, including VHL gene and exons 5–8 of the TP53 gene in 54 cases of sporadic ccRCC  tumours, all of which are either previously described pathogenic mutations or are predicted to be pathogenic. One tumour harboured both VHL and TP53 mutations, while the other four TP53 mutant tumours were wild-type for VHL. While methylation analyses for the VHL gene were not possible in these samples, it is likely that pVHL expression may be silenced in these tumours as they showed very high immunohistochemical staining for the HIFα target genes. In agreement with our data, the COSMIC database (http://cancer.sanger.ac.uk/cosmic) lists 30 of 209 (14.4%) tumours that display TP53 coding region mutations. Unfortunately the VHL mutation status of these tumours is in most cases unknown. Thus, TP53 is mutated in a significant fraction of sporadic ccRCCs.We sequenced the entire ic ccRCC . As expeVhl and Trp53 genes in proliferative control. Deletion of Vhl in primary and transformed MEFs induces proliferation arrest and/or the onset of premature senescence  as control or with adenovirus expressing a Cre-GFP fusion protein (Adeno-Cre) to delete the floxed genes. Vhl knockout MEFs rapidly lost proliferative capacity whereas Vhl/Trp53 double deletion allowed proliferation, albeit at a slower rate of proliferation than Trp53 deletion alone . None of the gene deletions allowed the formation of colonies in soft agar, demonstrating that the cells are immortalized but not transformed. Western blotting 4 days after infection confirmed the high efficiency of gene deletion but after approximately 20–30 days of continuous proliferation, a significant restoration of pVHL expression was evident in the Vhlfl/flTrp53fl/fl Cre-infected cell population .We first utilized primary MEFs to investigate potential cooperative interactions of the mouse ype MEFs . In cont−/− MEFs . Interes−/− MEFs , the ext/− cells , suggeston alone . Consistpulation , suggestVhl can be rescued by culturing cells at 2 or 5% oxygen  cells but these cells proliferated more slowly than control (Adeno-GFP treated) cells with Trp53 knockdown alone  has undergone Cre-mediated recombination have a proliferative advantage over the Vhl/Trp53 null cells, allowing them to accumulate over time in bulk populations.Given the strong selection against pVHL-expressing cells in bulk population experiments, we performed experiments using single cells to definitively address the question of whether d allele . All celp53 gene . Westernp53 gene . Thus, TVhl and Trp53 deletion in a disease-relevant cell type we cultured primary mouse renal epithelial cells from the various floxed mouse strains at 5% oxygen and deleted Vhl and/or Trp53 using Adeno-Cre or using Adeno-GFP as control , and did not grow in soft agar, demonstrating that these cells are not immortalized or transformed.To investigate the cooperative effects of combined  control . While lferation . Unlike Vhl causes a reduction of Mad2 protein expression. Loss of Trp53 function in MEFs also causes higher levels of aneuploidy and polyploidy and has been shown to result in aberrantly elevated expression levels of the checkpoint proteins Aurora A , 4–8 months  and 11–13 months . Littermate mice that were negative for the Ksp1.3-Cre transgene served as controls for all of these cohorts.VhlΔ/Δ mice developed normally and showed no histological abnormalities within 18 months of age . By 5 months of age small clusters of disorganized cells . The non-recombined Trp53 floxed and Vhl floxed alleles were also detected, likely due to presence of wild-type  stromal, inflammatory or vascular cells in these lesions.As previously described, kidneys of Trp53ed cells  or micro lesions  and mild lesions  while 50 lesions . Some la lesions . These l lesions  and cell lesions  or solid lesions  growth p lesions , similar lesions  whereas  lesions . It was  lesions  and neop lesions  displaye lesions  verifyinVhl mutant cystic lesions and neoplasms in the mouse retain expression of the epithelial marker E-cadherin and do not display the mesenchymal marker vimentin .Immunohistochemical staining using antibodies against NaPi2 , NCC , THP (thick ascending loop of Henle) and AQP2 (collecting ducts) to mark different tubule segments revealed that most simple cysts express one of these markers , demonstrating that cysts arise from different nephron segments. Very rarely, remnants of the glomerulus could be observed in simple cysts (data not shown), suggesting that these cysts had arisen from the tubular epithelium at the urinary pole of the glomerulus. However, atypical cysts and neoplasms were always negative for all of the tubular markers , preventing assessment of the tubular segment of origin of these lesions and suggesting that the transition to tumour formation involves some degree of de-differentiation. Unlike the findings reported for some precursor lesions in human VHL patient kidneys  but histological analysis of aged cohorts revealed that they displayed a qualitatively more severe phenotype of nuclear abnormalities than the Trp53Δ/Δ mice . At 11–13 months of age, the epididymides of all male VhlΔ/ΔTrp53Δ/Δ mice, but not of control or single mutant mice, displayed benign growths . These growths were predominantly due to squamous metaplasia  and extensive epithelial dysplasia . These lesions are histologically identical to those arising in VhlΔ/ΔPtenΔ/Δ mice , but not of VhlΔ/Δ or Trp53Δ/Δ mice (not shown), were malformed. In contrast to the normal single layered epithelium, vesicular glands of VhlΔ/ΔTrp53Δ/Δ mice displayed a disorganized epithelium characterized by multiple convoluted layers of epithelial cells and the formation of gland-like structures . This phenotype increased in severity with age and two mice exhibited carcinomas in the vesicular gland . The uterus in all genotypes developed normally  and displayed a normal organisation of lumenal and glandular endometrial epithelium . In older VhlΔ/ΔTrp53Δ/Δ mice, small foci of disorganized and multilayered epithelial cells could frequently be observed. Consistent with this, one mouse developed a high-grade carcinoma of the endometrium  and another a high-grade squamous carcinoma of the upper cervix . One mouse displayed a high-grade carcinoma that most likely arose in the urothelium of the renal pelvis and which had also metastasized to the lungs and liver .VhlVhl and Trp53 tumour suppressor genes ultimately causes dysregulation of epithelial cell proliferation and the evolution of dysplastic and malignant lesions in multiple tissues in mice.Collectively, these findings demonstrate that mutation of the Vhl/Trp53 double mutant mice displayed a similar loss of primary cilia. Visualising the primary cilium using an antibody against acetylated tubulin revealed that only 40% of cystic epithelial cells but almost 90% of cells in non-cystic tubules displayed a primary cilium  is consistent with our primary cell culture data showing that Vhl/Trp53 mutant mice develop an apparent spectrum of cystic lesions beginning with simple cysts lined by a single layer of epithelial cells, followed by atypical cysts that display micro-papillary epithelial growths that project into the lumen of the cyst and finally cysts that are almost entirely filled with neoplastic growth. About half of the neoplasms are a solid mass of cells, preventing assessment of whether they arise via a cystic precursor lesion or not. Vhl/Trp53 neoplastic lesions display several features of human ccRCC including clear cell-like changes, HIFα stabilisation and high rate of proliferation, but differ in that they exhibit a low nuclear grade and do not invade surrounding tissue. The lack of a capsule surrounding the neoplasms and absence of extra-renal metastases speaks against a malignant ccRCC lesion. Vhl/Trp53 mutant neoplasms also frequently grow in a micro-papillary pattern, akin to papillary renal cell carcinomas. While the precursor lesions of human renal carcinomas are poorly characterized in general, in papillary type I and type II tumours the size of the lesion is the sole definitive distinguishing criteria. Lesions smaller than 5 mm are classified as adenomas and larger lesions are carcinomas , implying that there may be other unidentified pathways that cooperate with pVHL in maintenance of the primary cilium. In contrast to simple cysts, atypical cysts and neoplasms display hyperactivation of mTORC1 signalling. Since both lesions are characterized by disorganized patterns of cellular growth it is noteworthy that mTORC1 activation has been shown to induce a translational program that promotes cellular invasion . In ccRCC, several mechanisms have been proposed to act to compromise p53 function, potentially alleviating the selective pressure for TP53 mutation or deletion during tumour formation. USP10 normally de-ubiquitinates p53 in response to DNA damage, opposing the action of Mdm2 and allowing p53 protein accumulation . Similarly, SETD2 is mutated in a small fraction of ccRCCs  were treated using Ventana Benchmark XT  or BOND-MAX  automated systems. Immunostainings for CAIX, GLUT1 and HIF1α were performed as recently described . The study was approved by the local ethics commission (reference number StV 38-2005). Haematoxylin and eosin stained sections of all paraffin embedded ccRCC specimens were reviewed by H.M. DNA extraction and Trp53−/− MEFs were a kind gift from Scott Lowe. Cells were cultured either in conventional cell culture incubators at atmospheric oxygen or at 5% oxygen or were cultured in a darkened oxygen glove-box incubator  at 5% oxygen in which medium and PBS were equilibrated for 2 h prior to splitting of cells to ensure that cells were exposed to constant oxygen tension throughout the experiment. For proliferation assays, cells were seeded at densities of either 2 × 105 or 3 × 105 cells per 6 cm dish in triplicate dishes and counted after 3 days before reseeding at the same density for the next passage. All proliferation assays shown in the Figures are representative of at least three independent experiments. Wild-type and Vhlfl/fl MEFs were transformed by transfection with a plasmid expressing SV40 large T-Antigen  and pools of cells that formed colonies after plating at low density were harvested to generate cell lines. Cells were infected with adenoviruses expressing GFP  or Cre-GFP , retroviruses (LMP) expressing non-silencing hairpin or miR30-shRNA against Trp53   containing the U6 promoter (derived from pENTR/pSM2 (U6), Addgene 17387) driving expression of a previously described  or expressing an empty (ns) miR30 backbone. Infections were followed after 48 h by puromycin selection (4 µg/ml) where appropriate. Genotyping for the floxed or recombined Vhl and Trp53 alleles were performed as described , loss of VHL function alone in humans and in mice is insufficient to cause kidney tumour formation. It is presumed that other genetic events must cooperate with loss of VHL to cause ccRCC but these cooperating mutations remain poorly understood.The cooperating genetic events that lead to the formation of clear cell renal cell carcinoma (ccRCC), the most frequent form of kidney cancer in humans, remain unclear. While the vast majority of familial and sporadic forms of ccRCC harbour biallelic inactivation of the von Hippel–Lindau tumour suppressor gene , Mad2 .Real-time PCR was performed as described , 34 pg/ml triiodothyronine, 5 µg/ml Apo-transferrin, 1.73 ng/ml sodium selenite and 18 ng/ml of hydrocortisone] and 25 ng/ml epidermal growth factor (EGF). Cells were counted and seeded at a density of 1 × 106 cells on standard 100 mm plastic tissue culture plates. After 5–6 days in culture, cells were infected with adenoviruses expressing GFP  or Cre-GFP . Sulforhodamine B (SRB) proliferation assay was performed in 96-well format as described  and OD was measured at 540 nm in a micro-plate reader.Kidneys were dissected from 2-month-old floxed mice. After removing the capsule under sterile conditions, kidneys were mashed with a razor blade on ice and digested in collagenase II (Gibco) and soya trypsin inhibitor (Gibco) solution at 37°C for 30 min. The cell suspension was filtered through a 70 µm cell strainer and washed in HBSS + 5% FCS. Erythrocytes were lysed for 1 min using standard ACK buffer. Cells were resuspended in complete K-1 culture medium [Dulbecco's modified Eagle's medium (DMEM):Hams F12] (50:50), supplemented with 0.5% foetal calf serum, hormone mix [5 µg/ml insulin, 1.25 ng/ml prostaglandin ECT antibody (Hergovich et al, Western blotting, immunohistochemistry or immunofluorescence were conducted using previously described methods (Frew et al,


