Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization

Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch's membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3(-/-) mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-beta2, and beta-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases.     

Evaluation of online extraction/mass spectrometry for in vivo cassette analysis

An online extraction/mass spectrometry technique was evaluated for direct analysis of plasma samples. A simple user-friendly online extraction system that consists of two pumps, an autosampler, a six-port switching valve and a mass spectrometer is described. The system was controlled by the LC-MS software (Masslynx 3.5, Waters Corporation, Beverly, MA). Various analytical conditions such as extraction column, mobile phases, run time and wash solvent were optimized to establish an analytical method that was simple, easy to set up and generic. Sample preparation effort was minimal, which included dilution of plasma with water and centrifugation conducted in 96-well plate format. The system was used to analyze in vivo plasma samples from rat n-in-one cassette dosing studies. Concentration and pharmacokinetic (PK) data obtained from the online extraction method were comparable with data obtained from the protein precipitation extraction method. Overall, the simple, robust online extraction system provides cost savings by minimizing sample preparation and method development time. The system was used to analyze compounds from different structural classes. These studies suggest that calculated lipophilicity of a compound can be used as a tool for pre-selection of extraction column, which would save method development time for early discovery studies.     

Estrogen, neutrophils and oxidation

The potential role of estrogens in the prevention of cardiovascular disease (CVD) is still under debate. Previous studies from our laboratory have shown that estradiol may act as a pro oxidant at physiological concentrations, enhancing peroxidase-mediated oxidation of low density lipoprotein (LDL). In the present study, we show that physiological concentrations of estradiol enhance fMLP-mediated neutrophil degranulation and oxidative stress markers. For example, 10 nM estradiol increased myeloperoxidase (MPO), elastase, and superoxide release by 19.9 +/- 9.6% (p = 0.006), 16.3 +/- 5.2% (p = 0.09), and 36.1 +/- 19.5% (p = 0.05), respectively. The enhancement of neutrophil degranulation by estradiol resulted in an increase in the formation of LDL oxidation markers such as conjugated dienes and thiobarbituric acid-reactive substances (20.7 +/- 7.2%, p = 0.04). Thus, estradiol can act as a pro oxidant, promoting neutrophil degranulation as well as reacting with MPO to enhance the oxidation of LDL. This mechanism supports our hypothesis that oxidative stress may be beneficial towards the prevention of CVD both by promoting plasma oxidation of LDL, with its subsequent clearance by the liver, as well as by inducing a threshold antioxidant defense in the arteries. Our study also suggests that estradiol by promoting oxidation in the plasma is beneficial in preventing CVD.     

Development and mapping of microsatellites from a microdissected BTA 11-specific DNA library

A chromosome-specific library was developed for Bos taurus autosome 11 by chromosome microdissection and microcloning using a bovine primary fibroblast culture, obtained from a t(X;23) heifer, that spontaneously developed a translocation chromosome involving bovine chromosome 11. The library was screened using (AC)12 oligos, positive clones selected, sequenced and primers developed to generate bovine chromosome 11-specific microsatellite markers. This study suggests that chromosome-specific libraries have great potential for development of microsatellite markers for the construction of marker-saturated linkage maps for each chromosome.     

Sexual selection accounts for the geographic reversal of sexual size dimorphism in the dung fly, sepsis punctum (Diptera: Sepsidae)

Sexual size dimorphism (SSD) varies widely across and within species. The differential equilibrium model of SSD explains dimorphism as the evolutionary outcome of consistent differences in natural and sexual selection between the sexes. Here, we comprehensively examine a unique cross-continental reversal in SSD in the dung fly, Sepsis punctum. Using common garden laboratory experiments, we establish that SSD is male-biased in Europe and female-biased in North America. When estimating sexual (pairing success) and fecundity selection (clutch size of female partner) on males under three operational sex ratios (OSRs), we find that the intensity of sexual selection is significantly stronger in European versus North American populations, increasing with male body size and OSR in the former only. Fecundity selection on female body size also increases strongly with egg number and weakly with egg volume, however, equally on both continents. Finally, viability selection on body size in terms of intrinsic (physiological) adult life span in the laboratory is overall nil and does not vary significantly across all seven populations. Although it is impossible to prove causality, our results confirm the differential equilibrium model of SSD in that differences in sexual selection intensity account for the reversal in SSD in European versus North American populations, presumably mediating the ongoing speciation process in S. punctum.     

