The hypoxic microenvironment of glioblastoma multiforme (GBM) is thought to increase resistance to cancer therapies. Recent evidence suggests that hypoxia induces protein phosphatase 2A (PP2A), a regulator of cell cycle and cell death. The effects of PP2A on GBM tumor cell proliferation and survival during hypoxic conditions have not been studied.

Tubular cell HIV-infection has been reported to manifest in the form of cellular hypertrophy and apoptosis. In the present study, we evaluated the role of mammalian target of rapamycin (mTOR) pathway in the HIV induction of tubular cell protein synthesis. Mouse proximal tubular epithelial cells (MPTECs) were transduced with either gag/pol-deleted NL4-3 (HIV/MPTEC) or empty vector (Vector/MPTEC). HIV/MPTEC showed enhanced DNA synthesis when compared with Vector/MPTECs by BRDU labeling studies. HIV/MPTECs also showed enhanced production of β-laminin and fibronection in addition to increased protein content per cell. In in vivo studies, renal cortical sections from HIV transgenic mice and HIVAN patients showed enhanced tubular cell phosphorylation of mTOR. Analysis of mTOR revealed increased expression of phospho (p)-mTOR in HIV/MPTECs when compared to vector/MPTECs. Further downstream analysis of mTOR pathway revealed enhanced phosphorylation of p70S6 kinase and associated diminished phosphorylation of eEF2 (eukaryotic translation elongation factor 2) in HIV/MPTECs; moreover, HIV/MPTECs displayed enhanced phosphorylation of eIF4B (eukaryotic translation initiation factor 4B) and 4EBP-1 (eukaryotic 4E binding protein). To confirm our hypothesis, we evaluated the effect of rapamycin on HIV-induced tubular cell downstream signaling. Rapamycin not only attenuated phosphorylation of p70S6 kinase and associated down stream signaling in HIV/MPTECs but also inhibited HIV-1 induced tubular cell protein synthesis. These findings suggest that mTOR pathway is activated in HIV-induced enhanced tubular cell protein synthesis and contributes to tubular cell hypertrophy.

Systemic inflammatory response syndrome is a fatal disease because of multiple organ failure. Acute kidney injury is a serious complication of systemic inflammatory response syndrome and its genesis is still unclear posing a difficulty for an effective treatment. Aldose reductase (AR) inhibitor is recently found to suppress lipopolysaccharide (LPS)-induced cardiac failure and its lethality. We studied the effects of AR inhibitor on LPS-induced acute kidney injury and its mechanism.

Enterohemorrhagic Escherichia coli serotype O157:H7 is a food borne enteric bacterial pathogen that causes significant morbidity and mortality in both developing and industrialized nations. E. coli O157:H7 infection of host epithelial cells inhibits the interferon gamma pro-inflammatory signaling pathway, which is important for host defense against microbial pathogens, through the inhibition of Stat-1 tyrosine phosphorylation. The aim of this study was to determine which bacterial factors are involved in the inhibition of Stat-1 tyrosine phosphorylation. Human epithelial cells were challenged with either live bacteria or bacterial-derived culture supernatants, stimulated with interferon-gamma, and epithelial cell protein extracts were then analyzed by immunoblotting. The results show that Stat-1 tyrosine phosphorylation was inhibited by E. coli O157:H7 secreted proteins. Using sequential anion exchange and size exclusion chromatography, YodA was identified, but not confirmed to mediate subversion of the Stat-1 signaling pathway using isogenic mutants. We conclude that E. coli O157:H7 subverts Stat-1 tyrosine phosphorylation in response to interferon-gamma through a still as yet unidentified secreted bacterial protein.

