HIV-1 Particle Release Mediated by Vpu Is Distinct from That Mediated by p6

AbstractVpu and the C-terminal peptide of Gag (p6) are both HIV-1-encoded proteins that augment the release of virus particles from cells. We examined the functional relationship between these proteins and their activities during particle release. Our results indicate that efficient HIV-1 particle release from HeLa and Jurkat cells depends on the presence of Vpu. However, Vpu is dispensable for efficient release from Cos cells. In contrast, p6 is required for efficient release from Cos cells but not from Jurkat or HeLa cells. These data suggest that Vpu and p6 have distinct activities in virus exit from different cell lines. Intracellular proteolytic processing of Gag precursor protein is more complete in Cos cells than in HeLa cells. However, this processing has little or no effect on Vpu- or p6-mediated particle release. p6 is required for incorporation of yet another virus protein (Vpr) into cells but our data suggest that Vpr plays no role in p6-dependent particle release. Vpu also facilitates the degradation of CD4 in virus producing cells but, in contrast to particle release, the ability of Vpu to facilitate the degradation of CD4 is not cell line-dependent

A Strategy for Adenovirus Vector Targeting with a Secreted Single Chain Antibody

BACKGROUND:Successful gene therapy will require targeted delivery vectors capable of self-directed localization. In this regard, the use of antibodies or single chain antibody fragments (scFv) in conjunction with adenovirus (Ad) vectors remains an attractive means to achieve cell-specific targeting. However, a longstanding barrier to the development of Ad vectors with genetically incorporated scFvs has been the biosynthetic incompatibility between Ad capsid proteins and antibody-derived species. Specifically, scFv require posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during protein synthesis and virion assembly. METHODOLOGY/PRINCIPAL FINDINGS:We have therefore sought to develop scFv-targeted Ad vectors using a secreted scFv that undergoes the requisite posttranslational modifications and is trafficked for secretion. Formation of the scFv-targeted Ad vector is achieved via highly specific association of the Ad virion and a targeting scFv employing synthetic leucine zipper-like dimerization domains (zippers) that have been optimized for structural compatibility with the Ad capsid and for association with the secreted scFv. Our results show that zipper-containing Ad fiber molecules trimerize and incorporate into mature virions and that zippers can be genetically fused to scFv without ablating target recognition. Most importantly, we show that zipper-tagged virions and scFv provide target-specific gene transfer. CONCLUSIONS/SIGNIFICANCE:This work describes a new approach to produce targeted Ad vectors using a secreted scFv molecule, thereby avoiding the problem of structural and biosynthetic incompatibility between Ad and a complex targeting ligand. This approach may facilitate Ad targeting using a wide variety of targeting ligands directed towards a variety of cellular receptors

Validation of a multiplex-tandem RT-PCR for the detection of bovine respiratory disease complex using Scottish bovine lung samples

The welfare and economic impact of bovine respiratory disease complex (BRDC), and its associated antibiotic usage, are major challenges to cattle rearing and beef cattle finishing industries. Accurate pathogen diagnosis is important to undertake appropriate treatment and long-term management strategies, such as vaccine selection. Conventional diagnostic approaches have several limitations including high costs, long turnaround times and difficulty in test interpretation, which could delay treatment decisions and lead to unnecessary animal losses. We describe the validation of a multiplex-tandem (MT) reverse transcription-polymerase chain reaction (RT-PCR) for the detection of seven common pathogens associated with BRDC. This test has the potential to advance pathogen identification and to overcome many of the limitations of current testing methods. It requires a single sample and results are obtained quickly and not influenced by prior antimicrobial therapy or overgrowth of contaminating organisms. We demonstrated a test specificity of 100% and sensitivity ranging from 93.5% to 100% for these seven common pathogens. This test will be a useful addition to advance BRDC investigation and diagnosis.</p

Prediction of congenital hypothyroidism based on initial screening thyroid-stimulating-hormone

Background: In thyroid-stimulating-hormone (TSH)-based newborn congenital hypothyroidism (CH) screening programs, the optimal screening-TSH cutoff level is critical to ensuring that true cases of CH are not missed. Screening-TSH results can also be used to predict the likelihood of CH and guide appropriate clinical management. The purpose of this study is to evaluate the predictive value of various screening-TSH levels in predicting a diagnosis of CH in the Ontario Newborn Screening Program (ONSP). Methods: The initial screening and follow-up data of 444,744 full term infants born in Ontario, Canada from April 1, 2006 to March 31, 2010 were analyzed. Confirmed CH cases were based on local endocrinologists\u27 report and initiation of thyroxine treatment. Results: There were a total of 541 positive screening tests (~1/822 live births) of which 296 were true positives (~1:1,500 live births). Subjects were further subdivided based on screening-TSH and positive predictive values (PPV) were calculated. Twenty four percent in the 17-19.9 mIU/L range were true positives. In the 17-30 mIU/L range, 29 % were true positives with a significantly higher PPV for those sampled after (43 %) rather than before (25 %) 28 h of age (p \u3c 0.02). Seventy three percent of neonates with an initial screening-TSH of ≥ 30 mIU/L and 97 % of those with ≥ 40 mIU/L were later confirmed to have CH. Conclusions: Infants with modestly elevated screening positive TSH levels between 17 and 19.9 mIU/L have a significant risk (24 %) of having CH. The very high frequency of true positives in term newborns with initial TSH values ≥ 30mIU/L suggests that this group should be referred directly to a pediatric endocrinologist in an effort to expedite further assessment and treatment. Screen positives with a modestly elevated TSH values (17-19.9 mIU/L) need to be examined in more detail with extended follow-up data to determine if they have transient or permanent CH