Development of a cardiomyopathy in diabetes mellitus is independent of traditional risk factors, with no clinical trials targeting specific therapeutic interventions. Myocardial fibrosis is one of the key mechanisms and aldosterone is a key mediator of myocardial fibrosis. We propose that aldosterone antagonism will improve cardiac function. We aim to evaluate the efficacy of selective aldosterone receptor antagonism with eplerenone added to optimal medical treatment in improving cardiac structure and function in diabetic cardiomyopathy. We will randomize 130 patients with type 2 diabetes mellitus, stable metabolic control and impaired left ventricular (LV) systolic or diastolic function, to either eplerenone (target dose 50mg) or matching placebo, in addition to optimal medical therapy for 12 months. The primary endpoints are changes in LV systolic and diastolic function, measured by echocardiographic 2-dimensional speckle tracking strain and strain rate and tissue Doppler imaging. The secondary endpoints include changes in echocardiographic markers and plasma biomarkers of collagen turnover; left atrial dimensions and function, incidence of atrial fibrillation and changes in exercise capacity and dyspnea score. The present study will assess whether specific aldosterone antagonism with eplerenone in addition to standard therapy will prevent progression or reverse cardiac dysfunction in diabetic cardiomyopathy using sensitive, robust and quantifiable echocardiographic measures that allow early detection of change. The study may offer a new direction in the management of this condition.ACTRN12610001063000 The incidence of obesity and type 2 diabetes mellitus (T2DM) are increasing towards epidemic levels in both developing and industrialized countries. Patients with T2DM are at increased risk of heart failure from diabetic cardiomyopathy , with thMyocardial fibrosis has been suggested as one of the key mechanisms underlying myocardial dysfunction in T2DM . CardiacOveractivation of the renin angiotensin-II aldosterone system (RAAS) occurs in diabetic cardiomyopathy resulting in cardiac insulin resistance and a cascade of abnormalities mediated by angiotensin II and aldosterone Figure . AldosteThere is paucity of data on the specific use of aldosterone antagonists in T2DM and this is a particular group who are prone to hyporeninemic hypoaldosteronism . As a coCirculating biomarkers of collagen synthesis and degradation provide a means of monitoring ECM turnover and the balance between collagen synthesis and degradation -22. LeveCardiac imaging modalities such as echocardiography and cardiac magnetic resonance imaging can be used to quantify myocardial interstitial fibrosis. Histologically quantified collagen accumulation has been shown to be linearly related to the magnitude of ultrasound backscatter, a marker of myocardial reflectivity, in experimental ,24 as weThe aim of the current study is to investigate whether treatment with eplerenone, a specific aldosterone antagonist, for 12 months, in addition to ACEI or ARB, would improve myocardial structural and functional abnormalities in patients with diabetic cardiomyopathy without advanced heart failure or severe LV systolic dysfunction.This study was designed as a prospective, investigator-initiated, multi-centre, randomized, double blind, placebo controlled study to evaluate the impact of treatment with eplerenone for 12 months on myocardial structure and function in patients with T2DM and LV diastolic or systolic dysfunction, in the absence of class III-IV symptoms with severe LV systolic dysfunction. The study was conducted in accordance with the NHMRC guidelines and was approved by Sydney Southwest Area Health Service Human Research Ethics committee. Figure Eligible subjects include male and female adults that meet the following criteria: 1) An established diagnosis of T2DM of any duration and severity. 2) Presence of LV diastolic or systolic dysfunction. 3) Stable metabolic control , blood glucose levels (HbA1c ≤ 8.0%)) such that minimal or no dose changes of other medications, such as ACEI, ARB, lipid lowering and blood glucose lowering medications are anticipated during the course of the study. 4) NYHA functional class I or II. 5) Treatment with optimal target or maximal tolerated dose of ACEI or ARB and ß-blocker, unless contraindicated. 6) Serum potassium ≤5.0 mmol/L within 1 week prior to randomization.2. 7) Estimated glomerular filtration rate (eGFR) > 30 ml/min/1.73m 8) Subjects who received previous treatment with aldosterone antagonists for more than 7 consecutive days must meet the following additional criteria:a. No history of clinically significant hyperkalemia or renal impairment during previous aldosterone antagonist treatment.a. Aldosterone antagonist treatment has been discontinued for at least 3 months prior to randomization.Subjects are not eligible for enrolment if they present any of the following criteria: 1) Non-sinus rhythm. 2) Pregnancy. 3) Severe heart failure . 4) Significant CAD (patients who have not undergone recent coronary angiography will have significant CAD excluded by stress echocardiography).Further exclusion criteria pertain specifically to the use of aldosterone antagonists, and include: 1) Serum potassium >5.0 mmol/L.2. 2) eGFR < 30 ml/min/1.73m 3) Concomitant potassium sparing diuretic. 4) Combination of ACEI with ARB. 5) Addison’s disease. 6) Acute renal insufficiency. 7) Severe hepatic insufficiency.Preliminary screening for trial eligibility is based on review of medical records by clinical trials staff at or before the baseline visit. After written informed consent, eligible subjects are randomly assigned by the clinical trial pharmacist to treatment with eplerenone or matching placebo. Randomization is by permuted blocks and is conducted via an electronic data management system.All patients will undergo resting echocardiography with strain and strain rate imaging for evaluation of LV and left atrial (LA) function. Myocardial calibrated IB will be performed on grey scale 2-dimensional (2D) images to assess the presence and extent of myocardial fibrosis.All transthoracic echocardiograms will be performed with Vivid E9 . All standard echo and Doppler parameters of LV systolic and diastolic function including tissue Doppler (TDI) will be measured. Maximum, pre-P, and minimum LA volumes will be measured using Simpson’s biplane method of discs.Longitudinal