Virus-induced apoptosis and viral mechanisms that regulate this cell death program are key issues in understanding virus-host interactions and viral pathogenesis. Like many other human and animal viruses, coronavirus infection of mammalian cells induces apoptosis. In this study, the global gene expression profiles are first determined in IBV-infected Vero cells at 24 hours post-infection by Affymetrix array, using avian coronavirus infectious bronchitis virus (IBV) as a model system. It reveals an up-regulation at the transcriptional level of both pro-apoptotic Bak and pro-survival myeloid cell leukemia-1 (Mcl-1). These results were further confirmed both in vivo and in vitro, in IBV-infected embryonated chicken eggs, chicken fibroblast cells and mammalian cells at transcriptional and translational levels, respectively. Interestingly, the onset of apoptosis occurred earlier in IBV-infected mammalian cells silenced with short interfering RNA targeting Mcl-1 (siMcl-1), and was delayed in cells silenced with siBak. IBV progeny production and release were increased in infected Mcl-1 knockdown cells compared to similarly infected control cells, while the contrary was observed in infected Bak knockdown cells. Furthermore, IBV infection-induced up-regulation of GADD153 regulated the expression of Mcl-1. Inhibition of the mitogen-activated protein/extracellular signal-regulated kinase (MEK/ERK) and phosphoinositide 3-kinase (PI3K/Akt) signaling pathways by chemical inhibitors and knockdown of GADD153 by siRNA demonstrated the involvement of ER-stress response in regulation of IBV-induced Mcl-1 expression. These results illustrate the sophisticated regulatory strategies evolved by a coronavirus to modulate both virus-induced apoptosis and viral replication during its replication cycle.

Lysosomal exocytosis and fusion to cellular membrane is critical in the oxidative stress formation of endothelium under apoptotic stimulus. We investigated the role therein of it in hyperglycaemia-induced endothelial dysfunction. The lysosome-membrane fusion was shown by the expression of lamp1, the lysosomal membrane marker, on cellular membrane and the transportation of lysosomal symbolic enzymes into cultural medium. We also examined the ceramide production, lipid rafts (LRs) clustering, colocalization of gp91(phox), a NADPH oxidase subunit (NOX) to LRs clusters, superoxide (O₂·⁻) formation and nitric oxide (NO) content in human umbilical vein endothelial cells (HUVEC) and the endothelium-dependent NO-mediated vasodilation in isolated rat aorta. As compared to normal glucose (5.6 mmol/l, Ctrl) incubation, high glucose (22 mmol/l, HG) exposure facilitated the lysosome-membrane fusion in HUVEC shown by significantly increased quantity of lamp1 protein on cellular membrane and enhanced activity of lysosomal symbolized enzymes in cultural medium. HG incubation also elicited ceramide generation, LRs clustering and gp91(phox) colocalization to LRs clusters which were proved to mediate the HG induced O₂·⁻ formation and NO depletion in HUVEC. Functionally, the endothelium-dependent NO-mediated vasodilation in aorta was blunted substantially after HG incubation. Moreover, the HG-induced effect including ceramide production, LRs clustering, gp91(phox) colocalization to LRs clusters, O₂·⁻ formation and endothelial dysfunction could be blocked significantly by the inhibition of lysosome-membrane fusion. We propose that hyperglycaemia-induced endothelial impairment is closely related to the lysosome-membrane fusion and the following LRs clustering, LRs-NOX platforms formation and O₂·⁻ production.

Chagas disease is endemic in the rural areas of southern Peru and a growing urban problem in the regional capital of Arequipa, population ∼860,000. It is unclear how to implement cost-effective screening programs across a large urban and periurban environment.

A novel influenza A (H1N1) virus detected in April 2009 rapidly spread around the world. North American provincial and state laboratories have well-defined roles and responsibilities, including providing accurate, timely test results for patients and information for regional public health and other decision makers. We used the multidisciplinary response and rapid implementation of process changes based on Lean methods at the provincial public health laboratory in British Columbia, Canada, to improve laboratory surge capacity in the 2009 influenza pandemic. Observed and computer simulating evaluation results from rapid processes changes showed that use of Lean tools successfully expanded surge capacity, which enabled response to the 10-fold increase in testing demands.

Prostaglandins are important for female reproduction. Prostaglandin-E2 acts via four different receptor subtypes, EP1, EP2, EP3 and EP4 whereas prostaglandin-F2alpha acts through FP. The functions of prostaglandins depend on the expression of their receptors in different uterine cell types. Our aim was to investigate the expression of EPs and FP in rat uterus and to identify the regulation by estradiol, progesterone and estrogen receptor (ER) selective agonists.

