Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease , demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers.Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5–5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations  using whole-exome sequence data, in order to understand how an individual from a bottlenecked population might differ from an individual from an out-bred population. We provide empirical evidence that there are more rare and low-frequency deleterious alleles in Finns compared to NFEs, such that an average Finn has almost twice as many low-frequency complete knockouts of a gene. As such, we hypothesized that some of these low-frequency loss-of-function variants might have important medical consequences in humans and genotyped 83 of these variants in 36,000 Finns. In doing so, we discovered that completely knocking out the  After widespread success with genome-wide association studies (GWAS) of common variants, several studies have recently begun to identify rare  and low-frequency (0.5–5%) variants in complex diseases and traits such as triglycerides To explore this question, we used exome sequencing to characterize the allelic architecture of the Finnish population compared with a set of non-Finnish Europeans (NFEs) from the United States, Great Britain, Germany and Sweden. We demonstrate that Finns carry a significant enrichment of low-frequency (0.5–5%) LoF variation, defined here as nonsense and essential splice sites that are rare in NFEs. In addition to the isolate population structure, Finland has nationwide health records that provide decades of follow-up data that can be linked to epidemiological studies. The availability of nationwide health records in a population isolate structure triggered us to study the impact of low-frequency variants on risk factors and disease outcomes and their risk factors. The Sequencing Initiative Suomi (The SISu project) aims to combine these resources and build knowledge and tools for genome health initiatives. We genotyped 83 LoF variants discovered through our exome sequencing, in several large well-phenotyped population-based cohorts comprised of 36,262 Finns and tested for association to 60 quantitative traits and used data from the 13 disease outcomes assessed using the National Health Registers. We demonstrate that 5 of these variants have significant associations with clinically relevant phenotypes, illustrating the general value of the Finnish population for the study of low-frequency variants studies in complex as well as Mendelian diseases. We further confirm two LoF variants that significantly reduce lipoprotein(a) levels are associated with protection from cardiovascular disease.−6). On the other hand, there was an excess of low-frequency variants in Finns versus NFEs , collectively suggesting that while most rare variants did not survive the bottleneck, the variants that did have become substantially elevated in frequency As part of the SISu Project, we assembled 3,000 whole-exome sequences from Finns in projects including GoT2D, ENGAGE, migraine, METSIM and the 1000 Genomes Project along with 3,000 whole exome-sequences of NFEs from GoT2D, ESP, NIMH and 1000 Genomes project using the same data generation and processing pipelines . The rawWe then stratified the variants according to their functional annotations – LoF variants, missense variants and synonymous variants. We found a higher proportion of LoF variants in Finns compared to NFEs across the rare and low-frequency allelic spectrum  and for www.findis.org) variants and their population frequencies. The extent to which such variants contribute to more common diseases, either through highly-penetrant recessive subtypes or modest risk to carriers, will correspond to advantages in rare and low-frequency association studies in isolated populations.Despite the reduced overall variation in the isolated population, the existence of a greater number of low frequency LoF variants results in an average Finn harboring 0.16 homozygous LoF variants compared to only 0.095 in an average NFE, driven primarily by homozygosity in the 0.5 to 5% allele frequency range . These fSERPINA10, LPA and FANCM) contained two LoF variants each; we combined these pairs and tested them as single composite LoF variants, resulting in a total of 80 independent LoF variants tested in this study. These 83 variants were genotyped in a total of 36,262 individuals from three population cohorts: FINRISK Given our empirical observations of proportionally more LoF variants in the 0.5–5% allele frequency range in Finns, we next conducted a test of this hypothesis that some of the Finnish-enriched low-frequency LoF variants might have strong phenotypic effects. We successfully genotyped 83 low-frequency LoF variants  enriched in Finns based on their ability to multiplex in four Sequenom MALDI-TOF genotyping pools . Of thesTranslation Elongation Factor, Mitochondrial gene (TSFM) that is present at 1.2% allele frequency in Finns and absent in NFEs, was not found in a homozygous state in >36,000 Finns (Hardy Weinberg Equilibrium (HWE) P = 0.0077). This suggests that complete loss of TSFM might result in embryonic lethality, severe childhood diseases in humans, or that the individuals might not have been ascertained by the studies employed, i.e. if the individuals are too sick to be included in the studies. A lookup of this variant in another 25,237 Finnish samples in exome chip genotyping data from the GoT2D studies confirmed that the variant is present at 1.2% in Finns, but again with no homozygotes observed (combined HWE P = 1.6×10−4). Recessive missense variants in TSFM have been reported to result in mitochondrial translation deficiency TSFM)  - lending strong evidence to the hypothesis that complete loss of this gene is not tolerated in humans. Neither did we observe strong associations for the TSFM Q246X heterozygotes across major diseases  arising from within population substructure. A nonsense variant (Q246X) in the diseases .Fanconi anemia complementation group M gene (FANCM) was initially discovered in one family with Fanconi anemia FANCM from our dataset , which we would typically observe for a disease causing recessive variant. Furthermore, examination of the hospital discharge records did not provide any evidence for blood diseases, increased cancer events or any other chronic diseases in these individuals with homozygous LoFs in FANCM. We also had blood counts for two homozygote individuals. Both of them had normal hemoglobin, erythrocyte size and counts as well as leukocyte and thrombocyte counts. Singh et al. reported that the initial case that led to the association of FANCM with Fanconi anemia also harbor biallelic, functional mutations in FANCA, a well-established Fanconi anemia gene et al. do not support the hypothesis that FANCM is a Fanconi anemia gene but rather suggest that the initial FANCM association was not causative. In addition to FANCM, we further evaluated evidence for two other genes COL9A2 and DPYD that were previously implicated in other Mendelian diseases (Supplementary Methods).Several other LoF variants occur in genes where recessive mutations have been noted to cause severe Mendelian diseases from the Online Mendelian Inheritance in Man database (OMIM) −4 – that is, a value where we would expect only one chance observation in the entire study. In total, we observed 41 associations that exceeded this significance threshold  gene (LPA) with lipoprotein(a) measurements in plasma  with increased Vitamin B12 levels −26 or 43 pg/mL per allele, Citrate Lyase Beta Like gene (CLYBL) with decreased Vitamin B12 levels −5 or −43 pg/mL per allele, Three of these association have been previously reported and represent positive controls for our approach: a strong association for the 2 splice variants (c.4974-2A>G and c.4289+1G>A) in the LPA variant was found to protect against coronary heart disease , demonstrating that lowering lipoprotein(a) levels are likely to confer protection for cardiovascular diseases. We adjusted the association for the composite LPA variant with a previously published risk variant (rs3798220) LPA.In addition to a strong correlation between circulating lipoprotein(a) levels and cardiovascular disease, it has been previously reported that genetic variants that elevate circulating lipoprotein(a) levels are cardiovascular risk factors LPA variants confer cardioprotective effect . After meta-analyzing all the datasets, the final odds ratio was found to be 0.84  is well-tolerated and might constitute a potential drug target for cardiovascular diseases. A survey across other diseases showed potential association between the LPA variants with acute coronary disease and myocardial infarction but not Type 2 Diabetes (LPA variants across other cardiovascular risk factors and observed that the LPA variants were associated with mildly increased glucose levels but not high-density lipoproteins (HDL), low-density lipoproteins (LDL) or triglycerides , which collectively replicated the observation that the Diabetes . In addiycerides .FGL1, MS4A2 and ATP2C2 variants. The 1-bp c.545_546insA frameshift in the Fibrinogen-like 1 gene (FGL1) was associated with increased D-dimer levels . If replicated, this would be consistent with modest risk increase for stroke that other variants associated to circulating D-dimer levels, such as reported for variants in coagulation Factor V, Factor III and FGAIn addition, we observed novel associations for the membrane-spanning 4-domains, subfamily A, member 2 gene (MS4A2) with triglycerides  was associated with increased systolic blood pressure  or blood pressure lowering medication (−5), ATP2C2 is predicted to catalyze the hydrolysis of ATP coupled with calcium transport. Interestingly, the ATP2C2 c.2482-2A>C variant is also significantly associated to several highly correlated immune markers, such as granulocyte colony-stimulating factor (−7), interleukin-4 (−6), interferon-γ (−6) and interleukin-6 (−6).The c.2482-2A>C splice variant in the et al. first observed that there were proportionally more deleterious variants in European American individuals compared to African American individuals et al. showed conflicting results suggesting that there are similar burdens of deleterious alleles in Europeans and West Africans and that demography is unlikely to contribute to the proportions of deleterious alleles in human populations The empirical data of this study sheds light on an active debate in population genetics theory whether or not bottlenecked populations have an excess burden of deleterious alleles. Lohmueller The comparison of Finns, with a well-documented bottleneck, with non-Finnish Europeans here provides strong empirical data on these questions. While the distribution of common alleles, both synonymous and non-synonymous, is as expected unchanged by the bottleneck, when exploring the rare and low-frequency allelic spectrum where the Finns and NFEs demonstrate distinct distributions, we indeed observe a significant excess of deleterious variants in the Finns – despite the considerable deficit in variable sites in the population overall. This suggests that negative selection has had insufficient time to suppress the frequency of deleterious alleles dramatically elevated in frequency through the founding bottleneck, an observation that generalizes the intuitive understanding of the existence of characteristic and unusually common Mendelian recessive disorders in Finland. However, we note that while we observe a strong influence of the founding bottleneck, the observed results, particularly the proportional enrichment of rare deleterious variants, are also influenced by other elements in the unique history of the Finnish population and will not necessarily apply to all populations influenced by a bottleneck.TSFM  suggests that low-frequency variants in Finns, beyond those already identified in Mendelian disease, do include more unusually strong acting alleles than in non-founder populations. In this study, both replicated results and novel associations demonstrate the association of low-frequency LoF variants with various complex traits and diseases. In addition, we discovered a novel cardiovascular protective effect from splice variants in the LPA gene, suggesting that knocking down levels of circulating lipoprotein(a), or Lp(a), can confer a protection from