The trabecular vBMD GWA meta-analysis  followed by replication  identified one locus reaching genome-wide significance . High-resolution pQCT analyses, giving information about bone microstructure, were available in a subset of the GOOD cohort (n = 729). rs1021188 was significantly associated with cortical porosity while rs9287237 was significantly associated with trabecular bone fraction. The genetic variant in the FMN2/GREM2 locus was associated with fracture risk in the MrOS Sweden cohort  and GREM2 expression in human osteoblasts. In conclusion, five genetic loci associated with trabecular or cortical vBMD were identified. Two of these (FMN2/GREM2 and LOC285735) are novel bone-related loci, while the other three have previously been reported to be associated with aBMD. The genetic variants associated with cortical and trabecular bone parameters differed, underscoring the complexity of the genetics of bone parameters. We propose that a genetic variant in the RANKL locus influences cortical vBMD, at least partly, via effects on cortical porosity, and that a genetic variant in the FMN2/GREM2 locus influences GREM2 expression in osteoblasts and thereby trabecular number and thickness as well as fracture risk.Most previous genetic epidemiology studies within the field of osteoporosis have focused on the genetics of the complex trait areal bone mineral density (aBMD), not being able to differentiate genetic determinants of cortical volumetric BMD (vBMD), trabecular vBMD, and bone microstructural traits. The objective of this study was to separately identify genetic determinants of these bone traits as analysed by peripheral quantitative computed tomography (pQCT). Separate GWA meta-analyses for cortical and trabecular vBMDs were performed. The cortical vBMD GWA meta-analysis  and deteriorated bone microstructure, resulting in an increased risk of fracture. Most previous genetic epidemiology studies have focused on the genetics of the complex trait BMD, not being able to separate genetic determinants of the trabecular and cortical bone compartments and bone microstructure. The trabecular and cortical BMDs can be analysed separately by computed tomography. Therefore, we performed separate genome-wide association studies for trabecular and cortical BMDs, demonstrating that the genetic determinants of cortical and trabecular BMDs differ. Genetic variants in the  Meta-analyses of genome-wide association studies (GWAS) have identified a large number of loci associated with areal bone mineral density (aBMD) s 0.11 in the young adult men of the GOOD cohort; s 0.04) or moderate (trabecular vBMD rs 0.65), suggesting that cortical and trabecular vBMDs are at least partly influenced by genetic determinants not possible to identify by a GWAS of aBMD Standard peripheral QCT (pQCT) with a resolution of 500 µm has the advantage of being able to separately analyse trabecular and cortical vBMDs. The correlation between trabecular and cortical vBMDs is low  identified a genetic variant in the ex vivo μCT imaging (resolution 20 µm or better) High resolution pQCT (HRpQCT) not only allows the separation of the trabecular and cortical bone compartments but also the assessment of bone microstructure. HRpQCT has an isotrophic voxel size of 82 µm and shows excellent correlation with The objective of the present study was to identify genetic determinants of vBMDs and bone microstructure parameters separately for the cortical and trabecular bone compartments as analyzed by pQCT and HRpQCT. As our assembled discovery cohort was larger for the pQCT measurements  than for the HRpQCT measurements (n = 729), we aimed to first identify genome-wide significant genetic variants for cortical and trabecular vBMDs separately and then to evaluate the impact of the identified variants on trabecular and cortical bone microstructure parameters in the HRpQCT cohort.GOOD baseline r = 0.11 GOOD five year follow-up r = −0.01). Separate GWA meta-analyses for cortical and trabecular vBMD were performed including all three discovery cohorts for cortical vBMD while trabecular vBMD was available in the YFS and GOOD cohorts.−12) on chromosome 13, slightly upstream of the RANKL gene  is a novel bone-related locus, located on chromosome 6, upstream of LOC285735 (−9) was located on chromosome 8, upstream of OPG  was located on chromosome 6, in C6orf97 upstream and close to estrogen receptor-α ), but a marked deviation from the null distribution amongst the lowest observed p-values . We idenTNFSF11; . The secOC285735 . The thi-α ESR1; .RANKL region (rs1021188) an additional suggestive signal  appeared, but did not achieve genome-wide significance (−5).We selected our top four regions and carried out analyses conditional on the most associated SNPs in each region. When conditioning on the most significant SNP in the ificance . Using sRANKL, OPG and ESR1 regions have earlier been reported to be associated with aBMD in large scale GWA meta-analyses RANKL region, rs1021188 and rs17638544 were conditioned on the known aBMD hit rs9533090; OPG region, rs7839059 was conditioned on the known aBMD hit rs2062377; ESR1 region, rs6909279 was conditioned on known aBMD hits rs7751941 and rs4869742; RANKL signals (rs1021188 and rs17638544) were distinct from the previously reported aBMD signal  with rs9533090  is independent from one of the previous reported aBMD signals (rs7751941) while the other reported independent aBMD SNP in this region (rs4869742 2 with rs6909279 (r2 = 0.60)  . However = 0.60) .−8) on chromosome 1, in the formin 2 gene .In the trabecular vBMD GWA meta-analysis there was little systematic inflation of test statistics ), but a significant deviation from the null distribution amongst the lowest observed p-values . We idenOPG displayed a modest association with trabecular vBMD  in the same direction as seen for the genome-wide significant association with cortical vBMD . The four GWA significant cortical vBMD SNPs rs1021188, rs271170, rs7839059 and rs6909279 explained 0.6%, 0.4%, 0.6% and 0.3%, respectively of the variation in cortical vBMD in the elderly men of the MrOS Sweden replication cohort. When including all four SNPs together, 1.5% of the variation in cortical vBMD was explained in the MrOS Sweden replication cohort. There was evidence for heterogeneity in the meta-analyses of the associations with rs1021188 (het p = 0.028) and rs17638544 (het p = 0.011). Random effects meta-analysis results for rs1021188 and rs17638544 were 0.18 SD per allele  and 0.14 SD per allele , respectively.Five SNPs  were selected for replication in the MrOS Sweden cohort. In this replication analysis all five SNPs were significantly associated with cortical vBMD in the same direction and with similar effect sizes as found in the discovery GWA meta-analysis . rs9287237 explained 0.7% of the variation in trabecular vBMD in the MrOS Sweden replication cohort.One SNP (rs9287237) was replicated in the MrOS Sweden cohort and it was significantly associated with trabecular vBMD also in the replication cohort . The sex difference may explain the heterogeneity observed in the unstratified analyses for rs1021188 (both sexes have a het p>0.05). Significant heterogeneity is still observed in the males for rs17638544 (het p = 0.016).Sex-specific analyses demonstrated that all five cortical vBMD SNPs were significantly associated with cortical vBMD in both men and women with effects in the same direction. However, the magnitude of the effect sizes differed significantly according to sex for three of the identified hits (The genetic variant associated with trabecular vBMD (rs9287237) was significantly associated with trabecular vBMD in men  but, as expected, in the inverse direction compared with the association with cortical vBMD  cortical but not trabecular vBMD at the five year follow-up visit of the GOOD cohort and their effect sizes for cortical vBMD were of similar magnitude and direction as seen for the GOOD cohort at the baseline visit . Interescal vBMD .−6) for trabecular vBMD was of similar magnitude and direction as seen for the GOOD cohort at the baseline visit (−5) while it was not significantly associated with cortical porosity (−2), trabecular thickness  and trabecular spacing  All five genome-wide significant vBMD SNPs were nominally significantly associated (p<0.05) with both femoral neck and lumbar spine aBMD as provided in the public data release from the discovery phase  of the recent aBMD analyses from the GEFOS consortium  with expression of the nearby GREM2 gene. No significant effects on gene expression were noted at the additional four loci  data from resting (i.e. untreated) and induced  primary human osteoblasts Overall, 388 men had at least one validated incident fracture after an average follow-up of 5.4 years in the MrOS Sweden cohort . The traIn two centers of MrOS Sweden, Gothenburg and Malmö, overall, 1445 men had a spine X-ray at baseline, which identified 225 men with prevalent vertebral fractures 15.6%; . The traRANKL, LOC285735, OPG and ESR1 loci were associated with cortical vBMD while a genetic variant in the FMN2/GREM2 locus was associated with trabecular vBMD. Follow-up analyses in a HRpQCT cohort providing bone microstructural traits revealed that a genetic variant in the RANKL locus is associated with not only cortical vBMD but also with cortical porosity. The FMN2/GREM2 locus, on the other hand, is robustly associated with not only trabecular vBMD but also trabecular number and thickness as well as GREM2 expression in human osteoblasts and fracture risk.Osteoporosis is a common highly heritable skeletal disease characterized by reduced aBMD and deteriorated bone microstructure, resulting