Identification of novel subgroup a variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus

<b>BACKGROUND:</b> The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C.<p></p> <b>RESULTS:</b> Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C.<p></p> <b>CONCLUSIONS:</b> Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established

Classification of patients with knee osteoarthritis in clinical phenotypes: data from the osteoarthritis initiative

<div><p>Objectives</p><p>The existence of phenotypes has been hypothesized to explain the large heterogeneity characterizing the knee osteoarthritis. In a previous systematic review of the literature, six main phenotypes were identified: Minimal Joint Disease (MJD), Malaligned Biomechanical (MB), Chronic Pain (CP), Inflammatory (I), Metabolic Syndrome (MS) and Bone and Cartilage Metabolism (BCM). The purpose of this study was to classify a sample of individuals with knee osteoarthritis (KOA) into pre-defined groups characterized by specific variables that can be linked to different disease mechanisms, and compare these phenotypes for demographic and health outcomes.</p><p>Methods</p><p>599 patients were selected from the OAI database FNIH at 24 months’ time to conduct the study. For each phenotype, cut offs of key variables were identified matching the results from previous studies in the field and the data available for the sample. The selection process consisted of 3 steps. At the end of each step, the subjects classified were excluded from the further classification stages. Patients meeting the criteria for more than one phenotype were classified separately into a ‘complex KOA’ group.</p><p>Results</p><p>Phenotype allocation (including complex KOA) was successful for 84% of cases with an overlap of 20%. Disease duration was shorter in the MJD while the CP phenotype included a larger number of Women (81%). A significant effect of phenotypes on WOMAC pain (F = 16.736 p <0.001) and WOMAC physical function (F = 14.676, p < 0.001) was identified after controlling for disease duration.</p><p>Conclusion</p><p>This study signifies the feasibility of a classification of KOA subjects in distinct phenotypes based on subgroup-specific characteristics.</p></div

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

X-Ray Spectroscopy of Stars

(abridged) Non-degenerate stars of essentially all spectral classes are soft X-ray sources. Low-mass stars on the cooler part of the main sequence and their pre-main sequence predecessors define the dominant stellar population in the galaxy by number. Their X-ray spectra are reminiscent, in the broadest sense, of X-ray spectra from the solar corona. X-ray emission from cool stars is indeed ascribed to magnetically trapped hot gas analogous to the solar coronal plasma. Coronal structure, its thermal stratification and geometric extent can be interpreted based on various spectral diagnostics. New features have been identified in pre-main sequence stars; some of these may be related to accretion shocks on the stellar surface, fluorescence on circumstellar disks due to X-ray irradiation, or shock heating in stellar outflows. Massive, hot stars clearly dominate the interaction with the galactic interstellar medium: they are the main sources of ionizing radiation, mechanical energy and chemical enrichment in galaxies. High-energy emission permits to probe some of the most important processes at work in these stars, and put constraints on their most peculiar feature: the stellar wind. Here, we review recent advances in our understanding of cool and hot stars through the study of X-ray spectra, in particular high-resolution spectra now available from XMM-Newton and Chandra. We address issues related to coronal structure, flares, the composition of coronal plasma, X-ray production in accretion streams and outflows, X-rays from single OB-type stars, massive binaries, magnetic hot objects and evolved WR stars.Comment: accepted for Astron. Astrophys. Rev., 98 journal pages, 30 figures (partly multiple); some corrections made after proof stag

Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the bla[subscript NDM] gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies.Kinship Foundation. Searle Scholars ProgramMassachusetts Institute of Technology. Department of Chemistr

<em>Enterococcus faecalis</em> Infection Causes Inflammation, Intracellular Oxphos-Independent ROS Production, and DNA Damage in Human Gastric Cancer Cells

Background: Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. Methods: To separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA). Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Mitochondrial mutations were determined by sequencing. Results: Infection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. Conclusions: Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-kappa B inflammatory response as well as impaired DNA damage response and cell cycle control gene expression