strain and strain rates of the left ventricle Figure  will be Left atrial longitudinal strain and strain rate will be similarly obtained in 2 annular and 2 mid segments of the apical 4- and 2-chamber views using velocity vector imaging   and short axis  views.The IB curves Figure  will be Biomarker levels will be taken prior to commencement of the study medication, as well as after 12 months treatment. Peripheral venous blood will be obtained between 8am and 9am after 15 minutes of supine rest. Serum P1NP and P3NP will be measured by radioimmunoassay ; serum P1CP will be measured by enzyme-linked immunoadsorbent assay ; plasma MMP-1 and MMP-9 will be measured by immunoassay ; plasma TIMP-1 levels will be measured by enzyme-linked immunoadsorbent assay ; serum TGF-ß1 will be measured using an enzyme-linked immunoadsorbent assay ; and serum direct renin and aldosterone levels will be quantified by radioimmunoassay . For quality control and validation purposes, all assays will be performed twice.A 6-minute walk test will be performed and Medical Research Council (MRC) dyspnea score recorded at randomization and after 12 months of therapy.Eplerenone and placebo tablets and packaging are identical in appearance and are manufactured as 25mg tablets by Pfizer . Subjects are commenced on 25mg eplerenone or matching placebo (1 × 25mg tablet) orally once daily, and may have their dose up-titrated to 50mg (2 × 25mg tablet) after 4 weeks in the absence of worsening renal function or hyperkalemia. In accordance with guidelines for management of heart failure ,30, seruThe primary endpoints are changes in global mean peak LV systolic strain and strain rate, peak early diastolic myocardial tissue Doppler velocities at the basal septal LV wall (e’), the ratio of mitral inflow early diastolic velocity to peak early diastolic mitral annular velocity (E/e’), LV calibrated IB, LV mass index and LA strain and strain rate at ventricular systole and early and late ventricular diastole, and LA phasic volumes. Secondary endpoints include changes in collagen turnover measured as changes in serum levels of procollagen type I aminoterminal peptide (PINP), PIIINP, PICP, matrix metalloproteinases 1 and 9 , tissue inhibitor of metalloproteinase-1 (TIMP-1), renin, and aldosterone levels). Other secondary endpoint measurements include MRC dyspnea score, exercise tolerance, as measured by 6-minute walk test distance and incidence of atrial fibrillation.Serious adverse events (SAE) including life-threatening hyperkalemia (>7mmol/L), acute renal failure, hypotension and death will be monitored. 1. Aldosterone antagonism with eplerenone in diabetic cardiomyopathy leads to an improvement in LV systolic and diastolic function, measured by 2D speckle tracking strain and strain rate imaging. 2. Myocardial fibrosis, as measured by echocardiographic calibrated IB and serum collagen biomarkers is reduced by eplerenone. 3. Eplerenone improves LA function and phasic volumes. 4. Changes in LV and LA function with eplerenone are correlated with changes in the degree of myocardial fibrosis.At the time of study design and commencement, there were no studies that examined the impact of aldosterone antagonism with eplerenone on cardiac function as measured by strain and strain rate imaging and cardiac fibrosis in patients with diabetic cardiomyopathy. Therefore, sample size calculation was based on previous studies that examined the effects of spironolactone on strain and strain rate in patients with diastolic heart failure from dilated and hypertensive cardiomyopathies, changes in myocardial calibrated IB in these patients, as well as differences in IB between patients with early diabetic cardiomyopathy and normal controls ,13,28,31Since commencement of this study, Kosmala and colleagues  examinedThese sample size calculations are based on a two-sample t-test and are therefore conservative. As analysis of covariance (ANCOVA) adjusted for the baseline measurement will be the method of analysis, the power will be higher or the minimal detectable effect smaller for the same power target. The gain in efficiency is linked to the correlation (r) between the baseline and on-treatment measurements which is currently unknown for the endpoints involved, but expected to be high .A data safety and monitoring board (DSMB) comprising of personnel with sufficient cardiological, endocrinological and statistical experience will be formed to review the interim results and oversee the conduct of the trial. The DSMB and ethics committee will review all unblinded data on clinical endpoint events and determine whether events meet pre-specified criteria. The DSMB will monitor safety and efficacy of the trial and periodically assess whether the trial should continue.Interim analyses for safety and efficacy are incorporated into this trial. The Haybittle-Peto rule with two interim efficacy analyses detailed in a statistical analysis plan are planned after approximately 30% and 60% of the patients have been followed up at 12 months. Early termination for the study will be recommended for evidence of overwhelming benefit (2-sided p < 0.003 favouring eplerenone) or substantive harm (2-sided p < 0.01 against eplerenone).The intention-to-treat principle will be applied to the analysis. Baseline and demographic characteristics will also be summarized by treatment group. All changes from baseline, including the primary and secondary end-points will be analysed by means of ANCOVA, adjusted for the baseline measurement.2-test or Fisher’s exact test if the number of events is small. All hypotheses are two-sided. A p-value of <0.05 is considered statistically significant.Descriptive statistics will be provided for safety data. The number of patients reporting any SAE, the occurrence of specific SAEs and discontinuation due to SAEs will be tabulated. The effect of treatment on the number of patients experiencing at least one SAE will be tested using a χThere are features of this trial design that deserve particular emphasis:● Patients of all symptomatology and degrees of myocardial abnormalities ranging from grade 1–3 diastolic dysfunction to mild to moderate systolic dysfunction are enrolled. Including patients with more severe grades of baseline diastolic or systolic dysfunction may allow greater improvement on eplerenone. Furthermore, patients with milder degrees of systolic/diastolic dysfunction randomized to placebo may experience disease progression during the course of the study.