in an increased risk of fragility fracture WNT16 gene to be associated with cortical bone thickness Wnt16 inactivated mice had reduced cortical bone thickness, providing strong evidence that WNT16 is an important regulator of cortical bone thickness. In the present discovery GWAS-meta-analysis, we identified genome-wide significant cortical vBMD signals in four loci and a trabecular vBMD signal in one locus and all these signals were significantly replicated in the MrOS Sweden cohort. One of the identified cortical vBMD loci (LOC285735) and the identified trabecular vBMD locus (FMN2) are novel bone-related loci while the remaining three identified cortical vBMD loci have been previously reported to be associated with aBMD. WNT16 locus recently identified to be associated with cortical bone thickness Using the same pQCT cohorts as evaluated in the present study, we have recently identified a missense variant in the TNFRSF11B locus) was also nominally (p<0.05) significantly associated with trabecular vBMD, although with less pronounced effect size, suggesting that this SNP does not exclusively have an impact on cortical bone. The present report describing two independent RANKL signals and one OPG signal with an impact on cortical vBMD provides further evidence that the RANK/RANKL/OPG axis affects the skeleton at least in part by influencing volumetric apparent density of cortical bone. It is tempting to speculate that changes in cortical vBMD contribute to the recent observations that the RANKL inhibitor denosumab reduces fracture risk Conditional analysis revealed a secondary signal in the RANKL locus and both these cortical vBMD signals were independent of the previously reported aBMD signal , 93.4 kb upstream of FMN2 gene. The combined effect size of this signal was substantial with an increase of 0.19 SD per T allele. FMN2 is a gene that is expressed in oocytes and is required for progression through metaphase of meiosis 1 but it is not previously reported to influence the skeleton FMN2 has been associated with coronary heart disease GREM2 ( = PRDC), which is an extracellular antagonist of bone morphogenetic proteins (BMPs) and it inhibits osteoblastic differentiation GREM2 gene, indicating that GREM2 is a strong candidate for mediating the trabecular vBMD association at rs9287237. However, further targeted sequencing efforts and functional studies are required to characterize this signal.A significant signal (rs9287237) for trabecular vBMD was identified on chromosome 1 located in the intron region of the RANKL and ESR1 signals were more than three (0.21 SD vs. 0.06 SD) and two (0.12 vs. 0.05 SD) times larger, respectively, in men than in women. The smaller effect within females observed for rs1021188 in the RANKL region is mainly driven by ALSPAC, and there may be other reasons (such as younger age) why this study showed a smaller effect. However, the consistent results between ALSPAC and the YFS provide some evidence against the differences being driven primarily by age. The relative strong ESR1 signal in men supports experimental and clinical studies, demonstrating that estrogens are crucial for male bone health There are known sex differences in bone traits in mice We examined genetic effects across cohorts encompassing a relatively broad age range, including 15 year old participants from ALSPAC who were still attaining peak bone mass, to older men from MrOS Sweden starting to show age-related bone loss. Inclusion of an older cohort had the advantage of providing an opportunity to study relationships with fracture risk. However, this design may have reduced the power to detect genetic associations by introducing greater heterogeneity. In contrast to aBMD, vBMD has been reported to change relatively little from adolescence to mid-life suggesting that analyses combining cohorts of different ages might be more informative when based on this trait RANKL region with the strongest association with cortical vBMD was also significantly associated with cortical porosity but, as expected, in the reverse direction. This finding suggests that a genetic variant in the RANKL locus influences cortical vBMD, at least partly, via effects on cortical porosity. Importantly, this signal in the RANKL region was independent from the previously reported aBMD signal in the same region FMN2/GREM2 locus was significantly associated with several trabecular but not cortical bone microstructure parameters. When evaluated in the five-year follow-up visit in the GOOD cohort, each T allele of this SNP resulted in a substantial increase in trabecular vBMD (0.32 SD), trabecular bone fraction (BV/TV 0.29 SD), trabecular number (0.15 SD), and trabecular thickness (0.18 SD). Thus, a genetic variant in the FMN2/GREM2 locus influences trabecular vBMD via substantial effects on both trabecular number and thickness. Although, the present study is the first to report on genetic variants associated with microstructural bone-parameters, the analyses were candidate-based as a follow-up of our initial cortical and trabecular vBMD GWA meta-analyses. In order to identify novel genetic loci for bone microstructural parameters in a hypothesis-free manner, well-powered HRpQCT cohorts with genome-wide genotype data available need to be established.Age-related changes in bone include microstructural deterioration, such as trabecular perforation, thinning, and loss of connectivity, as well as increased cortical porosity We believe that our study provides strong evidence that previous large-scale GWA meta-analyses of the complex bone trait aBMD did not have the capability to identify a number of important loci with an impact on aspects of micro-architecture which may have important effects on fracture risk but be poorly reflected by overall aBMD measurements. We, therefore, propose that future well-powered pQCT and HRpQCT GWA meta-analyses of these specific bone structural traits will add useful information and might result in the identification of novel osteoporosis drug targets and provide novel aBMD-independent genetic markers for the prediction of fracture risk.FMN2/GREM2 locus helps to explain this type of presentation.The implication of our results suggesting that cortical and trabecular bone compartments are under distinct genetic control is consistent with the fact that patients with idiopathic osteoporosis may present with a predominantly trabecular or cortical bone phenotype FMN2/GREM2 locus was associated with fracture risk and prevalent X-ray verified vertebral fractures in the MrOS Sweden cohort. However, further large-scale studies are required to replicate the fracture findings of this SNP. Collectively our data demonstrate that each extra T allele of rs9287237 is associated with decreased expression of the BMP antagonist GREM2 in osteoblasts, increased trabecular vBMD and decreased fracture risk. As previous in vitro studies have demonstrated that GREM2 inhibits osteoblast differentiation, we propose that rs9287237 is involved in the regulation of GREM2 expression in osteoblasts, which in turn regulates osteoblast differentiation and thereby the amount of trabecular bone and fracture risk The genetic variant in the FMN2/GREM2 and LOC285735) are novel bone-related loci while the other three have previously been reported to be associated with aBMD. The genetic variants reported to be associated with cortical and trabecular bone parameters differed and were also distinct from the WNT16 locus recently identified to be associated with cortical thickness RANKL locus influences cortical vBMD, at least partly, via effects on cortical porosity, and that a genetic variant in the FMN2/GREM2 locus influences trabecular vBMD and fracture risk via substantial effects on both trabecular thickness and number.In conclusion, we identified five genetic loci associated with trabecular or cortical vBMD. Two of these  study was initiated to determine both environmental and genetic factors involved in the regulation of bone and fat mass 3 as cortical bone. Trabecular vBMD (mg/cm3) was measured using a scan through the metaphysis  of the tibia. Tibia length was measured from the medial malleolus to the medial condyle. The CVs were <1% for all pQCT measurements. Both pQCT measurements and genotype information were available for 938 study subjects.Cortical volumetric BMD (not including the bone marrow) was measured on a single tibial diaphyseal slice  using the Stratec XCT2000 (Germany) at both the baseline visit and the five-year follow-up visit in the GOOD cohort −1), trabecular thickness  and trabecular separation . For the cortical porosity, we used the manufacturer's software, which uses an approach published recently by several groups A high-resolution 3D pQCT device  was used to scan the ultradistal tibia of the nondominant leg at the five-year follow-up visit in the GOOD cohort −6) using Mach 1.0, Markov Chain Haplotyping, giving a total of 2,608,508 SNPs.Genotyping was performed with Illumina HumanHap610 arrays at the Genetic Laboratory, Department of Internal Medicine, Erasmus Medical Center, Rotterdam, the Netherlands. Genotypes were called using the BeadStudio calling algorithm. Genotypes from 938 individuals passed the sample quality control criteria , duplicates and/or first degree relatives identified using IBS probabilities (>97%), ethnic outliers (3 SD away from the population mean) using multi-dimensional scaling analysis with four principal coordinates). Across 22 duplicate samples, genotype concordance exceeded 99.9%. We carried out imputation to HapMap   is an ongoing multi-centre follow-up of atherosclerosis risk factors in young Finns of Caucasian origin 3 as cortical bone. Trabecular vBMD (mg/cm3) was measured using a scan through the metaphysis  of the tibia. Tibia length was measured from the medial malleolus to the medial condyle. The CVs were <2.6% for all pQCT measurements. Both pQCT measurements and genotype information were available for 1558 study subjects.Cortical volumetric BMD was measured on a single tibia diaphyseal slice  using the Stratec XCT2000 (Germany). A threshold routine was used for defining cortical bone, which specified a voxel with a density >710 mg/cm−6), 7,746 SNPs failed a missingness test  and 34,596 SNPs failed frequency test (MAF<0.01). After quality control there were 2,442 samples and 546,677 genotyped SNPs available for further analysis. Genotype imputation was performed using MACH 1.0 and HapMap II CEU  samples as the reference set.Genomic DNA was extracted from peripheral blood leukocytes using a commercially available kit and Qiagen BioRobot M48 Workstation according to the manufacturer's instructions . Genotyping was done for 2556 samples using custom build Illumina Human 670 k BeadChip at the Welcome Trust Sanger Institute. Genotypes were called using the Illuminus clustering algorithm. 