Analysis of two hypervariable human cytomegalovirus genes, UL146 and UL139

Abstract Human cytomegalovirus (HCMV) is a highly host-specific, ubiquitous herpesvirus that results in asymptomatic infection for the majority of those infected. However, it produces serious clinical disease in neonates and immunocompromised individuals such as transplant recipients and AIDS patients. The majority of the 236 kbp genome is highly conserved, but there are a number of highly variable regions, coding and non-coding, scattered throughout the genome. Numerous studies have been published investigating the genotypes of hypervariable genes, most focussed on potential associations between genotype and clinical disease or tropism. In general, no convincing connections between genotype and disease have been found. The present study investigated two hypervariable HCMV genes, UL146 and UL139, in a large number of clinical samples (179) from a number of locations worldwide in Europe, Africa, Asia and Australia. A total of 14 UL146 genotypes (G1-G14) were detected, which agrees with previous findings based on many fewer samples. For UL139, eight genotypes were detected, three of them (G5, G7 and G8) novel. The genotypes of both genes appear to have evolved under constraint rather than positive selection. Possible bias in the geographical distribution of the UL146 and UL139 genotypes was investigated. In general, all genotypes were found in all areas and any variation from the expected distribution was probably a result of small sample numbers from certain regions, specifically Asia and Australia. This general finding is in agreement with that of a previously published study on gene UL73. No evidence for linkage disequilibrium between UL146 and UL139 genotypes was found. This is in accordance with a previously published study of linkage disequilibrium among six other genes (UL55, UL74, UL75, UL115, US9 and US28), and is consistent with the theory that recombination has played a role in HCMV evolution. The absence of linkage between highly variable genes complicates attempts to examine associations between genotype and disease, as many combinations of genotypes are possible. Investigation of transcriptional expression of UL146 and UL139 from HCMV strain Merlin in fibroblast cell culture revealed that UL146 is expressed with late kinetics and UL139 with early-late kinetics. Northern blot and RACE data suggested that UL146 is 3’-coterminally expressed with UL147, UL147A, UL148 and UL132, and that UL139 is 3’-coterminally expressed with UL140 and UL141. To determine whether the high degree of sequence divergence corresponds to structural divergence, the UL146 genotypes were homology modelled on the related human chemokines IL-8, gro-a and IF9S. All 14 genotypes were predicted to be structurally very similar, which suggests they may also be functionally similar. However, small differences between the structures of human chemokines are known to result in slightly differing binding affinities for cellular receptors, and therefore even small differences between UL146 genotypes could conceivably confer functional differences. UL139 has been predicted to encode a type 1 membrane glycoprotein. No information has been published regarding UL139 function, although a short region of similarity with the cellular signal transducer CD24 has been noted previously, tentatively suggesting an immunomodulatory role. Preliminary experiments to characterise UL139 were performed utilising recombinant adenovirus vectors expressing tagged UL139 variants from three genotypes (G1, G5 and G7). The tagged UL139 variants expressed proteins that were considerably larger in mass than predicted from amino acid sequences. This extra mass may be attributable to glycosylation as well as other forms of post translational modification. Mixed infections of HCMV strains in immunocompromised individuals, such as transplant recipients, have been associated with enhanced pathogenesis and increased risk of transplant rejection. The presence of mixed infections also further complicates attempts to establish connections between genotype and disease outcome. In the analysis of UL146 and UL139 genotypes, multiple genotypes were detected in 14% of samples and in 29% when repeated experimental results were included, and even these values may be underestimations. The utility of a QPCR-based assay using genotype-specific primers was assessed as a means of more accurately determining the occurrence of mixed infections, and showed promise. Passage of HCMV strains in cell culture has been shown to result in various mutations. AD169, a commonly used laboratory strain, lacks 15 kbp sequence that includes UL146 and UL139. An alternative stock of AD169 (AD169varUC) was obtained that was thought to contain most or all of the deleted region and, indeed, both UL146 and UL139 were detected. Further sequencing confirmed that this stock is derived from AD169 and revealed that it contains all but 3.2 kbp of the 15 kbp absent from commonly used AD169 stocks. The 3.2 kbp deletion affects UL144, UL142, UL141 and UL140. This propensity of HCMV to undergo mutation during cell culture highlights the importance of studying characterised strains that are as close to wild type virus as possible

