The sperm proteome of the Pacific oyster <i>Crassostrea gigas</i> and immunolocalization of heat shock proteins

<div><p>The Pacific oyster <i>Crassostrea gigas</i> is a potential model organism of bivalve mollusks. Comprehensive studies on the proteome of its sperm are necessary to expand our understanding on its reproduction and development, which however are still poor currently. In this study, to improve the situation, we conducted a proteomic analysis on the sperm based on two-dimensional electrophoresis combined with protein identification through mass spectra data. Fifty-six protein spots with constant and relatively high expression levels were selected for protein identification. Among them, 36 were identified (corresponding to 31 proteins), including cytoskeletal proteins, proteins involved in energy supply, protein modifiers, signal regulators, antioxidant proteins, and others. We proposed that these proteins might play important roles in sperm motility, gamete interaction, and oxidation resistance. In particular, we observed several heat shock proteins that were proved to play essential roles in animal sperms. A further immunofluorescence experiment revealed a mitochondria localization of Hsp60s and wide distributions of Hsp70s, indicating these proteins might function in various processes such as mitochondrial function, gamete interaction, and regulation of receptor activity. Our data will provide fundamental supports for the studies on the mechanisms of fertilization and contribute to expand the understandings on reproduction and development of bivalve mollusks.</p></div

miR-homoHSV of Singapore Grouper Iridovirus (SGIV) Inhibits Expression of the SGIV Pro-apoptotic Factor LITAF and Attenuates Cell Death

<div><p>Growing evidence demonstrates that various large DNA viruses could encode microRNAs (miRNAs) that regulate host and viral genes to achieve immune evasion. In this study, we report that miR-homoHSV, an miRNA encoded by Singapore grouper iridovirus (SGIV), can attenuate SGIV-induced cell death. Mechanistically, SGIV miR-homoHSV targets SGIV ORF136R, a viral gene that encodes the pro-apoptotic lipopolysaccharide-induced TNF-α (LITAF)-like factor. miR-homoHSV suppressed exogenous and endogenous SGIV LITAF expression, and thus inhibited SGIV LITAF-induced apoptosis. Meanwhile, miR-homoHSV expression was able to attenuate cell death induced by viral infection, presumably facilitating viral replication through the down-regulation of the pro-apoptotic gene SGIV LITAF. Together, our data suggest miR-homoHSV may serve as a feedback regulator of cell death during viral infection. The findings of this study provide a better understanding of SGIV replication and pathogenesis.</p> </div

si-LITAF specifically blocks SGIV-induced apoptosis.

<p>(A) si-LITAF efficiently suppresses endogenous SGIV LITAF mRNA (left) and protein (right) expression in FHM cells. (B and C) Morphology (B) and nuclear morphology (C) of FHM cells 48 h after infection with SGIV. FHM cells were transfected with si-con (left) or si-LITAF (right), and infected with SGIV 18 h later. Arrows (bottom panels) indicate apoptotic bodies. Many apoptotic bodies were observed in si-con transfected FHM cells. (D) si-LITAF blocks SGIV-induced apoptosis. PI staining and fluorescence-activated cell sorter analysis of FHM cells 48 h after infection (up). Statistical results of the proportion of PI-stained positive cells 48 h after infection with SGIV (bottom). Data are means of at least three independent experiments; error bars indicate SEM. *<i>P</i><0.05. Flow cytometric analysis of apoptotic cells in FHM cells treated as described for panel B. </p

Funnel plots of MACE/MACCE for the comparison of high versus standard maintenance-dose clopidogrel.

