Vascular Smooth Muscle Cell Stiffness and Adhesion to Collagen I Modified by Vasoactive Agonists

In vascular smooth muscle cells (VSMCs) integrin-mediated adhesion to extracellular matrix (ECM) proteins play important roles in sustaining vascular tone and resistance. The main goal of this study was to determine whether VSMCs adhesion to type I collagen (COL-I) was altered in parallel with the changes in the VSMCs contractile state induced by vasoconstrictors and vasodilators. VSMCs were isolated from rat cremaster skeletal muscle arterioles and maintained in primary culture without passage. Cell adhesion and cell E-modulus were assessed using atomic force microscopy (AFM) by repetitive nano-indentation of the AFM probe on the cell surface at 0.1 Hz sampling frequency and 3200 nm Z-piezo travelling distance (approach and retraction). AFM probes were tipped with a 5 μm diameter microbead functionalized with COL-I (1mg\ml). Results showed that the vasoconstrictor angiotensin II (ANG-II; 10−6 ) significantly increased (p<0.05) VSMC E-modulus and adhesion probability to COL-I by approximately 35% and 33%, respectively. In contrast, the vasodilator adenosine (ADO; 10−4 ) significantly decreased (p<0.05) VSMC E-modulus and adhesion probability by approximately −33% and −17%, respectively. Similarly, the NO donor (PANOate, 10−6 M), a potent vasodilator, also significantly decreased (p<0.05) the VSMC E-modulus and COL-I adhesion probability by −38% and −35%, respectively. These observations support the hypothesis that integrin-mediated VSMC adhesion to the ECM protein COL-I is dynamically regulated in parallel with VSMC contractile activation. These data suggest that the signal transduction pathways modulating VSMC contractile activation and relaxation, in addition to ECM adhesion, interact during regulation of contractile state

Does Applicability Domain Exist in Microarray-Based Genomic Research?

Constructing an accurate predictive model for clinical decision-making on the basis of a relatively small number of tumor samples with high-dimensional microarray data remains a very challenging problem. The validity of such models has been seriously questioned due to their failure in clinical validation using independent samples. Besides the statistical issues such as selection bias, some studies further implied the probable reason was improper sample selection that did not resemble the genomic space defined by the training population. Assuming that predictions would be more reliable for interpolation than extrapolation, we set to investigate the impact of applicability domain (AD) on model performance in microarray-based genomic research by evaluating and comparing model performance for samples with different extrapolation degrees. We found that the issue of applicability domain may not exist in microarray-based genomic research for clinical applications. Therefore, it is not practicable to improve model validity based on applicability domain

The Mechanism of Enhanced Insulin Amyloid Fibril Formation by NaCl Is Better Explained by a Conformational Change Model

The high propensity of insulin to fibrillate causes severe biomedical and biotechnological complications. Insulin fibrillation studies attain significant importance considering the prevalence of diabetes and the requirement of functional insulin in each dose. Although studied since the early years of the 20th century, elucidation of the mechanism of insulin fibrillation has not been understood completely. We have previously, through several studies, shown that insulin hexamer dissociates into monomer that undergoes partial unfolding before converting into mature fibrils. In this study we have established that NaCl enhances insulin fibrillation mainly due to subtle structural changes and is not a mere salt effect. We have carried out studies both in the presence and absence of urea and Gdn.HCl and compared the relationship between conformation of insulin induced by urea and Gdn.HCl with respect to NaCl at both pH 7.4 (hexamer) and pH 2 (monomer). Fibril formation was followed with a Thioflavin T assay and structural changes were monitored by circular dichroism and size-exclusion chromatography. The results show salt-insulin interactions are difficult to classify as commonly accepted Debye-Hückel or Hofmeister series interactions but instead a strong correlation between the association states and conformational states of insulin and their propensity to fibrillate is evident

Identification of PLCL1 Gene for Hip Bone Size Variation in Females in a Genome-Wide Association Study

Osteoporosis, the most prevalent metabolic bone disease among older people, increases risk for low trauma hip fractures (HF) that are associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of the key measurable risk factors for HF. Although hip BS is highly genetically determined, genetic factors underlying the trait are still poorly defined. Here, we performed the first genome-wide association study (GWAS) of hip BS interrogating ∼380,000 SNPs on the Affymetrix platform in 1,000 homogeneous unrelated Caucasian subjects, including 501 females and 499 males. We identified a gene, PLCL1 (phospholipase c-like 1), that had four SNPs associated with hip BS at, or approaching, a genome-wide significance level in our female subjects; the most significant SNP, rs7595412, achieved a p value of 3.72×10−7. The gene's importance to hip BS was replicated using the Illumina genotyping platform in an independent UK cohort containing 1,216 Caucasian females. Two SNPs of the PLCL1 gene, rs892515 and rs9789480, surrounded by the four SNPs identified in our GWAS, achieved p values of 8.62×10−3 and 2.44×10−3, respectively, for association with hip BS. Imputation analyses on our GWAS and the UK samples further confirmed the replication signals; eight SNPs of the gene achieved combined imputed p values<10−5 in the two samples. The PLCL1 gene's relevance to HF was also observed in a Chinese sample containing 403 females, including 266 with HF and 177 control subjects. A SNP of the PLCL1 gene, rs3771362 that is only ∼0.6 kb apart from the most significant SNP detected in our GWAS (rs7595412), achieved a p value of 7.66×10−3 (odds ratio = 0.26) for association with HF. Additional biological support for the role of PLCL1 in BS comes from previous demonstrations that the PLCL1 protein inhibits IP3 (inositol 1,4,5-trisphosphate)-mediated calcium signaling, an important pathway regulating mechanical sensing of bone cells. Our findings suggest that PLCL1 is a novel gene associated with variation in hip BS, and provide new insights into the pathogenesis of HF

