Identification of secretory granule phosphatidylinositol 4,5-bisphosphateinteracting proteins using an affinity pulldown strategy

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P-2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P-2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; alpha-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P-2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P-2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P-2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P-2 binding, are important for chromaffin granule exocytosis

Impact of delayed initiation of erythropoietin in critically ill patients

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to evaluate the impact of recombinant human erythropoietin (rHuEPO) use for anemia of critical illness at a practice site where delayed initiation is common.</p> <p>Methods</p> <p>Retrospective medical record review involving patients treated with rHuEPO for anemia of critical illness. Those patients given rHuEPO or diagnosed with end-stage renal disease (ESRD) prior to ICU admission were excluded. The primary endpoints were rHuEPO use and RBC transfusion patterns.</p> <p>Results</p> <p>Complete data were collected for consecutive admissions of 126 patients. Average age (SD) and APACHE II score were 56.5 (18.6) years and 25 (7.8), respectively. The median ICU (IQR) and hospital length of stay (LOS) were 24 (11.25, 39) and 29 (17, 44.75) days, respectively. Treatment with rHuEPO was started an average of 12.5 +/- 10.5 days after ICU admission and given for 3.8 +/- 3.8 doses. Eighty percent of patients were transfused with an average total of 5.42 +/- 5.08 units received. RBC exposure inversely correlated with a lower mean hemoglobin response to rHuEPO. ICU LOS (p < 0.0001), hemoglobin at 24 hours (p = 0.055), transfusion within 48 hours of admit (p < 0.0001), and postoperative status (p = 0.019) were the best predictors of transfusion requirements (r²= 0.37).</p> <p>Conclusion</p> <p>Delayed initiation of rHuEPO for anemia of critical illness resulted in comparable hemoglobin and transfusion benefits. Future studies are needed to establish clinical benefit and role in therapy. RBC exposure may blunt the erythropoietic effects of rHuEPO, potentially frustrating benefits to those of greatest apparent need.</p

Phytosterols and phytostanols and the hallmarks of cancer in model organisms: a systematic review and meta-analysis

Phytosterols and phytostanols are natural products present in vegetable oils, nuts, and seeds, or added to consumer food products whose intake is inversely associated with incidence and prognosis of several cancers. Randomized cancer prevention trials in humans are unfeasible due to time and cost yet the cellular processes and signaling cascades that underpin anti-cancer effects of these phytochemicals have been explored extensively in vitro and in preclinical in vivo models. Here we have performed an original systematic review, meta-analysis, and qualitative interpretation of literature published up to June 2020. MEDLINE, Scopus, and hand-searching identified 408 unique records that were screened leading to 32 original articles that had investigated the effects of phytosterols or phytostanols on cancer biology in preclinical models. Data was extracted from 22 publications for meta-analysis. Phytosterols were most commonly studied and found to reduce primary and metastatic tumor burden in all cancer sites evaluated. Expression of pAKT, and markers of metastasis (alkaline phosphatase, matrix metalloproteases, epithelial to mesenchymal transcription factors, lung and brain colonization), angiogenesis (vascular endothelial growth factor, CD31), and proliferation (Ki67, proliferating cell nuclear antigen) were consistently reduced by phytosterol treatment in breast and colorectal cancer. Very high dose treatment (equivalent to 0.2–1 g/kg body weight not easily achievable through diet or supplementation in humans) was associated with adverse events including poor gut health and intestinal adenoma development. Phytosterols and phytostanols are already clinically recommended for cardiovascular disease risk reduction, and represent promising anti-cancer agents that could be delivered in clinic and to the general population at low cost, with a well understood safety profile, and now with a robust understanding of mechanism-of-action

Biochemical Effects of Carbohydrate Supplementation in a Simulated Competition of Short Terrestrial Duathlon

The purpose of the present study was to investigate the biochemical effects of carbohydrate supplementation in a simulated competition of short terrestrial duathlon. Ten duathletes participated in a simulated competition of short terrestrial duathlon 30 minutes after the ingestion of a 6% (30 g/500 ml) maltodextrin solution (MALT) or a placebo (PLA). This solution was also ingested every 15 minutes during the competition (12 g/200 ml); and immediately after the competition (18 g/300 ml). Samples of blood were collected at 3 time points: 1) at rest 1 hour before the beginning of the competition; 2) during the competition (approximately 1 hour and 45 minutes after the 1st collection); 3) immediately after the competition. Blood was analyzed for blood glucose, lactate, insulin and cortisol. Significant differences were observed in relation to blood glucose levels between MALT and PLA in the post-competition phase. There was also a significant difference in the lactate levels observed between MALT and PLA during the competition phase. Similarly, a significant difference in the cortisol concentrations during and after the competition phases (MALT and PLA) were observed. We conclude that maltodextrin supplementation appears to be beneficial during short terrestrial duathlon competition as evidenced by biochemical markers

Q methodology and a Delphi poll: a useful approach to researching a narrative approach to therapy

Q methodology and a Delphi poll combined qualitative and quantitative methods to explore definitions of White and Epston's (1990) narrative approach to therapy among a group of UK practitioners. A Delphi poll was used to generate statements about narrative therapy. The piloting of statements by the Delphi panel identified agreement about theoretical ideas underpinning narrative therapy and certain key practices. A wider group of practitioners ranked the statements in a Q sort and made qualitative comments about their sorting. Quantitative methods (principal components analysis) were used to extract eight accounts of narrative therapy, five of which are qualitatively analysed in this paper. Agreement and differences were identified across a range of issues, including the social construction of narratives, privileging a political stance or narrative techniques and the relationship with other therapies, specifically systemic psychotherapy. Q methodology, combined with the Delphi poll, was a unique and innovative feature of this study

