Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-beta, IL-2, IL-6, IL-10, IL-12, and TNF-alpha in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.     

Calibration of ADHD Assessments Across Studies: A Meta-Analysis Tool

When analyzed separately, data from small studies provide only limited information with limited clinical generalizability, due to small sample size, differing assessments, and limited scope. In this methodological paper we outline a theoretical framework for performing meta-analysis of data obtained from disparate studies using disparate tests, based on calibration of the data from such studies and tests into a unified probability scale. We apply this method to combine the data from five studies examining the diagnostic abilities of different assessments of Attention Deficit/Hyperactivity Disorder (ADHD), including behavioral rating scales and EEG assessments. The studies enrolled a total of 111 subjects, 56 ADHD and 55 controls. Each individual study had a small sample focused on a specific age/gender group, for example 8 boys ages 6–10, and generally had insufficient power to detect statistically significant differences. No gender, or age comparisons were possible within any single study. However, when calibrated and combined, the data resulted in a clear separation between ADHD versus non-ADHD groups in males below the age of 16 (p < 0.001), males above the age of 16, (p = 0.015), females below the age of 16, (p = 0.0014), and females above the age of 16, (p = 0.0022).We conclude that if data from various studies using various tests are made comparable, the resulting combined sample size and the increased diversity of the combined sample lead to increased significance of the statistical tests and allow for cross-sectional comparisons, which are not possible within each individual study.     

Natural occurrence of microbial sulphur oxidation by long-range electron transport in the seafloor

Recently, a novel mode of sulphur oxidation was described in marine sediments, in which sulphide oxidation in deeper anoxic layers was electrically coupled to oxygen reduction at the sediment surface. Subsequent experimental evidence identified that long filamentous bacteria belonging to the family Desulfobulbaceae likely mediated the electron transport across the centimetre-scale distances. Such long-range electron transfer challenges some long-held views in microbial ecology and could have profound implications for sulphur cycling in marine sediments. But, so far, this process of electrogenic sulphur oxidation has been documented only in laboratory experiments and so its imprint on the seafloor remains unknown. Here we show that the geochemical signature of electrogenic sulphur oxidation occurs in a variety of coastal sediment environments, including a salt marsh, a seasonally hypoxic basin, and a subtidal coastal mud plain. In all cases, electrogenic sulphur oxidation was detected together with an abundance of Desulfobulbaceae filaments. Complementary laboratory experiments in intertidal sands demonstrated that mechanical disturbance by bioturbating fauna destroys the electrogenic sulphur oxidation signal. A survey of published geochemical data and 16S rRNA gene sequences identified that electrogenic sulphide oxidation is likely present in a variety of marine sediments with high sulphide generation and restricted bioturbation, such as mangrove swamps, aquaculture areas, seasonally hypoxic basins, cold sulphide seeps and possibly hydrothermal vent environments. This study shows for the first time that electrogenic sulphur oxidation occurs in a wide range of marine sediments and that bioturbation may exert a dominant control on its natural distribution.     

SR-A deficiency reduces myocardial ischemia/reperfusion injury; involvement of increased microRNA-125b expression in macrophages