<p>MACE: major adverse cardiac events; MACCE, major adverse cardiac and cerebrovascular events.</p

High-Maintenance-Dose Clopidogrel in Patients Undergoing Percutaneous Coronary Intervention: A Systematic Review and Meta-Analysis

<div><p>Background</p><p>Despite routine use of clopidogrel, adverse cardiovascular events recur among some patients undergoing percutaneous coronary intervention (PCI). To optimize antiplatelet therapies, we performed a meta-analysis to quantify the efficacy of high versus standard-maintenance-dose clopidogrel in these patients.</p> <p>Methods</p><p>Randomized controlled trials (RCTs) comparing high (>75 mg) and standard maintenance doses of clopidogrel in patients undergoing PCI were included. The primary efficacy and safety end-points were major adverse cardiovascular/cerebrovascular events (MACE/MACCE) and major bleeding. The secondary end-points were other ischemic and bleeding adverse effects. The pooled odds ratio (OR) for each outcome was estimated.</p> <p>Results</p><p>14 RCTs with 4424 patients were included. Compared with standard-maintenance-dose clopidogrel, high-maintenance-dose clopidogrel significantly reduced the incidence of MACE/MACCE (OR 0.60; 95% CI 0.43 to 0.83), stent thrombosis (OR 0.56; 95% CI 0.32 to 0.99) and target vessel revascularization (OR 0.38; 95% CI 0.20 to 0.74), without significant decrease of the risk of cardiovascular death (OR 0.92; 95% CI 0.74 to 1.13) and myocardial infarction (OR 0.83; 95% CI 0.51 to 1.33). For safety outcomes, it did not significantly increase the risk of major bleeding (OR 0.73; 95% CI 0.41 to 1.32), minor bleeding (OR 1.29; 95% CI 1.00 to 1.66) and any bleeding (OR 1.14; 95% CI 0.91 to 1.43).</p> <p>Conclusion</p><p>High-maintenance-dose clopidogrel reduces the recurrence of most ischemic events in patients post-PCI without increasing the risk of bleeding complications.</p> </div

Expression profiles of endogenous miR-homoHSV and SGIV LITAF.

<p>The relative expression level of miR-homoHSV rose rapidly reaching its peak 6 h p.i. and then decreased (blue). Relative expression level of miR-homoHSV 6 h p.i. was set to 1 for comparison. The U6 gene was used as an internal control. The amount of SGIV LITAF mRNA accumulated stably from 6 to 48 h p.i. (red). Relative expression level of SGIV LITAF 6 h p.i. was set to 1 for comparison. β-Actin was used as an internal control. Data are means from rapidly at least three independent experiments done in duplicate; error bars indicate SEM.</p

Comparisons of high versus standard maintenance-dose clopidogrel on MACE/MACCE, ST and TVR.

<p>A: MACE/MACCE; B: ST; C: TVR. MACE: major adverse cardiac events; MACCE, major adverse cardiac and cerebrovascular events; ST: stent thrombosis; TVR: target vessel revascularization.</p

Flow chart of study selection.

<p>Flow chart of study selection.</p

Comparisons of high versus standard maintenance-dose clopidogrel on bleeding complications.

<p>A: major bleeding; B: minor bleeding; C: any bleeding.</p

miR-homoHSV inhibits SGIV LITAF-induced apoptosis.

<p>(A) Morphology of FHM cells 48 h after transfection with the empty vector pLL3.7, pLL-homoHSV, pLL3.7 and pEGFP-LITAF, and pLL-homoHSV and pEGFP-LITAF, from left to right. (B) Nuclear morphology of FHM cells 48 h after transfection with the same vectors above. Arrows indicate apoptotic bodies. Many apoptotic bodies were observed in cells co-transfected with pLL3.7 and pEGFP-LITAF. (C) Flow cytometric analysis of dead cells in FHM cells transfected with the vectors above 48 h after transfection. (D) Statistical results for the proportion of PI-stained positive cells 24 h and 48 h after transfection with empty vector pLL3.7, pLL-homoHSV (left panel), pLL3.7 and pEGFP-LITAF, and pLL-homoHSV and pEGFP-LITAF (right panel). Data are means from at least three independent experiments. Error bars indicate SEM. *<i>P</i><0.05.</p