Summary Data Report of the 2009-2010 Annual Survey of Divisions of General Practice

The Primary Health Care Research and Information Service (PHCRIS) conducts the Annual Survey of Divisions (ASD) on behalf of the Australian Government Department of Health and Ageing. As part of their contractual obligations, all Divisions of General Practice are required to complete the Survey which includes questions about their membership, activities (including population health) and infrastructure for the previous financial year. Consequently, the results provide a comprehensive overview of Divisions and summarise the broad range of activities that they are involved in

It Cannot Be Helped: Racial Stratification in Jerome and Rohwer, Arkansas During World War II

Between March 1942 and 1945 the War Relocation Authority (WRA) forced over one hundred and twenty thousand Japanese Americans into internment camps hastily constructed across the country. Small communities outside of the rural towns of McGehee and Denson, Arkansas located in the historically racially charged Delta housed two of these internment camps: WRA Internment Camps Jerome and Rohwer. At these two internment camps Japanese-Americans from the far West entered into Jim Crow era Arkansas and into a hotbed of established racial stratification. This research project examined the modern history of this particular geographic region and placed the liminal status of the Japanese-American internees into perspective through analysis of oral histories, newspaper articles, and statistical data. The established racial hierarchy in Delta shaped the way that the local population treated and perceived the Japanese-American internees and conversely impacted the way that the Japanese-American internees perceived the local population

Enhancement of insulin-mediated rat muscle glucose uptake and microvascular perfusion by 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside

BACKGROUND: Insulin-induced microvascular recruitment is important for optimal muscle glucose uptake. 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, an activator of AMP-activated protein kinase), can also induce microvascular recruitment, at doses that do not acutely activate glucose transport in rat muscle. Whether low doses of AICAR can augment physiologic insulin action is unknown. In the present study we used the euglycemic hyperinsulinemic clamp to assess whether insulin action is augmented by low dose AICAR. METHODS: Anesthetized rats were studied during saline infusion or euglycemic insulin (3 mU/kg/min) clamp for 2 h in the absence or presence of AICAR for the last hour (5 mg bolus followed by 3.75 mg/kg/min). Muscle glucose uptake (R\u27g) was determined radioisotopically with (14)C-2-deoxyglucose and muscle microvascular perfusion by contrast-enhanced ultrasound with microbubbles. RESULTS: AICAR did not affect blood glucose, or lower leg R\u27g, although it significantly (p < 0.05) increased blood lactate levels and augmented muscle microvascular blood volume via a nitric oxide synthase dependent pathway. Insulin increased femoral blood flow, whole body glucose infusion rate (GIR), R\u27g, hindleg glucose uptake, and microvascular blood volume. Addition of AICAR during insulin infusion increased lactate production, further increased R\u27g in Type IIA (fast twitch oxidative) and IIB (fast twitch glycolytic) fiber containing muscles, and hindleg glucose uptake, but decreased R\u27g in the Type I (slow twitch oxidative) fiber muscle. AICAR also decreased GIR due to inhibition of insulin-mediated suppression of hepatic glucose output. AICAR augmented insulin-mediated microvascular perfusion. CONCLUSIONS: AICAR, at levels that have no direct effect on muscle glucose uptake, augments insulin-mediated microvascular blood flow and glucose uptake in white fiber type muscles. Agents targeted to endothelial AMPK activation are promising insulin sensitizers, however, the decrease in GIR and the propensity to increase blood lactate cautions against AICAR as an acute insulin sensitizer