Defining the Plasticity of Transcription Factor Binding Sites by Deconstructing DNA Consensus Sequences: The PhoP-Binding Sites among Gamma/Enterobacteria

Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs) using a machine learning method inspired by the “Divide & Conquer” strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target genes and/or the promoter architectures resulting from the interaction of those binding sites with the RNA polymerase

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Wolbachia Bacteria Reside in Host Golgi-Related Vesicles Whose Position Is Regulated by Polarity Proteins

Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration

Transcriptional Portrait of Actinobacillus pleuropneumoniae during Acute Disease - Potential Strategies for Survival and Persistence in the Host

BACKGROUND: Gene expression profiles of bacteria in their natural hosts can provide novel insight into the host-pathogen interactions and molecular determinants of bacterial infections. In the present study, the transcriptional profile of the porcine lung pathogen Actinobacillus pleuropneumoniae was monitored during the acute phase of infection in its natural host. METHODOLOGY/PRINCIPAL FINDINGS: Bacterial expression profiles of A. pleuropneumoniae isolated from lung lesions of 25 infected pigs were compared in samples taken 6, 12, 24 and 48 hours post experimental challenge. Within 6 hours, focal, fibrino hemorrhagic lesions could be observed in the pig lungs, indicating that A. pleuropneumoniae had managed to establish itself successfully in the host. We identified 237 differentially regulated genes likely to encode functions required by the bacteria for colonization and survival in the host. This group was dominated by genes involved in various aspects of energy metabolism, especially anaerobic respiration and carbohydrate metabolism. Remodeling of the bacterial envelope and modifications of posttranslational processing of proteins also appeared to be of importance during early infection. The results suggested that A. pleuropneumoniae is using various strategies to increase its fitness, such as applying Na+ pumps as an alternative way of gaining energy. Furthermore, the transcriptional data provided potential clues as to how A. pleuropneumoniae is able to circumvent host immune factors and survive within the hostile environment of host macrophages. This persistence within macrophages may be related to urease activity, mobilization of various stress responses and active evasion of the host defenses by cell surface sialylation. CONCLUSIONS/SIGNIFICANCE: The data presented here highlight the importance of metabolic adjustments to host conditions as virulence factors of infecting microorganisms and help to provide insight into the mechanisms behind the efficient colonization and persistence of A. pleuropneumoniae during acute disease

A Network-Based Approach to Prioritize Results from Genome-Wide Association Studies

Genome-wide association studies (GWAS) are a valuable approach to understanding the genetic basis of complex traits. One of the challenges of GWAS is the translation of genetic association results into biological hypotheses suitable for further investigation in the laboratory. To address this challenge, we introduce Network Interface Miner for Multigenic Interactions (NIMMI), a network-based method that combines GWAS data with human protein-protein interaction data (PPI). NIMMI builds biological networks weighted by connectivity, which is estimated by use of a modification of the Google PageRank algorithm. These weights are then combined with genetic association p-values derived from GWAS, producing what we call ‘trait prioritized sub-networks.’ As a proof of principle, NIMMI was tested on three GWAS datasets previously analyzed for height, a classical polygenic trait. Despite differences in sample size and ancestry, NIMMI captured 95% of the known height associated genes within the top 20% of ranked sub-networks, far better than what could be achieved by a single-locus approach. The top 2% of NIMMI height-prioritized sub-networks were significantly enriched for genes involved in transcription, signal transduction, transport, and gene expression, as well as nucleic acid, phosphate, protein, and zinc metabolism. All of these sub-networks were ranked near the top across all three height GWAS datasets we tested. We also tested NIMMI on a categorical phenotype, Crohn’s disease. NIMMI prioritized sub-networks involved in B- and T-cell receptor, chemokine, interleukin, and other pathways consistent with the known autoimmune nature of Crohn’s disease. NIMMI is a simple, user-friendly, open-source software tool that efficiently combines genetic association data with biological networks, translating GWAS findings into biological hypotheses