56 samples failed Sanger genotyping pipeline QC criteria . From the remaining 2500 samples one sample failed gender check, three were removed due to low genotyping call rate (<0.95) and 54 samples for possible relatedness (pi-hat>0.2). 11,766 SNPs were excluded based on HWE test . Both mothers and children have been extensively followed from the 8th gestational week onwards using a combination of self-reported questionnaires, medical records and physical examinations. Ethical approval was obtained from the ALSPAC Law and Ethics committee and relevant local ethics committees, written informed consent was provided by all parents. Blood samples were taken and DNA extracted as previously described The Avon Longitudinal Study of Parents and their Children (ALSPAC) is a geographically based birth cohort study investigating factors influencing the health, growth, and development of mainly Caucasian children 3 as cortical bone. Of the 4500 pQCT scans obtained in ALSPAC 89 were rejected as being of insufficient quality. The CVs for cortical volumetric BMD based on 139 ALSPAC subjects scanned a mean of 31 days apart, was 1.3%.pQCT scans were performed on approximately 4,500 children when they attended the age 15 research clinic. Cortical vBMD was measured on a single slice at the mid tibia (50%) using the Stratec XCT2000L (Germany). A threshold routine was used for defining cortical bone, which specified a voxel with a density >650 mg/cm−7) were excluded from further analysis. We also excluded any individuals who did not cluster with the CEU individuals in multidimensional scaling analysis, who had >3% missing data, minimal or excessive heterozygosity (>34.5% or <32% for the Sanger data and >33% or <31% for the LabCorp data), evidence of cryptic relatedness (>10% IBD) and any individuals with incorrect gender assignments. After data cleaning we were left with 8365 unrelated individuals with genome-wide genotyping, and 500,541 SNPs. We carried out imputation using MACH 1.0.16, Markov Chain Haplotyping, using CEPH individuals from phase 2 of the HapMap project as a reference set . Of the 8,365 individuals with imputed genotype data, 3,382 also had pQCT data.9,912 ALSPAC individuals were genotyped using the Illumina HumanHap550 quad genome-wide SNP genotyping platform by 23andMe, subcontracting the Wellcome Trust Sanger Institute, Cambridge, UK and the Laboratory Corporation of America, Burlington, NC, US. Markers with <1% minor allele frequency >5% missing genotypes or which failed an exact test of Hardy-Weinberg equilibrium (HWE) (p<5×10The Osteoporotic Fractures in Men (MrOS) study is a prospective multicenter study including older Caucasian men in Sweden , Hong Kong , and the United States . In the present study, associations between candidate polymorphisms and skeletal parameters were investigated in the Swedish cohort, which consists of three sub-cohorts from three different Swedish cities  3 as cortical bone. Trabecular vBMD (mg/cm3) was measured using a scan through the metaphysis  of the tibia. Tibia length was measured from the medial malleolus to the medial condyle. The CVs were <1% for all pQCT measurements. Adjustments for study centre were performed.Cortical volumetric BMD (not including the bone marrow) was measured on a single tibial diaphyseal slice  using the Stratec XCT2000 (Germany) Genotyping of SNPs identified in the GWAS meta-analysis was carried out at KBioscience using a competitive allele specific PCR (KASP) genotyping chemistry. The genotyping call rate was >97%.SNPs associated with vBMD at the genome-wide significance level as reported here were tested for association with resting or induced gene expression of neighbouring gene transcripts, in primary human osteoblasts derived from 113  unrelated Swedish donors. Detailed cell culture and analysis methods have been described in detail Participants were followed for 5.4 years on average after the baseline examination. The follow-up time was recorded from the date of the baseline visit to the date of the first fracture, the date of death, or end of the present follow-up interval. When a subject sustained a first fracture at different sites during the follow-up, the various fractures and the follow-up time for each respective first fracture type were included in the analyses. Complete follow-up was possible because central registers covering all Swedish citizens were used to identify the subjects and the time of death for all subjects who died during the study, and these analyses were performed after the time of fracture validation. At the time of fracture evaluation, the computerized X-ray archives in Malmö, Göteborg, and Uppsala were searched for new fractures occurring after the baseline visit, using the unique personal registration number, which all Swedish citizens have. All fractures reported by the study subject after the baseline visit were confirmed by physician review of radiology reports. Fractures reported by the study subject, but not possible to confirm by X-ray analyses report, were not included in this study. All validated fractures and hip fractures were evaluated. Information about the type of trauma associated with the incident fractures was not available. Fracture rates were expressed as the number of subjects with first fractures per 1000 person-years .In Gothenburg and Malmö, 1445 men had an X-ray of the lateral thoracic and lumbar spine at baseline. All vertebral fractures were evaluated by an expert radiologist. If the vertebral body had a reduced height of 3 mm or more compared with the vertebra above, it was classified as a vertebral fracture 2 imputation quality score of >0.3 in all 3 sets . We carried out genome-wide association analyses for cortical and trabecular vBMDs using additive linear regression in Mach2QTL for ALSPAC, ProbABEL −8. We also repeated the analyses in each of the three discovery cohorts, conditional on these top SNPs, to identify any additional independent associations in the regions. We selected one SNP for replication in the MrOS Sweden cohort from each independent region that had a p<5×10−8 as well as a secondary SNP from the RANKL region which appeared to influence cortical vBMD. Additive linear regression analyses were carried out for the associations between these SNPs and cortical and trabecular vBMDs in SPSS Statistics 17.0 for MrOS Sweden, using age, sex, height and weight(ln) as covariates. The results of all four cohorts were combined using a fixed effects inverse-variance meta-analysis in Stata (version 11.2). The SNPs showing evidence for heterogeneity (as assessed by a chi-squared test) were also meta-analysed using the DerSimonian & Laird random effects method. Correlations between bone traits in the GOOD cohort were tested and presented as Spearman's rank correlation coefficients (rho). The difference of the allelic association effects between males and females was tested using a two sample z-test.The ALSPAC (n = 3382), YFS (n = 1558) and GOOD (n = 938) discovery cohorts contributed to the cortical vBMD genome-wide meta-analysis while the YFS and GOOD discovery cohorts contributed to the trabecular vBMD genome-wide meta-analysis. We analyzed only those imputed SNPs which had a minor allele frequency of >0.01 and an rCox proportional hazards models were used to study the associations between SNPs and incident fractures. Prevalent vertebral fractures were analyzed using binary logistic regression models.In order to estimate the genetic correlation between cortical and trabecular vBMD we ran a bivariate REML analysis using the GCTA software package in the GOOD cohort, having both cortical and trabecular vBMDs measurements available Table S1Discovery meta-analysis of cortical and trabecular vBMDs.(PDF)Click here for additional data file.Table S2Cortical vBMD SNPs conditioned on known aBMD SNPs.(PDF)Click here for additional data file.Table S3Association of top cortical and trabecular vBMD signals with pQCT and HRpQCT parameters in the GOOD cohort at the five-year follow-up visit.(PDF)Click here for additional data file.Table S4Associations with cortical and trabecular vBMD for 64 reported genome-wide significant aBMD SNPs.(PDF)Click here for additional data file.Table S5eQTL analysis in human osteoblasts.(PDF)Click here for additional data file.Table S6Characteristics of the MrOS Sweden fracture cohort.(PDF)Click here for additional data file.Table S7Associations between cortical and trabecular vBMD SNPs and fractures in MrOS Sweden.(PDF)Click here for additional data file.


