Phosphorylation of the WH2 domain in yeast Las17/WASP regulates G-actin binding and protein function during endocytosis.

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. WH2 domains are short sequence motifs found in many different actin binding proteins including WASP family proteins which regulate the actin nucleating complex Arp2/3. In this study we reveal a phosphorylation site, Serine 554, within the WH2 domain of the yeast WASP homologue Las17. Both phosphorylation and a phospho-mimetic mutation reduce actin monomer binding affinity while an alanine mutation, generated to mimic the non-phosphorylated state, increases actin binding affinity. The effect of these mutations on the Las17-dependent process of endocytosis in vivo was analysed and leads us to propose that switching of Las17 phosphorylation states may allow progression through distinct phases of endocytosis from site assembly through to the final scission stage. While the study is focused on Las17, the sole WASP family protein in yeast, our results have broad implications for our understanding of how a key residue in this conserved motif can underpin the many different actin regulatory roles with which WH2 domains have been associated.

The role of Kono-S anastomosis and mesenteric resection in reducing recurrence after surgery for Crohn's disease: a systematic review.

AIM: Recurrence after surgery for Crohn's disease is common. Anastomotic configuration may influence recurrence and the mesentery may be key. Recently the Kono-S anastomosis and radical mesenteric excision have been proposed as methods of reducing recurrence. We analysed the literature pertaining to these novel techniques. METHOD: We searched MEDLINE, Embase and the Cochrane Library for, and selected, studies evaluating Kono-S anastomosis and/or radical mesenteric excision in Crohn's disease. We assessed methodological quality and risk of bias using the Cochrane risk of bias tool for randomized controlled trials and the Joanna Briggs Institute tool for nonrandomized trials. A narrative synthesis was used to summarize the findings. RESULTS: Nine studies (896 patients) were identified. Apart from one randomized controlled trial with a low risk of bias the overall level of evidence was poor (Grade IV). The Kono-S anastomosis was associated with a lower incidence of endoscopic and surgical recurrence (0%-3.4% vs 15%-24.4% respectively). Complications, particularly anastomotic leak rate, were also lower (1.8% vs 9.3% respectively). Evidence from a single poor quality study suggested that mesenteric excision may reduce surgical recurrence rates compared with mesentery preservation. CONCLUSION: The existing literature suggests that the Kono-S anastomosis is safe and may reduce endoscopic and surgical recurrence, but level of evidence is mainly poor. One element of the Kono-S technique, preservation of the mesentery, may be detrimental to recurrence. Further, higher quality, studies are required to investigate these techniques. Such studies should consider the impact of the degree of mesenteric resection in addition to the anastomosis on disease recurrence.

Crassulacean acid metabolism in the Basellaceae (Caryophyllales).

C4 and crassulacean acid metabolism (CAM) have evolved in the order Caryophyllales many times but neither C4 nor CAM have been recorded for the Basellaceae, a small family in the CAM-rich sub-order Portulacineae. 24 h gas exchange and day-night changes in titratable acidity were measured in leaves of Anredera baselloides exposed to wet-dry-wet cycles. While net CO2 uptake was restricted to the light period in well-watered plants, net CO2 fixation in the dark, accompanied by significant nocturnal increases in leaf acidity, developed in droughted plants. Plants reverted to solely C3 photosynthesis upon rewatering. The reversible induction of nocturnal net CO2 uptake by drought stress indicates that this species is able to exhibit CAM in a facultative manner. This is the first report of CAM in a member of the Basellaceae.

Chemical composition, true nutrient digestibility, and true metabolizable energy of novel pet food protein sources using the precision-fed cecectomized rooster assay.

