Illustrated Workbook for the Detection of Plant Viruses Infecting Cucurbits: Demonstration of the Use of Direct Immunoblotting Assays

There are at least 5 viruses of cucurbitaceous plants (squash, melons, cucumber, etc.) that frequently cause severe to complete economic loss for cucurbit producers in the tropics. These viruses are: zucchini yellow mosaic virus (ZYMV), papaya ringspot virus watermelon isolate (PRSV-W, formerly watermelon mosaic virus 1 or WMV I), watermelon mosaic virus 2 (WMV 2), cucumber mosaic virus (CMV) and squash mosaic virus (SqMV). To accurately diagnose virus infections you must use a detection technique. Many techniques are available, including host plant range analysis and serological and molecular analysis. The purpose of our workshop and this illustrated workbook is to demonstrate the use of a serological technique called direct immunoblotting. The advantage of this technique is that it requires very little special equipment, the assay is relatively easy to perform and the assay has some characteristics that make it ideal for diagnosing problems associated with surveying island communities.Funded through the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service Grant 99-38826-785

Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. © 2013 Kaabinejadian et al

In silico toxicology protocols

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information

OSR1-sensitive renal tubular phosphate reabsorption

Background: The oxidative stress-responsive kinase 1 (OSR1) participates in the WNK-(with no K) kinase dependent regulation of renal salt excretion and blood pressure. Little is known, however, about the role of OSR1 in the regulation of further renal transport systems. The present study analyzed the effect of OSR1 on NaPiIIa, the major renal tubular phosphate transporter. Methods: Immunohistochemistry and confocal microscopy were employed to determine renal localization of OSR1 and NaPiIIa. To elucidate the effect of OSR on NaPiIIa activity, cRNA encoding NaPiIIa was injected into Xenopus oocytes with or without additional injection of cRNA encoding OSR1, and phosphate transport was estimated from phosphateinduced currents determined with dual electrode voltage clamp. To elucidate the in vivo significance of OSR1 serum phosphate and hormone concentrations as well as urinary phosphate output of mice carrying one allele of WNK-resistant OSR1 (osr1(tg/+)) were compared to the respective values of wild type mice (osr1(+/+)). Results: NaPiIIa and OSR1 were both expressed in proximal renal tubule cells. Coexpression of OSR1 significantly up-regulated phosphate-induced currents in NaPiIIa-expressing Xenopus oocytes. Despite decreased serum phosphate concentration urinary phosphate excretion was significantly increased and NaPiIIa protein abundance in the brush border membrane significantly reduced in osr1(tg/+) mice as compared to osr1(+/+) mice. Serum PTH and calcitriol levels were similar in osr1(tg/+) mice and in osr1(+/+) mice, serum FGF23 concentration was, however, significantly higher in osr1(tg/+) mice than in osr1(+/+) mice. Conclusions: OSR1 is expressed in proximal renal tubules and participates in the regulation of FGF23 release and renal tubular phosphate transport

Chromosome Xq23 is associated with lower atherogenic lipid concentrations and favorable cardiometabolic indices

Abstract Autosomal genetic analyses of blood lipids have yielded key insights for coronary heart disease (CHD). However, X chromosome genetic variation is understudied for blood lipids in large sample sizes. We now analyze genetic and blood lipid data in a high-coverage whole X chromosome sequencing study of 65,322 multi-ancestry participants and perform replication among 456,893 European participants. Common alleles on chromosome Xq23 are strongly associated with reduced total cholesterol, LDL cholesterol, and triglycerides (min P = 8.5 × 10−72), with similar effects for males and females. Chromosome Xq23 lipid-lowering alleles are associated with reduced odds for CHD among 42,545 cases and 591,247 controls (P = 1.7 × 10−4), and reduced odds for diabetes mellitus type 2 among 54,095 cases and 573,885 controls (P = 1.4 × 10−5). Although we observe an association with increased BMI, waist-to-hip ratio adjusted for BMI is reduced, bioimpedance analyses indicate increased gluteofemoral fat, and abdominal MRI analyses indicate reduced visceral adiposity. Co-localization analyses strongly correlate increased CHRDL1 gene expression, particularly in adipose tissue, with reduced concentrations of blood lipids