To explore Bangladesh's ability to detect novel influenza, we examined a series of laboratory-confirmed pandemic (H1N1) 2009 cases. During June-July 2009, event-based surveillance identified 30 case-patients (57% travelers); starting July 29, sentinel sites identified 252 case-patients (1% travelers). Surveillance facilitated response weeks before the spread of pandemic (H1N1) 2009 infection to the general population.

Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein actively shuttles between the nucleus, where it interacts with transcriptional and splicing regulatory proteins, and the cytoplasm, where it activates NF-κB. Posttranslational modifications of Tax such as ubiquitination regulate its subcellular localization and hence its function; however, the regulation of Tax trafficking and NF-κB activation by host factors is poorly understood. By screening a deubiquitinating (DUB) enzyme small interfering RNA (siRNA) library, we identified the metalloprotease STAM-binding protein-like 1 (STAMBPL1) as a positive regulator of Tax-mediated NF-κB activation. Overexpression of wild-type STAMBPL1, but not a catalytically inactive mutant, enhanced Tax-mediated NF-κB activation, whereas silencing of STAMBPL1 with siRNA impaired Tax activation of both the canonical and noncanonical NF-κB signaling pathways. STAMBPL1 regulated Tax-induced NF-κB signaling indirectly by controlling Tax nuclear/cytoplasmic transport and was required for DNA damage-induced Tax nuclear export. Together, these results reveal that the deubiquitinase STAMBPL1 is a key regulator of Tax trafficking and function.

Ubiquitin is important for the budding of many retroviruses and other enveloped viruses, but the precise role of ubiquitin in virus budding remains unclear. Here, we characterized the ubiquitination of the matrix (M) protein of a paramyxovirus, parainfluenza virus 5 (PIV5). The PIV5 M protein (but not the PIV5 nucleocapsid protein) was found to be targeted for monoubiquitination in transfected mammalian cells. Major sites of ubiquitin attachment identified by mass spectrometry analysis were lysine residues at amino acid positions 79/80, 130, and 247. The cumulative mutation of lysine residues 79, 80, and 130 to arginines led to an altered pattern of M protein ubiquitination and impaired viruslike particle (VLP) production. However, the cumulative mutation of lysine residues 79, 80, 130, and 247 to arginines restored M protein ubiquitination and VLP production, suggesting that ubiquitin is attached to alternative sites on the M protein when the primary ones have been removed. Additional lysine residues were targeted for mutagenesis based on the UbiPred algorithm. An M protein with seven lysine residues changed to arginines exhibited altered ubiquitination and poor VLP production. A recombinant virus encoding an M protein with seven lysines mutated was generated, and this virus exhibited a 6-fold-reduced maximum titer, with the defect being attributed mainly to the budding of noninfectious particles. The recombinant virus was assembly deficient, as judged by the redistribution of viral M and hemagglutinin-neuraminidase proteins in infected cells. Similar assembly defects were observed for the wild-type (wt) virus after treatment with a proteasome inhibitor. Collectively, these findings suggest that the monoubiquitination of the PIV5 M protein is important for proper virus assembly and for the budding of infectious particles.

Varicella-zoster virus (VZV) is a member of the alphaherpesvirus family and the causative agent of chickenpox and shingles. To determine the utility of cynomolgus macaques (Macaca fascicularis) as a nonhuman primate model to evaluate VZV-based simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) vaccines, we experimentally inoculated 10 animals with the parental Oka (Oka-P) strain of VZV derived from MeWo or Telo-RF cells. VZV DNA could be detected in the lungs as late as 4 days postinfection, with replicating virus detected by shell vial culture assay in one case. Infection did not result in any overt clinical symptoms but was characterized by humoral and cell-mediated immunity in a time frame and at a magnitude similar to those observed following VZV vaccination in humans. The cell line source of VZV inoculum influenced both the magnitude and polyfunctionality of cell-mediated immunity. Animals mounted a vigorous anamnestic antibody response following a second inoculation 12 weeks later. Inoculations resulted in transient increases in CD4(+) T-cell activation and proliferation, as well as a sustained increase in CD4(+) T cells coexpressing CCR5 and α4β7 integrin. In contrast to previous failed attempts to successfully utilize attenuated VZV-Oka as an SIV vaccine vector in rhesus macaques due to suboptimal infectivity and cellular immunogenicity, the ability to infect cynomolgus macaques with Oka-P VZV should provide a valuable tool for evaluating VZV-vectored SIV/HIV vaccines.

