Transcription Mapping and Characterization of 284R and 121R Proteins Produced from Early Region 3 of Bovine Adenovirus Type 3

AbstractWe established the transcription map of early region (E) 3 of bovine adenovirus 3 (BAV-3) by Northern blot, S1 nuclease protection assays, cDNA sequencing, and RT-PCR analysis. Five major classes of mRNAs were identified, which shared the 3′ ends. Four classes of mRNAs transcribed from the E3 promoter also shared the 5′ end, while one major class of mRNA transcribed from the major late promoter contained a tripartite leader sequence at the 5′ end. These five transcripts have the potential to encode four proteins, namely 284R, 121R, 86R, and 82R. To identify the proteins, rabbit antiserum was prepared using a bacterial fusion protein encoding 284R or 121R protein. Serum against 284R immunoprecipitated protein of 26–32 kDa in in vitro translated and transcribed mRNA and three proteins of 48, 67, and 125 kDa from BAV-3-infected cells. Western blots and enzymatic digestions confirmed that the 284R protein is a glycoprotein, which contains only N-linked oligosaccharides, both high mannose (48 kDa) and complex types (67 kDa). Serum against 121R immunoprecipitated a protein of 14.5 kDa from in vitro translated and transcribed mRNA and BAV-3-infected cells. Although 121R protein shows limited sequence similarity to a 14.7-kDa protein of human adenovirus 5, the 284R protein appears to be unique to BAV-3. Since proteins encoded by the E3 region appear to influence adenovirus pathogenesis, the 284R protein may contribute to the unique pathogenic properties of BAV-3

Pathogenesis and Immunogenicity of Bovine Adenovirus Type 3 in Cotton Rats (Sigmodon hispidus)

AbstractIntranasal inoculation of cotton rats (Sigmodon hispidus) with 108 PFU of bovine adenovirus type 3 (BAd3) resulted in limited virus replication in the lung and trachea. Histopathological changes in the lungs were characterized by necrosis and hyperplasia of bronchiolar epithelium, eosinophilic intranuclear inclusions, pneumocyte type II hyperplasia in the alveoli, and mild peribronchiolar and perivascular lymphocytic infiltration. Immunohistochemically, viral antigens were observed more frequently in bronchiolar epithelial cells than in alveolar cells in cotton rat lung sections stained using a rabbit anti-BAd3 serum. Bronchiolar epithelial changes, intranuclear inclusion bodies, type II pneumocyte proliferation, peribronchiolar infiltration, and immunohistological staining were maximum at Day 3 or Day 4 postinoculation, whereas perivascular infiltration was first observed at Day 8 postinoculation. In addition to the histological study of the pathogenesis of BAd3 infection, we monitored the BAd3-specific immune response in cotton rats. Anti-BAd3 IgG and virus neutralizing antibodies were detected in sera, whereas anti-BAd3 IgA antibodies were found in the sera, lung, and nasal washes. Our results suggest that the cotton rat can serve as a useful small-animal model for investigating the pathogenesis of BAd3 infection, as well as immune responses to BAd3 recombinant virus vaccines

Construction and Characterization of E3-Deleted Bovine Adenovirus Type 3 Expressing Full-Length and Truncated Form of Bovine Herpesvirus Type 1 Glycoprotein gD

AbstractUsing the homologous recombination machinery ofE. coli,a 1.245-kb deletion was introduced in the E3 region of bovine adenovirus 3 (BAV3) genomic DNA cloned in a plasmid. Transfection of the restriction enzyme-excised, linear E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3.E3d), suggesting that all the E3-specific open reading frames are nonessential for virus replicationin vitro. Using a similar approach, we constructed replication-competent (BAV3.E3gD and BAV3.E3gDt) BAV3 recombinant expressing full-length (gD) or truncated (gDt) glycoprotein of bovine herpes virus 1. Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt, respectively, were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in bovine herpes virus 1-infected cells. Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses. These results suggest that replication-competent bovine adenovirus 3-based vectors have potential for the delivery of vaccine antigens to the mucosal surfaces of animals

Cholera toxin, zonula occludens toxin and accessary cholera enterotoxin gene-negative Vibrio cholerae non-01 strains produce the new cholera toxin

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Decitabine impact on the endocytosis regulator RhoA, the folate carriers RFC1 and FOLR1, and the glucose transporter GLUT4 in human tumors.

