Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription

ABSTRACT Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression. IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates

How portuguese and american teachers plan for literacy instruction

This study explored American and Portuguese elementary teachers' preferences in planning for literacy instruction using the Language Arts Activity Grid (LAAG; Cunningham, Zibulsky, Stanovich, & Stanovich, 2009), on which teachers described their preferred instructional activities for a hypothetical 2-h language arts block. Portuguese teachers (N = 186) completed Portuguese versions of a background questionnaire and LAAG electronically, in Survey Monkey; American teachers (N = 102) completed identical English measures using paper and pencil. Results showed that teachers in both groups usually addressed comprehension and reading fluency on their LAAGs and that they also allocated the most time to these two areas. However, American teachers were more likely to include teacher-directed fluency activities, whereas Portuguese teachers were more likely to include fluency activities that were not teacher directed. Significantly more American than Portuguese teachers addressed phonics in their planning, whereas significantly more Portuguese than American teachers addressed writing processes such as revision. Both groups of educators demonstrated large variability in planning, with many teachers omitting important components of literacy identified by researchers, for writing as well as reading. The study highlights the importance of providing teachers with comprehensive, research-based core literacy curricula as well as professional development on key components of literacy. Study findings also suggest significant relationships between orthographic transparency and teachers' instructional planning.This research was supported by a 2-year grant from the Foundation Francisco Manuel dos Santos in Portugal as well as by a Connecticut State University research grant in the U.S.A. We would like to express our sincere gratitude to these funding agencies as well as to the teachers and school districts who participated in the study and sent messages of interest about our research. In addition, warm thanks to our research assistants for their help with data collection, coding, and analysis, and to Anne Cunningham for providing us with inspiration as well as guidance in this work.info:eu-repo/semantics/publishedVersio

Quantitative Detection of Schistosoma japonicum Cercariae in Water by Real-Time PCR

In China alone, an estimated 30 million people are at risk of schistosomiasis, caused by the Schistosoma japonicum parasite. Disease has re-emerged in several regions that had previously attained transmission control, reinforcing the need for active surveillance. The environmental stage of the parasite is known to exhibit high spatial and temporal variability, and current detection techniques rely on a sentinel mouse method which has serious limitations in obtaining data in both time and space. Here we describe a real-time PCR assay to quantitatively detect S. japonicum cercariae in laboratory samples and in natural water that has been spiked with known numbers of S. japonicum. Multiple primers were designed and assessed, and the best performing set, along with a TaqMan probe, was used to quantify S. japonicum. The resulting assay was selective, with no amplification detected for Schistosoma mansoni, Schistosoma haematobium, avian schistosomes nor organisms present in non-endemic surface water samples. Repeated samples containing various concentrations of S. japonicum cercariae showed that the real-time PCR method had a strong linear correlation (R2 = 0.921) with light microscopy counts, and the detection limit was below the DNA equivalent of half of one cercaria. Various cercarial concentrations spiked in 1 liter of natural water followed by a filtration process produced positive detection from 93% of samples analyzed. The real-time PCR method performed well quantifying the relative concentrations of various spiked samples, although the absolute concentration estimates exhibited high variance across replicated samples. Overall, the method has the potential to be applied to environmental water samples to produce a rapid, reliable assay for cercarial location in endemic areas

Sexual Size Dimorphism and Body Condition in the Australasian Gannet

Funding: The research was financially supported by the Holsworth Wildlife Research Endowment. Acknowledgments We thank the Victorian Marine Science Consortium, Sea All Dolphin Swim, Parks Victoria, and the Point Danger Management Committee for logistical support. We are grateful for the assistance of the many field volunteers involved in the study.Peer reviewedPublisher PD

Detection of Progeny Immune Responses after Intravenous Administration of DNA Vaccine to Pregnant Mice

A number of factors influence the development of tolerance, including the nature, concentration and mode of antigen presentation to the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant mice with liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding antigens from HIV-1 and influenza virus) were administered intravenously to pregnant mice. At 9.5 days post conception with cationic liposomes, injected plasmid was present in the tissues of the fetus, consistent with trans-placental transfer. When the offspring of vaccinated dams were immunized with DNA vaccine, they mounted stronger antigen-specific immune responses than controls and were protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA vaccinated mothers confer the antigen-specific immunity to their progeny. Here we describe the methods in detail as they relate to our previously published work

Environmental Enrichment Preceding Early Adulthood Methylphenidate Treatment Leads to Long Term Increase of Corticosterone and Testosterone in the Rat

Attention-deficit/hyperactivity disorder (ADD/ADHD) has been emerging as a world-wide psychiatric disorder. There appears to be an increasing rate of stimulant drug abuse, specifically methylphenidate (MPH) which is the most common treatment for ADHD, among individuals who do not meet the criteria for ADHD and particularly for cognitive enhancement among university students. However, the long term effects of exposure to MPH are unknown. Thus, in light of a developmental approach in humans, we aimed to test the effects of adolescence exposure to enriched environment (EE) followed by MPH administration during early adulthood, on reactions to stress in adulthood. Specifically, at approximate adolescence [post natal days (PND) 30–60] rats were reared in EE and were treated with MPH during early adulthood (PND 60–90). Adult (PND 90–92) rats were exposed to mild stress and starting at PND 110, the behavioral and endocrine effects of the combined drug and environmental conditions were assessed. Following adolescence EE, long term exposure to MPH led to decreased locomotor activity and increased sucrose preference. EE had a beneficial effect on PPI (attentive abilities), which was impaired by long term exposure to MPH. Finally, the interaction between EE and, exposure to MPH led to long-term elevated corticosterone and testosterone levels. In view of the marked increase in MPH consumption over the past decade, vigilance is crucial in order to prevent potential drug abuse and its long term detrimental consequences

