Studies on the contribution of the left unique region of the genome to Murine Gammaherpesvirus-68 pathogenesis

Members of the gamma subgroup of the herpesvirus family are characterised by their ability to establish lifelong latent infection in lymphocytes, and their association with lymphoproliferative disease and a range of malignant tumours. Members include the Epstein-Barr virus (EBV) and Kaposi's sarcomaassociated herpesvirus (KSHV), which are linked with clinically important human diseases, as well as alcelaphine herpesvirus 1 (A1HV-1) and ovine herpesvirus 2 (OvHV-2), which are of veterinary importance. Due to the severe limitations on the study of gammaherpesviruses presented by their species specificity and restricted growth in vitro, infection of laboratory mice with the naturally occurring virus murine gammaherpesvirus 68 (MHV-68) has become an excellent model system for the study of gammaherpesvirus pathogenesis.This project involved the characterisation of murine herpesvirus 76 (MHV-76), another of the murine herpesviruses isolated at the same time as MHV-68. Molecular analysis revealed that MHV-76 is a deletion mutant of MHV-68, that lacks four genes unique to MHV-68 (Ml, M2, M3 and M4) and eight viral tRNA-like genes that are all present at the left end of the MHV-68 genome. Biological characterisation of MHV-76, including the generation and study of rescue viruses which restored the deleted sequence into MHV-76, showed that loss of these genes leads to enhanced clearance of virus from the lungs due to a vigorous inflammatory response. There was also a significant reduction in splenomegaly and the amount of latent virus detectable in the spleen. These results show that the genes present at the left end of the unique region of the MHV-68 genome play a critical role in the viral immune evasion strategy.Detailed studies on one of these unique MHV-68 genes, M2, show that it is a 30kDa protein that has localised homology with the Gab2 and semaphorin protein families. Expression of M2 in mammalian cells reveals that it co-localises to the plasma membrane, as well as being present in diffuse areas of the nucleus and cytoplasm. Generation of a recombinant virus with a disruption in the M2 ORE showed that the M2 gene plays a critical role in the establishment of latency and the regulation of the numbers of latently infected cells in the spleen. The M2 gene is not involved in the genesis of splenomegaly. These studies uncover some of the genetic elements involved in gammaherpesvirus pathogenesis

ECONOMIC ANALYSIS OF USING A BORDER TREATMENT FOR REDUCING ORGANOPHOSPHATE USE IN SEED POTATO PRODUCTION

Recent research shows initial colonization of potato fields by winged green peach aphid is concentrated at field edges. This suggests that insecticides applied only to field margins during initial colonization would largely eliminate a colonizing aphid population, conserve natural enemies in the field center, and reduce insecticide use. To better understand the costs and benefits of reducing organophosphate use, the six participating growers were interviewed to ascertain their reason for participating and their satisfaction with the border only treatment method as well as their estimated net economic benefits. Five of the farms ranked cost reduction as the most important reason for participating. The sixth farm ranked reducing virus spread as the most important reason with cost reduction as their second most important reason. The average cost savings over all 28 participating fields of using the border treatment is estimated to be $23.85 per acre for the entire field-a 93% savings. Almost all the farmers found the border treatment method to be successful at aphid control. None of the farmers observed any impact on the physical yield of seed potato. All the fields were certified during the summer except for one of Farmer F's fields that was lost because of off type. In conclusion, the border treatment method seems likely to be adopted by many farmers since the potential cost saving is large and farmers dislike Monitor. However, some farmers may resist the method due to scouting requirements and costs. Also, farmers with fields that do not meet the uniformity requirements of the border treatment will not be successful in their use of the border method.Crop Production/Industries,

DOLORS: Versatile Strategy for Internal Labeling and Domain Localization in Electron Microscopy

SummarySingle-particle electron microscopy (EM) is a powerful tool for studying the structures of large biological molecules. However, the achievable resolution does not always allow for direct recognition of individual protein domains. Labels that can be visualized by EM have been developed for protein termini, but tagging internal domains remains a challenge. We describe a robust strategy for determining the position of internal sites within EM maps, termed domain localization by RCT sampling (DOLORS). DOLORS uses monovalent streptavidin added posttranslationally to tagged sites in the target protein. Internal labels generally display less conformational flexibility than terminal labels, providing more precise positional information. Automated methods are used to rapidly generate assemblies of unique 3D models allowing the attachment sites of labeled domains to be accurately identified and thus provide an overall architectural map of the molecule

Single-pot enzymatic synthesis of Dicer-substrate siRNAs

We describe an inexpensive and efficient method for generating functional pools of Dicer-substrate small interfering RNAs (siRNAs) in a single reaction tube. The method exploits a highly active form of the enzyme Dicer from Giardia lamblia, which is capable of accurately processing double-stranded RNA (dsRNA) into 25–27 nt RNA pools during in vitro transcription. The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells. The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes

WebCSD: the online portal to the Cambridge Structural Database

The new web-based application WebCSD is introduced, which provides a range of facilities for searching the Cambridge Structural Database within a standard web browser. Search options within WebCSD include two-dimensional substructure, molecular similarity, text/numeric and reduced cell searching

Deducing chemical structure from crystallographically determined atomic coordinates

An improved algorithm has been written for assigning chemical structures to incoming entries to the Cambridge Structural Database

p53 regulates mitochondrial dynamics in vascular smooth muscle cell calcification.

Arterial calcification is an important characteristic of cardiovascular disease. It has key parallels with skeletal mineralization; however, the underlying cellular mechanisms responsible are not fully understood. Mitochondrial dynamics regulate both bone and vascular function. In this study, we therefore examined mitochondrial function in vascular smooth muscle cell (VSMC) calcification. Phosphate (Pi)-induced VSMC calcification was associated with elongated mitochondria (1.6-fold increase, p p p p p p p p p p p p p p < 0.001) was also observed upon p53 knockdown in calcifying VSMCs. In summary, we demonstrate that VSMC calcification promotes notable mitochondrial elongation and cellular senescence via DRP1 phosphorylation. Furthermore, our work indicates that p53-induced mitochondrial fusion underpins cellular senescence by reducing mitochondrial function

Little evidence for association between the TGFBR1*6A variant and colorectal cancer: a family-based association study on non-syndromic family members from Australia and Spain

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Genome-wide linkage studies have identified the 9q22 chromosomal region as linked with colorectal cancer (CRC) predisposition. A candidate gene in this region is transforming growth factor β receptor 1 (TGFBR1). Investigation of TGFBR1 has focused on the common genetic variant rs11466445, a short exonic deletion of nine base pairs which results in truncation of a stretch of nine alanine residues to six alanine residues in the gene product. While the six alanine (*6A) allele has been reported to be associated with increased risk of CRC in some population based study groups this association remains the subject of robust debate. To date, reports have been limited to population-based case–control association studies, or case–control studies of CRC families selecting one affected individual per family. No study has yet taken advantage of all the genetic information provided by multiplex CRC families