Diabetes is a major cause of chronic kidney disease, and oral antidiabetic drugs are the mainstay of therapy for most patients with Type 2 diabetes. Here we evaluated their role on renal outcomes by using a national Veterans Administration database to assemble a retrospective cohort of 93,577 diabetic patients who filled an incident oral antidiabetic drug prescription for metformin, sulfonylurea, or rosiglitazone, and had an estimated glomerular filtration rate (eGFR) of 60 ml/min or better. The primary composite outcome was a persistent decline in eGFR from baseline of 25% or more (eGFR event) or a diagnosis of end-stage renal disease (ESRD). The secondary outcome was an eGFR event, ESRD, or death. Sensitivity analyses included using a more stringent definition of the eGFR event requiring an eGFR <60 ml/min per 1.73 m(2) in addition to the 25% or more decline; controlling for baseline proteinuria thereby restricting data to 15,065 patients; and not requiring persistent treatment with the initial oral antidiabetic drug. Compared to patients using metformin, sulfonylurea users had an increased risk for both the primary and the secondary outcome, each with an adjusted hazard ratio of 1.20. Results of sensitivity analyses were consistent with the main findings. The risk associated with rosiglitazone was similar to metformin for both outcomes. Thus, compared to metformin, oral antidiabetic drug treatment with sulfonylureas increased the risk of a decline in eGFR, ESRD, or death.

B-cell lymphoma 2 (Bcl-2) homology 3 (BH3)-only proteins represent a class of pro-apoptotic factors that neutralize pro-survival Bcl-2 proteins, and, in some cases, directly activate Bax. The mechanisms of control and the role of BH3-only proteins, such as Bcl-2 like protein 11 extra large and Bad are well studied. By contrast, relatively little is known about the regulation and role of Bcl-2 modifying factor (Bmf). The B-RAF oncogene is mutated in ∼8% of human tumors. We have previously shown that Bmf is upregulated at the transcript level and is required for apoptosis induced by targeting B-RAF signaling in tumor cells harboring mutant B-RAF. In this study, we show that Bmf is regulated at the post-translational level by mutant B-RAF-MEK-ERK2 signaling. Extracellular signal-regulated kinase (ERK2) directly phosphorylates Bmf on serine 74 and serine 77 residues with serine 77 being the predominant site. In addition, serine 77 phosphorylation reduces Bmf pro-apoptotic activity likely through a mechanism independent of altering Bmf localization to the mitochondria and/or interactions with dynein light chain 2 and the pro-survival proteins, B-cell lymphoma extra large, Bcl-2 and Mcl-1. These data identify a novel mode of regulation in Bmf that modulates its pro-apoptotic activity in mutant B-RAF tumor cells.

We have recently found that primary rat embryonic fibroblasts (REFs) could be immortalized by overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2). A derived cell line, designated 18IM, expressed the embryonic stem cell markers SSEA-1 and Sox2. Upon inoculation into severe combined immunodeficiency mice, 18IM cells differentiated to express pan-keratin. They were not tumorigenic. Here we report the gene profiling of 18IM, compared with REF cells. Pathways involved in oxidative phosphorylation, ubiquinone (Coenzyme Q 10) biosynthesis, fatty acid elongation in mitochondria, PI3K/AKT signaling, a characteristic of rapidly proliferating cells, were upregulated in 18IM. Genes involved in the transcription/translation machinery and redox reactions, like elongation factors, ATP synthases, NADH dehydrogenases, mitogen activated kinases were upregulated as well. 18IM cells produced more pyruvate, indicating enhanced ATP synthesis. The expression of Oct4, Sox2, and Nanog that can contribute to the experimental induction of pluripotency in primary fibroblasts was also elevated, in contrast to Klf4 and C-myc that were downregulated. Subsequently, three new immortalized cell lines were produced by S18-2 overexpression in order to check the representativeness of 18IM. All of them showed anchorage-independent growth pattern. Two of three clones lost vimentin and smooth muscle actin, and expressed Sox2 and Oct4. We suggest that S18-2 is involved in the developmental regulation.