cardiovascular diseases. Given that we detected numerous individuals in these adult population cohorts, healthy and in the expected Hardy-Weinberg proportions, carrying a complete knockout of LPA (homozygous or compound heterozygous for the 2 splice variants), this suggests that knocking out the gene in humans does not result in severe medical consequences. As such, this study provides data suggesting that LPA may be an effective target for therapeutic purposes.This excess of presumably deleterious variants motivated the subsequent association study and indeed, the absence of homozygotes at As more Finnish samples are being sequenced, these enriched variants can also be imputed with high precision to the large number of existing samples with array-based GWAS genotypes. This advantage is likely to be more pronounced for the much larger pool of missense variation – while one can presume all LoF variants in a gene might have a comparable effect on phenotype (and thereby burden tests of LoF variants in an out-bred sample is not at a great disadvantage compared to isolated populations), it is evident that many rare missense variants within the same gene will not all have the same impact on gene function. Thus the ability to assess single low-frequency variants conclusively, especially since they will include an excess of damaging variants enriched through a bottleneck, rather than perform burden tests on heterogeneous sets of extremely rare variants, will offer substantial ongoing advantage to isolated population studies as indicated by these and other recent findings.All research involving human participants have been approved by the Hospital District of Helsinki and Uusimaa Coordinating Ethical Committee, and all clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.http://useast.ensembl.org/info/docs/tools/vep/). We modified it to produce custom annotation tags and additional loss-of-function annotations. The additional annotations were applied to variants that were annotated as STOP_GAINED, SPLICE_DONOR_VARIANT, SPLICE_ACCEPTOR_VARIANT, and FRAME_SHIFT and the variants were flagged if any filters failed. A loss-of-function variant was predicted as high confidence if there is one transcript that passes all filters, otherwise it is predicted as low confidence. In our genotyping study, we had used loss-of-function variants that were predicted to be high confidence. For quality control, we required all variants to pass the basic GATK filters and required all genotypes to have a quality score of ≥30, read depth of ≥10 and allele balance of between 0.3 and 0.7 for heterozygous calls and <0.1 for homozygous calls. Allele counts and frequencies were calculated within the 3,000 individuals for Finns and NFEs respectively.Raw Binary Sequence Alignment/Map (BAM) files from the various projects were jointly processed at the Broad Institute and joint variant calling was performed on all exomes to minimize batch differences. Functional annotation was performed using the Variant Effect Predictor (VEP v2.5) tool from Ensembl . The high concordance between the allele frequencies in two independent NFE datasets ensures that the variants are unlikely to arise from alignment or sequencing artifacts and that these variants are unlikely to reside in a region of the exome that is difficult to sequence or genotype, which can result in highly variable allele frequencies from different experiments.All frameshifts and loss-of-function single nucleotide variants with allele frequencies of 0.5–5% in Finns and at least 2-fold enriched in Finns compared to NFEs were selected for genotyping. To minimize the false positives in our variant selection, we performed Fisher's Exact Test for each variant between two independent NFE datasets and kept variants whose allele frequencies were highly concordant between the two NFE datasets (P>1×102 and 1 U HotStar Taq (Qiagen). Reactions were heated to 94°C for 15 min followed by 45 cycles at 94°C for 20 s, 56°C for 30 s and 72°C for 1 min, then a final extension at 72°C for 3 min. Unincorporated dNTPs were SAP digested prior to iPLEX Gold allele specific extension with mass-modified ddNTPs using an iPLEX Gold reagent kit (Sequenom Inc.). SAP digestion and extension were performed according to the manufacturer's instructions with reaction extension primer concentrations adjusted to between 0.7–1.8 µM, dependent upon primer mass. Extension products were desalted and dispensed onto a SpectroCHIP using a MassARRAY Nanodispenser prior to MALDI-TOF analysis with a MassARRAY Analyzer Compact mass spectrometer. Genotypes were automatically assigned and manually confirmed using MassARRAY TyperAnalyzer software version 4.0 (Sequenom Inc.). The genotyped variants were then checked for concordance in allele frequencies with the exome sequencing data.Genotyping was performed using the iPLEX Gold Assay (Sequenom Inc.). Assays for all SNPs were designed using the eXTEND suite and MassARRAY Assay Design software version 3.1 (Sequenom Inc.). Amplification was performed in a total volume of 5 µL containing ∼10 ng genomic DNA, 100 nM of each PCR primer, 500 µM of each dNTP, 1.25× PCR buffer (Qiagen), 1.625 mM MgClData on disease status from National Health registers , Cause of Death Register, Statistics Finland and Prescription Medication Register, THL) for FINRISK, Health2000 and the Young Finns Study participants of this study were collected and curated. A description of each cohort is provided in the Supplement.http://www.broadinstitute.org/gtex/) et al.−5.To analyze the effects of the LoF variants on gene expression, we used RNA sequencing data from two major studies: the GEUVADIS project 2 as covariates. Linear regression was then performed on the normalized Z-scores using R to obtain the statistics for the associations. We tested the correlations between the quantitative measurements and disease outcomes using two one-tailed t-tests to assess the significance of observing higher levels of the quantitative measurements in cases  versus controls , as well as lower levels of the quantitative measurements in cases versus controls. To test the association of the variants with the prevalent disease outcomes, we performed a logistic regression in R to obtain the reported statistics. In addition, a Fisher's Exact Test on the homozygous counts in cases and controls were performed to test for association with the homozygotes. The results for the LPA with cardiovascular disease association from MIGen ExA and the Estonian Biobank were meta-analyzed using METAL Inverse rank-based normalization was performed on the quantitative measurements in males and females separately, with linear regression residuals using age and age2-normalised gene probe expression of individual i was regressed on the LoF genotype, which was encoded as iX = 0, 1 or 2 for the LoF genotypes −/−, +/− or +/+ respectively and association analysis of MS4A2 gene expression and triglycerides was performed as previously reported MS4A2 c.637-1G>A and triglycerides using a 2-sided t-test.We fit a linear model in which the logFigure S1Ratio of the number of missense variants predicted by PolyPhen2 found in Finns versus NFEs. (A) The ratios for probably damaging missense variants highlighted in red text and the ratios for benign missense variants in black. The p-values represent the binomial probabilities of the variants being enriched in Finns and similarly, the p-values in red represent the probabilities for the probably damaging missense variants and the p-values in black represent the probabilities for the benign missense variants. (B) Percentage of variants that are missense variants across the allele frequency spectrum.(DOCX)Click here for additional data file.Figure S2Allele frequency distribution in 3,000 Finns compared to 3,000 Swedes. The ratios for LoF variants highlighted in red text and the ratios for synonymous variants in black.(DOCX)Click here for additional data file.Figure S3Distribution of LoF variants per individual. (A) Number of LoF variants in an average Finn vs NFE individual. (B) Number of homozygous LoF variants in Finns vs NFEs per individual.(DOCX)Click here for additional data file.Figure S4Simulations for a set of variants  with complete recessive lethality. The red line indicates the expected allele frequencies in present-day Finns (where the Finnish bottleneck occurred ∼100 generations ago) and the blue line indicates the expected allele frequencies in Finns 1,000 generations after the Finnish bottleneck, similar to the out-of-Africa bottleneck which occurred >1,000 generations ago.(DOCX)Click here for additional data file.Figure S5Boxplots for the known and novel associations.(DOCX)Click here for additional data file.Figure S6MS4A2 gene expression.Correlation between triglycerides and (DOCX)Click here for additional data file.Table S1LPA variants from the different studies was performed on the exome chip genotyping platform.Exomes collected from ongoing studies. All the Finnish and NFE exome sequences were captured using the Agilent SureSelect v2 kit. The replication data for the (XLSX)Click here for additional data file.Table S2The number of variants in each category in Finns and NFEs.(XLSX)Click here for additional data file.Table S3Allele frequencies of variants discovered from the FinDis database.(XLSX)Click here for additional data file.Table S4Final list of variants from Sequenom genotyping in 36,262 Finns. The cohorts used in this study are from FINRISK 1992, FINRISK 1997, FINRISK 2002, FINRISK 2007, Health 2000 and Young Finns studies (83 variants + 3 composite variants).(XLSX)Click here for additional data file.Table S5TSFM Q246X heterozygotes and various disease states, as well as various neurological and muscular diseases from the medical record system (ICD 9/10) with >30 cases.Associations between (XLSX)Click here for additional data file.Table S6List of 60 blood pressure measures and biochemical assays from plasma/serum of fasting subjects.(XLSX)Click here for additional data file.Table S7LPA variant and various disease states. The rows with significant correlation between the levels of the biomarker and disease status (P<1×10−3) are shaded in blue and the rows with significant association (P≤0.05) between the variant and disease status  are highlighted in red text.Correlations between the combined (XLSX)Click here for additional data file.Table S8FUT2 W154X and various disease states. The rows with significant correlation between the levels of the biomarker and disease status (P<1×10−3) are shaded in blue and the rows with significant association (P≤0.05) between the variant and disease status  are highlighted in red text.Correlations between (XLSX)Click here for additional data file.Table S9CLYBL R225X and various disease states. The rows with significant correlation between the levels of the biomarker and disease status (P<1×10−3) are shaded in blue and the rows with significant association (P≤0.05) between the variant and disease status  are highlighted in red text.Correlations between (XLSX)Click here for additional data file.Table S10LPA variant with different diseases.Association of the composite (XLSX)Click here for additional data file.Table S11LPA composite variant with other potential cardiovascular risk factors.Association of (XLSX)Click here for additional data file.Table S12FGL1 c.545_546insA and various disease states. The rows with significant correlation between the levels of the biomarker and disease status (P<1×10−3) are shaded in blue and the rows with significant association (P≤0.05) between the variant and disease status  are highlighted in red text.Correlations between (XLSX)Click here for additional data file.Table S13MS4A2 c.637-1G>A and various disease states. The rows with significant correlation between the levels of the biomarker and disease status (P<1×10−3) are shaded in blue and the rows with significant association (P≤0.05) between the variant and disease status  are highlighted in red text.Correlations between (XLSX)Click here for additional data file.Table S14ATP2C2 c.2482-2A>C and various disease states. The rows with significant correlation between the levels of the biomarker and disease status (P<1×10−3) are shaded in blue and the rows with significant association (P≤0.05) between the variant and disease status  are highlighted in red text.Correlations between (XLSX)Click here for additional data file.


Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion  Vocal folds are covered by a specialized, multilayered epithelium which protects the underlying tissue from environmental and mechanical insults. To maintain an intact epithelium, epithelial cells undergo constant turnover across the lifespan. Under homeostatic conditions, epithelial renewal is likely achieved by turnover of a small number of cells in the basal layer of the vocal fold epithelium in vitro; EGF stimulates canine fibroblast proliferation in vitro scratch assays in vivo awaits investigation.Various growth factors have been implicated in the regeneration of a structurally and functionally intact epithelium after injury. Here, we focus on two endogenous growth factors that are known to play key roles in epithelial proliferation throughout the body, epidermal growth factor (EGF) and transforming growth factor beta (TGFβ1). EGF is secreted by various cell types involved in wound healing including epithelial cells in vitroin vivo in its role as a profibrogenic factor in vitro, indicating a role for the growth factor in scar formation. In vivo experiments have shown that exogenous TGFβ1 and TGFβ3 reduce vocal fold scar formation post-injury The role of TGFβ has been better explored in vocal fold wound healing; however, its effects are not fully understood. TGFβ is secreted by epithelial cells, fibroblasts, macrophages, and platelets in vivoBoth EGF and TGFβ mediate their effects through activation of the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases; EGF activates EGFR by binding directly to the receptor while TGFβ1 activates or inhibits EGFR indirectly via a signaling pathway Based on findings in other airway epithelia, epithelial regeneration following vocal fold injury likely follows three overlapping steps: cell adhesion and migration, proliferation and stratification, and differentiation Fifteen three-month-old male Sprague Dawley rats were used in this study. The animal protocol was implemented with prior approval from the School of Medicine and Public Health Institutional Animal Care and Use Committee of the University of Wisconsin-Madison (Permit number: M02459). All animals underwent unilateral vocal fold surgery. The surgical procedure has been described previously elsewhere Larynges were fixed in 10% neutral phosphate-buffered formalin (pH of 7.0) overnight at room temperature, processed, and embedded in paraffin. Serial coronal sections, 5 µm thick, were cut along the length of the membranous vocal folds. Routine hematoxylin and eosin (H&E) staining was performed on sections at 100 µm increments. Standard immunohistochemistry was completed. Antigen retrieval was achieved using heat-activated 10 mM sodium citrate buffer (pH 6). Sections were incubated for one hour at room temperature using antibodies directed against the proliferation marker, Ki67 ; transforming growth factor beta 1 ; epidermal growth factor ; phosphorylated epidermal growth factor receptor ; and keratin 14, a marker of basal layer cells . Species-specific isotype controls were used as negative controls. Endogenous peroxidase activity was quenched by incubation of sections in 3% hydrogen peroxide. For anti-EGF and anti-TGFβ1 antibodies, sections were treated with a goat anti-rabbit biotinylated secondary antibody  followed by incubation in a universal prediluted streptavidin conjugated to horseradish peroxidase (Vector Labs). For the remaining antibodies, sections were treated with goat anti-rabbit prediluted Impress secondary antibodies (Vector Labs). All sections were visualized with a diaminobenzidine (DAB) chromagen and counterstained with hematoxylin. They were viewed using a Nikon E600 microscope and photographed using an Olympus DP71 microscope digital camera.Primary epithelial cells were derived from pig larynges obtained from an abattoir  using a protocol described elsewhere Pig epithelial cells were grown to approximately 80% confluency before being harvested for PCR. Messenger RNA from was obtained using standard methods. Total RNA was isolated using an RNA extraction kit . First strand cDNA was generated from 2 µg of total RNA using a QuantiTect Reverse Transcription Kit (Qiagen). Primer pairs reported elsewhere were used for EGF In the present study, the vocal fold epithelium and lamina propria were removed, exposing the underlying thyroarytenoid (TA) muscle. Injury to the depth of the TA was confirmed by observation of disorganization of the most medial muscle fibers and infiltration of cells around these fiber bundles one day post-injury . EpithelThe epithelial cells demonstrated positive staining for K14, a marker of basal cells in uninjured epithelium, throughout all epithelial layers at days 3 and 5 post-injury . By day Positive staining for Ki67, a marker of cell proliferation, was found primarily in the lamina propria at day 1 post-injury . While eAt day 1, EGF was found throughout the lamina propria, both associated with cells and in the extracellular matrix . By day Staining for TGFβ1 was noted in the basal layer of the epithelium at the wound margins, adjacent to the site of injury, and throughout the lamina propria at day 1 post-injury . By day EGFR activation was noted in epithelial cells adjacent to the site of injury one day post-injury . AbundanVocal fold healing post-injury is likely dictated by the interactions between various cells types, growth factors and components of the extracellular matrix. Here, we focused on establishing a timeline for epithelial restoration and presence of two growth factors likely to play an important role in wound healing in a rat model. Through histological and immunohistological assays, we demonstrated that epithelium follows the expected temporospatial sequence of wound healing observed in other airway epithelia. We further demonstrated that epithelial cells are active participants in the wound healing process as evidenced by secretion of growth factors critical for wound healing, EGF and TGFβ1, as well as activation of EGFR.Based on findings in other airway epithelia and studies of vocal fold mucosal repair, we hypothesized that epithelial regeneration following vocal fold injury would follow three steps: cell adhesion and migration, proliferation and stratification, and differentiation Epithelial cells showed positive staining for the growth factors, EGF and TGFβ1 during the early phase of wound healing. Further, the cytoplasmic and intercellular staining observed for both EGF and TGFβ1 is consistent with a role for the epithelial cells in synthesizing and secreting these growth factors. These findings suggest that vocal fold epithelial cells may participate in autocrine and paracrine signaling in wound healing. Presence of positive staining for growth factors in epithelium during the proliferation and migration phases of epithelial regeneration post-injury strongly support our hypothesis that epithelial cells participate in all phases of epithelial regeneration through autocrine signaling. EGF and TGFβ1 showed a similar, time-dependent pattern of staining across early time points post-injury. Both showed diffuse staining throughout the epithelium at day 3 and strongest staining in the superficial layer of the epithelium at day 5. Presence of growth factors in the superficial epithelial layers in the days following injury is consistent with a role for them in guiding proliferation and differentiation of these metabolically active but typically quiescent cells EGFR activation was observed during the acute phase of wound healing. Concomitant staining for EGFR and the growth factors, EGF and TGFβ1, in epithelial cells suggest growth factor-mediated autocrine signaling drives EGFR regulation of wound repair. Presence of activated EGFR in the proliferating, Ki-67-positive, epithelium is consistent with the receptor playing a key role in cell growth and wound repair. Our findings are consistent with a correlation between EGFR and Ki67 expression observed in benign and malignant laryngeal lesions Growth factors were also noted in the lamina propria, particularly during the earliest time points post-injury, day 1 and 3. These observations were expected as both EGF and TGFβ1 are secreted by fibroblasts, macrophages, and platelets We acknowledge two weaknesses in this study. First, dynamic reciprocity between epithelium and lamina propria likely molds vocal fold healing. Here, we focused exclusively examining on epithelial cell protein expression as a first step to elucidating the role of epithelial cells in mediating wound healing. Understanding the interaction of epithelial cells and fibroblasts, as well as other cells, in the lamina propria awaits controlled, in vitro studies of vocal fold healing. Second, we did not quantify EGF and TGFβ1 gene and protein expression in rats post-injury. We used immunohistochemistry (IHC) for the in vivo portion of the study. While IHC did not permit quantification of protein expression, it allowed visualization of the cells that expressed EGF and TGFβ1. TGFβ gene expression has been quantified during the acute and chronic phases of wound healing in vocal fold mucosa in vitro environment. Knowledge of these mechanisms of epithelial wound healing may help to inform development of growth factor driven interventions over time. It may also provide a benchmark against which the ability of in vitro models of vocal fold injury under development to mimic in vivo injury To date, studies have focused on analyzing changes in composition and organization of the lamina propria and their impact on vocal fold physiology following damage, with few exceptions S1 FigureEGF staining in the acute phase of healing. Positive EGF staining (brown) was observed in epithelium at 1 (A), 3 (B), and 5 (C) days post-injury in representative animals. Scale bars: 50 and 100 µm.(TIF)Click here for additional data file.S2 FigureTGFβ1 staining in the acute phase of healing. Positive TGFβ1 staining (brown) was observed in epithelium at 1 (A), 3 (B), and 5 (C) days post-injury in representative animals. Scale bars: 50 (insert) and 100 µm.(TIF)Click here for additional data file.


Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes were identified including the statins, TGF-βRI inhibitors, and GSK-3 inhibitors. The function of each class was also shown to be distinct, either to promote both the neuronal differentiation and mDA neuron specification, or selectively the latter, or promote the former but suppress the latter. We then carried out initial investigation on the possible mechanisms underlying, and demonstrated their applications on NPCs derived from human pluripotent stem cells (PSCs). Our study revealed the potential of several small molecule compounds for use in the directed differentiation of human NPCs. The screening result also provided insight into the signaling network regulating the differentiation of human NPCs. We first collected more than 5,000 such compounds , the most commonly used biomarker for mDA neuronsSimilarly, high-content screen was carried out to test the compounds’ effects on promoting the mDA neuron specification of ReNCell VM cells, which is potentially valuable for the study of Parkinson’s diseaseIn summary through the bioactive compound high-content screen approach we identified three major pharmacological classes, the GSK-3 inhibitors, statins and TGF-βRI inhibitors, to cause the significant enrichment of neuronal and/or mDA neuronal populations in ReNCell VM cell differentiation. The enrichment can possibly be attributed to the compounds’ promoting effects on signaling pathways driving the neuronal differentiation, or the proliferation/survival selection on the neuronal and non-neuronal cell types. To distinguish the mechanism experiments were carried out to test their effects on cell proliferation, apoptosis and lineage specification.During the screen we consistently observed changes of total cell numbers in the GSK-3 inhibitor and statin treated wells. To confirm the observation we seeded equal numbers of ReNCell VM cells in 96-well plates and incubated with individual compound selected from the three pharmacological classes, and counted the total cell numbers after differentiation for two weeks. While the TGF-βRI inhibitor LY364947 had no effect, the GSK-3 inhibitor kenpaullone significant increased the total cell number and the statin compound mevastatin decreased it . To distExperiments were also carried out for cell survival measurement using the TUNEL assay, and the result showed that mevastatin elevated apoptosis rate while the other two compounds had no effect . NotablyNPCs are tripotent cells which can differentiate into neurons, astrocytes and oligodendrocytes. In order to test whether the differentiation inducing capability of the compounds are selective to the neuronal lineage, experiments were carried to determine whether other lineages of cell differentiation were also altered by the compound treatment, using primary cultured NPCs isolated from the fetal rat ventral mesencephalon. The result showed that neither the astrocyte nor the oligodendrocyte differentiation was changed by the three compounds. Cells retained the differentiation capabilities and the glial cell differentiation rates remained the same as untreated cells . Taking Next we tested how the known signaling pathways controlling NPC differentiation were affected by the compounds. Previously GSK-3 inhibitors14The western blot results indicated that there might be common mediators, e.g., β-catenin and LMX1a, for the promoting effects of the different bioactive compounds identified in our screen. However, the actions of different compound classes on NPCs are also distinct as indicated by their differential effects on the neuronal differentiation and mDA neuron specification, as well as on the cell proliferation and survival. Therefore through complementary mechanisms, their effects may be additive or synergistic. In both screens two major pharmacological classes were identified, we thus examined whether higher efficiency could be achieved through the combinations of them. Significant further enhancement was observed when the two neuronal differentiation promoting compounds kenpaullone and mevastatin were applied together . When thThe GSK-3 inhibitors were identified in the neuronal differentiation screen but not the mDA neuron specification screen, and increase in LMX1a expression was not detected in the GSK-3 inhibitor treated cells . We carrin vitro assay and clarify the role of GSK-3 inhibition and Wnt signaling, we first directly tested the effect of Wnt-1 and showed that the recombinant protein significantly enhanced the number of TH+ cell differentiated from ReNCell VM cells  and induced pluripotent stem cells (iPSCs), hold great promise as unlimited transplantation source material for treating neurological disordersPreviously studies have been carried out for identifying compounds inducing the neuronal differentiation from rodent SCs1426In both neuronal differentiation and mDA neuron specification screens statins were identified to be effective. Since consistent result was observed for several different statin members, including mevastatin, fluvastatin and pravastatin, the promoting effect is highly likely to be associated with the specific bioactivity of statins instead of an individual compound off-target. Statins are best known as HMG-CoA reductase inhibitors which inhibit the cholesterogenesis33Of the statin molecules identified in our screening, mevastatin showed the outstanding effects in both promoting the neuronal lineage differentiation and mDA neuron specification, on both the NPCs from human ventral mesencephalon and human PSCs. Therefore it may have potential in the directed differentiation of NPCs for the study of Parkinson’s Disease. In addition, differentiation inducing compounds may also find its application in cancer therapy, by targeting the brain tumors that are initiated by cancer stem cellsThe discovery of GSK-3 inhibitors to promote the neurogenesis is consistent with literature reports14in vitro and in vivoTwo findings in this study were surprising and seemingly contradictory to existing evidences. The first is the result that GSK-3 inhibitors promoted DCX+ neuronal differentiation but inhibited TH+ mDA neuron specification, as GSK-3 inhibition enhances Wnt signaling which is known to play a critical role in the neurogenesis in the midbrain floor plate22Human NPC line ReNCell VM was purchased from Millipore , which were derived from the ventral mesencephalon of human fetal brain and immortalized with the myc oncogene43The H9 (WA09) human ESC derived NPCs was obtained from Life Technologies The dopaminergic neuron differentiation was carried out following a procedure modified from published protocolThe compounds libraries used for this study included: the LOPAC library consisting of 1,280 pharmacologically active compounds ; the Tocriscreen Mini library consisting of 1,120 biologically active compounds ; the Spectrum Collection consisting of 2,320 compounds ; the Prestwick Chemical library consisting of 1,200 compounds ; the Prestwick Phytochemicals Library consisting of 320 phytochemical compounds ; the Prestwick Natural Compounds Library consisting of 240 natural compounds .4 cells/cmUndifferentiated ReNCell VM cells were seeded at a density of 1.8 × 10Screening images were first subjected to a double blind manual analysis, in which each image was scored by an independent analyzer as 0 (absent of effect as untreated); or positive scores of 1 (minor enhancing effect), 2 (medium enhancing effect) or 3 (major enhancing effect); or negative scores of −1 (minor inhibiting effect), −2 (medium inhibiting effect) or –3 (major inhibiting effect). The results from all repeats and both analyzers were averaged and the compounds of scores ≥2 were selected to further test in 96-well plate format. Images acquired on the 96-well plates were quantified using the NeuriteIQ software3 cells/well (1.25 × 104 cells/cm2). Cells were treated with indicated compound or DMSO control for 48 hours before they are fixed and stained with Hoechst 33342 dye . The plate was scanned with an ImageXpress Micro microscope  using a 4× objective to take an image covering the whole well for each well. Cell numbers were counted using the ImageJ software with an automatic nuclei counter plug-in. Cell proliferation was also measured by BrdU assay. Cells were grown in laminin coated 96-well plates, and treated with indicated compound for 48 hours. After that cells were pulsed with 10 μM BrdU for 2 hours, then stained with BrdU antibody . For the survival assay, cells were differentiated in the presence of indicated compounds. Then the cells were fixed, permeabilized and assayed with the Roche in situ Cell Death detection kit following the manufacturer’s instruction. Cells were also blocked and stained with additional markers as indicated.For the cell proliferation assay, ReNCell VM cells were seeded into laminin coated 96-well plates at a density of 4 × 10The immunofluorescence staining of the cells were done with Alexa-fluor-488 or −568-conjugated secondary antibodies , and the cell nuclei were counterstained with DAPI. Antibodies against the following proteins were used at the following dilutions: DCX , GFAP , O4 , TH . Fluorescence images were taken using an IX81 inverted microscope . Image quantification was performed using the ImageJ software.For western blot, cells were lysed in RIPA buffer  in the presence of Xpert protease inhibitor cocktail and Xpert phosphatase inhibitor cocktail . Thirty microgram protein was loaded in each well and separated by 4–15% Mini-PROTEAN TGX precast gel  and transferred to a nitrocellulose membrane. The antibodies tested included β-catenin , Mash1 , Lmx1a , Sfrp1, Wnt1 , and actin .Animal experiments were carried out in accordance with the protocol approved by the Institutional Animal Care and Use Committee of Houston Methodist Research Institute. Groups of four C57BL/6 mice were injected intraperitoneally (i.p.) with 1 mg/kg brain permeable GSK-3 inhibitor BIO  or DMSO control daily for 4 days from E13 to E16. Forty-eight hours later the animals were sacrificed and brains were dissected and postfixed in 4% PFA overnight at 4 °C. Then the brains were dehydrated and embedded in paraffin. Sections were made from the midbrain at 10 μm thickness on a microtome. Slides from histologically comparable positions as confirmed by the H&E staining were used in the immunostainings. Immunohistochemistry was carried out with a Vectastain Elite ABC kit  following the manufacturer’s manual. The rabbit anti-TH antibody was purchased from Millipore .Statistical analyses were carried out using a two-tailed Student’s t-test. Data presented in the graphs are the mean values with the error bars representing the standard deviations (s.d.).How to cite this article: Rhim, J. et al. A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells. Sci. Rep.5, 16237; doi: 10.1038/srep16237 (2015).


