Functional characterization of the US3 serine/threonine kinase during BHV-1 infection

Bovine herpesvirus 1 (BHV-1) is a member of the Alphaherpesvirinae subfamily and is the prototype ruminant herpesvirus. BHV-1 causes a number of complications in cattle including upper respiratory tract disorders, conjunctivitis, genital disorders, abortions, and immune suppression. Like all herpesviruses, reactivation from latency can occur throughout the animal’s life. Of particular economic importance is the bovine respiratory disease complex (BRDC) or ‘shipping fever’, in which BHV-1 plays a major role. BRDC is an enormous economic concern as it costs the US cattle industry approximately one billion dollars annually. In order to generate improved gene-deleted vaccines against BHV-1, there is a need to understand the contributions of viral gene products during infection. US3 is a serine/threonine kinase present in BHV-1 and is thought to play major roles during viral infection. As in other herpesviruses, US3 in BHV-1 is expected to phosphorylate several cellular and/or viral proteins. We recently presented evidence that BHV-1 US3 phosphorylates both VP8 and VP22; however, further functional characteristics of BHV-1 US3 during viral infection have not been elucidated. The hypothesis of this project is that the deletion of the US3 gene leads to reduced BHV-1 fitness. To explore this hypothesis, we generated a US3-deleted (ΔUS3) and subsequent US3-rescued (US3R) BHV-1 virus. Using these viral mutants, we characterized the growth properties of the viruses, evaluated the effect of the US3 deletion on major structural BHV-1 proteins, characterized the protein composition of the mature virions, and, identified viral processes that were impaired in the deletion mutant. Initially, the ∆US3 virus was generated through a 3-step PCR strategy which replaced the gene of interest with an antibiotic resistance cassette. Following this, the US3 gene was rescued via a two-step en passant mutagenesis strategy which has been previously used to generate insertions, deletions, and substitutions in herpesvirus-containing bacterial artificial chromosome (BAC) DNA. In vitro characterization of ∆US3 BHV-1 has demonstrated that US3 deletion affects BHV-1 growth characteristics, expression kinetics of major structural proteins, mature virion composition, cell to cell spread, and the subcellular localization of key viral proteins during infection. Growth kinetics of ∆US3 BHV-1 were impaired compared to wild-type (WT) BHV-1, especially at late times post-infection. Plaque sizes formed by ∆US3 BHV-1 were significantly smaller than those formed by either WT or US3R BHV-1, demonstrating that US3 is important for cell to cell spread. The expression kinetics of major structural and regulatory BHV-1 proteins were different between cells infected with ∆US3 or WT BHV-1, and incorporation of these proteins into the mature viruses differed, demonstrating that US3 is instrumental in ensuring proper protein expression and mature virus composition in vitro. Of particular importance, glycoprotein B (gB), was shown to be expressed in higher quantities earlier during infection in the absence of US3, and that this protein was incorporated in significantly higher amounts in mature virions which lacked US3. Qualitative analysis of ∆US3 BHV-1 infected monolayers suggested the abolishment of cell to cell projections characteristic of WT BHV-1 infection. Finally, the disruption of gB in ∆US3 BHV-1 infected cells was confirmed by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Through confocal microscopy, evidence was provided that infection with ∆US3 BHV-1 possibly results in earlier expression of gB on the surface of cells and less intracellular accumulation of this protein during late stages of infection. The observed effect on the localization of intracellular gB in ∆US3 BHV-1 infected cells was quantified by flow cytometry. ∆US3 BHV-1 infected cells had approximately 25% higher gB expression on the surface of cells and a corresponding 25% decrease in intracellular gB. Although these differences have not yet been demonstrated to be statistically significant and not confirmed through infection with US3R BHV-1, this suggests that US3 may influence the synthesis and cellular trafficking of gB in vitro

The enhancement of the hyperglycemic effect of S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine by vitamin C in an animal model

