Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I.

The RER retains a specific subset of ER proteins, many of which have been shown to participate in the translocation of nascent secretory and membrane proteins. The mechanism of retention of RER specific membrane proteins is unknown. To study this phenomenon in yeast, where no RER-specific membrane proteins have yet been identified, we expressed the human RER-specific protein, ribophorin I. In all mammalian cell types examined, ribophorin I has been shown to be restricted to the membrane of the RER. Here we ascertain that yeast cells correctly target, assemble, and retain ribophorin I in their RER. Floatation experiments demonstrated that human ribophorin I, expressed in yeast, was membrane associated. Carbonate (pH = 11) washing and Triton X-114 cloud-point precipitations of yeast microsomes indicated that ribophorin I was integrated into the membrane bilayer. Both chromatography on Con A and digestion with endoglycosidase H were used to prove that ribophorin I was glycosylated once, consistent with its expression in mammalian cells. Proteolysis of microsomal membranes and subsequent immunoblotting showed ribophorin I to have assumed the correct transmembrane topology. Sucrose gradient centrifugation studies found ribophorin I to be included only in fractions containing rough membranes and excluded from smooth ones that, on the basis of the distribution of BiP, included smooth ER. Ribosome removal from rough membranes and subsequent isopycnic centrifugation resulted in a shift in the buoyant density of the ribophorin I-containing membranes. Furthermore, the rough and density-shifted fractions were the exclusive location of protein translocation activity. Based on these studies we conclude that sequestration of membrane proteins to rough domains of ER probably occurs in a like manner in yeast and mammalian cells

Transition from gas-like to liquid-like behavior in supercritical N2

We have studied in detail the transition from gas-like to rigid liquidlike behavior in supercritical N2 at 300 K (2.4 TC). Our study combines neutron diffraction and Raman spectroscopy with ab initio molecular dynamics simulations. We observe a narrow transition from gas-like to rigid liquid-like behavior at ca. 150 MPa, which we associate with the Frenkel line. Our findings allow us to reliably characterize the Frenkel line using both diffraction and spectroscopy methods, backed up by simulation, for the same substance. We clearly lay out what parameters change, and what parameters do not change, when the Frenkel line is crossed

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease.

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised

Oral Anti-Tumour Necrosis Factor Domain Antibody V565 Provides High Intestinal Concentrations, and Reduces Markers of Inflammation in Ulcerative Colitis Patients.

V565 is an engineered TNFα-neutralising single domain antibody formulated into enteric coated mini-tablets to enable release in the intestine after oral administration as a possible oral treatment for inflammatory bowel disease (IBD). Following oral administration, ileal recovery of V565 was investigated in four patients with terminal ileostomy. Intestinal and systemic pharmacokinetics were measured in six patients with Crohn's disease and evidence of target engagement assessed in five patients with ulcerative colitis. Following oral administration, V565 was detected at micromolar concentrations in ileal fluid from the ileostomy patients and in stools of the Crohn's patients. In four of the five ulcerative colitis patients, biopsies taken after 7d dosing demonstrated V565 in the lamina propria with co-immunostaining on CD3+ T-lymphocytes and CD14+ macrophages. Phosphorylation of signalling proteins in biopsies taken after 7d oral dosing was decreased by approximately 50%. In conclusion, enteric coating of V565 mini-tablets provided protection in the stomach with gradual release in intestinal regions affected by IBD. Immunostaining revealed V565 tissue penetration and association with inflammatory cells, while decreased phosphoproteins after 7d oral dosing was consistent with V565-TNFα engagement and neutralising activity. Overall these results are encouraging for the clinical utility of V565 in the treatment of IBD

Reduction of low- and high-grade cervical abnormalities associated with high uptake of the HPV bivalent vaccine in Scotland

In Scotland, a national HPV immunisation programme began in 2008 for 12-13 year olds, with a catch-up campaign from 2008-2011 for those under the age of 18. To monitor the impact of HPV immunisation on cervical disease at the population level, a programme of national surveillance was established.  We analysed colposcopy data from a cohort of women born between 1988-1992 who entered the Scottish Cervical Screening Programme (SCSP) and were aged 20-21 in 2008-2012.  By linking datasets from the SCSP and colposcopy services, we observed a significant reduction in diagnoses of cervical intraepithelial neoplasia 1 (CIN 1) (RR 0.71, 95% CI 0.58 to 0.87, p=0.0008), CIN 2 (RR 0.5, 95% CI 0.4, 0.63, p<0.0001) and CIN 3 (RR 0.45, 95% CI 0.35 to 0.58, p< 0.0001) for women who received 3 doses of vaccine compared with unvaccinated women.  To our knowledge, this is one of the first studies to show a reduction of low and high grade cervical intraepithelial neoplasia associated with high uptake of the HPV bivalent vaccine at the population level. These data are very encouraging for countries that have achieved high HPV vaccine uptake

Investigating and learning lessons from early experiences of implementing ePrescribing systems into NHS hospitals:a questionnaire study