A wide variety of animal protein-based ingredients is commonly used in the pet food products. The raw ingredients and processing procedures used may greatly affect protein quality. Testing the quality of alternative protein sources is necessary and contributes to the sustainability of pet foods. The objective of this study was to test the chemical composition of 8 protein sources intended for use in dog and cat foods (calamari meal, pork peptone, alligator meal, lamb meal, venison meal, chicken meal, and 2 duck meals), and evaluate their true nutrient digestibility and nitrogen-corrected true ME (TMEn) using the precision-fed cecectomized rooster assay. Calamari meal and pork peptone had lower ash (4.4 and 3.6% of DM, respectively) but greater CP (88.1 and 80.5% of DM, respectively) and either greater or similar GE (5.6 and 5.3 kcal/g of DM, respectively) compared with alligator, lamb, venison, chicken, and duck meals (11.8 to 24.5% ash, 58.7 to 65.9% CP, and 4.6 to 5.3 kcal GE/g). Acid-hydrolyzed fat (AHF) was lower in calamari meal (8.7% of DM) compared with the other proteins tested (15.5-22.1% of DM). True nutrient digestibility was variable among the protein sources (52 to 79% of DM, 60 to 83% of OM, 78 to 92% of AHF, and 70 to 89% of GE) with pork peptone having the highest DM, AHF, and GE digestibility and calamari meal having the highest OM digestibility. True indispensable AA digestibility was highest for calamari meal, with all AA having a digestibility greater than 90%. Except for histidine, all indispensable AA had a digestibility over 85% for pork peptone. In contrast, true indispensable AA digestibility was lowest for lamb meal, with histidine having digestibility less than 70% and the other entire indispensable AA having digestibility between 72 and 88%. The TMEn of calamari meal (4.82 kcal/g DM and 86.9% of GE) was greater ( < 0.05) than the other protein sources. The lamb meal had the lowest TMEn value (3.12 kcal/g DM and 66.9% of GE), with others being intermediate (3.46-4.09 kcal/g DM and 71.2-77.9% of GE). This study demonstrates the considerable variability that exists not only in the chemical composition but also in the true nutrient digestibility among protein sources intended for use in dog and cat foods and justifies further in vivo testing of novel protein sources.

Methane seep carbonates yield clumped isotope signatures out of equilibrium with formation temperatures.

Methane cold seep systems typically exhibit extensive buildups of authigenic carbonate minerals, resulting from local increases in alkalinity driven by methane oxidation. Here, we demonstrate that modern seep authigenic carbonates exhibit anomalously low clumped isotope values (Δ47), as much as ∼0.2‰ lower than expected values. In modern seeps, this range of disequilibrium translates into apparent temperatures that are always warmer than ambient temperatures, by up to 50 °C. We examine various mechanisms that may induce disequilibrium behaviour in modern seep carbonates, and suggest that the observed values result from several factors including kinetic isotopic effects during methane oxidation, mixing of inorganic carbon pools, pH effects and rapid precipitation. Ancient seep carbonates studied here also exhibit potential disequilibrium signals. Ultimately, these findings indicate the predominance of disequilibrium clumped isotope behaviour in modern cold seep carbonates that must be considered when characterizing environmental conditions in both modern and ancient cold seep settings.

Multicentre observational study of outcomes after drainage of acute perianal abscess.

INTRODUCTION: Management of perianal abscesses has remained largely unchanged for over 50 years. The evidence for postoperative wound packing is limited and may expose patients to painful procedures with no clinical benefit and at considerable increased cost. METHODS: Patients were recruited in 15 UK centres between December 2013 and October 2014. Outcome measures included number of dressing (pack) changes, healing, recurrence, return to work/normal function, postoperative fistula in ano and health utility scores (EQ-5D™). Pain was measured before, during and after dressing change on a visual analogue scale. RESULTS: Some 141 patients were recruited (median age 39 (range 18-86) years). The mean number of dressing changes in the first 3 weeks was 13 (range 0-21), equating to an annual cost to the National Health Service of €6 453 360 in England alone per annum. Some 43·8 per cent of wounds were healed by 8 weeks after surgery and 86 per cent of patients had returned to normal function. Some 7·6 per cent of abscesses had recurred and 26·7 per cent of patients developed a fistula in ano by 6 months following surgery. Patients reported a twofold to threefold increase in pain scores during and after dressing changes. CONCLUSION: Recurrent abscess is rare and fistula occurs in one-quarter of the patients. Packing is painful and costly.

New frontiers in pediatric Allo-SCT.

The inaugural meeting of 'New Frontiers in Pediatric Allogeneic Stem Cell Transplantation' organized by the Pediatric Blood and Transplant Consortium (PBMTC) was held at the American Society of Pediatric Hematology and Oncology Annual Meeting. This meeting provided an international platform for physicians and investigators active in the research and utilization of pediatric Allo-SCT in children and adolescents with malignant and non-malignant disease (NMD), to share information and develop future collaborative strategies. The primary objectives of the conference included: (1) to present advances in Allo-SCT in pediatric ALL and novel pre and post-transplant immunotherapy; (2) to highlight new strategies in alternative allogeneic stem cell donor sources for children and adolescents with non-malignant hematological disorders; (3) to discuss timing of immune reconstitution after Allo-SCT and methods of facilitating more rapid recovery of immunity; (4) to identify strategies of utilizing Allo-SCT in pediatric myeloproliferative disorders; (5) to develop diagnostic and therapeutic approaches to hematological complications post pediatric Allo-SCT; (6) to enhance the understanding of new novel cellular therapeutic approaches to pediatric malignant and non-malignant hematological disorders; and (7) to discuss optimizing drug therapy in pediatric recipients of Allo-SCT. This paper will provide a brief overview of the conference.

