Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis

More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3' terminus of the negative strand of the viral genome with a dissociation constant (K(d)) of approximately 3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development

Jejunal gene expression patterns correlate with severity of systemic infection in chicken

<p>Abstract</p> <p>Background</p> <p>Not much is known about the effect of <it>Salmonella enteritidis</it> on changes in the developmental processes occurring in the intestine of young chicken. Therefore we investigated the correlation of intestinal gene expression patterns with the severity of systemic Salmonella infections.</p> <p>Methods</p> <p>The number of Salmonella colony forming units (CFUs) in the liver of infected chicken were plotted against the average intestinal expression profiles of previously identified gene expression clusters. The functional properties of all the genes taken together present in 3 clusters exhibiting positive correlation at early time-points were compared with the functional properties of the genes displaying antagonistic correlations in 1 cluster. The top 5 ranking functional groups were analysed in further detail.</p> <p>Results</p> <p>Three clusters showed gene expression profiles which were positively correlated with the severity of systemic disease as measured by the number of Salmonella colony forming units in the liver. In these clusters, genes involved in morphological processes were predominantly present. One cluster had a profile that was negatively correlated with the severity of systemic disease, as measured by numbers of CFUs in the liver. The genes in the latter cluster were mostly involved in cell turn-over and metabolism.</p> <p>Conclusions</p> <p>In the developing jejunum of young chicken, both stimulatory and inhibitory gene expression mechanisms are correlated with the severity of systemic Salmonella infections.</p

Effect of ochratoxin A on the intestinal mucosa and mucosa-associated lymphoid tissues in broiler chickens

The immunotoxic effect of ochratoxin A (OTA) on the intestinal mucosa-associated lymphoid tissue and its cytotoxic action on the intestinal epithelium were studied in broiler chickens experimentally treated with the toxin. From the 7th day of life, 80 male broiler chickens (Ross 308) were randomly divided into four groups of 20 birds each. The three experimental groups (E1-3) were treated with OTA for 28 days (E1: 50 μg/kg body weight [bw]/day; E2: 20 μg/kg bw/day; E3: 1 μg/kg bw/day) and the fourth group served as control. Histological examination of the intestinal mucosa and immunohistochemical staining for identification of CD4+, CD8+, TCR1 and TCR2 lymphocytes in the duodenum, jejunum and ileocaecal junction were performed, and CD4+/CD8+ and TCR1/TCR2 ratios were calculated. OTA toxicity resulted in decreased body weight gain, poorer feed conversion ratio, lower leukocyte and lymphocyte count, and altered intestinal mucosa architecture. After 14 days of exposure to OTA, immunohistochemistry showed a significant reduction of the lymphocyte population in the intestinal epithelium and the lamina propria. After 28 days of exposure, an increase in the CD4+ and CD8+ values in both the duodenum and jejunum of chickens in Groups E1 and E2 was observed, but the TCR1 and TCR2 lymphocyte counts showed a significant reduction. No significant changes were observed in Group E3. The results indicate that OTA induced a decrease in leukocyte and lymphocyte counts and was cytotoxic to the intestinal epithelium and the mucosa-associated lymphoid tissue, altering the intestinal barrier and increasing susceptibility to various associated diseases

P19 H-Ras Induces G1/S Phase Delay Maintaining Cells in a Reversible Quiescence State

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Three functional c-ras genes, known as c-H-ras, c-K-ras, and c-N-ras, have been largely studied in mammalian cells with important insights into normal and tumorigenic cellular signal transduction events. Two K-Ras mRNAs are obtained from the same pre-mRNA by alternative splicing. H-Ras pre-mRNA can also be alternatively spliced in the IDX and 4A terminal exons, yielding the p19 and p21 proteins, respectively. However, despite the Ras gene family’s established role in tumorigenic cellular signal transduction events, little is known about p19 function. Previous results showed that p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multi-protein complexes in different signaling pathways (Cancer Res 2003, 63 p5178). This observation suggests that p19 and p21 play differential and complementary roles in the cell.[Principal Findings]: We found that p19 regulates telomerase activity through its interaction with p73a/b proteins. We also found that p19 overexpression induces G1/S phase delay; an observation that correlates with hypophosphorylation of both Akt and p70SK6. Similarly, we also observed that FOXO1 is upregulated when p19 is overexpressed. The three observations of (1) hypophosphorylation of Akt, (2) G1/S phase delay and (3) upregulation of FOXO1 lead us to conclude that p19 induces G1/S phase delay, thereby maintaining cells in a reversible quiescence state and preventing entry into apoptosis. We then assessed the effect of p19 RNAi on HeLa cell growth and found that p19 RNAi increases cell growth, thereby having the opposite effect of arrest of the G1/S phase or producing a cellular quiescence state.[Significance]: Interestingly, p19 induces FOXO1 that in combination with the G1/S phase delay and hypophosphorylation of both Akt and p70SK6 leads to maintenance of a reversible cellular quiescence state, thereby preventing entry into apoptosis.This work was supported by Fundacion de Investigacion Medica Mutua Madrileña Automovilista (Fundacion MMA), the Plan Nacional (MEC) BFU2005-00701 and the Fundacion Eugenio Rodriguez Pascual. M.C. was a recipient of a Fmed MMA fellowship.Peer reviewe