Sepsis is one of the highest causes of mortality in hospitalized people and a common complication in both surgical and clinical patients admitted to hospital for non-infectious reasons. Sepsis is especially common in older people and its incidence is likely to increase substantially as a population ages. Despite its increased prevalence and mortality in older people, immune responses in the elderly during septic shock appear similar to that in younger patients. The purpose of this study was to conduct a genome-wide gene expression analysis of circulating neutrophils from old and young septic patients to better understand how aged individuals respond to severe infectious insult. We detected several genes whose expression could be used to differentiate immune responses of the elderly from those of young people, including genes related to oxidative phosphorylation, mitochondrial dysfunction and TGF-β signaling, among others. Our results identify major molecular pathways that are particularly affected in the elderly during sepsis, which might have a pivotal role in worsening clinical outcomes compared with young people with sepsis. Sepsis is a complex syndrome triggered by infection and characterized by the massive deregulation of immunological networks . Septic Age-related defects in immunity are caused by major defects in cell-mediated and humoral effector functions . Aging cInnate immunity was previously considered to be well-preserved in the elderly , but recThe inflammatory response of the elderly following infection, however, remains under debate. Animal studies demonstrated that mortality, inflammation, hypothermia, apoptosis and disseminated intravascular coagulation are increased in aged animals subjected to experimental models of sepsis . It is iThe purpose of this study was to perform a genome-wide gene expression analysis of neutrophils obtained from elderly and young patients in septic shock, to investigate the potential differences in cell activation that might explain the altered status of immune and inflammatory systems in advanced aged patients with sepsis. We decided to focus on neutrophils because recent reports suggested their function was particularly altered in the elderly .The current study was a prospective cohort study, conducted in the Hospital das Clínicas Intensive Care Units . Blood samples were obtained from six aged septic patients (age range 65–78 years old), six young septic patients (age range 22–35 years old), six healthy aged volunteers (age range 60–82 years old) and six healthy younger individuals (age range 20–35 years old). Patients’ profiles are described in the The study protocol was approved by the Hospital das Clínicas Ethics Committee. Patients (or their close relatives) received detailed explanations and provided written consent prior to inclusion in the study (Protocol # 1207/09).All blood samples were sent to our laboratory for processing immediately after collection. The anticoagulant-treated blood was layered on the Ficoll-Paque PLUS solution  and centrifuged for a short period of time. Differential migration during centrifugation results in the formation of layers containing different cell types. The bottom layer contains erythrocytes that have been aggregated by the Ficoll and, therefore, sediment completely through the Ficoll-Paque PLUS. The layer immediately above the erythrocyte layer contains the granulocytes, which at the osmotic pressure of the Ficoll-Paque PLUS solution attain a density great enough to migrate through the Ficoll-Paque PLUS layer.g for 10 minutes at 4°C the pellet was resuspended in lysis buffer and kept on ice for additional 10 minutes. A new centrifugation step was performed at 2500 x g for 2 minutes at room temperature and the samples were washed with phosphate-buffered saline (PBS). Finally, the samples were centrifuged at 1500 x g for 2 minutes at room temperature and the pellet was resuspended in Trizol  and stored at -80°C.After Ficoll-Paque PLUS  density gradient centrifugation, we separated the second layer containing the granulocytes. This layer was transferred to new tubes, diluted in lysis buffer and kept on ice for 10 minutes. After centrifugation at 290 x Total RNA was isolated following the manufacturer’s protocol and its integrity and concentration were assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit . Expression levels of protein-coding genes and long noncoding RNAs (lncRNA) were evaluated using the SurePrint G3 Human Gene Expression 8x60K v2 Microarray (design ID # 039494) and the Low Input Quick Amp Labeling kit, following a two-color labeling protocol (Agilent Technologies). Cyanine-3 labeled RNA from each sample and cyanine-5 labeled reference RNA  were combined and hybridized to the microarrays following the manufacturer’s protocols.Microarrays were scanned using the SureScan Microarray Scanner (Agilent Technologies) and images were processed using the Feature Extraction Software v12 (Agilent Technologies) for quality control, determination of feature intensities and ratios, and for background correction. The LOWESS procedure was used for data normalization and transformation . We onlyA number of probes (8192) could not be assigned to the known or predicted lncRNA sequences annotated in public databases . These poorly annotated probes were categorized as “unannotated” and were not analyzed further. The raw and processed microarray datasets are deposited at the Gene Expression Omnibus (accession number GSE67652 associated to platform GPL16699).P-value of p ≤ 0.01 by both methods.We performed two sets of differential gene expression analyses. One analysis was to identify genes deregulated in young and elderly sepsis subjects compared with matched healthy controls. A second analysis searched for genes deregulated in septic or healthy elderly subjects compared with matched young controls. In each analysis, a gene was considered differentially expressed when it was identified by a combination of two statistical methods, namely Significance Analysis of Microarrays  and rankwww.qiagen.com/ingenuity). IPA Upstream Analysis was used to identify putative upstream regulators and to predict whether they were activated or inhibited, given the observed gene expression changes in the experimental dataset, and the expected causal effects, which were compiled from the literature in Ingenuity’s Knowledge Base.Lists of differentially expressed genes were annotated and investigated to identify the enrichment of particular gene categories or pathways using the Ingenuity Pathway Analysis suite  was used to determine the statistical significance of expression differences between the sample groups.To validate the microarray expression data, selected genes were subjected to quantitative Real Time-PCR using the TaqMan assay system (Life Technologies). Briefly, 100 ng of total RNA was subjected to reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) following the manufacturer’s protocol. Specific TaqMan Gene Expression Assays (Life Technologies) were used for detecting gene expression levels of TGFB1 (Hs00998133), SRC (Hs1082246), HDAC4 (Hs01041638), CREBBP (Hs00231733), NDUFA4 (Hs00800172), INHBB (Hs00173582), BMP7 Hs00233476) and SDHC (Hs01698067). PCR was carried out following the protocol recommended for the TaqMan Universal PCR Master Mix (Life Technologies) in an AB7500 thermocycler (Life Technologies). For each sample, the expression data of each candidate gene was normalized to 18S rRNA expression . For relative quantification we used the comparative ∆Ct method [76 and SDThe microarray in this study contained 58,717 probes that included protein-coding mRNAs 36,075), long noncoding RNAs , and a number of poorly annotated transcripts (8192) . Approxi,075, lonWe first compared gene expression profiles of neutrophils across the four groups of samples, grouped by septic status and age . S1 Tablp = 5.3 × 10−13) and mitochondrial dysfunction (p = 1.4 × 10−10) pathways were the most enriched in septic patients of advanced age when compared with the young septic group. Other pathways significantly enriched in the elderly were extracellular signal-regulated kinase 5 (ERK5) signaling (p = 9.4 × 10−4) and the NRF2-mediated oxidative stress response (p = 5.2 × 10−3)  . Table 3Next, we performed a pathway enrichment analysis of the genes differentially expressed in septic patients compared with healthy controls. Many pathways were identified, but none showed significantly different enrichment in the elderly compared with younger adults. Pathways that are modulated during sepsis such as Cdc42 signaling , phosphoTo favor the identification of molecular pathways preferentially affected in the elderly with sepsis, we performed a similar analysis using a subset of genes differentially expressed between elderly sepsis and healthy subjects, but not in younger adults  or inhibited  in septic elders, as well as compounds that may exert an antagonic regulatory role in these patients compared with healthy controls  between qPCR and DNA microarray measurements (p < 0.02). Although not statistically significant, TGFB1 and HDAC4 showed transcriptional changes in agreement with those observed in the microarray. Only CREBBP and SRC showed changes that were the reverse of those observed in the microarray, but these were not statistically significant, and therefore were inconclusive. Overall, these results support our observation that aged people had a reduced expression of genes related to respiratory chain activity and an increased activation of TGF-β-related genes during the course of overwhelming infection.To provide independent support of the DNA microarray data analysis, quantitative PCR experiments were performed for the following genes: 1) NDUFA4 and SDHC, to investigate respiratory chain activity and 2) INHBB, TGFB1 and CREBBP, to investigate TGF-β signaling. HDAC4  and SRC  were also investigated because these genes were significantly altered in elderly individuals with sepsis compared with healthy elderly controls, and were not expressed at all in young adults . In geneurements . NDUFA4,Transcriptomics is a powerful technique used to identify therapeutic targets and biomarker signatures during infectious disease , 44. A nCARD12, APAF1 and ELMOD2 in mononuclear cells from septic patients [Using whole blood-derived RNA from patients in septic shock, the global gene expression experiments of Shanley et al. agreed with these studies. However, they also observed the upregulation of integrin, IGF-1, GM-CSF and insulin receptor signaling . Similarpatients . Surprispatients .Sepsis affects very different groups of patients . Historically, it has been accepted that each specific subset of septic patients may have its own characteristic inflammatory immune response. In the course of sepsis, a transition from inflammation to immunosuppression has been suggested to explain the disappointing results obtained by clinical trials that investigated the use of anti-inflammatory drugs in this population –53. HighMost transcriptome studies in sepsis were performed in a pediatric population, and to our knowledge, our study is the first genome-wide expression analysis comparing septic shock patients of advanced age with young adults.Initially, our results confirmed that both the young and the elderly responded alike regarding the production of tumor necrosis factor α (TNFα), IL-6, IL-1β, TLRs and other classical markers of cell activation, following severe infectious stress. Some specific pathways, however, appeared to be more critically affected in the elderly with sepsis, explaining the worse prognosis observed in these patients. Based on the data shown here, it is our opinion that defects in mitochondrial function/oxidative phosphorylation, TGF-β, Wnt/β-catenin, nerve growth factor (NGF) and calcium signaling may play a major role in the clinical features of sepsis in elderly patients.We observed a marked decrease in the expression of genes encoding components of the mitochondrial respiratory chain in the septic elderly. Impairment of mitochondrial function significantly contributed