Facilitating medical withdrawal from opiates in rural Ontario.

CONTEXT: The abuse of oxycodone in Northwestern Ontario, Canada, has escalated at alarming rates raising concerns that opiate use has reached epidemic proportions, particularly among the First Nations communities. The authors were involved in establishing Ontario's first rural inpatient medical withdrawal unit to serve patients seeking abstinence. ISSUES: The development of the medical withdrawal support services (MWSS) required creative and adaptive strategies to respond to the geographical, cultural and institutional circumstances. LESSONS LEARNED: Key factors to support program efficacy and successful outcomes for clients during the inaugural eight months of operation are interprofessional and collaborative approaches with a cultural awareness.Key words: addiction, Canada, First Nation, medical withdrawal.

Association of caspase-9 and RUNX3 with inflammatory bowel disease.

Previous linkage studies have identified a region at 1p36 as the susceptibility locus (IBD7) of inflammatory bowel disease (IBD). The objective of this study was to investigate whether polymorphisms of caspase-9 (CASP9) gene and RUNX3 are associated with IBD susceptibility and clinical phenotypes. We studied 555 Crohn's disease (CD) and 651 ulcerative colitis (UC) patients recruited from a single UK center. A total of 964 healthy Caucasian subjects were recruited as controls from general practitioner well person clinics in Oxfordshire. Fourteen single nucleotide polymorphisms (SNPs) of CASP9 and 11 SNPs of RUNX3 were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (homogenous MassEXTEND, hME, Sequenom™, Sequenom Inc., San Diego, CA). Linkage disequilibrium (LD) and haplotype association analysis were performed using 2ld and phase v2.0 software. No association of individual SNPs of CASP9 or RUNX3 with UC or CD was identified. The rs1052571 of CASP9 was associated with severe UC [P = 0.0034, odds ratio (OR) = 1.957, 95% confidence interval (CI) = 1.240-3.088]. Significant haplotype associations between CASP9 and IBD were identified, while no association of RUNX3 haplotypes with either UC or CD was found. Our findings suggested that CASP9 gene might be another IBD susceptibility gene.

Changes in the management and outcome of rectal cancer over a 10-year period in Oxford.

AIM: The changes in the management and outcome of rectal cancer in Oxford were studied over a 10-year period. METHOD: Rectal cancer data using a prospectively collected data base were divided into curative (global) and palliative groups. The global curative group was further divided into those with and without (selected group) the following features: emergency cases, local excision, salvage surgery for recurrence or incomplete local excision, metastatic disease, perioperative death, hereditary cancer, inflammatory bowel disease-related cancer, and synchronous cancer. RESULTS: Between 1994 and 2003, 709 cases of rectal cancer were treated, 532 for cure and a selected group of 393 after removing patients with the aforementioned exclusions. For the selected group, the average follow-up was 51.2 months, overall survival 65.4% and cancer-specific survival 75.3%. There was no 2-year survival difference between each of the 10-year periods of study. Two-year local recurrence was 5.6% for the first 5-year period and 2.3% for the second (P = 0.11). MRI staging increased during the 10 years (0% in 1994; 66.7% in 2003) as did use of definitive chemoradiotherapy (dCRT) (0% in 1994; 64.7% in 2003). The anastomotic leakage rate was significantly higher in the second 5-year period (2.6%vs 9.6%; P = 0.01). CONCLUSION: Despite increasing use of MRI and dCRT, 2-year survival and local recurrence were not significantly different within the 10 years studied.

The genetics of NOD-like receptors in Crohn's disease.

The first Crohn's disease (CD) susceptibility gene identified was CARD15, which is a member of the emerging NOD-like receptor (NLR) family. These function as intracellular cystosolic pattern recognition receptors (PRRs) and play a central role in the innate immune response. We studied other members of the NLR family using a gene-wide haplotype tagging approach in a well-characterised collection of 547 CD patients and 465 controls. Four single nucleotide polymorphisms (SNPs) in NLRP3 had P values < 0.05 and are in high linkage disequilibrium (LD) with each other (r(2) > 0.90 for all four SNPs). rs4925648 and rs10925019 were the most strongly associated with CD susceptibility (P = 0.001, odds ratio (OR) 1.62, 95% CI 1.2-2.18; and P = 6.5 x 10(-4), OR 1.65, 95% CI 1.23-2.19, respectively). rs1363758 located in NLRP11 was associated with CD susceptibility [P = 0.002 (1.64, 1.19-2.25)], which was weakly confirmed in an independent case-cohort collection on joint analysis [P = 0.05, (1.28, 1-1.64)]. On sub-phenotype analysis, an interesting association between NLRP1 and skin extra-intestinal manifestations and colonic, inflammatory CD was identified. None of these results was replicated in the Wellcome Trust Case Control Consortium study and therefore need replication in a further large cohort.