Carbon monoxide (CO) poisoning leads to demyelination of cerebral white matter (CWM) fibers, causing chronic neuropsychiatric symptoms. To clarify whether fractional anisotropy (FA) from diffusion tensor imaging in the centrum semiovale can depict demyelination in the CWM during the subacute phase after CO inhalation, we examined correlations between FA in the centrum semiovale and myelin basic protein (MBP) in cerebrospinal fluid. Subjects comprised 26 adult CO-poisoned patients ≤60 years old. MBP concentration was examined for all patients at 2 weeks after CO inhalation. The mean FA of the centrum semiovale bilaterally at 2 weeks was also examined for all patients and 21 age-matched healthy volunteers as controls. After these examinations, the presence of chronic symptoms was checked at 6 weeks after CO poisoning. Seven patients displayed chronic symptoms, of whom six showed abnormal MBP concentrations. The remaining 19 patients presented no chronic symptoms and no abnormal MBP concentrations, with MBP concentrations undetectable in 16 patients. The MBP concentration differed significantly between patients with and without chronic symptoms. The mean FA was significantly lower in patients displaying chronic symptoms than in either patients without chronic symptoms or controls. After excluding the 16 patients with undetectable MBP concentrations, a significant correlation was identified between MBP concentration and FA in ten patients. The present results suggest that FA in the centrum semiovale offers a quantitative indicator of the extent of demyelination in damaged CWM during the subacute phase in CO-poisoned patients.

Genome-wide association studies for breast cancer have identified over 40 single-nucleotide polymorphisms (SNPs), a subset of which remains statistically significant after genome-wide correction. Improved strategies for mining of genome-wide association data have been suggested to address heritable component of genetic risk in breast cancer. In this study, we attempted a two-stage association design using markers from a genome-wide study (stage 1, Affymetrix Human SNP 6.0 array, cases=302, controls=321). We restricted our analysis to DNA repair/modifications/metabolism pathway related gene polymorphisms for their obvious role in carcinogenesis in general and for their known protein-protein interactions vis-à-vis, potential epistatic effects. We selected 22 SNPs based on linkage disequilibrium patterns and high statistical significance. Genotyping assays in an independent replication study of 1178 cases and 1314 controls were attempted using Sequenom iPLEX Gold platform (stage 2). Six SNPs (rs8094493, rs4041245, rs7614, rs13250873, rs1556459 and rs2297381) showed consistent and statistically significant associations with breast cancer risk in both stages, with allelic odds ratios (and P-values) of 0.85 (0.0021), 0.86 (0.0026), 0.86 (0.0041), 1.17 (0.0043), 1.20 (0.0103) and 1.13 (0.0154), respectively, in combined analysis (N=3115). Of these, three polymorphisms were located in methyl-CpG-binding domain protein 2 gene regions and were in strong linkage disequilibrium. The remaining three SNPs were in proximity to RAD21 homolog (S. pombe), O-6-methylguanine-DNA methyltransferase and RNA polymerase II-associated protein 1. The identified markers may be relevant to breast cancer susceptibility in populations if these findings are confirmed in independent cohorts.

Somatic cell genetic alterations are a hallmark of tumor development and progression. Although various technologies have been developed and utilized to identify genetic aberrations, identifying genetic translocations at the chromosomal level is still a challenging task. High density SNP microarrays are useful to measure DNA copy number variation (CNV) across the genome. Utilizing SNP array data of cancer cell lines and patient samples, we evaluated the CNV and copy number breakpoints for several known fusion genes implicated in tumorigenesis. This analysis demonstrated the potential utility of SNP array data for the prediction of genetic aberrations via translocations based on identifying copy number breakpoints within the target genes. Genome-wide analysis was also performed to identify genes harboring copy number breakpoints across 820 cancer cell lines. Candidate oncogenes were identified that are linked to potential translocations in specific cancer cell lines.