● Blood pressure independent effects of eplerenone will be examined by recruiting those with stable blood pressure control at baseline, and blood pressure will be continually monitored to ensure stability throughout the study. Furthermore, the dose of eplerenone will not exceed 50mg daily, minimizing the antihypertensive effects.● The impact of aldosterone antagonism on myocardial fibrosis will be measured both by cardiac imaging and serum levels of biomarkers. Furthermore, attempts will be made to correlate the changes in myocardial function with changes in the degree of myocardial fibrosis both on imaging as well as changes in biomarker levels.● In comparison to previous studies examining biomarkers of fibrosis, it is assumed that the beneficial effects of aldosterone antagonism come from more effective suppression of aldosterone, yet few have definitively shown that this beneficial effect is present despite a persistently elevated renin and aldosterone level after treatment. We will measure both renin and aldosterone level before and after treatment in our study.Diabetic cardiomyopathy is an increasingly prevalent disorder for which there is currently no specific therapeutic intervention. This trial, when successfully completed, will advance our understanding of diabetic cardiomyopathy and the underlying mechanisms. This may provide a new direction in the management of diabetes; the early treatment by eplerenone may prevent and reverse the progression of atrial and ventricular dysfunction and myocardial fibrosis to irreversible structural and symptomatic cardiac disease. Thus, the proposed research will have a favourable impact in turning the focus of treatment of this disease from secondary prevention and heart failure symptom management into an interventional approach at the preclinical stage. This may result in a reduction in morbidity and mortality for patients with T2DM, with improved outcomes and consequent substantial health cost savings for the community.2D: 2-dimensional; ACEI: Angiotensin converting enzyme inhibitors; AMI: Acute myocardial infarction; ARB: Angiotensin receptor blockers; CAD: Coronary artery disease; IB: Integrated backscatter; DSMB: Data safety and monitoring board; e’: Peak early diastolic velocity; E/e’: Ratio of mitral inflow early diastolic velocity to peak early diastolic mitral annular velocity; HbA1c: Glycosylated hemoglobin A1c; LA: Left atrial; LV: Left ventricular; MMPs: Matrix metalloproteinases; MMP-1: Matrix metalloproteinase 1; MMP-9: Matrix metalloproteinase 9; NYHA: New York Heart Association functional class; PICP: Procollagen type I carboxy-terminal propeptide; PIIINP: Procollagen type III amino-terminal propeptide; PINP: Procollagen type I aminoterminal peptide; RAAS: Renin angiotension-II aldosterone system; TGF-β: Transforming growth factor-β; TIMP-1: Tissue inhibitor of metalloproteinase 1; T2DM: Type 2 diabetes mellitus.Pfizer Inc. provided eplerenone and placebo tablets for this trial. However, Pfizer has no role in study design, data collection, analysis, interpretation of data, or manuscript preparation. ML has previously received a CardioVascular Lipid research grant support from the sponsor for an unrelated study but has no other financial conflicts.ML jointly conceived the study with DL, performed literature review, designed and implemented the trial, obtained ethics approval, have been involved in the recruitment of subjects, review of trial patients, data collection, and preparation of the manuscript. ML and SH conducted statistical trial planning, SH generated the random allocation sequence and concealment mechanism, reviewed and edited the manuscript, VW contributed towards recruitment and review of subjects, reviewed and edited the manuscript, AM reviewed and edited the manuscript. All authors read and approved the final manuscript.


Excessive exposure to alcohol prenatally has a myriad of detrimental effects on the health and well-being of the offspring. It is unknown whether chronic low-moderate exposure of alcohol prenatally has similar and lasting effects on the adult offspring’s health. Using our recently developed Sprague-Dawley rat model of 6% chronic prenatal ethanol exposure, this study aimed to determine if this modest level of exposure adversely affects glucose homeostasis in male and female offspring aged up to eight months. Plasma glucose concentrations were measured in late fetal and postnatal life. The pancreas of 30 day old offspring was analysed for β-cell mass. Glucose handling and insulin action was measured at four months using an intraperitoneal glucose tolerance test and insulin challenge, respectively. Body composition and metabolic gene expression were measured at eight months. Despite normoglycaemia in ethanol consuming dams, ethanol-exposed fetuses were hypoglycaemic at embryonic day 20. Ethanol-exposed offspring were normoglycaemic and normoinsulinaemic under basal fasting conditions and had normal pancreatic β-cell mass at postnatal day 30. However, during a glucose tolerance test, male ethanol-exposed offspring were hyperinsulinaemic with increased first phase insulin secretion. Female ethanol-exposed offspring displayed enhanced glucose clearance during an insulin challenge. Body composition and hepatic, muscle and adipose tissue metabolic gene expression levels at eight months were not altered by prenatal ethanol exposure. Low-moderate chronic prenatal ethanol exposure has subtle, sex specific effects on glucose homeostasis in the young adult rat. As aging is associated with glucose dysregulation, further studies will clarify the long lasting effects of prenatal ethanol exposure. It is well recognised that an adverse intrauterine environment can have long lasting effects on the subsequent health of the adult. This concept is known as the Developmental Origins of Health and Disease (DOHaD) hypothesis. One clinical marker for an adverse intrauterine environment is low birth weight, a result of decreased fetal growth, which has been associated with characteristics of the metabolic syndrome including impaired glucose tolerance, insulin resistance and type 2 diabetes in adult life In the last few decades it has become increasingly evident