Viperin Is Induced following Dengue Virus Type-2 (DENV-2) Infection and Has Anti-viral Actions Requiring the C-terminal End of Viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.     

In vivo DNA damage in gastric epithelial cells

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.     

The Need for Improved Identification and Accurate Classification of Stages 3–5 Chronic Kidney Disease in Primary Care: Retrospective Cohort Study

Background

Around ten percent of the population have been reported as having Chronic Kidney Disease (CKD), which is associated with increased cardiovascular mortality. Few previous studies have ascertained the chronicity of CKD. In the UK, a payment for performance (P4P) initiative incentivizes CKD (stages 3–5) recognition and management in primary care, but the impact of this has not been assessed.

Methods and Findings

Using data from 426 primary care practices (population 2,707,130), the age standardised prevalence of stages 3–5 CKD was identified using two consecutive estimated Glomerular Filtration Rates (eGFRs) seven days apart. Additionally the accuracy of practice CKD registers and the relationship between accurate identification of CKD and the achievement of P4P indicators was determined. Between 2005 and 2009, the prevalence of stages 3–5 CKD increased from 0.3% to 3.9%. In 2009, 30,440 patients (1.1% unadjusted) fulfilled biochemical criteria for CKD but were not on a practice CKD register (uncoded CKD) and 60,705 patients (2.2% unadjusted) were included on a practice CKD register but did not fulfil biochemical criteria (miscoded CKD). For patients with confirmed CKD, inclusion in a practice register was associated with increasing age, male sex, diabetes, hypertension, cardiovascular disease and increasing CKD stage (p<0.0001). Uncoded CKD patients compared to miscoded patients were less likely to achieve performance indicators for blood pressure (OR 0.84, 95% CI 0.82–0.86 p<0.001) or recorded albumin-creatinine ratio (OR 0.73, 0.70–0.76, p<0.001).

Conclusions

The prevalence of stages 3–5 CKD, using two laboratory reported eGFRs, was lower than estimates from previous studies. Clinically significant discrepancies were identified between biochemically defined CKD and appearance on practice registers, with misclassification associated with sub-optimal care for some people with CKD.     

Demonstration of rhodanese activity in polyacrylamide gels

Rhodanese activity from crude extracts of Thiobacillus sp. strain IV-85 was demonstrated in polyacrylamide gels after incubation in the reaction mixture by staining with dichloroindophenol in the presence of methylphenazonium methosulfate. The sensitivity of the staining system was found to be 8 × 10^-7 moles of sulfite.     

Additional glomangioma families link to chromosome 1p: no evidence for genetic heterogeneity

Venous malformations are a common abnormality of the vasculature that may occur sporadically or, more rarely, as an autosomal dominant trait. One familial form of venous malformations has previously been linked to chromosome 9p. Mutations in the gene encoding Tie2, an endothelial specific receptor tyrosine kinase, have been identified in four different families. Glomangiomas are a subtype of venous malformations with glomus cell involvement. These cutaneous lesions can be inherited as an autosomal dominant disease with reduced penetrance and variable expressivity. We present evidence of linkage to chromosome 1p21-1p22 using four new glomangioma families, with a combined maximum two-point lod score of 7.32 at marker D1S2804. Markers D1S2129 and D1S2881 define the 24-cM linkage interval determined by recombination within affected individuals. A recent report also showed linkage of the glomangioma locus to chromosome 1p. A total of 9 families now map to this region, suggesting a decreased likelihood of locus heterogenity in familial glomangiomas. Investigation of candidate genes within the interval should provide new insights into lesion formation in inherited venous malformations.     

Electrical, chemical, and topological addressing of mammalian cells with microfabricated systems

This communication describes our work in electrical, topological, and chemical micromodification of surfaces to modulate cellular form and function. We have addressed the surface physico-chemico-mechano properties of cell culture substrates that play a role in modulating cellular behavior. Single factorial model systems have been built using techniques adapted from microlithography. The tools and techniques of microfabrication, if harnessed and used correctly, can be enabling in elucidating the underlying principles and fundamental forces driving the cell-substrate interface. Additionally, the long-term practical applications of microfabrication in medicine and biomaterial/tissue engineering lie in enabling "communication" with living cells/tissues at the cellular and subcellular levels.     

Genomic patenting and the utility requirement

This paper analyses the ways in which genomic knowledge is portrayed as useful knowledge in gene patenting in order to fulfil the 'utility'/'industrial applicability' requirement for patentability. It gives examples of utility claims in gene patents and asks whether genomics (as opposed to genetics) changes our ideas about what is useful and what can be patented. It puts forward a provisional classification of different types of utility and argues that merely identifying the physiological function of a gene diverges radically from our commonsense understanding of what it is for an invention to be useful. Furthermore, social, political and ethical issues inevitably arise when discussing the utility requirement, because an invention cannot be useful in isolation from a social context.     

Developmental pattern of glucose-6-phosphatase activity in the small intestine of the mouse fetus

Summary The distribution of glucose-6-phosphatase (G6Pase) activity in the epithelium of the small intestine in mouse embryos (the last 4 days of gestation) was studied by electron microscope cytochemistry and by enzymatic assays. At 16 days, the lead phosphate deposited by the cytochemical reaction is localized on the rough endoplasmic reticulum (RER) and nuclear envelope of very few cells in the duodenum and jejunum. Positive cells are more frequently seen in the upper part of the developing villi. At 17 days of gestation, a tremendous burst in RER differentiation is noticed in all parts of the small intestine and concomitantly glycogen disappears. At 18 days of gestation all the principal cells of the intestinal mucosa show a well differentiated positive RER and the enzyme is also present in the smooth endoplasmic reticulum. Biochemically, G6Pase activity is detected in the proximal 2 thirds of the small intestine at 17 days of gestation and appears at 18 days in the last third. Afterwards the activity increases up until birth. These results suggest (1) that the endoplasmic reticulum differentiates very late in the intestinal mucosa of mouse embryos (2) that the differentiation with respect to G6Pase is asynchronous between the enterocytes, (3) that for a given cell all the cisternae of RER are involved in G6Pase synthesis at the same moment and (4) that the enterocytes of the duodenum differentiate sooner and faster that those of the jejunum and ileum.This work was supported by Grant No. 6069 from the MRC of CanadaMr. D. Malka was supported by a studentship from the F.C.A.C. of the province of QuebecDr. D. Ménard is a Chercheur boursier du Conseil de la Recherche en Santé du Québec