Confirmation of the novel association at the BTNL2 locus with ulcerative colitis.

Linkage in families and association in population case-control investigations have clearly shown that genes within the major histocompatibility complex region on chromosome 6p are relevant to the susceptibility and pathogenesis of ulcerative colitis (UC) and Crohn's disease. However, identifying the causative variants by fine mapping has not been conclusive. In this study using 58 single nucleotide polymorphisms (SNPs) with 616 UC cases, there was significant association with SNP rs2294881 of the (butyrophilin-like 2) BTNL2 gene with odds ratio (OR) = 2.80, confidence interval (CI) = 1.62-4.84 and P = 5.69 x 10(-4) (P(Bonferroni) = 3.3 x 10(-2)) and replication of SNP rs9268480. The missense SNP rs2076523 (K196E) showed novel association with a subset of UC cases with colectomy (n = 126), OR = 0.25, CI = 0.11-0.58 and P = 4.42 x 10(-4) (P(Bonferroni) = 2.56 x 10(-2)). These three associated variants within the BTNL2 gene were neither in linkage disequilibrium with each other nor correlated with the SNPs tagging the human leukocyte antigen (HLA)-DRB1*1502 and HLA-DRB1*0301 alleles.

Inflammatory bowel disease: the view of the surgeon.

Despite advances in medical therapy, surgery is required in approximately 30-40% of patients with ulcerative colitis (UC) and 70-80% of patients with Crohn's disease (CD) at some point during their lifetime. For patients with UC, surgery may be curative, whereas recurrence of CD following surgery is common due to the potentially pan-enteric distribution of the disease. As a result, the indications and surgical management of the disease may be quite different. For UC, the surgeon is involved in the identification of new cases, management of severe disease, recognition of dysplasia and restorative proctocolectomy. Most of the advances in surgery for UC have been in novel techniques relating to the ileal pouch-anal anastomosis, which can now be performed safely for UC with a 10% pouch failure rate long term. For CD, the surgeon is involved in the management of small bowel and ileo-colonic disease, Crohn's colitis and perianal disease. Advances in the surgical management of CD include strictureplasty for extensive small bowel disease, laparoscopic ileo-caecal resection and a combined medical and surgical approach to perianal disease. For both CD and UC close liaison between the gastroenterologist and colorectal surgeon is essential.

High-resolution genotyping of HLA-DQA1 in the GoKinD study and identification of novel alleles HLA-DQA1*040102, HLA-DQA1*0402 and HLA-DQA1*0404.

In order to achieve high-resolution HLA-DQA1 genotyping, it is necessary to identify polymorphisms in exons 1, 2 and 3. We present a high-resolution sequence-based typing (SBT) strategy for genotyping exons 1, 2 and 3 of the polymorphic HLA-DQA1 locus. This method is an improvement upon previously presented methods, because it utilizes the minimum number of SSP-PCR assays to obtain clear DNA sequence in both the forward and reverse directions of all three exons. All known HLA-DQA1 alleles are resolved with the exception of HLA-DQA1*010101 and HLA-DQA1*010102 for which the distinguishing polymorphism is located in exon 4 and does not result in an amino acid change. This method has enabled our laboratory to identify three new HLA-DQA1 alleles - HLA-DQA1*040102, HLA- DQA1*0402 and HLA-DQA1*0404 - in the Genetics of Kidneys in Diabetes (GoKinD) study population. Additionally, we present single-allele amplification methods, which identify the coding sequences of HLA-DQA1 exons 1, 2, 3, intron 2 and 300 bp of the HLA-DQA1 promoter (QAP). This study, also describes the QAP for most of the known HLA-DQA1 alleles, three HLA-DQA2 promoter sequences and the intron 2 sequences for HLA-DQA1*040101, HLA-DQA1*040102, HLA-DQA1*0402 and HLA-DQA1*0404.

"A little encouragement": health services and domestic violence.

The aim of this study was to identify those elements of health care which women who have experienced domestic violence consider to represent quality care and explore opportunities for an area health service to improve its service delivery to these women. To do this nine focus groups were conducted with six urban and three rural groups, recruited from Women's Support Services and Refuges, and 65 women participated. A trained facilitator conducted the focus groups with a representative from each participating organisation. Audiotapes were transcribed then coded using a thematic analysis approach. Analysis of the tapes revealed six opportunities for improvement of health services. Improve services to Aboriginal women; increase access to services; promote available services; improve the health service environment; educate service providers; and provide specialist health services. A major theme to emerge was the importance of a supportive environment where women were supported to make choices for their future.