The southern coastline of Australia forms part of the worlds' only northern boundary current system. The Bonney Upwelling occurs every austral summer along the south-eastern South Australian coastline, a region that hosts over 80% of the worlds population of an endangered endemic otariid, the Australian sea lion. We present the first data on the movement characteristics and foraging behaviour of adult male Australian sea lions across their South Australian range. Synthesizing telemetric, oceanographic and isotopic datasets collected from seven individuals enabled us to characterise individual foraging behaviour over an approximate two year time period. Data suggested seasonal variability in stable carbon and nitrogen isotopes that could not be otherwise explained by changes in animal movement patterns. Similarly, animals did not change their foraging patterns despite fine-scale spatial and temporal variability of the upwelling event. Individual males tended to return to the same colony at which they were tagged and utilized the same at-sea regions for foraging irrespective of oceanographic conditions or time of year. Our study contrasts current general assumptions that male otariid life history strategies should result in greater dispersal, with adult male Australian sea lions displaying central place foraging behaviour similar to males of other otariid species in the region. Arctocephalus tropicalisMiroungia angustirostris and M. leoninaOtaria flavescensPhocarctos hookeri feed on the sea floor Aptenodytes paptagonicusA. forsteriMegadyptes antipodesA. pusillus doriferusThe distribution and foraging patterns of marine mammal top predators are influenced by bottom-up processes affecting lower trophic levels, such as oceanic currents and coastal upwelling events. These predators use three broad foraging strategies in response to the challenges of locating sufficient food to survive and reproduce in changing environments: epipelagic foragers such as sub-Antarctic fur seals Investigating the foraging behaviour of air-breathing marine predators typically involves the use of expensive geospatial tracking devices to describe movements of large, wild, elusive predators during foraging trips to sea. Consequently, prohibitive costs and logistical difficulties often limits answering questions of seasonal and intersexual differences in foraging to those that may be answered using data from a small subsample of animals Otaria flavescens in South America also exhibit benthic and pelagic foraging behaviour, travelling twice the distance covered by conspecific adult females A. gazella display post-breeding seasonal migration into (presumably) richer foraging grounds ∼900 km from their breeding colonies on South Georgia Callorhinus ursinus move from shelf to oceanic waters outside the breeding season but do not travel as far as conspecific adult females A. forsteri in South Australia feed along a shelf-break and their dives are typically pelagic, but approximately 20% of their dives are benthic Our understanding of the foraging behaviour of adult male otariids comes from limited studies of only seven of the 16 extant species. California sea lion adult males display both benthic and pelagic foraging behaviour, leaving the Gulf of California at the end of the breeding season to migrate north as far as Oregon into more productive waters before returning late spring with the onset of upwelling 15N) from primary producers to the top of the food web. In marine ecosystems a decreasing stable carbon ratio (δ13C) gradient has been identified with distance from shore 13C and δ15N from metabolically active (blood) and inert  tissues in several seal and marine bird species Neophoca cinereaThe use of cost-effective stable isotope biogeochemistry has provided greater insights into foraging ecology across a range of marine mammals The biogeography and unique reproductive ecology of the Australian sea lion has made characterising its foraging ecology challenging. This endangered species The hypothesis that Australian sea lion life history and foraging strategies reflect a species adapted to an oligotrophic, aseasonal marine environment has been suggested 2-powered tranquilizer gun . Once immobilized, animals were anaesthetized using isoflurane delivered through a portable gas anesthesia machine . A Conductivity-Temperature-Depth Satellite-Relay Data Logger  weighing ∼540 g was attached to the pelage distal to the midpoint of the back of each individual using two-part epoxy glue . Prior to recovery from anesthesia procedure a whisker was clipped at the base of the muzzle from each individual for isotopic analysis.Movement and environmental data were collected using oceanographic sensors deployed on a single adult male Australian sea lion from each of seven breeding colonies spanning the South Australian range between October 2009 and June 2010 . Males wThe sensor platform within the CTD-SRDL comprised a Keller PA-7 pressure transducer (accuracy 2±0.3DBar), a custom-made temperature sensor  and an inductive coil to measure conductivity . Instruments were calibrated after assembly 13C and 15N were Pee Dee Belemnite and atmospheric nitrogen, respectively. Observed measurement error rates were recorded as <±0.1‰ (δ13C) and <±0.3‰ (δ15N).All vibrissae were cleaned as described in −1Raw ARGOS data were pre-processed to remove extreme outliers by removing locations with unclassified error estimates (LC- Z) and by using a swim-speed filter with a moving average of 2 msmaxFPT identified for each animal (herein referred to as ARS patch size). FPT values for this radii were then plotted against time for each track, with ARS behaviour being inferred at segments of tracks that took the longest time to traverse  2) for each trip of each individual, using a Brownian Bridge movement model (BBMM) described in To quantify space-use and the environment the animal experienced while foraging, two parameters were derived for each foraging trip. First Passage Time (FPT) was used to identify the scale at which Area Restricted Search  was conducted −3) at one metre intervals for each CTD profile using the Thermodynamic Equation of State of seawater −3 and define the surface σ ⊖ as the value calculated for the bottom of the first depth bin (4 m) to determine MLD at the location of each CTD profile −1) and MLD (m) were calculated and interpolated across the study area on a 1 km resolution grid using ODV4.5.1 Surface mixed layer depth (MLD) can be defined in terms of a critical density gradient or by a finite change in density from surface values 2 >0.83, p<0.001 in all cases). This exploratory process indicated adult male Australian sea lions exhibited a benthic diving strategy, suggesting environmental conditions at the surface may have been unimportant to foraging. Accordingly, in conjunction with static features (bathymetric slope and depth), we considered only temperature and salinity at the sea floor, MLD, transition layer thickness (the depth between the bottom of the MLD and the sea floor) and thermocline intensity as biologically-relevant, dynamic variables. The significance of temporal variability in core (UD-50) and home (UD-95) ranges, both between individuals and among trips for each individual, were characterised using Kruskal Wallis Χ2. Subsequently, for those individuals that expressed significant variability, we examine the influence of environmental conditions on individual core, home and ARS patch sizes using generalised additive mixed models (GAMM in R package ‘mgcv’). Accounting for the expected high degree of correlation between most oceanographic variables , models ranged in complexity from null models to the inclusion of all uncorrelated covariates (and their interactions). However all models included month-since-deployment as a fixed factor and individual foraging trip as a random factor   =  exp(β1X1 +β2X2 +β3X3 +...