The macrophage scavenger receptor class A (SR-A) participates in the innate immune and inflammatory responses. This study examined the role of macrophage SR-A in myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation (H/R)-induced cell damage. SR-A^?/? and WT mice were subjected to ischemia (45 min) followed by reperfusion for up to 7 days. SR-A^?/? mice showed smaller myocardial infarct size and better cardiac function than did WT I/R mice. SR-A deficiency attenuated I/R-induced myocardial apoptosis by preventing p53-mediated Bak-1 apoptotic signaling. The levels of microRNA-125b in SR-A^?/? heart were significantly greater than in WT myocardium. SR-A is predominantly expressed on macrophages. To investigate the role of SR-A macrophages in H/R-induced injury, we isolated peritoneal macrophages from SR-A deficient (SR-A^?/?) and wild type (WT) mice. Macrophages were subjected to hypoxia followed by reoxygenation. H/R markedly increased NF-κB binding activity as well as KC and MCP-1 production in WT macrophages but not in SR-A^?/? macrophages. H/R induced caspase-3/7 and -8 activities and cell death in WT macrophages, but not in SR-A^?/? macrophages. The levels of miR-125b in SR-A^?/? macrophages were significantly higher than in WT macrophages. Transfection of WT macrophages with miR-125b mimics attenuated H/R-induced caspase-3/7 and -8 activities and H/R-decreased viability, and prevented H/R-increased p-53, Bak-1 and Bax expression. The data suggest that SR-A deficiency attenuates myocardial I/R injury by targeting p53-mediated apoptotic signaling. SR-A^?/? macrophages contain high levels of miR-125b which may play a role in the protective effect of SR-A deficiency on myocardial I/R injury and H/R-induced cell damage.     

Computational and biological inference of gene regulatory networks of the LINE-1 retrotransposon

Computational approaches were used to define structural and functional determinants of a putative genetic regulatory network of murine LINE-1 (long interspersed nuclear element-1), an active mammalian retrotransposon that uses RNA intermediates to populate new sites throughout the genome. Polymerase (RNA) II polypeptide E AI845735 and mouse DNA homologous to Drosophila per fragment M12039 were identified as primary attractors. siRNA knockdown of the aryl hydrocarbon receptor NM_013464 modulated gene expression within the network, including LINE-1, Sgpl1, Sdcbp, and Mgst1. Genes within the network did not exhibit physical proximity and instead were dispersed throughout the genome. The potential impact of individual members of the network on the global dynamical behavior of LINE-1 was examined from a theoretical and empirical framework.     

Discovery of non-covalent dipeptidyl peptidase IV inhibitors which induce a conformational change in the active site

A series of non-covalent inhibitors of the serine protease dipeptidyl peptidase IV (DPP-IV) were found to adopt a U-shaped binding conformation in X-ray co-crystallization studies. Remarkably, Tyr547 undergoes a 70 degrees side-chain rotation to accommodate the inhibitor and allows access to a previously unexposed area of the protein backbone for hydrogen bonding.     

Glucans exhibit weak antioxidant activity, but stimulate macrophage free radical activity

Polymeric carbohydrates have been reported to modulate inflammatory responses in vitro and in vivo. Previous reports suggest that certain carbohydrate polymers, such as (1-->3)-beta-D-glucans, may possess free radical scavenging activity. If glucans are free radical scavengers then it might explain, in part, the ability of these ligands to modulate inflammatory responses. The present study examined the free radical scavenging activity of a variety of carbohydrate polymers and the effect of the polymers on free radical levels in a murine macrophage cell line. All of the carbohydrates exhibited concentration dependent antioxidant effects (EC(50) range = 807 to 43 microg/ml). However, the antioxidant activity for the carbohydrates was modest in comparison with PDTC (EC(50) = 0.13 microg/ml) and the carbohydrate concentration required for antioxidant activity was high (x EC(50) = 283 microg/ml). The antioxidant ability of the polymers was greater (p < .05) than their monosaccharide constituents, i.e., dextrose EC(50) = 807 vs. glucan sulfate EC(50) = 43 microg/ml. Coincubation of glucans with murine J774a.1 cells increased free radical levels when compared to controls. Therefore, the weak free radical scavenging activity of glucan polymers cannot explain their modulatory effect on inflammatory responses in tissue culture and/or disease models of inflammation.     

Reduced lentivirus susceptibility in sheep with TMEM154 mutations

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.     

Activation of Myocardial Phosphoinositide-3-Kinase p110α Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p<0.01) and 50% survived >30 days (p<0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110α expression. Cardiac high mobility group box–1 (HMGB-1) translocation was also inhibited by caPI3K p110α expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110α signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.