BACKGROUND: S-nitrosoglutathione (GSNO) and S-nitroso-N-acetlypenicillamine (SNAP) are two of the most common sources of nitric oxide (NO) in the biomedical field. Vitamin C has been known to accelerate the decomposition of GSNO and SNAP increasing the release and availability of NO which is cytotoxic at non-physiological concentrations. The study investigates any potential detrimental effect of vitamin C and GSNO, vitamin C and SNAP on glucose metabolism in normotensive and normoglycemic dogs. RESULTS: The results showed that administration of vitamin C (50 mg/kg) and GSNO (35 mg/kg & 50 mg/kg), or vitamin C (50 mg/kg) and SNAP (10 mg/kg) to overnight fasted dogs resulted in significant elevation of the blood glucose levels, attaining maximum level at the 2.0 or 2.5 h time point postprandially. The elevated blood glucose levels were due to significant reduction in plasma insulin levels in the dogs treated with vitamin C and GSNO, or vitamin C and SNAP (P < 0.05). The decreased insulin response was associated with significant elevation of nitric oxide produced from GSNO and SNAP co-administered with vitamin C, as assessed by plasma nitrate/nitrite levels. CONCLUSIONS: The results indicate that enhanced NO release by vitamin C affects postprandial blood glucose and plasma insulin levels and the reduced glucose tolerance is mainly due to impaired insulin release. The clinical relevance of the findings of this study suggest that hypertensive diabetic patients treated with GSNO or SNAP, who are on vitamin C supplements may be more predisposed to further decrease in their glycemic control

Decreased insulin binding to mononuclear leucocytes and erythrocytes from dogs after S-Nitroso-N-Acetypenicillamine administration

BACKGROUND: Nitric oxide (NO) and oxygen free-radicals play an important part in the destruction of beta-cells in auto- immune diabetes although the precise mechanism of interaction is still not known. This study was designed to examine any possible diabetogenic effect of NO by investigating any differences in cellular binding of insulin to its receptor on the cell membranes of erythrocytes and mononuclear leucocytes of dogs treated with the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) and controls treated with captopril. RESULTS: The result obtained showed decreased binding of insulin to its receptor on the cell membranes of erythrocytes and mononuclear leucocytes. Mononuclear leucocytes from SNAP-treated dogs had decreased ability to bind insulin (16.30 ± 1.24 %) when compared to mononuclear leucocytes from captopril-treated controls (20.30 ± 1.93 %). Similar results were obtained for erythrocytes from dogs treated with SNAP (27.20 ± 1.33 %) compared with dogs treated with captopril (34.70 ± 3.58 %). Scatchard analysis demonstrated that this decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with mononuclear leucocytes of SNAP-treated dogs having 55 % less insulin receptor sites per cell compared with those of captopril-treated controls (P < 0.05). Average affinity and kinetic analysis revealed a 35 % decrease in the average receptor affinity, with mononuclear leucocytes from captopril-treated controls having an empty site affinity of 12.36 ± 1.12 × 10(-8) M(-1) compared with 9.64 ± 0.11 × 10(-8) M(-1) in SNAP-treated dogs (P < 0.05). CONCLUSION: These results suggest that acute alteration of the insulin receptor on the membranes of mononuclear leucocytes and erythrocytes occurred in dogs treated with S-nitroso-N-acetylpenicillamine. These findings suggest the first evidence of the novel role of NO as a modulator of insulin binding and the involvement of NO in the aetiology of diabetes mellitus

Participatory Design of Paediatric Upper Limb Prostheses: qualitative methods and prototyping