+βpXp)h0(t), where h(t) is the risk (hazard) of an individual leaving an area (defined by the FPT across a predetermined radius) at time t, X represents a physical variable , β denotes the regression coefficient fitted to each variable during the modelling process and 0(t)h the baseline hazard function . In this context, a hazard ratio provides a quantitative means to assess how an animal responds to changes in environmental parameters c) We tested the effect of environmental variability on FPT using a series of individual Cox Proportional Hazard models (CPH) of the form: p,d,q) (AutoRegressive Integrated Moving Average) models to stable carbon (δ13C) and nitrogen (δ15N) isotope values implemented by a three-step Box Jenkins method p,d,q) models require the estimation of the number of autoregressive parameters (p), the differencing order (d) and the variance of the error term (‘moving average’) parameters (q). When data exploration suggested seasonality, additional parameters in the form s required estimation with ‘s’ representing the periodicity of seasonal influence. Thus, the minimum-parameter first order Seasonal ARIMA0 is essentially an aseasonal first-order autoregression that can be characterised as a temporally-correlated random walk, with the current value being influenced only by the value immediately preceding it. Conditional sum of squares  followed by exact maximum likelihood methods were employed to estimate each parameter Temporal variation in foraging behaviour was quantified using univariate ARIMA , upwelling occurred between December and March along the western Eyre Peninsula reaching as far as Cape Bauer near Streaky Bay . Mean benthic conditions also varied significantly for each individual throughout their respective tracking period with the exception of West Island . Thus, when upwelling was present, individuals that foraged in the deepest water experienced the thickest mixed layer and thickest transition layer .Using the threshold criteria of eaky Bay . Upwelleeaky Bay . Cross-seaky Bay . Althougeaky Bay . Betweensmooth = 7.6 and 5.1, respectively, P<0.05 in both cases). Conversely, despite exploiting at-sea regions close to those used by the West Island male, the individual at Nicholas Baudin did not appear to respond to changes in dynamic features, instead decreasing its core range at shallower depths (GAMM Fsmooth(bathymetry) = 44.6, P<0.001) and steeper slopes (GAMM Fsmooth(bathymetry) = 3.5, P<0.05). At Seal Bay the male started to increase its core range when bottom temperatures started to rise above 13°C (GAMM Fsmooth(benthic temperature) = 6.9, P<0.001; Generally, individual movement patterns did not change throughout the study period . HoweverIn terms of residency times along individual tracks, optimal CPH models failed to include any dynamic variable for any individual except for the West Island male, which showed an aversion to warmer bottom temperatures . The mal13C and δ15N ranged from -14.7 to -18.2‰ and 14.8 to 17.7‰, respectively. Mean carbon and nitrogen isotope ratios generally increased along a longitudinal cline  were best characterised by first-order ARIMA models with a seasonal periodicity ranging from 3–12 months  and are retained year-round, in contrast to phocid seals such as the gray seal and harbor seal 13C, a pattern commonly interpreted in other otariid and seabird species as representing seasonal migration There are several assumptions implicit to the interpretation of our isotopic data. Foremost, we had no species-specific whisker growth rates thus our assumption of 3mm representing an isotopic timeline of one month may be incorrect. Furthermore, the possibility that individuals undergo periodic whisker replacement may also have confounded our interpretation. Empirical estimates of whisker growth rates in otariids are restricted to Steller sea lions and Antarctic fur seals 13C and 15N detected in these eastern individuals probably reflects seasonal changes in the structure of prey guilds rather than changes in where individuals foraged. Thus we strongly recommend employing biotelemetry devices to put isotopic data into context, particularly in temperate latitudes or coastal marine environments where isoscapes are poorly defined or highly variable. Assuming our interpretation is correct, we also caution against generalising adult male Australian sea lion diet based on limited temporal or spatial sampling. The apparent long-term fidelity of adult male Australian sea lions to individual colonies we show does provide an opportunity to generate species-specific whisker growth rates. Biological markers such as isotopically-labelled glycine could be administered to individuals during the initial deployment of transmitters, creating a highly-visible marker that would be evident in whiskers removed at the end of a deployment Our study is a cautionary tale about relying solely on biogeochemical markers to infer geotrophic behaviour. The incorporation of prolonged time-series telemetry data and information about oceanography enables us to interpret our isotopic data in a more appropriate manner. There were no consistent trends in adult male at-sea movement that related to dynamic oceanographic variability and the manner in which individuals explored their environment. Adult males in the region that experienced the greatest environmental variation (Kangaroo Island) did not alter their movement patterns throughout the entire upwelling season (Dec – May). Indeed, the individual who foraged on the shelf break consistently occupied similar habitat to those recorded for adult male New Zealand fur seals If suitable prey exists year-round along the South Australian coastline then why do males not forage more broadly, particularly given they are not restricted by the need to provide parental care? Even though some individuals in our study could conceivably reach the same foraging grounds as males from neighboring colonies, they did not do so. This suggests that the parameters we measured were not sufficient to explain strong individual preferences for particular foraging habitat. However, tracking one male at each colony hampers our ability to generalise any behaviour. For example we do not know whether the strategies adopted by individuals we followed are representative of all males at a colony, or are one of several behaviours within a colony Our interpretations may be confounded by assuming that the location at which a male was initially captured represents its home colony. On more than one occasion the Nicholas Baudin male hauled out at nearby Olive Island, the fifth largest breeding colony of the species that at the time of instrumentation was over two months past a breeding episode. Conversely, the Liguanea male made several trips up the Thorny Passage into the southern Spencer Gulf, stopping at two colonies of which one was entering a breeding cycle during the tracking period  fully characterising the extent of individual male variability in foraging behaviour both within and between colonies, particularly in relation to breeding cycles of colonies, and 2) determining whether aseasonal foraging behaviour and an apparent lack of dispersal translates into reduced male-mediated geneflow.Table S1Core (UD-50) and home range (UD-95) estimates for individual adult male Australian sea lions tracked along the South Australian coast between November 2009 and May 2010. Males that travelled the furthest during foraging trips had the largest ARS, core and home range estimates. Conversely, the male at Seal Slide occupied the smallest mean home range utilizing less than 140 km2 of at-sea habitat.(DOCX)Click here for additional data file.Table S2The top four Generalised Additive Mixed Models (estimated using δAIC) which best fitted ARS patch size, UD-50 and UD-95 estimates for adult male Australian sea lions tracked along the South Australian coast. Dynamic and static environmental parameters failed to significantly explain variation in ARS patch size of any male. Similarly, core and home ranges for three males were unrelated to any estimated parameter. For the remaining males, the only dynamic environmental parameters retained in any model were temperature and salinity at the benthos (significant results highlighted in bold).(DOCX)Click here for additional data file.Table S3Cox Proportional Hazard models fitted to adult male Australian sea lion data, using First Passage Time (FPT) as the independent variable sensu Freitas et al. (2008). FPT variability of three individuals were most parsimoniously explained by a null model. Exponential coefficient values greater than +1 suggest an aversion to the associated environmental parameter. Thus, males at West Island, Liguanea and West Waldegrave stopped foraging in the presence of increasing benthic temperature and slope, respectively.(DOCX)Click here for additional data file.Table S413C) and nitrogen (δ15N) from the whiskers of seven adult male Australian sea lions instrumented with CTD-SRDL transmitters along the South Australian coastline.Seasonal Autoregressive Integrated Moving Average (SARIMA) model results for stable isotope ratios of carbon  that identify the order of autoregressive (AR) correlation (p), the differencing required for stationarity (d) and the moving average (MA) order (q). Considering stable isotope ratios as a time series, when a seasonal pattern is observed then a seasonal model is added to the ARIMA of the form , with P, D and Q reflecting the order of AR, differencing and MA component of the seasonal pattern. The number to the right of the seasonal model indicates the lag (in months) of the seasonal term – thus, ‘12’ indicates a seasonal periodicity of 12 months. Ljung-Box Q statistics and associated P values reflect the degree of autocorrelation in SARIMA model residuals, analogous to testing for patterns in the residuals of regression models. Seasonal AR(1) patterns were detected in all 13C and three 15N isotopic time series. ‘-’ denotes models could not be fitted, or were insignificant. Note, that while seasonal patterns may be detected, the model does not reflect the biological significance of these patterns. For example, although models from the western colonies (i.e West Island and Nicholas Baudin) showed significant patterns with an almost annual cycle, the visualisation of isotope ratios in (DOCX)Click here for additional data file.