Regulation of microRNA biogenesis and turnover by animals and their viruses

Item does not contain fulltextMicroRNAs (miRNAs) are a ubiquitous component of gene regulatory networks that modulate the precise amounts of proteins expressed in a cell. Despite their small size, miRNA genes contain various recognition elements that enable specificity in when, where and to what extent they are expressed. The importance of precise control of miRNA expression is underscored by functional studies in model organisms and by the association between miRNA mis-expression and disease. In the last decade, identification of the pathways by which miRNAs are produced, matured and turned-over has revealed many aspects of their biogenesis that are subject to regulation. Studies in viral systems have revealed a range of mechanisms by which viruses target these pathways through viral proteins or non-coding RNAs in order to regulate cellular gene expression. In parallel, a field of study has evolved around the activation and suppression of antiviral RNA interference (RNAi) by viruses. Virus encoded suppressors of RNAi can impact miRNA biogenesis in cases where miRNA and small interfering RNA pathways converge. Here we review the literature on the mechanisms by which miRNA biogenesis and turnover are regulated in animals and the diverse strategies that viruses use to subvert or inhibit these processes

Fatty acid activated PPARγ promotes tumorigenicity of prostate cancer cells by up regulating VEGF via PPAR responsive elements of the promoter.

In previous work, it is suggested that the excessive amount of fatty acids transported by FABP5 may facilitate the malignant progression of prostate cancer cells through a FABP5-PPARγ-VEGF signal transduction axis to increase angiogenesis. To further functionally characterise the FABP5-PPARγ-VEGF signal transduction pathway, we have, in this work, investigated the molecular mechanisms involved in its tumorigenicity promoting role in prostate cancer. Suppression of PPARγ in highly malignant prostate cancer cells produced a significant reduction (up to 53%) in their proliferation rate, invasiveness (up to 89%) and anchorage-independent growth (up to 94%) in vitro. Knockdown of PPARγ gene in PC3-M cells by siRNA significantly reduced the average size of tumours formed in nude mice by 99% and tumour incidence by 90%, and significantly prolonged the latent period by 3.5 fold. Results in this study combined with some previous results suggested that FABP5 promoted VEGF expression and angiogenesis through PPARγ which was activated by fatty acids transported by FABP5. Further investigations showed that PPARγ up-regulated VEGF expression through acting with the PPAR-responsive elements in the promoter region of VEGF gene in prostate cancer cells. Although androgen can modulate VEGF expression through Sp1/Sp3 binding site on VEGF promoter in androgen-dependent prostate cancer cells, this route, disappeared as the cells gradually lost their androgen dependency; was replaced by the FABP5-PPARγ-VEGF signalling pathway. These results suggested that the FABP5-PPARγ-VEGF signal transduction axis, rather than androgen modulated route, may be a more important novel therapeutic target for angiogenesis-suppression treatment of castration resistant prostate cancer

Identification of Selected Persistent Organic Pollutants in Agricultural Land by Carbon Nitride (C3N5) Based Nano Sensors

Efficient detection of selected persistent organic pollutants (POPs) is extremely important for the safety of humans and for the moderation of agriculture. This calls for the design of versatile nano sensors capable of sensing toxic POPs with high sensitivity and selectivity. Inspired by this, the sensing characteristics of carbon nitride (C3N5) monolayers toward selected POPs are reported, such as Dichlorodiphenyltrichloroethane (DDT), Methoxychlor (DMDT), Fenthion (FT), Fenitrothion (FNT), and Rennol (RL),employing density functional theory calculations. Analysis of results predicts adsorption energies of−0.93,−1.55,−1.44,−0.98, and−1.15 eV for DDT,DMDT, FT, FNT, and RM, respectively, on C3N5monolayers. Significant charge transfers among organic pollutants and C3N5lead to distinct electronic properties of the conjugated complexes, revealed by the density of states, electrostatic potential, and work function calculations. To detect the selected pollutants in high humidity, the effects due to aqueous medium are considered. Additionally, a statistical thermodynamic analysis utilizing the Langmuir adsorption model is utilized to explore the influence of temperature and pressure