Recombinant monoclonal antibody recognizes a unique epitope on varicella-zoster virus immediate-early 63 protein

We previously constructed a recombinant monoclonal antibody (rec-MAb 63P4) that detects immediate-early protein IE63 encoded by varicella-zoster virus (VZV) in the cytoplasm of productively infected cells. Here, we used ORF63 truncation mutants to map the rec-MAb 63P4 binding epitope to amino acids 141 to 150 of VZV IE63, a region not shared with other widely used anti-IE63 antibodies, and found that the recombinant antibody does not bind to the simian IE63 counterpart.     

Acute running stimulates hippocampal dopaminergic neurotransmission in rats, but has no influence on brain-derived neurotrophic factor

Hippocampal brain-derived neurotrophic factor (BDNF) protein is increased with exercise in rats. Monoamines seem to play a role in the regulation of BDNF, and monoamine neurotransmission is known to increase with exercise. The purpose of this study was to examine the influence of acute exercise on monoaminergic neurotransmission and BDNF protein concentrations. Hippocampal microdialysis was performed in rats that were subjected to 60 min of treadmill running at 20 m/min or rest. Two hours postexercise, the rats were killed, and the hippocampus was dissected. In experiments without microdialysis, hippocampus and serum samples were collected immediately after exercise. Exercise induced a twofold increase in hippocampal dopamine release. Noradrenaline and serotonin release were not affected. Hippocampal BDNF levels were not influenced, whether they were measured immediately or 2 h after the exercise protocol. Serum BDNF levels did not change either, but serum BDNF was negatively correlated to peripheral corticosterone concentrations, indicating a possible inhibitory reaction to the stress of running. Sixty minutes of exercise enhances dopamine release in the hippocampus of the rat in vivo. However, this increase is not associated with changes in BDNF protein levels immediately nor 2 h after the acute exercise bout. An increased corticosterone level might be the contributing factor for the absence of changes in BDNF.     

Novel colicin Fy of Yersinia frederiksenii inhibits pathogenic Yersinia strains via YiuR-mediated reception, TonB import, and cell membrane pore formation

A novel colicin type, designated colicin Fy, was found to be encoded and produced by the strain Yersinia frederiksenii Y27601. Colicin Fy was active against both pathogenic and nonpathogenic strains of the genus Yersinia. Plasmid YF27601 (5,574 bp) of Y. frederiksenii Y27601 was completely sequenced. The colicin Fy activity gene (cfyA) and the colicin Fy immunity gene (cfyI) were identified. The deduced amino acid sequence of colicin Fy was very similar in its C-terminal pore-forming domain to colicin Ib (69% identity in the last 178 amino acid residues), indicating pore forming as its lethal mode of action. Transposon mutagenesis of the colicin Fy-susceptible strain Yersinia kristensenii Y276 revealed the yiuR gene (ykris001_4440), which encodes the YiuR outer membrane protein with unknown function, as the colicin Fy receptor molecule. Introduction of the yiuR gene into the colicin Fy-resistant strain Y. kristensenii Y104 restored its susceptibility to colicin Fy. In contrast, the colicin Fy-resistant strain Escherichia coli TOP10F' acquired susceptibility to colicin Fy only when both the yiuR and tonB genes from Y. kristensenii Y276 were introduced. Similarities between colicins Fy and Ib, similarities between the Cir and YiuR receptors, and the detected partial cross-immunity of colicin Fy and colicin Ib producers suggest a common evolutionary origin of the colicin Fy-YiuR and colicin Ib-Cir systems.     

Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect?

Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3?mg?kg^?1 lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.     

Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme

The Drosophila γ-glutamyl carboxylase (dγC) has substrate recognition properties similar to that of the vertebrate γ-carboxylase (γC), and its carboxylated product yield, in vitro, was shown to be more than that obtained with the human enzyme. However, whether the Drosophila enzyme is able to γ-carboxylate the human vitamin K-dependent (VKD) proteins, such as the human coagulation factor IX (hFIX), as synthesized in cultured Drosophila cells was not known. To examine this possibility, the Drosophila Schnider (S2) cell line was transfected with a metallothionein promoter-regulated hFIX-expressing plasmid. After induction with copper ion, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assey (ELISA) and coagulation test on the culture supernatant of the transfected S2 cells during 72 h of postinduction. In comparison with Chinese hamster ovary cell line, S2 cells showed higher (≈ 12-fold) expression level of the hFIX. The γ-carboxylation of the Drosophila-derived hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate. The biological activity of the S2 cell-derived hFIX indicated the capability of S2 cells to fulfill the required γ-carboxylation of the expressed hFIX. Coexpression of the human γ-glutamyl carboxylases (hγC) was also shown to improve both expression and γ-carboxylation of the hFIX. This is the first in vivo data to describe the ability of the dγC to recognize the human-based propeptide as substrate, which is an essential step for production of biologically active γ-carboxylated VKD proteins.     

A heterogeneous computing environment to solve the 768-bit RSA challenge

In December 2009 the 768-bit, 232-digit number RSA-768 was factored using the number field sieve. Overall, the computational challenge would take more than 1700 years on a single, standard core. In the article we present the heterogeneous computing approach, involving different compute clusters and Grid computing environments, used to solve this problem.     