The cylindromatosis tumor suppressor (CYLD) is a deubiquitinating enzyme that has been implicated in various aspects of adaptive and innate immune responses. Nevertheless, the role of CYLD in the function of specific types of immune cells remains elusive. In this report we have used conditional gene targeting in mice to address the role of the deubiquitinating activity of CYLD in the myelomonocytic lineage. Truncation of the deubiquitinating domain of CYLD specifically in myelomonocytic cells impaired the development of lethal LPS-induced endotoxic shock and the accumulation of thioglycollate-elicited peritoneal macrophages. Our data establish CYLD as a regulator of monocyte-macrophage activation in response to inflammatory stimuli and identify it as a potential target for therapeutic intervention in relevant inflammatory disorders in humans. Cyld was identified as the predisposition gene for the tumor syndrome of familial cylindromatosis. It mediates the suppression of the NF-κB, JNK, p38 and Wnt pathways in a manner that depends on its deubiquitinating activity  and investigate a plausible implication of CYLD in the development and function of the myelomonocytic lineage. In accordance with previous studies no obvious hematopoietic defects were observed. However, Δ9Cyld mice exhibited increased resistance to LPS induced endotoxic shock. Moreover, in an in vivo model of induced peritonitis CYLD-deficient macrophages failed to accumulate to the inflammatory site. Overall, our data demonstrate that Cyld is dispensable for hematopoiesis but unravel a cell intrinsic function of Cyld in monocyte-macrophage response to inflammatory stimuli.In the present study we employed a conditional gene targeting approach to introduce a cell-specific deubiquitinating-domain-truncating mutation in the Cyld locus (flx9/flx9Cyld ), has been described elsewhere LysMcre transgenic mice we aimed to render murine CYLD catalytically inactive in myelomonocytic cells, by truncating its catalytic domain through Cre-mediated excision of exon 9flx9/flx9LysMCre-Cyld mice (termed Δ9M-Cyld mice from this point onwards), the effective excision of Cyld exon 9 was readily evident both in bone marrow purified progenitor cells  (+Gr1−) and neutrophils (CD11b+Gr1+) were dramatically decreased in Δ9M-Cyld mice in comparison to control mice (Δ9Cyld macrophages that were recruited to the inflammatory site exhibited similar expression pattern of the activation maker F4/80 (data not shown). At this point it is not clear whether Δ9Cyld macrophages cannot be recruited due to defects in the migration process itself as a result of microtubule reorganization problems Cyld-deficient macrophages at the site of inflammation reflects an increased propensity of these cells to death following their recruitment to the site of inflammation. Nevertheless, it is undisputable that in the context of chemically induced aseptic peritonitis Δ9Cyld macrophages are unable to respond properly.In order to investigate further the implication of 0000248) . Interesrol mice . NotablyΔ9M-Cyld mice permitted the functional evaluation of CYLD in the myelomonocytic lineage and revealed an anti-inflammatory element of the functional profile of this molecule. Our findings add to a growing list of evidence that highlight the discrete and in certain cases contradictory tissue-specific activities of CYLD. For example, inactivation of CYLD in thymocytes leads to excessive apoptosis and impaired development Cyld expression causes their hyperresponsiveness which can be associated with spontaneous autoimmune and inflammatory symptoms The development and characterization of In summary, our experiments identified a cell-intrinsic requirement of functional CYLD for the efficient response of macrophages to different inflammatory stimuli and raise the possibility of targeting CYLD with inhibitory molecules as a pharmacological approach to control certain cases of pathogenic inflammation.Experiments on live animals were approved by the Hellenic Ministry of Rural Development- and by Biomedical Sciences Research Center “Al. Fleming's Animal Research and Ethics Committee for compliance to FELASA regulations .flx9]/flx9Cyld  mice has been described elsewhere flx9]/flx9Cyld  mice were crossed with LysMcre mice Cyld in myelomonocytic cells. The mice were bred and maintained in the animal facilities of the Biomedical Sciences Research Centre “Alexander Fleming” under specific-pathogen free conditions.The generation of BMDM were generated as previously described Mice of 8 weeks age were intraperitoneally injected with a sublethal dose of 800 µg LPS (Sigma)/25 g animal weight and survival was monitored for 5 days.Nuclear extracts were prepared by BMDMs and EMSA was performed as previously described The sequence of the oligonucleotides used for NF-κB with two tandemly repeated NF-κB binding sites (underlined) were as follows:G GAC TTT CCG CTG GGG ACT TT- 3′5′ -ATC AGNF-κBf: CGG AAA GTC CCC AGC GGA AAG TCC CT-3′5′-NF-κBr: Δ9M-Cyld  mice were injected i.p. with 1 ml of 4% sterile thioglycollate (Becton Dickinson). 72 h later mice were sacrificed and the leukocyte number in the peritoneal lavage was assessed by a Coulter Counter. The percentage of the different cell types was assessed by Flow Cytometry.Control and Cell-associated fluorescence was analyzed by a FACS Aria or a FACSCantoII flow cytometer and the DIVA V6 software (Becton Dickinson). Sorting was performed with BD FACS Vantage SE II. Flow cytometry figures were prepared using the FlowJo software . Differences in populations were determined using the unpaired t test as calculated with Sigmaplot 9 statistical software.5 cells/well) were incubated with 100 ng/ml LPS  and 24 h later the culture supernatants were collected.For the TNFα  ELISA assay, BMDMs cultures , F4/80 (eBioscience), Lin- enriched fraction from bone marrow was prepared by staining with biotinylated Ter119, B220, Cd11b, Gr1, CD3 (eBioscience). Cylindromatosis and TFIIB were purchased from Santa Cruz. The anti-actin mouse monoclonal antibody was purchased from MP Biomedical Inc.