to organ failure in septic patients . Physiol+ T cell responses. TGF-β is a potent suppressor of Th1 and Th2 effector cell differentiation, a regulator of Foxp3+ regulatory T cells, and is a critical cytokine for the induction of Th9 and Th17 cells [+ cells [TGF-β has pleiotropic effects on adaptive immunity, especially for the regulation of effector and regulatory CD417 cells –68. TGF-[+ cells  and prom[+ cells . Thus, wThe Wnt/β-catenin pathway determines major developmental processes in the embryo and regulates maintenance, self-renewal and differentiation of adult mammalian tissue stem cells . Many orNerve growth factor (NGF) ensures the maintenance of phenotypic and functional characteristics of several populations of neurons and immune cells. The role of NGF has never been investigated in sepsis, but might be involved because neuropeptides possess a broad number of regulatory functions in immunity  and NGF Calcium has a myriad of cellular functions, including effects on hormone secretion, enzyme activity, nerve conduction, and muscle contraction with obvious repercussions in sepsis. Calcium influx is a potent cell activator and, together with phosphate, alters electrostatic fields and protein conformation, dictating the majority of signal transduction .The mechanisms that lead to the dysfunction of specific pathways in the immune responses of older people are complex and might involve multiple factors. We propose that environmental factors , 86, micOur data confirm previous reports that aging is accompanied by changes in the expression of genes related to immune responses , and furThere is great hope that high-throughput screening technology will result in a better understanding of the complexity of systemic inflammation, identifying new therapeutic targets and groups of patients that may benefit from specific interventions.Here, we identified major pathways preferentially deregulated in the elderly following severe infection, providing evidence that the systemic inflammatory response differs according to patient age. Thus, oxidative stress might have a major role in inducing multiple dysfunctions in the elderly. Furthermore, our study identified additional important genes and molecular pathways, thus helping to elucidate the mechanisms involved in signaling crosstalk during infection and in aging.S1 FigAfter background filtering (see “(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigGenes with comparable changes in gene expression are shown in green and with an inverted pattern in red. Statistically significant results are shown in dark green. Values that follow a trend, but do not reach statistical significance are shown in light green.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The endothelins are a family of three highly conserved and homologous vasoactive peptides that are expressed across all organ systems. Endothelin (Edn) dysregulation has been implicated in a number of pathophysiologies, including diabetes and diabetes-related complications. Here we examined Edn2 and endothelin receptor B (Endrb) expression in retinae of diabetic mouse models and measured serum Edn2 to assess its biomarker potential.Edn2 and Ednrb mRNA and Edn2 protein expression were assessed in young (8wk) and mature (24wk) C57Bl/6 , Kimba , Akita  and Akimba mice (T1D plus RNV) by qRT-PCR and immunohistochemistry. Edn2 protein concentration in serum was measured using ELISA.Fold-changes in Edn2 and Ednrb mRNA were seen only in young Kimba  and young Akimba  and in mature Kimba  and mature Akimba  mice. Co-localisation of Edn2 with Müller-cell-specific glutamine synthetase demonstrated Müller cells and photoreceptors as the major cell types for Edn2 expression in all animal models. Edn2 serum concentrations in young Kimba, Akita and Akimba mice were not elevated compared to wt. However, in mature mice, Edn2 serum concentration was increased in Akimba  compared to wt, Kimba and Akita mice .These results demonstrated that long-term hyperglycaemia in conjunction with VEGF-driven RNV increased Edn2 serum concentration suggesting Edn2 might be a candidate biomarker for vascular changes in diabetic retinopathy. Diabetic retinopathy (DR) is a microvascular complication of the eye, where prolonged hyperglycaemia leads to capillary injury following changes to resident neuronal cell populations. Vascular endothelial growth factor (VEGF) becomes upregulated over time to replace lost and damaged capillaries in the retina. However, this process of neovascularisation produces dysfunctional neovessels and ultimately leads to tissue damage in the retina causing vision impairment and even loss. All diabetes sufferers are at risk of developing DR, which occurs in several stages from background and mild non-proliferative DR (NPDR) to proliferative DR (PDR), diabetic macular oedema and vision threatening DR, but only a subset are at risk of progressing to the severe forms of the disease. This subset of patients is currently estimated to be in the order of 66 million out of a projected 347 million diabetes patients worldwide , 2.The gold standard of treatment for PDR has been laser photocoagulation (LPC) mainly to arrest and reverse neovascularisation , 4, despCurrent biomarkers such as duration of diabetes, glycated haemoglobin level (HbA1c), retinal examinations and albuminuria, for example, cannot detect early tissue damage . Data frMethods for biomarker discovery vary widely and are continuously evolving. Novel biomarkers can be derived from genomic studies, investigations of disease-specific proteomes and metabolomes as well as from animal models of human diseases, for example. Here, we are evaluating a potential marker originally identified in a genome-wide microarray study on retinae from Kimba mice, a model of retinal neovascularisation (RNV) driven by photoreceptor-specific overexpression of VEGF –15 whereWe examined Edn2 mRNA and protein expression across these three very distinct genotypes and phenotypes. The Kimba mouse is normoglycaemic, yet displays many of the retinal microvascular characteristics of human NPDR and PDR, including capillary drop-out, neovascularisation, microaneurysms, cotton wool spots and focal haemorrhages. The Akita mouse, with its spontaneous mutation in the insulin 2 gene, becomes hyperglycaemic at around 4 weeks of age, but does not develop any of the vascular changes associated with DR and RNV , 21. TheThe aims of the current study were 1: to assess the effect of hyperglycaemia on Edn2 differential expression; 2: to determine whether in Akimba mice the combination of advanced RNV and hyperglycaemia would increase Edn2 expression above that observed in Kimba mice; 3: to establish the cellular localisation of Edn2 in the retinae of these animals deriving further clues to the retinal pathologies in these models; and 4: to assess whether mRNA changes in the retina translate into changes in circulating serum Edn2 protein, an important attribute of a potential biomarker.Akita), Kimba (trVEGF029) and Akimba (trVEGF029xIns2Akita) mice, all on the C57Bl/6 genetic background, were used throughout this study. Wt littermates were used as controls to ensure uniformity in genetic background, age and parental influence. Mice were housed at a constant temperature of 22°C with a 12:12 hour light/dark cycle [ad libitum. Genotyping was carried out as previously described [Young (8wk) and mature (24wk) Akita  and anti-glial fibrillary acidic protein (GFAP) antibodies as previously described . All secBlood was collected via intracardiac puncture into a 1ml syringe  Pte Ltd, Singapore) with a 25G 1” needle  and allowed to coagulate at room temperature (RT) in 2ml glass BD Vacutainer® tubes . Samples were centrifuged for 15min at 1159rfc without braking, serum was transferred to a new tube for a second centrifugation at 1000rfc for 7min, both at RT, before being stored at -80°C. Total serum protein concentrations (mg/ml) were determined using the DC Protein Quantitation Assay  on the Beckman Coulter AD200 Absorbance Detector  to determine amount of Edn2 (pg) per mg of total serum protein.Six mice per group  were analysed using the Endothelin-2 ELISA Kit  as previously described .Following single factor ANOVA at α = 0.05, multiple comparisons were made using Tukey’s multiple comparison test. Effect of age and genotype on Edn2 serum concentrations was determined using two-factor ANOVA at α = 0.05. All data analyses were performed using Prism 5 . Data are presented as mean ± SEM.There was a clear demarcation across all physiological and metabolic parameters between normoglycaemic (wt and Kimba) and hyperglycaemic (Akita and Akimba) young (8wk) and mature (24wk) mice . In Akit165 transgene alone (Kimba) and the combination of transgene and hyperglycaemia in Akimba mice led to significant fold-change increases in Edn2 mRNA , the outer plexiform layer (OPL) and the photoreceptor inner segments PIS; . Co-staiThe expression pattern for both Edn2 and GS proteins did not change in mature wt and Akita mice compared to young mice, with the IPL and INL portions of Müller cells showing very little or no GS immunoreactivity ; arrows.The presence of RNV in Kimba and Akimba retinae did produce a different expression pattern for both Edn2 and GS compared to hyperglycaemic Akita retinae. In the diabetic Akita retina, Edn2 staining was most pronounced in Müller cell endfeet in the inner retina, Müller cell processes in the OPL and throughout the ONL, as well as the inner and outer segments of the photoreceptors , a patteWe also examined Müller cell gliosis in mature Akimba mice with severe retinal pathology in comparison to mature wt retinae. As expected, GFAP protein staining in mature wt retinae remained localised to astrocytes resident on the nerve fibre layer of the GCL —Fig 4H GSerum Edn2 protein concentrations decreased significantly with age in wt mice (p<0.05), but not in Kimba, Akita or Akimba mice . In matuA and ETB, with similar affinity, and hence little distinction has been made between the two isoforms [The endothelins are a family of three highly conserved and homologous vasoactive peptides that are expressed across all organ systems and their dysregulation has been implicated in a number of pathophysiologies , 24–31, isoforms . Howeverisoforms . Furtherisoforms –45. In eisoforms .In the present study we confirmed that Edn2 and Ednrb mRNAs were upregulated in both Kimba and Akimba mice. We have also shown that this upregulation persisted in mature mice, when vasculopathy was well advanced in Akimba mice. Interestingly, Ednrb mRNA expression changed in accordance with that of its ligand, Edn2. Thus, there is the potential for increased Edn2 affecting downstream metabolic processes, particularly in light of our previous demonstration of increased Edn2 protein in whole eye extracts . There wPhotoreceptors and Müller cells were the primary expression sites for Edn2 in retinae of young and mature Kimba, Akita and Akimba mice. The retinal architecture in the mature Akimba retina was markedly different to that of the mature Akita and young Akimba retina. Significant photoreceptor cell loss coupled  with red165 transgene expression commences upon photoreceptor maturation by postnatal day 7 and we know from studies on 10d old Kimba mice that even three days of transgene expression were sufficient to induce pathological changes in retinae of these mice, including Edn2 and Ednrb upregulation [Müller cells have been described as the core of a columnar ‘micro-unit’ of retinal neurons essentially providing structural as well as metabolic support to the photoreceptors to which they are anatomically connected . Müller gulation . Hyperglgulation , 49. ThiStudies on young normoglycaemic Kimba mice have shown that RNV also leads to increased GFAP expression and reactive Müller cell gliosis, including scar formation along the OLM . Based oThese results demonstrated that long-term hyperglycaemia in conjunction with VEGF-driven RNV increased Edn2 serum concentration suggesting Edn2 might be a candidate biomarker for vascular changes in diabetic retinopathy.