N-Acetylcholinesterase-Induced Apoptosis in Alzheimer's Disease

Background: Alzheimer’s disease (AD) involves loss of cholinergic neurons and Tau protein hyper-phosphorylation. Here, we report that overexpression of an N-terminally extended ‘‘synaptic’ ’ acetylcholinesterase variant, N-AChE-S is causally involved in both these phenomena. Methodology and Principal Findings: In transfected primary brain cultures, N-AChE-S induced cell death, morphological impairments and caspase 3 activation. Rapid internalization of fluorescently labeled fasciculin-2 to N-AChE-S transfected cells indicated membranal localization. In cultured cell lines, N-AChE-S transfection activated the Tau kinase GSK3, induced Tau hyper-phosphorylation and caused apoptosis. N-AChE-S-induced cell death was suppressible by inhibiting GSK3 or caspases, by enforced overexpression of the anti-apoptotic Bcl2 proteins, or by AChE inhibition or silencing. Moreover, inherent N-AChE-S was upregulated by stressors inducing protein misfolding and calcium imbalances, both characteristic of AD; and in cortical tissues from AD patients, N-AChE-S overexpression coincides with Tau hyper-phosphorylation. Conclusions: Together, these findings attribute an apoptogenic role to N-AChE-S and outline a potential value to ACh

Major reduction of malaria morbidity with combined vitamin A and zinc supplementation in young children in Burkina Faso: a randomized double blind trial

BACKGROUND: Vitamin A and zinc are crucial for normal immune function, and may play a synergistic role for reducing the risk of infection including malaria caused by Plasmodium falciparum. METHODS: A randomized, double-blind, placebo-controlled trial of a single dose of 200 000 IU of vitamin A with daily zinc supplementation was done in children of Sourkoudougou village, Burkina Faso. Children aged from 6 to 72 months were randomized to receive a single dose of 200 000 IU of vitamin A plus 10 mg elemental zinc, six days a week (n = 74) or placebo (n = 74) for a period of six months. Cross-sectional surveys were conducted at the beginning and the end of the study, and children were evaluated daily for fever. Microscopic examination of blood smear was done in the case of fever (temperature > or =37.5 degrees C) for malaria parasite detection. RESULTS: At the end of the study we observed a significant decrease in the prevalence malaria in the supplemented group (34%) compared to the placebo group (3.5%) (p < 0.001). Malaria episodes were lower in the supplemented group (p = 0.029), with a 30.2% reduction of malaria cases (p = 0.025). Time to first malaria episode was longer in the supplemented group (p = 0.015). The supplemented group also had 22% fewer fever episodes than the placebo group (p = 0.030). CONCLUSION: These results suggest that combined vitamin A plus zinc supplementation reduces the risk of fever and clinical malaria episodes among children, and thus may play a key role in malaria control strategies for children in Africa

RACK-1 Acts with Rac GTPase Signaling and UNC-115/abLIM in Caenorhabditis elegans Axon Pathfinding and Cell Migration

Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones

RACK1 Associates with Muscarinic Receptors and Regulates M2 Receptor Trafficking

Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M2 muscarinic acetylcholine receptor undergoes internalization via a poorly-defined clathrin-independent mechanism. We used isotope coded affinity tagging and mass spectrometry to identify the scaffolding protein, receptor for activated C kinase (RACK1) as a protein enriched in M2-immunoprecipitates from M2-expressing cells over those of non-M2 expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M2. We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M2 receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M2 receptor, but decreases the extent of subsequent down-regulation. These results suggest that RACK1 may both interfere with agonist-induced sequestration and be required for subsequent targeting of internalized M2 receptors to the degradative pathway