Objectives: The study aims to develop an understanding of the views of children and adolescents, parents, and professionals on upper limb prosthetic devices to develop and improve device design. Previous research has found that children are dissatisfied with prostheses but has relied heavily on parent proxy reports and quantitative measures (such as questionnaires) to explore their views.Methods: Thirty-four participants (eight children aged 8–15 years with upper limb difference, nine parents, eight prosthetists, and nine occupational therapists) contributed to the development of new devices through the BRIDGE methodology of participatory design, using focus groups and interviews.Results: The study identified areas for improving prostheses from the perspective of children and adolescents, developed prototypes based on these and gained feedback on the prototypes from the children and other stakeholders (parents and professionals) of paediatric upper limb prostheses. Future device development needs to focus on ease of use, versatility, appearance, and safety.Conclusions: This study has demonstrated that children and adolescents can and should be involved as equal partners in the development of daily living equipment and that rapid prototyping (three-dimensional printing or additive manufacturing), used within a participatory design framework, can be a useful tool for facilitating this

The Influence of Small Versus Large Pen Design on Health and Lesion Scores of the Grow-finisher Pig

The objective of this study was to determine the effects of raising pigs in small versus large pens during the grow-finish period on health and number of lesions of the finisher pig. The experiment was conducted from April to July, 2009. One wean to finish site within a large Midwestern commercial production system was used. There were four rooms on this site. A total of 3,162 pigs were used to compare health status and frequency of lesions. Within each room, one side of the aisle was set-up with the small pen treatment (SP; n = 45 pens; [34 pigs/pen]), while the other side was set-up with the large pen treatment (LP; n= 6 pens; [272 pigs/pen]). Therefore, both treatments were represented in each room. All pigs were kept in smaller pen configurations for 4 weeks and then the back gates of eight consecutive pens in the LP treatment were opened. Pens were mixed sexed and when the first market group of pigs reached targeted market weight the trial was terminated. One day prior to trial termination, a total of 316 pigs (10% of the population) were visually assessed by two observers for the frequency of lesions. Lesions were defined per the PQA Plus definition of skin lesions (NPB, 2007), as “…breaks that completely penetrate the skin, such as bites or other lesions that penetrate through the skin.” Lesion scores were analyzed using the PROC GLIMMIX procedure of SAS. When a pig was identified within their home pen as requiring medication, the drug type, number of pigs treated, the dose amount and cost per dose were recorded and this information will be presented descriptively. There were differences in lesion frequency with pigs housed in large pens having a higher (P \u3c 0.05) number of lesions compared to pigs in the small treatment. This was consistent across all locations on the pig. More pigs were treated in the large pen (n = 198) compared to the small pen (n = 158) and consequently a higher drug cost was noted for large pens ($127.63 vs.$95.47). Therefore in conclusion, larger pens had higher lesion frequency and higher drug treatment costs

The Influence of Changing Pen Design From a Small to Large Configuration on the Performance of the Grow-to-Finisher Pig

The objective of this study was to determine the effects of small versus large pens throughout the grow-finish period on growth performance of the pig. This experiment consisted of two replications. One wean to finish site within a large Midwestern commercial production system was used for both replications. The site consisted of four rooms. Within each room, one side of the aisle was set-up with the small pen treatment (SP; n = 96 pens [34 pigs/pen; 0.69 m2/pig]), while the other side was set-up with the large pen treatment (LP; n = 12 pens [272 pigs/pen; 0.69 m2/pig]). Pens were mixed sexed and when the first market group of pigs reached the targeted market weight in both treatments the trial was terminated. Starting and ending weights and average daily gain on a pen basis was recorded and calculated for a total of 6,528 crossbred pigs. Performance was analyzed using the PROC MIXED procedure of SAS. Small penned pigs had a higher ADG (P = 0.004) and overall gain (P = 0.05) than large penned pigs. In conclusion, pigs raised in small pens throughout the grow-finish period had a higher average daily gain and overall gain than pigs housed in large pens throughout the grow-finish period

Evidence of detrimental effects of prenatal alcohol exposure on offspring birthweight and neurodevelopment from a systematic review of quasi-experimental studies

Background: Systematic reviews of prenatal alcohol exposure effects generally only include conventional observational studies. However, estimates from such studi