Bacillus anthracis protease InhA regulates BslA-mediated adhesion in human endothelial cells

To achieve widespread dissemination in the host, Bacillus anthracis cells regulate their attachment to host endothelium during infection. Previous studies identified BslA (Bacillus anthracis S-layer Protein A), a virulence factor of B. anthracis, as necessary and sufficient for adhesion of vegetative cells to human endothelial cells. While some factors have been identified, bacteria-specific contributions to BslA mediated adhesion remain unclear. Using the attenuated vaccine Sterne 7702 strain of B. anthracis, we tested the hypothesis that InhA (immune inhibitor A), a B. anthracis protease, regulates BslA levels affecting the bacteria's ability to bind to endothelium. To test this, a combination of inhA mutant and complementation analysis in adhesion and invasion assays, Western blot and InhA inhibitor assays were employed. Results show InhA downregulates BslA activity reducing B. anthracis adhesion and invasion in human brain endothelial cells. BslA protein levels in ΔinhA bacteria were significantly higher than wild-type and complemented strains showing InhA levels and BslA expression are inversely related. BslA was sensitive to purified InhA degradation in a concentration- and time-dependent manner. Taken together these data support the role of InhA regulation of BslA-mediated vegetative cell adhesion and invasion.     

Identification of novel microRNAs in Xenopus laevis metaphase II arrested eggs

Using a combination of deep sequencing and bioinformatics approach, we for the first time identify miRNAs and their relative abundance in mature, metaphase II arrested eggs in Xenopus laevis. We characterize 115 miRNAs that have been described either in Xenopus tropicalis (85), X. laevis (9), or other vertebrate species (21) that also map to known Xenopus pre-miRNAs and to the X. tropicalis genome. In addition, 72 new X. laevis putative candidate miRNAs are identified based on mapping to X. tropicalis genome within regions that have the propensity to form hairpin loops. These data expand on the availability of genetic information in X. laevis and identify target miRNAs for future functional studies.     

Genetic association of KCNJ10 rs1130183 with seizure susceptibility and computational analysis of deleterious non-synonymous SNPs of KCNJ10 gene

Establishing genetic basis of Idiopathic generalized epilepsies (IGE) is challenging because of their complex inheritance pattern and genetic heterogeneity. Kir4.1 inwardly rectifying channel (KCNJ10) is one of the independent genes reported to be associated with seizure susceptibility. In the current study we have performed a comprehensive in silico analysis of genetic variants in KCNJ10gene at functional and structural level along with a case–control analysis for the association ofrs1130183 (R271C) polymorphism in Indian patients with IGE. Age and sex matched 108epileptic patients and normal healthy controls were examined. Genotyping of KCNJ10rs1130183variation was performed using PCR-RFLP method. The risk association was determined by using odds ratio and 95% confidence interval. Functional effects of non-synonymous SNPs (nsSNPs) in KCNJ10 gene were analyzed using SIFT PolyPhen-2, I-Mutant 2.0, PANTHER and FASTSNP. Subsequently, homology modeling of protein three dimensional (3D) structures was performed using Modeller tool (9.10v) and compared the native protein with mutant for assessment of structure and stability. SIFT, PolyPhen-2, I-Mutant 2.0 and PANTHER collectively showed rs1130183, rs1130182 and rs137853073 SNPs inKCNJ10 gene affect protein structure and function. There was a considerable variation in the Root Mean Square Deviation (RMSD) value between the native and mutant structure (1.17?). Association analysis indicate KCNJ10rs1130183 did not contribute to risk of seizure susceptibility in Indian patients with IGE (OR- 0.38; 95%CI, 0.07–2.05) and T allele frequency (0.02%) was in concordance with dbSNP reports. This study identifies potential SNPs that may contribute to seizure susceptibility and further studies with the selected SNPs in larger number of samples and their functional analysis is required for understanding the variants of KCNJ10with seizure susceptibility.