Season and vitamin D status are independently associated with glucose homeostasis in pregnancy

Background: Vitamin D status and season are intrinsically linked, and both have been proposed to be associated with glucose homeostasis in pregnancy, with conflicting results. We aimed to determine if exposure to winter and low maternal 25 hydroxyvitamin D (25OHD) in early pregnancy were associated with maternal glucose metabolism.Methods: This is a secondary data analysis of 334 pregnant women enrolled in the ROLO study, Dublin. Serum 25OHD, fasting glucose, insulin and insulin resistance (HOMA-IR) were measured in early (12 weeks' gestation) and late pregnancy (28 weeks' gestation). Season of first antenatal visit was categorised as extended winter (November–April) or extended summer (May–October). Multiple linear regression models, adjusted for confounders, were used for analysis.Results: Those who attended their first antenatal visit in extended winter had lower 25OHD compared to extended summer (32.9 nmol/L vs. 44.1 nmol/L, P < 0.001). Compared to those who attended their first antenatal visit during extended summer, extended winter was associated with increased HOMA-IR in early-pregnancy (46.7%) and late pregnancy (53.7%), independent of 25OHD <30 nmol/L and confounders. Early pregnancy 25OHD <30 nmol/L and extended winter were independently associated with significantly higher fasting glucose in late pregnancy (B = 0.15 and 0.13, respectively).Conclusions: Women who attended their first antenatal visit during the months of extended winter were more likely to have raised insulin resistance in early pregnancy, which had a lasting association to 28 weeks, and was independent of 25OHD. Our novel findings imply that seasonal variation in insulin resistance may not be fully explained by differences in vitamin D status. This could reflect circannual rhythm or seasonal lifestyle behaviours, and requires further exploration.Trial registration: ISRCTN registry, ISRCTN54392969, date of registration: 22/04/2009, retrospectively registered

Prediction of congenital hypothyroidism based on initial screening thyroid-stimulating-hormone

Background: In thyroid-stimulating-hormone (TSH)-based newborn congenital hypothyroidism (CH) screening programs, the optimal screening-TSH cutoff level is critical to ensuring that true cases of CH are not missed. Screening-TSH results can also be used to predict the likelihood of CH and guide appropriate clinical management. The purpose of this study is to evaluate the predictive value of various screening-TSH levels in predicting a diagnosis of CH in the Ontario Newborn Screening Program (ONSP). Methods: The initial screening and follow-up data of 444,744 full term infants born in Ontario, Canada from April 1, 2006 to March 31, 2010 were analyzed. Confirmed CH cases were based on local endocrinologists\u27 report and initiation of thyroxine treatment. Results: There were a total of 541 positive screening tests (~1/822 live births) of which 296 were true positives (~1:1,500 live births). Subjects were further subdivided based on screening-TSH and positive predictive values (PPV) were calculated. Twenty four percent in the 17-19.9 mIU/L range were true positives. In the 17-30 mIU/L range, 29 % were true positives with a significantly higher PPV for those sampled after (43 %) rather than before (25 %) 28 h of age (p \u3c 0.02). Seventy three percent of neonates with an initial screening-TSH of ≥ 30 mIU/L and 97 % of those with ≥ 40 mIU/L were later confirmed to have CH. Conclusions: Infants with modestly elevated screening positive TSH levels between 17 and 19.9 mIU/L have a significant risk (24 %) of having CH. The very high frequency of true positives in term newborns with initial TSH values ≥ 30mIU/L suggests that this group should be referred directly to a pediatric endocrinologist in an effort to expedite further assessment and treatment. Screen positives with a modestly elevated TSH values (17-19.9 mIU/L) need to be examined in more detail with extended follow-up data to determine if they have transient or permanent CH

Comparing usage of a web and app stress management intervention : an observational study

Choices in the design and delivery of digital health behaviour interventions may have a direct influence on subsequent usage and engagement. Few studies have been able to make direct, detailed comparisons of differences in usage between interventions that are delivered via web or app. This study compared the usage of two versions of a digital stress management intervention, one delivered via a website (Healthy Paths) and the other delivered via an app (Healthy Mind). Design modifications were introduced within Healthy Mind to take account of reported differences in how individuals engage with websites compared to apps and mobile phones. Data were collected as part of an observational study nested within a broader exploratory trial of Healthy Mind. Objective usage of Healthy Paths and Healthy Mind were automatically recorded, including frequency and duration of logins, access to specific components within the intervention and order of page/screen visits. Usage was compared for a two week period following initial registration. In total, 381 participants completed the registration process for Healthy Paths (web) and 162 participants completed the registration process for Healthy Mind (app). App users logged in twice as often (Mdn = 2.00) as web users (Mdn = 1.00), U = 13,059.50, p ≤ 0.001, but spent half as much time (Mdn = 5.23 min) on the intervention compared to web users (Mdn = 10.52 min), U = 19,740.00, p ≤ 0.001. Visual exploration of usage patterns over time revealed that a significantly higher proportion of app users (n = 126, 82.35%) accessed both types of support available within the intervention (i.e. awareness and change-focused tools) compared to web users (n = 92, 40.17%), χ2(1, n = 382) = 66.60, p < 0.001. This study suggests that the digital platform used to deliver an intervention (i.e. web versus app) and specific design choices (e.g. navigation, length and volume of content) may be associated with differences in how the intervention content is used. Broad summative usage data (e.g. total time spent on the intervention) may mask important differences in how an intervention is used by different user groups if it is not complemented by more fine-grained analyses of usage patterns over time. Trial registration number: ISRCTN67177737