Short communication: Muscle protein synthetic response to microparticulated whey protein in middle-aged men.

Whey protein concentrate (WPC) is a high-quality dairy ingredient that is often included in formulated food products designed to stimulate muscle anabolism. Whey protein concentrate can be affected by UHT processing, and its sensory properties are not compatible with some formulated food products. Microparticulated WPC (mWPC) is a novel ingredient that is resistant to heat treatment and has enhanced sensory properties. When 16 healthy middle-aged men consumed 20 g of either WPC or mWPC, both proteins increased plasma essential AA and leucine concentrations with no detectable difference in curve kinetics. Myofibrillar protein synthesis was increased in both groups for 90 min after ingestion with no difference between groups. Ingestion of mWPC resulted in a muscle anabolic response that was equivalent to that of WPC over the full 210-min measurement period. Formulated products incorporating mWPC or standard WPC would provoke equivalent anabolic responses.

Differential expression of key regulators of Toll-like receptors in ulcerative colitis and Crohn's disease: a role for Tollip and peroxisome proliferator-activated receptor gamma?

The innate immune system is currently seen as the probable initiator of events which culminate in the development of inflammatory bowel disease (IBD) with Toll-like receptors (TLRs) known to be involved in this disease process. Many regulators of TLRs have been described, and dysregulation of these may also be important in the pathogenesis of IBD. The aim of this study was to perform a co-ordinated analysis of the expression levels of both key intestinal TLRs and their inhibitory proteins in the same IBD cohorts, both ulcerative colitis (UC) and Crohn's disease (CD), in order to evaluate the potential roles of these proteins in the pathogenesis of IBD. Of the six TLRs (TLRs 1, 2, 4, 5, 6 and 9) examined, only TLR-4 was increased significantly in IBD, specifically in active UC. In contrast, differential alterations in expression of TLR inhibitory proteins were observed. A20 and suppressor of cytokine signalling 1 (SOCS1) were increased only in active UC while interleukin-1 receptor-associated kinase 1 (IRAK-m) and B cell lymphoma 3 protein (Bcl-3) were increased in both active UC and CD. In contrast, expression of both peroxisome proliferator-activated receptor gamma (PPARγ) and Toll interacting protein (Tollip) was decreased in both active and inactive UC and CD and at both mRNA and protein levels. In addition, expression of both PPARγ and A20 expression was increased by stimulation of a colonic epithelial cell line Caco-2 with both TLR ligands and commensal bacterial strains. These data suggest that IBD may be associated with distinctive changes in TLR-4 and TLR inhibitory proteins, implying that alterations in these may contribute to the pathogenesis of IBD.

Myosin 1e is a component of the glomerular slit diaphragm complex that regulates actin reorganization during cell-cell contact formation in podocytes.

Glomerular visceral epithelial cells, also known as podocytes, are critical to both normal kidney function and the development of kidney disease. Podocyte actin cytoskeleton and their highly specialized cell-cell junctions (also called slit diaphragm complexes) play key roles in controlling glomerular filtration. Myosin 1e (myo1e) is an actin-based molecular motor that is expressed in renal glomeruli. Disruption of the Myo1e gene in mice and humans promotes podocyte injury and results in the loss of the integrity of the glomerular filtration barrier. Here, we have used biochemical and microscopic approaches to determine whether myo1e is associated with the slit diaphragm complexes in glomerular podocytes. Myo1e was consistently enriched in the slit diaphragm fraction during subcellular fractionation of renal glomeruli and colocalized with the slit diaphragm markers in mouse kidney. Live cell imaging studies showed that myo1e was recruited to the newly formed cell-cell junctions in cultured podocytes, where it colocalized with the actin filament cables aligned with the nascent contacts. Myo1e-null podocytes expressing FSGS-associated myo1e mutant (A159P) did not efficiently assemble actin cables along new cell-cell junctions. We have mapped domains in myo1e that were critical for its localization to cell-cell junctions and determined that the SH3 domain of myo1e tail interacts with ZO-1, a component of the slit diaphragm complex and tight junctions. These findings suggest that myo1e represents a component of the slit diaphragm complex and may contribute to regulating junctional integrity in kidney podocytes.

Protein modules as organizers of membrane structure.

