Stroma Cell-Derived Factor-1α Signaling Enhances Calcium Transients and Beating Frequency in Rat Neonatal Cardiomyocytes

Stroma cell-derived factor-1α (SDF-1α) is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency.Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP₃R) blocker, but not with a ryanodine receptor (RyR) antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP₃R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax.These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP₃-gated fashion leading to a positive chronotropic and inotropic effect.     

Opposite Effect of Mast Cell Stabilizers Ketotifen and Tranilast on the Vasoconstrictor Response to Electrical Field Stimulation in Rat Mesenteric Artery

Objectives

We analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries.

Methods

Electrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O₂ ^.-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed.

Results

EFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O₂ ^- release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA.

Conclusion

While both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.     

Epistatic Interaction between BANK1 and BLK in Rheumatoid Arthritis: Results from a Large Trans-Ethnic Meta-Analysis

Background

BANK1 and BLK belong to the pleiotropic autoimmune genes; recently, epistasis between BANK1 and BLK was detected in systemic lupus erythematosus. Although BLK has been reproducibly identified as a risk factor in rheumatoid arthritis (RA), reports are conflicting about the contribution of BANK1 to RA susceptibility. To ascertain the real impact of BANK1 on RA genetic susceptibility, we performed a large meta-analysis including our original data and tested for an epistatic interaction between BANK1 and BLK in RA susceptibility.

Patients and Methods

We investigated data for 1,915 RA patients and 1,915 ethnically matched healthy controls genotyped for BANK1 rs10516487 and rs3733197 and BLK rs13277113. The association of each SNP and RA was tested by logistic regression. Multivariate analysis was then used with an interaction term to test for an epistatic interaction between the SNPs in the 2 genes.

Results

None of the SNPs tested individually was significantly associated with RA in the genotyped samples. However, we detected an epistatic interaction between BANK1 rs3733197 and BLK rs13277113 (P_interaction = 0.037). In individuals carrying the BLK rs13277113 GG genotype, presence of the BANK1 rs3733197 G allele increased the risk of RA (odds ratio 1.21 [95% confidence interval 1.04–1.41], P = 0.015. Combining our results with those of all other studies in a large trans-ethnic meta-analysis revealed an association of the BANK1 rs3733197 G allele and RA (1.11 [1.02–1.21], P = 0.012).

Conclusion

This study confirms BANK1 as an RA susceptibility gene and for the first time provides evidence for epistasis between BANK1 and BLK in RA. Our results illustrate the concept of pleiotropic epistatic interaction, suggesting that BANK1 and BLK might play a role in RA pathogenesis.     

Discovery of the Inhibitory Effect of a Phosphatidylinositol Derivative on P-Glycoprotein by Virtual Screening Followed by In Vitro Cellular Studies

P-glycoprotein is capable of effluxing a broad range of cytosolic and membrane penetrating xenobiotic substrates, thus leading to multi-drug resistance and posing a threat for the therapeutic treatment of several diseases, including cancer and central nervous disorders. Herein, a virtual screening campaign followed by experimental validation in Caco-2, MDKCII, and MDKCII mdr1 transfected cell lines has been conducted for the identification of novel phospholipids with P-gp transportation inhibitory activity. Phosphatidylinositol-(1,2-dioctanoyl)-sodium salt (8∶0 PI) was found to significantly inhibit transmembrane P-gp transportation in vitro in a reproducible-, cell line-, and substrate-independent manner. Further tests are needed to determine whether this and other phosphatidylinositols could be co-administered with oral drugs to successfully increase their bioavailability. Moreover, as phosphatidylinositols and phosphoinositides are present in the human diet and are known to play an important role in signal transduction and cell motility, our finding could be of substantial interest for nutrition science as well.     

Characteristics of Randomized Trials Published in Latin America and the Caribbean According to Funding Source

Introduction

Few studies have assessed the nature and quality of randomized controlled trials (RCTs) in Latin America and the Caribbean (LAC).