that prenatal alcohol (ethanol – EtOH) exposure may also alter glucose homeostasis and increase the risk of type 2 diabetes In rats, maternal ingestion of 25% weight/volume (w/v) EtOH (via the drinking water) for the entirety of pregnancy is associated with decreased fetal body and liver weights ad libitum feeding regimen produces a plasma EtOH concentration of ∼0.03% at 30 min after the onset of feeding, without causing maternal undernutrition or altering pregnancy outcomes . Using this model, the aim of the present study was to determine if chronic low-moderate prenatal EtOH exposure altered glucose homeostasis in rat offspring aged up to 8 months. We hypothesised that chronic low-moderate prenatal EtOH exposure induces sex specific alterations in glucose homeostasis that are less severe than those reported following high dose prenatal EtOH exposure Recently we have developed a model of chronic low-moderate prenatal EtOH exposure whereby pregnant rats are fed a liquid diet containing 6% EtOH for the entirety of gestation This study was carried out in strict accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The protocol was approved by the Animal Welfare Unit of The University of Queensland . All efforts were made to minimize suffering.ad libitum for 21 hours per day at the onset of the dark cycle (midday), with water being provided for the remaining three hours per day. Upon spontaneous delivery of the pups, the liquid diet was removed and the dams provided a standard laboratory rat chow diet with water ad libitum. All dams nursed their own pups until weaning at postnatal (PN) day 28 at which time male and female offspring were separated and housed with same-sex littermates.Treatment of dams to produce ethanol (EtOH)-exposed and control rats has been reported in detail previously On day E8 (±2 days), 30 minutes after the fresh diet was provided, a mixed blood  sample was collected from the tail of a subset of control and EtOH-fed dams (n = 7 per group). Whole blood glucose concentration was measured using an Accu-check Go glucometer .A subset of dams  were killed at E20 as described previously The day of delivery was defined as postnatal (PN) day 0. A subset of offspring  were killed at PN30 via cardiac puncture following anaesthesia (as described above) and pancreata collected and fixed whole in 10% buffered formalin for pancreatic histomorphology as described below. A second subset was fasted overnight and a mixed  blood sample collected from the tail via tail slice Formalin-fixed pancreata taken from PN30 male and female Control and EtOH-exposed offspring were processed from 10% buffered formalin to paraffin before being embedded whole in paraffin. Pancreata  were sectioned exhaustively at 6 µm thickness using a Microm HM325 rotary microtome , with sections collected every 200 µm. Sections  were mounted onto Superfrost microscope slides  and stained with aldehyde fuchsin. Using point counting, the number of pancreatic islets and β-cells were estimated with light microscopy  with a 20× objective. For each section, 25 fields of view were analysed by the same person, who was ‘blinded’ to the experimental group. The density of β-cells and islets was calculated as the ratio of β-cells/islets to pancreatic tissue and expressed as a percentage; β-cell/islet mass was determined as the product of density and pancreatic weight.At 4 months of age offspring underwent both an IPGTT  and an insulin challenge  in a similar manner to that described previously Glucose concentration was determined via colorimetric reaction using Infinity™ Glucose Oxidase Reagent  and measured at 500 nm . Plasma insulin was determined using a high sensitivity Rat Insulin Radioimmunoassay kit  following the manufacturer supplied protocol. For insulin, intra- and inter-assay coefficients of variation were 3.2% and 9.2% respectively for a quality control of 2.9 ng/mL. Total area under the glucose curve and area under the insulin curve  curves were calculated using PRISM GraphPad PRISM 5 . First phase insulin secretion was defined as the first 5 minutes after glucose administration with second phase insulin secretion being from 5 to 45 minutes The homeostasis model assessment for insulin resistance (HOMA-IR) was calculated as published previously Triglyceride concentrations were analysed via colorimetric reaction (at 500 nm) with a Cobas Mirra Chemistry Analyzer  and using Infinity™ Triglycerides reagents .GLUT2; assay ID Mn00446224_m1), GLUT4 (assay ID Rn00562597_m1), insulin receptor , forkhead box 01 , peroxisome proliferator-activated receptor gamma, co-activator 1α , phosphoenolpyruvate carboxykinase 1 , glucose-6-phosphate dehydrogenase , glucokinase , resistin (assay ID Rn00595224_m1) and adiponectin (assay ID Rn00595250_m1). Each gene of interest was multiplexed with the house-keeping gene, 18s rRNA  as reported previously Relative gene expression was measured by real-time PCR in the liver, abdominal adipose tissue and gastrocnemius of offspring killed at 8 months of age . Total RNA was extracted from tissues using an RNeasy Mini Kit , and reversed transcribed into cDNA using the Taqman reverse transcription reagents kit . Real-time PCR was performed in the StepOne™ Real-Time PCR system  and relative gene expression (in arbitrary units (AU)) analysed using the cycle of threshold fluorescence (Ct) method as reported previously post hoc analysis. Values are expressed as mean±SEM. The level of significance was taken as P<0.05.For all statistical analyses, comparisons were made between Control and EtOH-exposed offspring of the same sex using the statistical package SigmaStat . Means were compared using a two-tailed unpaired Students t-test or, in the case of a failed normality test, a Mann-Whitney Rank Sum test, or a two-way ANOVA with prenatal exposure and age as factors and a Tukey Maternal parameters and gestational outcome have been reported previously The amount of EtOH consumed (g/kg BW) within each period of dietary consumption recorded is shown in At both E8 and E20 there were no differences in plasma glucose concentrations between Control and EtOH-fed dams . In contFor the subset of offspring killed at PN30, there was no difference in body weight, pancreas weight or pancreas weight relative to body weight within male or female offspring groups . SimilarLongitudinal analyses of