Treatment with tumour necrosis factor (TNF) antagonists may lead to enhanced susceptibility to certain malignancies. In particular, an association is seen emerging between TNF antagonists and development of squamous cell carcinomas (SCCs) of the skin (in association with psoriasis), the oral cavity, and in the anogenital areas (possibly related to prior human papilloma virus infection). We present here a case of a 53-year old woman with a history of severe rheumatoid arthritis (RA), most recently treated with the TNF antagonist etanercept plus methotrexate, presented to our service after several months of increasing left pelvis and buttock pain. Evaluation with a computerised tomography (CT)-directed biopsy of a pelvic side wall mass revealed a metastatic SCC. On a fluorodeoxyglucose (FDG) positron-emission tomography (PET) an additional area of uptake was identified in the left posterior rectum corresponding to a 1 cm nodule palpable on digital exam. Colonoscopic biopsy revealed a basaloid SCC of the rectum as the likely primary site. Immunosuppression following TNF antagonist therapy may have given arise to this unrestrained neoplastic growth. It thereby underscores the need for an initial baseline study of risk factors and identification of patients who are at higher risk for development of a malignancy, in order to achieve a diagnosis at an early stage. However, some reports suggest there is no obvious link between HPV and squamous cell carcinoma of the rectum [Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that plays an important role in immune response and is implicated in the pathogenesis of many inflammatory conditions –4. Etanee rectum –11. StudA 53-year old woman with severe rheumatoid arthritis (RA) presented to her primary care physician in March 2014 with increasing left pelvis and buttock pain over several months. Treatment for her RA dated back to 1995 and included several regimens including steroids, methotrexate alone, and for the past six years she appeared to have benefitted from the addition of etanercept to methotrexate (50 mg weekly). In addition to the pelvic pain, she was experiencing fatigue but there were no neurologic signs, no weight loss, fevers, or night sweats. A computed tomography (CT) scan of the abdomen and pelvis revealed a lobular, infiltrative 3.1 x 6.1 x 4.4 cm soft tissue mass extending along left obturator space with encasement of the inferior branch of left internal iliac vessels and likely involvement of the sciatic bundle with extension to the left vaginal wall.CT-guided core biopsy was consistent with squamous cell carcinoma (SCC), poorly differentiated. A fluorodeoxyglucose (FDG) positron-emission/computerised tomography (PET/CT) scan demonstrated a markedly FDG-avid left femoral lymph node and intense uptake in the left posterior rectum corresponding to a 1 cm nodule palpable on digital exam that was concerning for malignancy  in additRadiation consisted of treatment to the pelvis with a 3D-conformal four-field set-up with a dose of 4320 cGy in 24 fractions using a mixed 16/6 MV photon beam. Treatment to the pelvis was sequentially followed by a boost to the pelvic mass to a dose of 1000 cGy in five fractions using a 3D-conformal anterior-posterior field set-up with a 16 MV photon beam. The patient experienced a gradual response with eventual full relief of symptoms. At completion, she relocated her care to Florida where she was consolidated with seven cycles of carboplatin and paclitaxel. She was followed until September 2015 but details of her disease status are unknown.The association of TNF antagonists and development of SCCs has come to light in several reports –21. Patiet al of nine randomised placebo-controlled trials of TNF antagonists in RA, found a malignancy of 0.2% in controls who received only placebo versus 0.8% in patients receiving anti-TNF medications for their RA. The number needed to harm was 154  for one additional malignancy within a treatment period of 6–12 months [In fact, one such study of the Swedish inpatient registry compared 53,067 RA patients admitted to hospital between 1990–2003 to patients treated with TNF antagonists for RA during 1999–2003 (n = 4160). Results demonstrated an increased occurrence of non-melanoma skin cancers (standardised incidence ratio = 3.6) but no rise in solid tumours frequency in the TNF antagonist-treated group. In fact, patients treated with TNF antagonists actually had a 20% decreased risk for breast cancer that echoed those of the inpatient registry as well as historical RA cohorts. However, the comparator RA cohort did have a 25% reduction in colorectal cancer relative to other admissions that was not seen in the TNF antagonist treated patients . Patient2 months . These lA case report by Smith and Skelton (2001) described seven patients with RA who experienced one or more rapidly growing cSCC that developed shortly after the initiation of etanercept therapy . Similaret al (2014) described a case of a 45-year-old man with RA who was started on etanercept and within two months developed oropharyngeal pain and ulcerations and was diagnosed with tonsillar carcinoma [SCCs of the oral cavity have also been reported with TNF antagonist therapy , 18, 21.et al 20 describeIn conclusion, our report of the rare occurrence of a basaloid SCC of the rectum with positive p16 expression occurring after treatment of RA arthritis with etanercept and methotrexate also raises questions about enhanced susceptibility to human papilloma virus (HPV) as a risk factor for secondary malignancies following TNF inhibitor use. Of possible relevance, TNF antagonists have been noted to exacerbate genital HPV recurrences . TherefoThe authors have no conflicts of interest to declare.