BackgroundIn 31 solid tumor patients treated with the demethylating agent decitabine, we performed tumor biopsies before and after the first cycle of decitabine and used immunohistochemistry (IHC) to assess whether decitabine increased expression of various membrane transporters. Resistance to chemotherapy may arise due to promoter methylation/downregulation of expression of transporters required for drug uptake, and decitabine can reverse resistance in vitro. The endocytosis regulator RhoA, the folate carriers FOLR1 and RFC1, and the glucose transporter GLUT4 were assessed.ResultsPre-decitabine RhoA was higher in patients who had received their last therapy >3 months previously than in patients with more recent prior therapy (P = 0.02), and varied inversely with global DNA methylation as assessed by LINE1 methylation (r = -0.58, P = 0.006). Tumor RhoA scores increased with decitabine (P = 0.03), and RFC1 also increased in patients with pre-decitabine scores ≤150 (P = 0.004). Change in LINE1 methylation with decitabine did not correlate significantly with change in IHC scores for any transporter assessed. We also assessed methylation of the RFC1 gene (alias SLC19A1). SLC19A1 methylation correlated with tumor LINE1 methylation (r = 0.45, P = 0.02). There was a small (statistically insignificant) decrease in SLC19A1 methylation with decitabine, and there was a trend towards change in SLC19A1 methylation with decitabine correlating with change in LINE1 methylation (r = 0.47, P <0.15). While SLC19A1 methylation did not correlate with RFC1 scores, there was a trend towards an inverse correlation between change in SLC19A1 methylation and change in RFC1 expression (r = -0.45, P = 0.19).ConclusionsIn conclusion, after decitabine administration, there was increased expression of some (but not other) transporters that may play a role in chemotherapy uptake. Larger patient numbers will be needed to define the extent to which this increased expression is associated with changes in DNA methylation

Partial loss of actin nucleator actin-related protein 2/3 activity triggers blebbing in primary T lymphocytes

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin‐related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F‐actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3‐knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon‐like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three‐dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3‐dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities

The effects of 10 to >160 GPa shock on the magnetic properties of basalt and diabase

© 2016. American Geophysical Union. All Rights Reserved.Hypervelocity impacts within the solar system affect both the magnetic remanence and bulk magnetic properties of planetary materials. Spherical shock experiments are a novel way to simulate shock events that enable materials to reach high shock pressures with a variable pressure profile across a single sample (ranging between ∼10 and >160 GPa). Here we present spherical shock experiments on basaltic lava flow and diabase dike samples from the Osler Volcanic Group whose ferromagnetic mineralogy is dominated by pseudo-single-domain (titano)magnetite. Our experiments reveal shock-induced changes in rock magnetic properties including a significant increase in remanent coercivity. Electron and magnetic force microscopy support the interpretation that this coercivity increase is the result of grain fracturing and associated domain wall pinning in multidomain grains. We introduce a method to discriminate between mechanical and thermal effects of shock on magnetic properties. Our approach involves conducting vacuum-heating experiments on untreated specimens and comparing the hysteresis properties of heated and shocked specimens. First-order reversal curve (FORC) experiments on untreated, heated, and shocked specimens demonstrate that shock and heating effects are fundamentally different for these samples: shock has a magnetic hardening effect that does not alter the intrinsic shape of FORC distributions, while heating alters the magnetic mineralogy as evident from significant changes in the shape of FORC contours. These experiments contextualize paleomagnetic and rock magnetic data of naturally shocked materials from terrestrial and extraterrestrial impact craters

TRAIP/RNF206 is required for recruitment of RAP80 to sites of DNA damage

RAP80 localizes to sites of DNA insults to enhance the DNA-damage responses. Here we identify TRAIP/RNF206 as a novel RAP80-interacting protein and find that TRAIP is necessary for translocation of RAP80 to DNA lesions. Depletion of TRAIP results in impaired accumulation of RAP80 and functional downstream partners, including BRCA1, at DNA lesions. Conversely, accumulation of TRAIP is normal in RAP80-depleted cells, implying that TRAIP acts upstream of RAP80 recruitment to DNA lesions. TRAIP localizes to sites of DNA damage and cells lacking TRAIP exhibit classical DNA-damage response-defect phenotypes. Biochemical analysis reveals that the N terminus of TRAIP is crucial for RAP80 interaction, while the C terminus of TRAIP is required for TRAIP localization to sites of DNA damage through a direct interaction with RNF20-RNF40. Taken together, our findings demonstrate that the novel RAP80-binding partner TRAIP regulates recruitment of the damage signalling machinery and promotes homologous recombinationopen

Peste des petits ruminants virus tissue tropism and pathogenesis in sheep and goats following experimental infection

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.A Canadian International Food Security Research Fund (CIFSRF) grant (no. 106930: Livestock vaccines against viral diseases for sub-Saharan Africa) by the Canadian International Development Research Centre (IDRC) and Canadian International Development Agency (CIDA).http://www.plosone.orgam201