Rap2A links intestinal cell polarity to brush border formation

The microvillus brush border at the apex of the highly polarized enterocyte allows the regulated uptake of nutrients from the intestinal lumen. Here, we identify the small G protein Rap2A as a molecular link that couples the formation of microvilli directly to the preceding cell polarization. Establishment of apicobasal polarity, which can be triggered by the kinase LKB1 in single, isolated colon cells, results in enrichment of PtdIns(4,5)P(2) at the apical membrane. The subsequent recruitment of phospholipase D1 allows polarized accumulation of phosphatidic acid, which provides a local cue for successive signalling by the guanine nucleotide exchange factor PDZGEF, the small G protein Rap2A, its effector TNIK, the kinase MST4 and, ultimately, the actin-binding protein Ezrin. Thus, epithelial cell polarization is translated directly into the acquisition of brush borders through a small G protein signalling module whose action is positioned by a cortical lipid cue.     

Effect of Sanitation on Soil-Transmitted Helminth Infection: Systematic Review and Meta-Analysis

Background

In countries of high endemicity of the soil-transmitted helminth parasites Ascaris lumbricoides, Trichuris trichiura, and hookworm, preventive chemotherapy (i.e., repeated administration of anthelmintic drugs to at-risk populations) is the main strategy to control morbidity. However, rapid reinfection of humans occurs after successful deworming, and therefore effective preventive measures are required to achieve public health goals with optimal efficiency and sustainability.

Methods and Findings

We conducted a systematic review and meta-analysis to assess the effect of sanitation (i.e., access and use of facilities for the safe disposal of human urine and feces) on infection with soil-transmitted helminths. PubMed, Embase, ISI Web of Science, and the World Health Organization Library Database were searched without language restrictions and year of publication (search performed until December 31, 2010). Bibliographies of identified articles were hand-searched. All types of studies reporting data on sanitation availability (i.e., having access at own household or living in close proximity to sanitation facility), or usage, and soil-transmitted helminth infections at the individual level were considered. Reported odds ratios (ORs) of the protective effect of sanitation on soil-transmitted helminth infections were extracted from the papers or calculated from reported numbers. The quality of published studies was assessed with a panel of criteria developed by the authors. Random effects meta-analyses were used to account for observed heterogeneity. Thirty-six publications, consisting of 39 datasets, met our inclusion criteria. Availability of sanitation facilities was associated with significant protection against infection with soil-transmitted helminths (OR  =  0.46 to 0.58). Regarding the use of sanitation, ORs of 0.54 (95% confidence interval [CI] 0.28–1.02), 0.63 (95% CI 0.37–1.05), and 0.78 (95% CI 0.60–1.00) were determined for T. trichiura, hookworm, and A. lumbricoides, respectively. The overall ORs, combining sanitation availability and use, were 0.51 (95% CI 0.44–0.61) for the three soil-transmitted helminths combined, 0.54 (95% CI 0.43–0.69) for A. lumbricoides, 0.58 (95% CI 0.45–0.75) for T. trichiura, and 0.60 (95% CI 0.48–0.75) for hookworm.

Conclusions

Despite a number of limitations (e.g., most studies used a cross-sectional design and were of low quality, with potential biases and considerable heterogeneity), our results reveal that sanitation is associated with a reduced risk of transmission of helminthiases to humans. Access to improved sanitation should be prioritized alongside preventive chemotherapy and health education to achieve a durable reduction of the burden of helminthiases. Please see later in the article for the Editors'' Summary     

Scavenger receptor BI facilitates the metabolism of VLDL lipoproteins in vivo

Scavenger receptor class B type I (SR-BI) functions as an HDL receptor that promotes the selective uptake of cholesteryl esters (CEs). The physiological role of SR-BI in VLDL metabolism, however, is largely unknown. SR-BI deficiency resulted in elevated VLDL cholesterol levels, both on chow diet and upon challenge with high-cholesterol diets. To specifically elucidate the role of SR-BI in VLDL metabolism, the plasma clearance and hepatic uptake of (125)I-beta-VLDL were studied in SR-BI(+/+) and SR-BI(-/-) mice. At 20 min after injection, 66 +/- 2% of the injected dose was taken up by the liver in SR-BI(+/+) mice, as compared with only 22 +/- 4% (P = 0.0007) in SR-BI(-/-) mice. In vitro studies established that the B(max) of (125)I-beta-VLDL binding was reduced from 469 +/- 30 ng/mg in SR-BI(+/+) hepatocytes to 305 +/- 20 ng/mg (P = 0.01) in SR-BI(-/-) hepatocytes. Both in vivo and in vitro, limited to no selective uptake of CEs from beta-VLDL was found. Interestingly, HDL effectively competed for the association of beta-VLDL in the presence as well as in the absence of SR-BI, indicating a second common recognition site. In conclusion, SR-BI plays an important physiological role in the metabolism of VLDL (remnants).     

Amphibian malformations and inbreeding

Inbreeding may lead to morphological malformations in a wide variety of taxa. We used genetic markers to evaluate whether malformed urodeles were more inbred and/or had less genetic diversity than normal salamanders. We captured 687 adult and 1259 larval tiger salamanders (Ambystoma tigrinum tigrinum), assessed each individual for gross malformations, and surveyed genetic variation among malformed and normal individuals using both cytoplasmic and nuclear markers. The most common malformations in both adults and larvae were brachydactyly, ectrodactyly and polyphalangy. The overall frequency of adults with malformations was 0.078 compared to 0.081 in larval samples. Genetic diversity was high in both normal and malformed salamanders, and there were no significant difference in measures of inbreeding (f and F), allele frequencies, mean individual heterozygosity or mean internal relatedness. Environmental contaminants or other extrinsic factors may lead to genome alternations that ultimately cause malformations, but our data indicate that inbreeding is not a causal mechanism.