plasma glucose  and insuAt seven months of age triglyceride levels were 18% lower (P = 0.105) in male EtOH-exposed offspring (0.71±0.05 mM) than their control counterparts (0.87±0.07 mM). In contrast, plasma triglyceride levels were similar for female control and EtOH-exposed offspring (0.69±0.05 mM versus 0.64±0.04 mM).The response to i.p. glucose administration is shown in The response to i.p. insulin administration is presented in Lean muscle mass at eight months of age was not different between male Control and EtOH-exposed offspring or female Control and EtOH-exposed offspring. Similarly, there was no difference between male or female groups in fat-free mass, fat mass, per cent body fat or total body mass .Prenatal EtOH exposure did not alter the expression of metabolic genes within the abdominal adipose tissue, gastrocnemius muscle or liver .Using a rodent model of chronic low-moderate dose maternal EtOH consumption, we have shown that this type of fetal EtOH exposure induces fetal hypoglycaemia that is independent of maternal glucose status. In addition, prenatal EtOH exposure has differential effects on glucose homeostasis of male and female offspring. Although pancreatic histomorphometry at PN30 was not significantly altered by low-moderate chronic prenatal EtOH exposure, four month old male EtOH-exposed offspring were hyperinsulinaemic and displayed elevated first phase insulin secretion in a glucose challenge, suggesting some degree of insulin resistance. In contrast, at this same age female EtOH-exposed offspring appeared to be hypoinsulinaemic under basal conditions and had a lower area under the glucose curve during the insulin challenge, suggesting increased insulin sensitivity. These results indicate that chronic low-moderate dose prenatal EtOH exposure may alter the programming of glucose homeostasis and induce metabolic abnormalities in postnatal life in a sex specific manner.As EtOH is known to induce hypoglycaemia in both humans In this study, it was expected that EtOH exposed offspring would have abnormal or delayed pancreatic development, however this was not observed at PN30. It is possible that fetuses in our study were only acutely hypoglycaemic at the time of measurement in late gestation, a period that coincides with both rapid fetal growth per se, but suggest it reflects a stress response to the unique housing conditions for the GTT. This hypothesis could be confirmed in future experiments by measuring circulating cortisol concentrations. Also observed at this age were subtle alterations in glucose clearance, glucose tolerance and insulin resistance when glucose homeostasis was challenged through the glucose tolerance test and insulin challenge. These alterations were sexually dimorphic with male EtOH-exposed offspring having some insulin resistance  and female EtOH-exposed offspring having increased insulin sensitivity. Such sexual dimorphism has also been reported in Wistar rats chronically fed a high dose EtOH diet postnatally Although no difference was seen between Control and EtOH-exposed groups in fasting basal glucose and insulin concentrations during the longitudinal analyses, basal glucose concentrations increased with age in females but not males. For both sexes, basal plasma insulin concentrations (and HOMA-IR) were elevated at four months compared to the other ages. It must be remembered that plasma insulin concentration at four months of age was determined from the basal blood sample collected during the GTT. Therefore we do not believe the increased insulin concentration is of biological significance to glucose homeostasis The changes observed in the present study were similar, yet more subtle, than the overt changes seen in offspring following high dose prenatal EtOH exposure At first glance it appears in the present study that chronic low-moderate level prenatal EtOH exposure in females may be beneficial (due to the enhanced glucose clearance observed) but this may in fact be detrimental to the long term health of the offspring; improved glucose clearance may lead to increased conversion of glucose into triglycerides and increased fat deposition. There are strong links between poor fetal nutrition (indicated by low birth weight), elevated triglycerides postnatally and, ultimately, obesity in males and females that can be detected as early as in childhood (reviewed in et. al. found that GLUT4 mRNA levels within the gastrocnemius were not altered by high-dose prenatal EtOH exposure when rats were in the fed state GLUT4 mRNA within the gastrocnemius Gene expression levels were measured in the liver, gastrocnemius muscle and abdominal adipose tissue of eight month old animals in an attempt to detect central and/or peripheral insulin resistance. Despite exploring genes involved in glucose clearance, glucose breakdown and gluconeogenesis, we did not detect differences in gene expression of male or female EtOH-exposed offspring. As the lack of gene changes concurred with the normoglycaemic and normoinsulinaemic status of EtOH-exposed offspring during monthly fasting conditions, we had no evidence to suggest regulation of glucose homeostatic feedback pathways at the protein level and therefore we did not measure protein expression. In contrast, our gene expression results do not concur with the alterations in insulin secretion or glucose tolerance that were observed in male EtOH-exposed offspring during the glucose tolerance test. Thus, we were unable to reveal any molecular mechanisms. Previously Chen In conclusion, this is the first study to explore the effect of chronic low-moderate prenatal EtOH exposure on glucose homeostasis in rats. Whilst this treatment did not cause changes in basal glucose and insulin concentrations, subtle differential alterations were seen in EtOH-exposed offspring during challenges, with four month old males displaying insulin resistance and females displaying enhanced glucose clearance. Further longitudinal studies need to be performed using aged rats to determine if this chronic, low-moderate prenatal insult induces more overt alterations in glucose homeostasis with age. Despite this, results of the current study suggest even modest amounts of alcohol during pregnancy can induce long term alterations in glucose regulation in offspring, thereby highlighting the need for abstinence in pregnant women.