Background: ePrescribing systems have significant potential to improve the safety and efficiency of healthcare, but they need to be carefully selected and implemented to maximise benefits. Implementations in English hospitals are in the early stages and there is a lack of standards guiding the procurement, functional specifications, and expected benefits. We sought to provide an updated overview of the current picture in relation to implementation of ePrescribing systems, explore existing strategies, and identify early lessons learned.Methods: a descriptive questionnaire-based study, which included closed and free text questions and involved both quantitative and qualitative analysis of the data generated.Results: we obtained responses from 85 of 108 NHS staff (78.7% response rate). At least 6% (n = 10) of the 168 English NHS Trusts have already implemented ePrescribing systems, 2% (n = 4) have no plans of implementing, and 34% (n = 55) are planning to implement with intended rapid implementation timelines driven by high expectations surrounding improved safety and efficiency of care. The majority are unclear as to which system to choose, but integration with existing systems and sophisticated decision support functionality are important decisive factors. Participants highlighted the need for increased guidance in relation to implementation strategy, system choice and standards, as well as the need for top-level management support to adequately resource the project. Although some early benefits were reported by hospitals that had already implemented, the hoped for benefits relating to improved efficiency and cost-savings remain elusive due to a lack of system maturity.Conclusions: whilst few have begun implementation, there is considerable interest in ePrescribing systems with ambitious timelines amongst those hospitals that are planning implementations. In order to ensure maximum chances of realising benefits, there is a need for increased guidance in relation to implementation strategy, system choice and standards, as well as increased financial resources to fund local activitie

Comparing the immune response to a novel intranasal nanoparticle PLGA vaccine and a commercial BPI3V vaccine in dairy calves

peer-reviewedBackground There is a need to improve vaccination against respiratory pathogens in calves by stimulation of local immunity at the site of pathogen entry at an early stage in life. Ideally such a vaccine preparation would not be inhibited by the maternally derived antibodies. Additionally, localized immune response at the site of infection is also crucial to control infection at the site of entry of virus. The present study investigated the response to an intranasal bovine parainfluenza 3 virus (BPI3V) antigen preparation encapsulated in PLGA (poly dl-lactic-co-glycolide) nanoparticles in the presence of pre-existing anti-BPI3V antibodies in young calves and comparing it to a commercially available BPI3V respiratory vaccine. Results There was a significant (P < 0.05) increase in BPI3V-specific IgA in the nasal mucus of the BPI3V nanoparticle vaccine group alone. Following administration of the nanoparticle vaccine an early immune response was induced that continued to grow until the end of study and was not observed in the other treatment groups. Virus specific serum IgG response to both the nanoparticle vaccine and commercial live attenuated vaccine showed a significant (P < 0.05) rise over the period of study. However, the cell mediated immune response observed didn’t show any significant rise in any of the treatment groups. Conclusion Calves administered the intranasal nanoparticle vaccine induced significantly greater mucosal IgA responses, compared to the other treatment groups. This suggests an enhanced, sustained mucosal-based immunological response to the BPI3V nanoparticle vaccine in the face of pre-existing antibodies to BPI3V, which are encouraging and potentially useful characteristics of a candidate vaccine. However, ability of nanoparticle vaccine in eliciting cell mediated immune response needs further investigation. More sustained local mucosal immunity induced by nanoparticle vaccine has obvious potential if it translates into enhanced protective immunity in the face of virus outbreak

A prospective observational study to assess the diagnostic accuracy of clinical decision rules for children presenting to emergency departments after head injuries (protocol): The Australasian Paediatric Head Injury Rules Study (APHIRST)

Background: Head injuries in children are responsible for a large number of emergency department visits. Failure to identify a clinically significant intracranial injury in a timely fashion may result in long term neurodisability and death. Whilst cranial computed tomography (CT) provides rapid and definitive identification of intracranial injuries, it is resource intensive and associated with radiation induced cancer. Evidence based head injury clinical decision rules have been derived to aid physicians in identifying patients at risk of having a clinically significant intracranial injury. Three rules have been identified as being of high quality and accuracy: the Canadian Assessment of Tomography for Childhood Head Injury (CATCH) from Canada, the Children's Head Injury Algorithm for the Prediction of Important Clinical Events (CHALICE) from the UK, and the prediction rule for the identification of children at very low risk of clinically important traumatic brain injury developed by the Pediatric Emergency Care Applied Research Network (PECARN) from the USA. This study aims to prospectively validate and compare the performance accuracy of these three clinical decision rules when applied outside the derivation setting.Methods/design: This study is a prospective observational study of children aged 0 to less than 18 years presenting to 10 emergency departments within the Paediatric Research in Emergency Departments International Collaborative (PREDICT) research network in Australia and New Zealand after head injuries of any severity. Predictor variables identified in CATCH, CHALICE and PECARN clinical decision rules will be collected. Patients will be managed as per the treating clinicians at the participating hospitals. All patients not undergoing cranial CT will receive a follow up call 14 to 90 days after the injury. Outcome data collected will include results of cranial CTs (if performed) and details of admission, intubation, neurosurgery and death. The performance accuracy of each of the rules will be assessed using rule specific outcomes and inclusion and exclusion criteria.Discussion: This study will allow the simultaneous comparative application and validation of three major paediatric head injury clinical decision rules outside their derivation setting.Trial registration: The study is registered with the Australian New Zealand Clinical Trials Registry (ANZCTR)- ACTRN12614000463673 (registered 2 May 2014). © 2014 Babl et al.; licensee BioMed Central Ltd