It has been suggested that skeletal muscle mitochondria play a key role in high fat (HF) diet induced insulin resistance (IR). Two opposite views are debated on mechanisms by which mitochondrial function could be involved in skeletal muscle IR. In one theory, mitochondrial dysfunction is suggested to cause intramyocellular lipid accumulation leading to IR. In the second theory, excess fuel within mitochondria in the absence of increased energy demand stimulates mitochondrial oxidant production and emission, ultimately leading to the development of IR. Noteworthy, mitochondrial bioenergetics is strictly associated with the maintenance of normal mitochondrial morphology by maintaining the balance between the fusion and fission processes. A shift toward mitochondrial fission with reduction of fusion protein, mainly mitofusin 2, has been associated with reduced insulin sensitivity and inflammation in obesity and IR development. However, dietary fat source during chronic overfeeding differently affects mitochondrial morphology. Saturated fatty acids induce skeletal muscle IR and inflammation associated with fission phenotype, whereas ω-3 polyunsaturated fatty acids improve skeletal muscle insulin sensitivity and inflammation, associated with a shift toward mitochondrial fusion phenotype. The present minireview focuses on mitochondrial bioenergetics and morphology in skeletal muscle IR, with particular attention to the effect of different dietary fat sources on skeletal muscle mitochondria morphology and fusion/fission balance. Skeletal muscle seems to play a central role in whole body insulin resistance (IR) and metabolic syndrome associated with high fat (HF) feeding, obesity and aging  metabolites, such as diacylglycerols (DAG) and/or Ceramides,  by which mitochondrial bioenergetics  emission and a shift to a more oxidized intracellular redox environment  production -induced obese mice by Liu et al. . Notablyu et al.  also shou et al. .It has been suggested that diverse dietary fat sources have different effects on obesity and associated diseases development. Saturated FA are well known to induce both obesity and related disease, whereas omega 3 polyunsaturated FA (ω-3 PUFA) from fish oil have been shown in many studies to protect against these metabolic diseases (Xin et al., The finding on the effects of different dietary FA on skeletal muscle mitochondrial fusion/fission proteins may be associated with effects on inflammatory processes involved in IR development. Indeed, Bach et al.  suggesteThe pro-fusion effect of ω-3 PUFAs dietary fat on skeletal muscle mitochondria is in agreement with the results found in liver mitochondria, where a shift toward mitochondrial fusion phenotype was also suggested (Zhang et al., The mechanism underlying fish oil/ω-3 PUFAs mitochondrial fusion stimulation may involve receptor-mediated signaling and/or lipid membrane composition, among other factors. Indeed, ω-3 PUFAs are incorporated into cellular membranes and may affect lipid-protein interactions as well as membrane fluidity, and therefore the function of embedded proteins. Recently, it has been suggested a role for ω-3 PUFAs in reorganizing the composition of the mitochondrial membrane, while promoting improvements in ADP sensitivity, determined as mitochondrial responses during ADP titration (Herbst et al., in vivo mechanistic studies to confirm the associational relationship between mitochondrial morphology/dynamics and IR development.Recent evidence highlighted an association between mitochondrial morphology and IR development in skeletal muscle. Few works on different dietary fat source started to underline the different effect of saturated and ω-3 PUFAs on skeletal muscle IR and mitochondrial protein involved in dynamics behavior, suggesting an association between beneficial protective effect of ω-3 PUFAs toward IR development and mitochondrial fusion phenotype Figure . HoweverThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Collections from Hawaii to the Eastern Indian Ocean comprise one large, undifferentiated population. This pattern of limited genetic subdivision across reefs of the central Indo-Pacific has been observed in a number of large-range reef fishes. Conversely, small-range fishes are often deeply structured over the same area. These findings demonstrate population connectivity differences among species at biogeographic and evolutionary timescales, which likely translates into differences in dispersal ability at ecological and demographic timescales. While interspecific differences in population connectivity complicate the design of management strategies, the integration of multiscale connectivity patterns into marine resource planning will help ensure long-term ecosystem stability by preserving functionally diverse communities.Recent reports of localized larval recruitment in predominately small-range fishes are countered by studies that show high genetic connectivity across large oceanic distances. This discrepancy may result from the different timescales over which genetic and demographic processes operate or rather may indicate regular long-distance dispersal in some species. Here, we contribute an analysis of mtDNA cytochrome  The recent dramatic decline of marine ecosystems –3 has leMost near-shore marine species exhibit an early pelagic larval phase  andRecent reports emphasizing the influence of species ecology on dispersal and connectivity –30 may oChaetodon vagabundus), direct tests of larval dispersal have only been applied to fishes with small geographic ranges  that are restricted to either the tropical West Pacific [Whether employing induced otolith tags or multi locus parentage assignment, direct larval tracking has consistently revealed unexpectedly high levels of local larval retention , 35–38.  Pacific , 36, 37, Pacific . Convers Pacific , indicatDispersal ability is thought to play an important role in establishing and maintaining large geographic ranges  across a range that stretches from the east coast of Africa to Hawaii and Pitcairn Island in the Central Pacific [ST =−0.006, P = .752 [To further investigate the relationship between species range size and patterns of population connectivity, we assessed mitochondrial DNA (mtDNA) diversity in the Brown Surgeonfish ( Pacific . The Bro Pacific –62. The  Pacific ). A prevP = .752 ), a pattSimilar phylogeographic comparisons have provided valuable insights into species’ life history , 66, ecoN = 281 [N = 279; b was amplified using the heavy strand primer (5’-GTGACTTGAAAAACCACCGTTG-3’) from [2, 0.26 µM of each primer, 50 nM dNTPs, 1.0 units Taq DNA polymerase and 2.0 µL of 10x PCR buffer  in 20 µL total volume. PCR thermal cycling consisted of an initial denaturing step at 94° C for 1 min, followed by 35 cycles of 94, 55, and 72°C for 30 s each, with a final extension of 72°C for 10 min. Sequencing reactions with fluorescently labeled dideoxy terminators were analyzed on an ABI 3100 automated sequencer  in the EPSCoR genetic analysis facility at the Hawaii Institute of Marine Biology. Only rare and questionable haplotypes were sequenced in both directions. Newly derived unique haplotypes have been deposited in Gen Bank (accession numbers HQ157717–157797). Resulting sequences were aligned in Mafft 6.62 [Tissue collections from Hawaii (N = 281 ) were suN = 279; , and a 63’) from  and ligh3’) from . In briefft 6.62 .h) and nucleotide (π) diversities for each collection site were calculated in Arlequin 3.11 [T-test. A statistical parsimony network of haplotypes was constructed using TCS 1.2.1 [Haplotype  framework (Arlequin 3.11) to identify maximally differentiated groupings without a priori assumptions of group identity. To ensure validity of the maximally differentiated groupings, the simulated annealing process was repeated 100 times from a different initial partition of samples into K groups. The configuration with the largest, statistically significant estimate of among group differentiations (Φct) is retained as the best sample grouping. Samova was run with values of K = 2 to 10 to identify the most likely number of populations. Because Samova cannot be run for K = 1, a separate AMOVA analysis was conducted with all collections combined into a single group. Deviations from random expectations were tested with 20,000 permutations. Patterns of pairwise genetic differentiation among individual sampling sites (Φst) were estimated in Arlequin with the mutational model of Tamura and Nei [odeltest [Dest . Samova and Nei , which wodeltest , 87. We n 13 see ).amova-defined populations.A Mantel test with 10,000 simulations was used to test for an isolation-by-distance (IBD) signature . TosF [rlequin with 90,000 simulated samples. Fu [sF is particularly sensitive to deviations from a constant population size, with population expansion resulting in a significant, negative sF. If there was evidence of population expansion, we estimated ancestral and contemporary female effective population sizes (Nef) from the equation: θ = Nef2 μt, with μ being the estimated annual fragment mutation rate and t is the estimated generation time. Estimates of pre- and post expansion θ were derived from the sudden expansion model of the mismatch distributions. Population ages in years were estimated from the population age parameter (τ), with τ = 2 μT, where T is the time since the most recent bottleneck. We provisionally applied a generation time of 5 years for the Brown Surgeonfish [−8 per year [We tested for a signature of population expansion with Fu’s Fs  and by cFs  in Arleqples. Fu  noted thgeonfish  and a wiper year .b sequencing revealed 110 closely related haplotypes  is similar to other peripheral collections  but is significantly lower than central Indo-Pacific collections .Cytochrome The statistical parsimony network demonstrates both the high diversity and low differentiation of haplotypes collected from sites distributed across two ocean basins . Abundanamova identified three maximally differentiated groupings, with significant population differentiation occurring only at eastern and western edges of the range  dropped incrementally with the addition of K > 3 populations (data not shown) indicating a lack of further population subdivision. Pairwise Φst and Dest highlight both the isolation of Moorea as well as a pattern of increasing divergence in the Indian Ocean with distance from the IPB . Pairwise estimates of Φst within the Samova-defined central Indo-Pacific population were near zero. Nonetheless, there was a clear IBD signature across this broad region  even though Christmas Island, located in the western Indian Ocean, was only marginally different from Hawaiian collections  and was genetically indistinguishable from Kiritibati, Fiji, and Palau.S the IPB . Both thamova populations. While a significant deviation between simulated and observed mismatch distributions was observed only in the Moorean collection , Fu’s sF was significantly negative in all three populations , with sGenetic evidence of postglacial population expansion is common in reef fishes and indicates broad ecosystem level responses to global climate change . These pLutjanus kasmira), which differs only in having an additional genetic break between Moorea and the Marquesas [The population structure of the Brown Surgeonfish is remarkably similar to the widely distributed Bluestriped Snapper (arquesas . NotablyAcanthurus triostegus) revealed little to no genetic subdivision within the Central and West Pacific [Naso unicornis) from the West Indian Ocean to French Polynesia in the South Pacific [Aulostomus chinensis,) from West Australia to Panama [Gymnothorax flavimarginatus) from East Africa to the East Pacific [The lack of pronounced mtDNA genetic structure across the central Indo-Pacific in both the Brown Surgeonfish and Bluestriped Snapper has been observed in a growing number of widely distributed reef fishes. Of the six published genetic surveys of reef fishes with geographic ranges extending from Africa to the East Pacific , all but the Convict Surgeonfish , Pyntropyge , 107), Tlostomus ), Unicorus [Naso , 108), Mnothorax ), and atLutjanus ) and Suranthurus , 109) haFor widely distributed species, genetic homogeneity across much of the Indo-Pacific is largely concordant with biogeographic provinces and barriers, previously defined based on species distributions. For example, the region stretching from the eastern Indian Ocean to the Central Pacific is recognized by biogeographers as the Indo-Polynesian province –112. HobPlectropomus leopardus) is restricted to reefs from West Australia to Fiji, and a survey of mtDNA control region diversity indicated deep genetic partitions, with a minimum of six highly differentiated populations  and the restricted-range Wrasse,  revealed concordant morphological and genetic differentiation, and indicates evolutionary level partitions among the closely linked reefs of the West Pacific [Amphiprion [Chrysiptera [Dascyllus [Pomacentrus [Epinephelus [Plectropomus [This pattern of widely distributed reef fishes successfully bridging long-distances contrasts starkly with the remarkably complex patterns of population structure commonly observed in smaller range fishes. The leopard coral grouper ( to 0.94 ). Likewi Pacific . Small rphiprion –117; genysiptera , 118; geascyllus , 119; genephelus , 114; geFor species occurring within the central Indo-Pacific, broad genetic homogeneity should be facilitated by stepping stone gene flow across the regions relatively abundant reefs. Indeed, dispersal can be accomplished across this vast region without having to traverse more than 800 km of open ocean , yet eveIt has been noted that mtDNA markers may not accurately reflect population histories, due to either direct selection on the marker or adjacent DNA segments . HoweverHow do these findings translate into policies for ecosystem-level management of reef communities? The debate continues over the extent to which genetic connectivity relates to demographic connectivity . FurtherWhile interspecific differences in realized dispersal would appear to complicate the development of effective marine reserves, reserve design may be simplified by focusing on the species that show the finest scale of genetic isolation , 131. Em