ZNF804a was identified by a genome-wide association study (GWAS) in which a single nucleotide polymorphism (SNP rs1344706) in ZNF804a reached genome-wide statistical significance for association with a combined diagnosis of schizophrenia (SZ) and bipolar disorder. Although the molecular function of ZNF804a is unknown, the amino acid sequence is predicted to contain a C2H2-type zinc-finger domain and suggests ZNF804a plays a role in DNA binding and transcription. Here, we confirm that ZNF804a directly contributes to transcriptional control by regulating the expression of several SZ associated genes and directly interacts with chromatin proximal to the promoter regions of PRSS16 and COMT, the two genes we find upregulated by ZNF804a. Using immunochemistry we establish that ZNF804a is localized to the nucleus of rat neural progenitor cells in culture and in vivo. We demonstrate that expression of ZNF804a results in a significant increase in transcript levels of PRSS16 and COMT, relative to GFP transfected controls, and a statistically significant decrease in transcript levels of PDE4B and DRD2. Furthermore, we show using chromatin immunoprecipitation assays (ChIP) that both epitope-tagged and endogenous ZNF804a directly interacts with the promoter regions of PRSS16 and COMT, suggesting a direct upregulation of transcription by ZNF804a on the expression of these genes. These results are the first to confirm that ZNF804a regulates transcription levels of four SZ associated genes, and binds to chromatin proximal to promoters of two SZ genes. These results suggest a model where ZNF804a may modulate a transcriptional network of SZ associated genes. Schizophrenia (SZ) is a heritable disorder having no single gene or environmental factor that accounts for a majority of cases. An emerging hypothesis to explain SZ etiology suggests that epistatic interactions between multiple susceptibility genes all converge onto a set of biological networks important for SZ pathology Genome-wide association studies (GWAS) are a powerful method to identify single nucleotide polymorphisms (SNPs) that are associated with a diagnosis. The first gene to reach genome-wide significance for psychosis was ZNF804a when SNP rs1344706 was significantly associated when schizophrenia and bipolar disorder diagnoses are combined In many cases, SNPs significantly associated with a particular diagnosis are located within intronic regions and therefore do not alter the coding sequence of proteins, but rather may affect the binding of transcription factors or alter the post-transcriptional splicing of nearby genes. The rs1344706 SNP has been correlated with increased ZNF804a transcript levels in adult tissue SZ is a neurodevelopmental disorder with symptoms associated with defects in cortical circuitry www.szgene.org/TopResults.asp; as of May 2011) We next asked if overexpression of ZNF804a in our neural progenitor cell cultures could alter the transcript levels of other SZ associated genes. We designed primers for the top 37 SZ associated genes highlighted on the SZgene.org website . These results indicate that overexpressed ZNF804a directly interacts with the promoter regions of PRSS16 and COMT and is consistent with ZNF804a having a direct positive effect on the transcription of these two SZ associated genes.To examine if ZNF804a expression may directly regulate transcription, we performed ChIP to determine whether ZNF804a directly interacts with genomic regions proximal to the promoters of COMT, PRSS16, PDE4B, and DRD2, the four regulated genes indentified in our qRT-PCR assays. Chromatin samples were isolated from progenitor cultures transfected with pCAG-ZNF804a. Proteins were cross-linked to DNA prior to isolation and then sheered, chromatin was immunoprecipitated with an anti-myc antibody, crosslinks reversed, and isolated DNA subjected to qRT-PCR with a set of tiled primers spanning 2 kb upstream of the annotated transcription start sites for PRSS16, COMT, PDE4B, and DRD2 that included a GC-rich region . For ourTo rule out the possibility that overexpression of ZNF804a results in artificial binding to DNA upstream for PRSS16 and COMT, we determined whether endogenous ZNF804a protein interacts with the same regions of PRSS16 and COMT. We performed ChIP assays using anti-ZNF804a antibody in untransfected neural progenitor cell cultures. Similar to ChIP results for the overexpressed ZNF804a protein ChIP for endogenous ZNF804a protein enriched DNA sequence within the PRSS16  class I region of the genome. A SNP in this region of the genome (6p22.1) reached genome-wide association for SZ Given the intronic location of SNP rs1233706 within the ZNF804a gene, altered expression or splice variation may confer SZ susceptibility. The alleles of SNP rs1344706 are shown to differentially bind nuclear proteins and suggest a molecular mechanism for altering ZNF804a expression Beyond developmental expression, understanding the function and determining the genes that are regulated by ZNF804a either directly through promoter/enhancer occupancy or indirectly through downstream molecular pathways is relevant to our understanding of the function of this gene and how it may associate with SZ. Regardless of the expression pattern observed in SZ, our data and others suggest that transcription downstream of ZNF804a is sensitive to the expression level of ZNF804a All studies were conducted in accordance with protocols that were approved by the University of Connecticut Institutional Animal Care and Use Committee . The facilities at the University of Connecticut are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC).Nuclei were extracted from confluent neural progenitors (∼90%) using the CelLytic Nuclear extraction Kit from Sigma (NXTRACT). Briefly, cells were swelled with a hypotonic buffer. They were then disrupted and cytoplasmic extract was removed. The nuclear extract was isolated from the remaining lysate with a high salt buffer.Chromatin recovered by myc-tag or IgG immunoprecipitation from rat neural progenitors was resolved by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, probed with a ZNF804a antibody (Santa Cruz Biotechnology), and visualized on an Odyssey Infrared Imager.Brains were dissected from E13 rat embryos. Paraffin embedded brains were cut on a cryostat. Cortical sections were taken and immunohistochemistry was performed. Sections were stained using antibodies against ZNF804A  with a nuclear counter stain TO-PRO . Anti-rabbit 488 secondary antibody  was used for fluorescent imaging. Images were taken with a Leica TCS SP2 confocal microscope.http://frodo.wi.mit.edu/primer3/) and tested for efficiency and specificity by serial dilutions and melt curve analysis. Sybr Green mix (ABI) was used to amplify cDNA. Primer sequences are in RNA (1 µg) was reverse-transcribed into cDNA using oligo-dT primers and reverse-transcriptase. RNA was then hydrolyzed, and re-suspended in nuclease-free water. Gene-specific primers were designed using Primer 3 software (6) was sonicated into 200 to 500 base pair (bp) fragments. Resulting samples were immunoprecipitated with 5 µg myc-tag antibody , 10 µg ZNF804a antibody (Santa Cruz Biotechnology), or 5 µg normal rabbit immunoglobulin G (IgG) (Sigma I8140). Immunoprecipitated DNA was probed by qRT-PCR using promoter specific tiling primers across the promoters of PRSS16, COMT, DRD2 and PDE4B. Primer sequences are in Chromatin immunoprecipitations were performed as described with several modifications (below). Briefly, chromatin isolated from rat neural progenitors  and wild type transfected with ZNF804A (ZNF) followed by the first delta Ct after Gapdh normalization. Fold Regulation was calculated and a standard error of the mean for each condition.(TIF)Click here for additional data file.Table S2Tiling primers used in ChIP experiments. Table of tiling primers of promoters for qRT-PCR combined with chromatin immunoprecipitation. All primers are ordered by distance from the transcription start site (TSS) of each gene starting with 1 (2 Kb away) to 8 (on the TSS).(TIF)Click here for additional data file.