Comparison of breast and bowel cancer screening uptake patterns in a common cohort of South Asian women in England

Background: Inequalities in uptake of cancer screening by ethnic minority populations are well documented in a number of international studies. However, most studies to date have explored screening uptake for a single cancer only. This paper compares breast and bowel cancer screening uptake for a cohort of South Asian women invited to undertake both, and similarly investigates these women's breast cancer screening behaviour over a period of fifteen years. Methods: Screening data for rounds 1, 2 and 5 (1989-2004) of the NHS breast cancer screening programme and for round 1 of the NHS bowel screening pilot (2000-2002) were obtained for women aged 50-69 resident in the English bowel screening pilot site, Coventry and Warwickshire, who had been invited to undertake breast and bowel cancer screening in the period 2000-2002. Breast and bowel cancer screening uptake levels were calculated and compared using the chi-squared test. Results: 72,566 women were invited to breast and bowel cancer screening after exclusions. Of these, 3,539 were South Asian and 69,027 non-Asian; 18,730 had been invited to mammography over the previous fifteen years (rounds 1 to 5). South Asian women were significantly less likely to undertake both breast and bowel cancer screening; 29.9% (n = 1,057) compared to 59.4% (n = 40,969) for non-Asians (p < 0.001). Women in both groups who consistently chose to undertake breast cancer screening in rounds 1, 2 and 5 were more likely to complete round 1 bowel cancer screening. However, the likelihood of completion of bowel cancer screening was still significantly lower for South Asians; 49.5% vs. 82.3% for non-Asians, p < 0.001. South Asian women who undertook breast cancer screening in only one round were no more likely to complete bowel cancer screening than those who decided against breast cancer screening in all three rounds. In contrast, similar women in the non-Asian population had an increased likelihood of completing the new bowel cancer screening test. The likelihood of continued uptake of mammography after undertaking screening in round 1 differed between South Asian religio-linguistic groups. Noticeably, women in the Muslim population were less likely to continue to participate in mammography than those in other South Asian groups. Conclusions: Culturally appropriate targeted interventions are required to reduce observed disparities in cancer screening uptakes

Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility

A concept for major incident triage: full-scaled simulation feasibility study

<p>Abstract</p> <p>Background</p> <p>Efficient management of major incidents involves triage, treatment and transport. In the absence of a standardised interdisciplinary major incident management approach, the Norwegian Air Ambulance Foundation developed Interdisciplinary Emergency Service Cooperation Course (TAS). The TAS-program was established in 1998 and by 2009, approximately 15 500 emergency service professionals have participated in one of more than 500 no-cost courses. The TAS-triage concept is based on the established triage Sieve and Paediatric Triage Tape models but modified with slap-wrap reflective triage tags and paediatric triage stretchers. We evaluated the feasibility and accuracy of the TAS-triage concept in full-scale simulated major incidents.</p> <p>Methods</p> <p>The learners participated in two standardised bus crash simulations: without and with competence of TAS-triage and access to TAS-triage equipment. The instructors calculated triage accuracy and measured time consumption while the learners participated in a self-reported before-after study. Each question was scored on a 7-point Likert scale with points labelled "Did not work" (1) through "Worked excellent" (7).</p> <p>Results</p> <p>Among the 93 (85%) participating emergency service professionals, 48% confirmed the existence of a major incident triage system in their service, whereas 27% had access to triage tags. The simulations without TAS-triage resulted in a mean over- and undertriage of 12%. When TAS-Triage was used, no mistriage was found. The average time from "scene secured to all patients triaged" was 22 minutes (range 15-32) without TAS-triage vs. 10 minutes (range 5-21) with TAS-triage. The participants replied to "How did interdisciplinary cooperation of triage work?" with mean 4,9 (95% CI 4,7-5,2) before the course vs. mean 5,8 (95% CI 5,6-6,0) after the course, p < 0,001.</p> <p>Conclusions</p> <p>Our modified triage Sieve tool is feasible, time-efficient and accurate in allocating priority during simulated bus accidents and may serve as a candidate for a future national standard for major incident triage.</p

Nutritional correlates of koala persistence in a low-density population

It is widely postulated that nutritional factors drive bottom-up, resource-based patterns in herbivore ecology and distribution. There is, however, much controversy over the roles of different plant constituents and how these influence individual herbivores and herbivore populations. The density of koala (Phascolarctos cinereus) populations varies widely and many attribute population trends to variation in the nutritional quality of the eucalypt leaves of their diet, but there is little evidence to support this hypothesis. We used a nested design that involved sampling of trees at two spatial scales to investigate how leaf chemistry influences free-living koalas from a low-density population in south east New South Wales, Australia. Using koala faecal pellets as a proxy for koala visitation to trees, we found an interaction between toxins and nutrients in leaves at a small spatial scale, whereby koalas preferred trees with leaves of higher concentrations of available nitrogen but lower concentrations of sideroxylonals (secondary metabolites found exclusively in eucalypts) compared to neighbouring trees of the same species. We argue that taxonomic and phenotypic diversity is likely to be important when foraging in habitats of low nutritional quality in providing diet choice to tradeoff nutrients and toxins and minimise movement costs. Our findings suggest that immediate nutritional concerns are an important priority of folivores in low-quality habitats and imply that nutritional limitations play an important role in constraining folivore populations. We show that, with a careful experimental design, it is possible to make inferences about populations of herbivores that exist at extremely low densities and thus achieve a better understanding about how plant composition influences herbivore ecology and persistence.IW and WF received a grant from New South Wales (NSW) Department of Environment, Climate Change & Water