The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca2+ signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca2+]cyt in PASMC is increased by Ca2+ influx through Ca2+ channels in the plasma membrane and by Ca2+ release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca2+ entry pathways, voltage-dependent Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) and voltage-independent Ca2+ influx through store-operated Ca2+ channels (SOC) and receptor-operated Ca2+ channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH.Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling,  The only organ in the body to receive the entire cardiac output (CO) at one time is the lung. To receive a high flow of volume from the entire CO, the pulmonary circulatory system must maintain a low-resistance and low-pressure system to carry blood to the pulmonary capillaries. Deoxygenated venous blood flows through the pulmonary artery to the pulmonary capillaries where oxygen and carbon dioxide gas exchange occurs. Pulmonary hypertension (PH) is a severe chronic disorder that affects the pulmonary circulatory system. This disorder is often a deadly hemodynamic irregularity that may be idiopathic, heritable, or secondary to other diseases such as chronic obstructive pulmonary disease (COPD). Pulmonary arteries are thin and have low myogenic tone compared to systemic arteries. Therefore, pulmonary arteries rely on arterial distension and recruitment for decreasing pulmonary vascular resistance (PVR) after increased blood flow or increased CO . In the lungs, increased PVR is the major cause for the development of PH. An increased PVR results in increased RV afterload which leads to right heart failure and eventually death . arteries + PVRcapillaries + PVRveins) . , 5. 4, 5There are 15 orders of branching in the pulmonary arteries between the main pulmonary artery and the capillaries in the human lung . The pulin situ thrombosis, and arterial wall stiffening, together these attributes increase PVR leading to right heart failure cyt in PASMC, which leads to smooth muscle contraction by both Ca2+-dependent and Ca2+-independent mechanisms. Ca2+ directly activates myosin light chain kinase (MLCK) leading to contraction and migration. Additionally, an increase in [Ca2+]cyt stimulates Ca2+-dependent signal transduction proteins. In a healthy individual, the pulmonary circulatory system has low resistance and easily dilates to accommodate the entire CO with a marginal change in pressure. In contrast to the systemic circulatory system, which has higher resistance and pressure, hypoxia causes the pulmonary arteries to constrict while the systemic arteries to dilate [2+]cyt in PASMC, increased contractile elements in lung tissue, and enhanced sensitivity of contractile proteins. Several studies in the past have demonstrated that the amplitude of contraction can be affected by any change in the ratio of myosin light chain kinase (MLCK): MLCP activity. MLCK activity is dependent on Ca2+ calmodulin; hence, the rise in [Ca2+]cyt is the primary determinant of smooth muscle contraction. The increase in sensitivity of smooth muscle contractility to [Ca2+]cyt  may be caused by inhibition of MLCP activity. It has been found that the predominant pathological cause in idiopathic pulmonary arterial hypertension (IPAH) in patients is a loss of vascular compliance and an increase in PVR due to pulmonary vascular remodeling and vasoconstriction cyt in PASMC. Vascular smooth muscle contraction decreases the radius of pulmonary blood vessels, leading to sustained vasoconstriction and to increased PVR. The mechanism of contraction in pulmonary vascular smooth muscle is different than that of striated cardiac or skeletal muscles. Skeletal muscles undergo fast contractions, which involve Ca2+ binding to troponin. However, in PASMC slow, tonic contractions are initiated through changes in [Ca2+]cyt that cause a Ca2+-activated phosphorylation of myosin. When [Ca2+]cyt rises, Ca2+ binds to calmodulin (CaM) and activates myosin light chain kinase (MLCK) which then phosphorylates the myosin light chain. Phosphorylation of MLC increases myosin ATPase activity that hydrolyzes ATP to release energy. The subsequent cycling of the myosin cross-bridges produces a sliding motion of myosin-actin filaments and results in contraction of the smooth muscles (2+ blocks the high K+-induced and phenylephrine- (PE-) induced contraction in isolated rat pulmonary arterial rings, illustrating that Ca2+ influx is necessary for smooth muscle contraction  and transcription factors  can stimulate cell proliferation (2+]cyt is an important stimulus for cellular proliferation (2+ in the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) is vital for cell growth cyt is essential for cell proliferation.In addition to causing PASMC contraction, increased .–30.2+]cyferation  31].In .In 2+]cyl growth . It has d growth . Ca2+ incle (G1) . Thus, t2+-dependent pathways play an important role in the regulation of numerous physiological and pathophysiological processes in PASMC such as contraction, proliferation, and migration cyt, and contractile Ca2+ sensitivity, altogether contributing to vasoconstriction, and thus leading to PH. Ca2+ influx in PASMC is mainly regulated by voltage-dependent Ca2+ channels and voltage-independent nonselective cation channels. Intracellular Caigration . Studies2+ channels (VDCC)  have established roles in the regulation of blood pressure; and dihydropyridine Ca2+ channel blockers have been clinically used for the treatment of hypertension. The Ca2+ influx mechanisms in PASMC via VDCC are influenced by changes in the membrane potential, an electrochemical driving force for Ca2+ entry. PASMCs maintain a resting membrane potential (EM) at about −40 to −60 mV, which is significantly less negative than the equilibrium potential for K+ (EK is about −85 mV), suggesting that resting EM is regulated not only by K+ currents, but also background cation  currents through voltage-independent cation channels and/or nonselective cation channels . At rest, the membrane is more permeable to K+, so background K+ currents through voltage-dependent and voltage-independent K+ channels are the major determinants for the resting EM, although Na+, Cl−, and Ca2+ currents also contribute to the regulation of the resting EM. The activity of various K+ channels in the plasma membrane contributes to maintaining the EM of PASMC. Inhibition of K+ channels causes membrane depolarization, whereas activation of K+ channels causes membrane repolarization and hyperpolarization. Among more than 50 different types of K+ channels, voltage-gated K+ (KV) channels are shown to be ubiquitously expressed in smooth muscle cells cyt , or capacitative Ca2+ entry (CCE), is identified as one of the important mechanisms that regulate [Ca2+]cyt, as addressed by Casteels and Droogmans in early 80s. The groups of channels that mediate SOCE are termed as store-operated Ca2+ channels (SOC) cyt and thus produces vasoconstriction, promotes cell cycle progression, and ultimately leads to PASMC proliferation and vascular remodeling cyt in a membrane-delimited fashion. TRPC6 is insensitive to PLC-IP3 activation at the cellular level cyt may presumably induce BKCa (like TRPV4) and regulate membrane potential depolarization. Thus, TRPM4-mediated depolarization may support Ca2+ influx. These findings were further substantiated by diminishing the expression of TRPM4 in the cerebral vasculature, which resulted in a loss of autoregulation of cerebral blood flow in response to changes in perfusion pressure cyt. Variations in the expression and function of the ion channels necessary for Ca2+ influx are key features in the development and pathogenesis of PAH. In PAH, a rise in [Ca2+]cyt in PASMC due to enhanced SOCE/ROCE (due to upregulated SOC/SOC components) and voltage-dependent Ca2+ entry in PASMC from IPAH patients also activates many signal transduction proteins  and transcription factors  thus influencing gene expression and promoting cell proliferation. Experiments have therefore shown that Ca2+ channels and significant signaling pathways are possible sites for therapeutic targets. In order to understand the pathogenic mechanisms of PAH, it is important to consider CaFuture experiments detailing the specific downstream effects of enhanced SOCE and ROCE will shed light on important therapeutic strategies for PAH. More specifically, understanding the role of other proteins such as transcription factors, signal transduction proteins, and nuclear transporters and their effect on vasoconstriction and proliferation could provide future pharmacological blockades of SOC/ROC. Additionally, downregulation of these proteins could reveal therapeutic effects for PH. Application of new experimental methods along with further use of well-established methods is crucial to the advancement of the field of pulmonary hypertension.