Glycosaminoglycan Interactions in Murine Gammaherpesvirus-68 Infection

Glycosaminoglycans (GAGs) commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces

Differences in lateral gene transfer in hypersaline versus thermal environments

<p>Abstract</p> <p>Background</p> <p>The role of lateral gene transfer (LGT) in the evolution of microorganisms is only beginning to be understood. While most LGT events occur between closely related individuals, inter-phylum and inter-domain LGT events are not uncommon. These distant transfer events offer potentially greater fitness advantages and it is for this reason that these "long distance" LGT events may have significantly impacted the evolution of microbes. One mechanism driving distant LGT events is microbial transformation. Theoretically, transformative events can occur between any two species provided that the DNA of one enters the habitat of the other. Two categories of microorganisms that are well-known for LGT are the thermophiles and halophiles.</p> <p>Results</p> <p>We identified potential inter-class LGT events into both a thermophilic class of Archaea (Thermoprotei) and a halophilic class of Archaea (Halobacteria). We then categorized these LGT genes as originating in thermophiles and halophiles respectively. While more than 68% of transfer events into Thermoprotei taxa originated in other thermophiles, less than 11% of transfer events into Halobacteria taxa originated in other halophiles.</p> <p>Conclusions</p> <p>Our results suggest that there is a fundamental difference between LGT in thermophiles and halophiles. We theorize that the difference lies in the different natures of the environments. While DNA degrades rapidly in thermal environments due to temperature-driven denaturization, hypersaline environments are adept at preserving DNA. Furthermore, most hypersaline environments, as topographical minima, are natural collectors of cellular debris. Thus halophiles would in theory be exposed to a greater diversity and quantity of extracellular DNA than thermophiles.</p

Caveats of chronic exogenous corticosterone treatments in adolescent rats and effects on anxiety-like and depressive behavior and hypothalamic-pituitary-adrenal (HPA) axis function

<p>Abstract</p> <p>Background</p> <p>Administration of exogenous corticosterone is an effective preclinical model of depression, but its use has involved primarily adult rodents. Using two different procedures of administration drawn from the literature, we explored the possibility of exogenous corticosterone models in adolescence, a time of heightened risk for mood disorders in humans.</p> <p>Methods</p> <p>In experiment 1, rats were injected with 40 mg/kg corticosterone or vehicle from postnatal days 30 to 45 and compared with no injection controls on behavior in the elevated plus maze (EPM) and the forced swim test (FST). Experiment 2 consisted of three treatments administered to rats from postnatal days 30 to 45 or as adults (days 70 to 85): either corticosterone (400 μg/ml) administered in the drinking water along with 2.5% ethanol, 2.5% ethanol or water only. In addition to testing on EPM, blood samples after the FST were obtained to measure plasma corticosterone. Analysis of variance (ANOVA) and alpha level of <it>P </it>< 0.05 were used to determine statistical significance.</p> <p>Results</p> <p>In experiment 1, corticosterone treatment of adolescent rats increased anxiety in the EPM and decreased immobility in the FST compared to no injection control rats. However, vehicle injected rats were similar to corticosterone injected rats, suggesting that adolescent rats may be highly vulnerable to stress of injection. In experiment 2, the intake of treated water, and thus doses delivered, differed for adolescents and adults, but there were no effects of treatment on behavior in the EPM or FST. Rats that had ingested corticosterone had reduced corticosterone release after the FST. Ethanol vehicle also affected corticosterone release compared to those ingesting water only, but differently for adolescents than for adults.</p> <p>Conclusions</p> <p>The results indicate that several challenges must be overcome before the exogenous corticosterone model can be used effectively in adolescents.</p

Deciphering Biosignatures in Planetary Contexts

Microbial life permeates Earth's critical zone and has likely inhabited nearly all our planet's surface and near subsurface since before the beginning of the sedimentary rock record. Given the vast time that Earth has been teeming with life, do astrobiologists truly understand what geological features untouched by biological processes would look like? In the search for extraterrestrial life in the Universe, it is critical to determine what constitutes a biosignature across multiple scales, and how this compares with “abiosignatures” formed by nonliving processes. Developing standards for abiotic and biotic characteristics would provide quantitative metrics for comparison across different data types and observational time frames. The evidence for life detection falls into three categories of biosignatures: (1) substances, such as elemental abundances, isotopes, molecules, allotropes, enantiomers, minerals, and their associated properties; (2) objects that are physical features such as mats, fossils including trace-fossils and microbialites (stromatolites), and concretions; and (3) patterns, such as physical three-dimensional or conceptual n-dimensional relationships of physical or chemical phenomena, including patterns of intermolecular abundances of organic homologues, and patterns of stable isotopic abundances between and within compounds. Five key challenges that warrant future exploration by the astrobiology community include the following: (1) examining phenomena at the “right” spatial scales because biosignatures may elude us if not examined with the appropriate instrumentation or modeling approach at that specific scale; (2) identifying the precise context across multiple spatial and temporal scales to understand how tangible biosignatures may or may not be preserved; (3) increasing capability to mine big data sets to reveal relationships, for example, how Earth's mineral diversity may have evolved in conjunction with life; (4) leveraging cyberinfrastructure for data management of biosignature types, characteristics, and classifications; and (5) using three-dimensional to n-D representations of biotic and abiotic models overlain on multiple overlapping spatial and temporal relationships to provide new insights