Methods and Findings

The aims of this systematic review are to evaluate the characteristics (including the risk of bias assessment) of RCT conducted in LAC according to funding source. A review of RCTs published in 2010 in which the author''s affiliation was from LAC was performed in PubMed and LILACS. Two reviewers independently extracted data and assessed the risk of bias. The primary outcomes were risk of bias assessment and funding source. A total of 1,695 references were found in PubMed and LILACS databases, of which 526 were RCTs (N = 73.513 participants). English was the dominant publication language (93%) and most of the RCTs were published in non-LAC journals (84.2%). Only five of the 19 identified countries accounted for nearly 95% of all RCTs conducted in the region (Brazil 70.9%, Mexico 10.1%, Argentina 5.9%, Colombia 3.8%, and Chile 3.4%). Few RCTs covered priority areas related with Millennium Development Goals like maternal health (6.7%) or high priority infectious diseases (3.8%). Regarding children, 3.6% and 0.4% RCT evaluated nutrition and diarrhea interventions respectively but none pneumonia. As a comparison, aesthetic and sport related interventions account for 4.6% of all trials. A random sample of RCTs (n = 358) was assessed for funding source: exclusively public (33.8%); private (e.g. pharmaceutical company) (15.3%); other (e.g. mixed, NGO) (15.1%); no funding (35.8%). Overall assessments for risk of bias showed no statistically significant differences between RCTs and type of funding source. Statistically significant differences favoring private and others type of funding was found when assessing trial registration and conflict of interest reporting.

Conclusion

Findings of this study could be used to provide more direction for future research to facilitate innovation, improve health outcomes or address priority health problems.     

Focused Examination of the Intestinal Epithelium Reveals Transcriptional Signatures Consistent with Disturbances in Enterocyte Maturation and Differentiation during the Course of SIV Infection

The Gastrointestinal (GI) tract plays a pivotal role in AIDS pathogenesis as it is the primary site for viral transmission, replication and CD4⁺ T cell destruction. Accordingly, GI disease (enteropathy) has become a well-known complication and a driver of AIDS progression. To better understand the molecular mechanisms underlying GI disease we analyzed global gene expression profiles sequentially in the intestinal epithelium of the same animals before SIV infection and at 21 and 90 days post infection (DPI). More importantly we obtained sequential excisional intestinal biopsies and examined distinct mucosal components (epithelium. intraepithelial lymphocytes, lamina propria lymphocytes, fibrovascular stroma) separately. Here we report data pertaining to the epithelium. Overall genes associated with epithelial cell renewal/proliferation/differentiation, permeability and adhesion were significantly down regulated (<1.5–7 fold) at 21 and 90DPI. Genes regulating focal adhesions (n = 6), gap junctions (n = 3), ErbB (n = 3) and Wnt signaling (n = 4) were markedly down at 21DPI and the number of genes in each of these groups that were down regulated doubled between 21 and 90DPI. Notable genes included FAK, ITGA6, PDGF, TGFβ3, Ezrin, FZD6, WNT10A, and TCF7L2. In addition, at 90DPI genes regulating ECM-receptor interactions (laminins and ITGB1), epithelial cell gene expression (PDX1, KLF6), polarity/tight junction formation (PARD3B&6B) and histone demethylase (JMJD3) were also down regulated. In contrast, expression of NOTCH3, notch target genes (HES4, HES7) and EZH2 (histone methyltransferase) were significantly increased at 90DPI. The altered expression of genes linked to Wnt signaling together with decreased expression of PDX1, PARD3B, PARD6B and SDK1 suggests marked perturbations in intestinal epithelial function and homeostasis leading to breakdown of the mucosal barrier. More importantly, the divergent expression patterns of EZH2 and JMJD3 suggests that an epigenetic mechanism involving histone modifications may contribute to the massive decrease in gene expression at 90DPI leading to defects in enterocyte maturation and differentiation.     

Genome-wide association mapping of sexual incompatibility genes in cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.)

Sexual compatibility limits the production of cacao plantations, being an important selection criterion in breeding programs. However, the current method for characterizing compatibility, based on the frequency of flower setting after controlled pollination, is time consuming, requiring a long time to identify self-compatible individuals. The identification of molecular markers in genomic regions can be an alternative to allow early selection of self-compatible plants. The present study aimed to identify SNP markers associated with sexual compatibility in cacao, by utilizing genome-wide association (GWAS) mapping. A population of 295 individuals mostly from third-generation breeding populations, but also founder clones, was used. This population was phenotypically characterized by hand pollinating 8199 flowers and evaluating the flower retention 15 days after pollination. In addition, leaf samples of each individual were collected and DNA extracted for genotyping by sequencing, generating 5301 SNP markers after cleaning. Genome-wide association mapping analysis was performed using Synbreed, GCTA, and TASSEL softwares. Significant markers associated to incompatibility, likely in strong linkage disequilibrium, were found within a region of 196 kb, in the proximal end of chromosome 4, suggesting the existence of a major gene in that region. However, this result should be validated in a larger population, considering that only 295 trees were used here. When the SNP effects were treated as random in the estimation process, many other regions in the genome appears to be involved with sexual incompatibility in cacao. Candidate genes were found not only in the proximal end of chromosome 4 but also spread in several other regions of the genome.