The development of forward genetic screens in human haploid cells has the potential to transform our understanding of the genetic basis of cellular processes unique to man. So far, this approach has been limited mostly to the identification of genes that mediate cell death in response to a lethal agent, likely due to the ease with which this phenotype can be observed. Here, we perform the first reporter screen in the near-haploid KBM7 cell line to identify constitutive inhibitors of NF-κB. CYLD was the only currently known negative regulator of NF-κB to be identified, thus uniquely distinguishing this gene. Also identified were three genes with no previous known connection to NF-κB. Our results demonstrate that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors. Caenorhabditis elegansDrosophila,  and other genetic model organisms to identify new gene functions. Application of this method to human cultured cells allows the dissection of pathways that are dissimilar or even absent in other model organisms. It may also enable the discovery of novel drug targets to treat disease. Genetic screens in human cells have been limited by the difficulties inherent in revealing recessive phenotypes in diploid cells. While RNAi screens have been an important advance, they are complicated by off-target effects and often do not completely eliminate the relevant gene product. The recent isolation of human cells lines that are nearly or completely haploid  has revolutionized human forward genetic screens and led to the identification of numerous human host factors required for infection by pathogens and intoxication by bacterial toxins [Forward genetic screens are a powerful means to decipher a biological process without any prior knowledge or assumptions. Typically such screens are performed in yeast, The majority of human haploid screens reported to date have involved the selection of mutants that are resistant to an agent that is lethal to wild-type cells. The one exception is a recent screen that used fluorescence activated-cell sorting (FACS) to identify genes involved in MHC (major histocompatibility complex) class I antigen presentation by sorting for mutants that were defective in surface expression of MHC-1 . We sougTranscription factors often lie at the terminus of complex signaling pathways and control gene transcription programs that regulate diverse processes, ranging from proliferation, differentiation, apoptosis, immune response, to metabolism. Given the importance of transcription factors in facilitating vital aspects of cell biology, mutations in -or aberrant regulation of-transcription factors have been associated with human disease ,10. The We chose to screen for inhibitors of NF-κB, a family of transcription factors that in mammals plays a central role in regulating immune responses, development, cell proliferation, and survival . The NF-Here we performed the first reporter screen in KBM7 cells to identify constitutive inhibitors of NF-κB. The identification of CYLD, a known negative regulator of NF-κB, demonstrates the utility of using human haploid cells to dissect a variety of biological processes.BSR) from Bacillus cereus. Thus, insertional inactivation of genes that normally repress activation of NF-κB would render the reporter cells resistant to blasticidin and provide an easy means to distinguish them from wild-type cells. To ensure that the selected clonal reporter cell line had intact NF-κB regulation, we stimulated both KBM7 cells and the NF-κB reporter cell line with TNF , respectively    and 5.CYLD encodes a deubiquitylase (DUB) that targets NF-κB signaling factors and is known to negatively regulate NF-κB activation [CYLD in NF-κB reporter cells  domains, a protein fold found in a variety of proteins including the four that its name derives from. HEATR7A is located on chromosome 8, the only chromosome to be present in two copies in KBM7 cells suggesting that the mutations caused by the gene trap insertions in HEATR7A are of a dominant nature.LRRC8D gene. While the LRRC8D mutant cell lines were notably resistant to blasticidin, examination by immunoblotting revealed no obvious impact of LRRC8D deficiency on IκBα and p100 degradation (data not shown). Our attempts to demonstrate a possible effect of the LRRC8D disruptions on blasticidin import as an explanation for the observed level of resistance were inconclusive, as the fluorescently labeled or biotinylated versions of blasticidin we prepared were themselves inactive on KBM7 cells. Thus we can neither exclude the possibility that resistance is conferred by differences in intracellular blasticidin levels, nor discount the possibility of alternative modes of NF-κB activation not accounted for by degradation of IκBα and p100 [LRRC8A and LRRC8D represent two of five members of the leucine-rich repeat-containing 8 (LRRC8) family of proteins that are composed of four transmembrane domains at the N-terminus, followed by up to 17 leucine-rich repeats . Their fand p100 .Most genetic screens performed in human haploid cells have sought to identify components in pathways required for cell death in response to a lethal insult. Here we have demonstrated that KBM7 cells can be modified with genetically encoded transcriptional reporters to study more diverse cellular processes. While we chose to screen for regulators -specifically, inhibitors-of NF-κB, our method could presumably be applied to study the approximately 1,391 human sequence specific DNA binding transcription factors, many of whose binding site profiles have recently been described .By using resistance to blasticidin as our reporter read-out, we were able to perform a selection -a genetic screen where only mutants of interest survive-to identify mutants that constitutively turned on the reporter. In principle, one could perform a screen in a reversed fashion, in which only mutants that fail to turn on the reporter survive, for example by exploiting thymidine kinase or some other protein whose expression could induce cell death. Identification of positive regulators of transcription factors should thus be possible. Fluorescent reporters could likewise be used in screens for both positive and negative transcriptional regulators. Since stringency in this type of screen would be more adjustable than in a lethal screen, mutations that result in intermediate phenotypes might be more easily recovered.While the identification of CYLD validated our approach, we were unable to identify mutations in other known negative regulators of NF-κB. Perhaps the selection we performed was particularly stringent. Presumably, only mutations that resulted in constitutive activation of NF-κB could be recovered. Thus, inhibitors whose function or expression is induced by NF-κB in a negative feedback loop, such as A20 and Cezanne, may not meet that criterion. In addition, loss-of-function mutations in dominant-negative adaptors such as MyD88s, IRAK-M, and SARM would not result in constitutive activation of NF-κB. We did not recover mutations in IκBs, possibly because there is some redundancy in function in KBM7 cells and removal of just one IκB is not sufficient for constitutive activation of NF-κB. In contrast, CYLD qualifies as a constitutively active inhibitor that prevents spontaneous ubiquitylation of its targets . CYLD muOur haploid reporter screen confirms the absolute requirement of CYLD function for proper regulation of NF-κB and further supports constitutive NF-κB activity as the mechanism underlying the development of human diseases associated with CYLD mutations. Our screen identified genes not previously known to be involved with NF-κB regulation. Their exact role remains to be determined. The ability to perform haploid reporter screens in human cultured cells opens up many new cellular processes for investigation.Bacillus cereus BSR-Thosea asigna virus 2A sequence-XbaI-Rabbit CYP4B1. Lentiviral shRNAs were obtained from The RNAi Consortium (TRC) collection of the Broad Institute. The TRC numbers for the shRNAs used are TRCN0000072245 (shLuc) and TRCN0000039632 (shCYLD).The NF-κB reporter was created by digesting pTRH1-NF-κB-dscGFP (System Biosciences) with SpeI and SalI to remove the dscGFP. The same restriction sites were used to insert a synthesized DNA fragment encoding mCMV-NheI-Kozak sequence-BamHI-KBM7 cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) with 10% heat-inactivated fetal serum (IFS) ,26. A NFThe screening procedure has been described previously –6. BriefThe mapping of the insertion sites was done as previously described –6. In shTwo million cells were used per condition. Cells were washed once in ice-cold PBS and then lysed in buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 mM EDTA, protease inhibitors (Roche) and 1% (v/v) NP40. Protein concentrations of lysates were determined by Bio-Rad Protein Assay and then normalized across samples. Proteins from total lysates were resolved by 10% SDS-PAGE and analyzed by immunoblotting with primary antibodies: mouse anti-IκBα (BD #610690) at 1:500 dilution and mouse anti-actin (BD #612656) at 1:10,000. Horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (GE NXA931) was used at 1:5,000 dilution. Restore PLUS Western Blot Stripping Buffer (Thermo) was used to strip the membranes between probing for IκBα and actin.CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to quantify cell viability. 200,000 cells were seeded per well in Optilux clear-bottom 96-well plates  in IMDM or IMDM supplemented with varying concentrations of Blasticidin S (Invivogen), TNF-α (Invivogen), FSL-1 (Invivogen) for 24 hours before reading on a Luminoskan Ascent luminometer (Thermo Scientific).CYLD were 5’ GGTAATCCGTTGGATCGGTCAGC 3’ and 5’ TGCAAACCTAGAGTCAGGCCTGC 3’. Primers used for GAPDH were 5’ ACCCACTCCTCCACCTTTGACG 3’ and 5’ CACCCTGTTGCTGTAGCCAAATTCG 3’.RNA was extracted using a RNAeasy kit (QIAGEN) followed by on-column DNase I (QIAGEN) digestion. SuperScript III First-Strand Synthesis System (Invitrogen) was used for the reverse transcription reaction using Oligo dT primers. SYBR Green PCR Master Mix (Applied Biosystems) was used according to manufacturer’s instructions. Real-time PCR reactions were run on an ABI 7900HT machine. PCR volume was 20 µL (96-well plate), and data values were derived from three replicates using the comparative Ct method. Primers used for


To perform 3D imaging of the left and right coronary vessel walls in a single interleaved acquisition.Vessel wall imaging is commonly gated to alternate R-waves, in order to increase SNR and minimise artefacts due to RR-interval variation. In previous work with 2D carotid artery wall imaging, the resulting redundant RR-interval has been used to acquire additional parallel slices. We postA free-breathing 3D spiral coronary artery wall imaging sequence was modified in order to acquire two high resolution 3D volumes , acquisition duration 600 cardiac cycles assuming 100% respiratory efficiency). The first and second volumes were imaged on odd and even cardiac cycles respectively (figure Acquisitions were performed in healthy subjects using a Siemens Avanto 1.5T scanner. The first and second volumes were positioned to obtain cross-sectional left and right coronary images respectively. The first reinversion slab was positioned to selectively reinvert the left coronary artery whilst avoiding reinversion of aortic blood and minimising reinversion of the tissue imaged in the right coronary artery volume. The second reinversion slab was similarly positioned for the right coronary artery. Inversion time was 400ms which is optimal for single R-wave gating at 60beats/minute.Left and right coronary artery wall images obtained simultaneously in 629 cardiac cycles are shown in figure We have demonstrated simultaneous left and right coronary artery wall imaging in the duration required to image a single artery. Used in conjunction with B2B-RMC, this has permitted high resolution 3D imaging of both the left and right coronary walls in approximately 10minutes. Future work will include a comparison with conventional alternate R-wave gating acquisitions and an investigation of the robustness of the technique to changes in RR interval.