ADAMTS7 Promotes Smooth Muscle Foam Cell Expansion in Atherosclerosis.

Human genetic studies have repeatedly associated ADAMTS7 with atherosclerotic cardiovascular disease. Subsequent investigations in mice demonstrated that ADAMTS7 is proatherogenic and induced in response to vascular injury and that the proatherogenicity of ADAMTS7, a secreted protein, is due to its catalytic activity. However, the cell-specific mechanisms governing ADAMTS7 proatherogenicity remain unclear.

On the benefits of the tryptophan metabolite 3-hydroxyanthranilic acid in Caenorhabditis elegans and mouse aging.

Tryptophan metabolism through the kynurenine pathway influences molecular processes critical to healthy aging including immune signaling, redox homeostasis, and energy production. Aberrant kynurenine metabolism occurs during normal aging and is implicated in many age-associated pathologies including chronic inflammation, atherosclerosis, neurodegeneration, and cancer. We and others previously identified three kynurenine pathway genes-tdo-2, kynu-1, and acsd-1-for which decreasing expression extends lifespan in invertebrates.

Single-nucleus resolution mapping of the adult C. elegans and its application to elucidate inter- and trans-generational response to alcohol.

Single-cell transcriptomic platforms provide an opportunity to map an organism's response to environmental cues with high resolution. Here, we applied single-nucleus RNA sequencing (snRNA-seq) to establish the tissue and cell type-resolved transcriptome of the adult C. elegans and characterize the inter- and trans-generational transcriptional impact of ethanol.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices.

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates.

Intrahost Selection Pressure Drives Equine Arteritis Virus Evolution during Persistent Infection in the Stallion Reproductive Tract.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event.

Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus.

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the gene () and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs).

Allelic Variation in CXCL16 Determines CD3+ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10-70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus.

Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope(®)) and assessment of their performance in tissues from aborted equine fetuses.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope(®) ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques.

Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples.

Further evaluation and validation of a commercially available competitive ELISA (cELISA) for the detection of antibodies specific to equine arteritis virus (EAV).

The purpose of this study was to further evaluate and validate two commercially available equine arteritis virus (EAV) competitive ELISAs (original and enhanced cELISAs) using archived equine sera from experimentally inoculated animals and field sera submitted for laboratory diagnosis. First, the original and subsequently enhanced cELISAs were compared with the virus neutralisation test (VNT) using a panel of archived serum samples from experimentally inoculated animals. Then, the enhanced cELISA was compared with the VNT using a large panel of archived serum samples.

Complete Genome Sequence of Noncytopathic Bovine Viral Diarrhea Virus 1 Contaminating a High-Passage RK-13 Cell Line.

A high-passage rabbit kidney RK-13 cell line (HP-RK-13[KY], originally derived from the ATCC CCL-37 cell line) used in certain laboratories worldwide is contaminated with noncytopathic bovine viral diarrhea virus (ncpBVDV). On complete genome sequence analysis, the virus strain was found to belong to BVDV group 1b.

Development and model testing of antemortem screening methodology to predict required drug withholds in heifers.

A simple, cow-side test for the presence of drug residues in live animal fluids would provide useful information for tissue drug residue avoidance programs. This work describes adaptation and evaluation of rapid screening tests to detect drug residues in serum and urine. Medicated heifers had urine, serum, and tissue biopsy samples taken while on drug treatment.

Validation of an improved competitive enzyme-linked immunosorbent assay to detect Equine arteritis virus antibody.

The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B7(9) using the World Organization for Animal Health (OIE)-recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis.

Comparison of an improved competitive enzyme-linked immunosorbent assay with the World Organization for Animal Health-prescribed serum neutralization assay for detection of antibody to Equine arteritis virus.

Equine arteritis virus (EAV) causes contagious equine viral arteritis, characterized by fever, anorexia, conjunctivitis, nasal discharge, dependent edema, abortion, infrequent death in foals, and establishment of the carrier state in stallions. The World Organization for Animal Health (OIE) defines a horse as seropositive if the serum neutralization (SN) antibody titer is ≥1:4 to EAV. However, determining the SN titer is time-consuming and requires specific laboratory facilities, equipment, and technical expertise to perform.

Single-layer centrifugation reduces equine arteritis virus titre in the semen of shedding stallions.

Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission.

Evidence that in vitro susceptibility of CD3+ T lymphocytes to equine arteritis virus infection reflects genetic predisposition of naturally infected stallions to become carriers of the virus.

We investigated the correlation between in vitro susceptibility of CD3(+) T lymphocytes to equine arteritis virus (EAV) infection and establishment of persistent infection among 14 stallions following natural infections. The data showed that carrier stallions with a CD3(+) T lymphocyte susceptibility phenotype to in vitro EAV infection may be at higher risk of becoming carriers than those that lack this phenotype (P = 0.0002).

Evaluation of two magnetic-bead-based viral nucleic acid purification kits and three real-time reverse transcription-PCR reagent systems in two TaqMan assays for equine arteritis virus detection in semen.

This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.

Molecular epidemiology and genetic characterization of equine arteritis virus isolates associated with the 2006-2007 multi-state disease occurrence in the USA.

In 2006-2007, equine viral arteritis (EVA) was confirmed for the first time in Quarter Horses in multiple states in the USA. The entire genome of an equine arteritis virus (EAV) isolate from the index premises in New Mexico was 12 731 nt in length and possessed a previously unrecorded unique 15 nt insertion in the nsp2-coding region in ORF1a and a 12 nt insertion in ORF3. Sequence analysis of additional isolates made during this disease occurrence revealed that all isolates from New Mexico, Utah, Kansas, Oklahoma and Idaho had 98.

Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid.

Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument.

Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture.

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S.

Analysis of the pore structure of the influenza A virus M(2) ion channel by the substituted-cysteine accessibility method.

The M(2) ion channel of influenza A virus is a small integral membrane protein whose active form is a homotetramer with each polypeptide chain containing 96-amino-acid residues. To identify residues of the transmembrane (TM) domain that line the presumed central ion-conducting pore, a set of mutants was generated in which each residue of the TM domain (residues 25 to 44) was replaced by cysteine. The accessibility of the cysteine mutants to modification by the sulfhydryl-specific reagents methane thiosulfonate ethylammonium (MTSEA) and MTS tetraethylammonium (MTSET) was tested.