Investigations conducted over the past 18 months have shed new light on how modular protein-binding domains, in particular PDZ domains, co-ordinate the assembly of functional plasma membrane domains. Members of the MAGUK (membrane-associated guanylate kinase) protein family, like PSD-95, use multiple domains to cluster ion channels, receptors, adhesion molecules and cytosolic signaling proteins at synapses, cellular junctions, and polarized membrane domains. Other PDZ proteins, like the Drosophila protein INAD and the epithelial Na(+)/H(+) regulatory factor (NHERF), organize cellular signaling by localizing transmembrane and cytosolic components to specific membrane domains and assembling these components into functional complexes. The organization of these proteins into discreet structures has functional consequences for downstream signaling.

The structure and regulation of tight junctions.

Tight junctions create a paracellular barrier between both epithelial and endothelial cells. Recent advances have helped define their molecular composition and regulation. Studies in cultured cell lines provide new insights into how assembly and barrier properties may be controlled by signal transduction cascades.

Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease.

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)-α, a known agonist of the mitogen-activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho-p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex- and age-matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF-α, interleukin (IL)-1ß and IL-6 were measured in the organ and cell culture supernatants by enzyme-linked immunosorbent assay. We found higher levels of phospho-p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF-α, IL-1ß and IL-6 from IBD LPMCs and biopsies. Activated p38α MAPK is up-regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down-regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.

Colonic microbiota is associated with inflammation and host epigenomic alterations in inflammatory bowel disease.

Studies of inflammatory bowel disease (IBD) have been inconclusive in relating microbiota with distribution of inflammation. We report microbiota, host transcriptomics, epigenomics and genetics from matched inflamed and non-inflamed colonic mucosa [50 Crohn's disease (CD); 80 ulcerative colitis (UC); 31 controls]. Changes in community-wide and within-patient microbiota are linked with inflammation, but we find no evidence for a distinct microbial diagnostic signature, probably due to heterogeneous host-microbe interactions, and show only marginal microbiota associations with habitual diet. Epithelial DNA methylation improves disease classification and is associated with both inflammation and microbiota composition. Microbiota sub-groups are driven by dominant Enterbacteriaceae and Bacteroides species, representative strains of which are pro-inflammatory in vitro, are also associated with immune-related epigenetic markers. In conclusion, inflamed and non-inflamed colonic segments in both CD and UC differ in microbiota composition and epigenetic profiles.

Connexin-occludin chimeras containing the ZO-binding domain of occludin localize at MDCK tight junctions and NRK cell contacts.

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.

Recognition of unique carboxyl-terminal motifs by distinct PDZ domains.

The oriented peptide library technique was used to investigate the peptide-binding specificities of nine PDZ domains. Each PDZ domain selected peptides with hydrophobic residues at the carboxyl terminus. Individual PDZ domains selected unique optimal motifs defined primarily by the carboxyl terminal three to seven residues of the peptides. One family of PDZ domains, including those of the Discs Large protein, selected peptides with the consensus motif Glu-(Ser/Thr)-Xxx-(Val/Ile) (where Xxx represents any amino acid) at the carboxyl terminus. In contrast, another family of PDZ domains, including those of LIN-2, p55, and Tiam-1, selected peptides with hydrophobic or aromatic side chains at the carboxyl terminal three residues. On the basis of crystal structures of the PSD-95-3 PDZ domain, the specificities observed with the peptide library can be rationalized.

Performance measurement of a Canadian provincial tuberculosis programme: Manitoba, 2008-2012.

BACKGROUND: Performance measurement assists tuberculosis (TB) programmes in understanding areas of strength and weakness, and planning for improvements. Canada currently does not have a national comprehensive system for the measurement and analysis of TB programme performance. OBJECTIVE: To analyse the performance of a Canadian provincial TB programme using measures and targets based on those published by the US Centers for Disease Prevention and Control for 2015. DESIGN: Using provincial surveillance data from the Canadian province of Manitoba, we analysed key programme performance outcome measures (treatment completion, early detection, human immunodeficiency virus [HIV] testing, paediatric TB, retreatment, and contact elicitation and assessment) for people diagnosed with TB between 2008 and 2010. RESULTS: Significant outcome variation was found between Indigenous and non-Indigenous populations as well as within populations. The reporting rate of HIV testing was low. High rates of paediatric TB among Indigenous populations, particularly in rural areas, were found. Significantly better performance in HIV testing and reporting as well as in contact investigation was found for rural compared with urban Indigenous populations. Foreign-born persons had the lowest contact assessment rate. CONCLUSION: This study of TB programme performance in Manitoba demonstrates the viability of the approach in the Canadian context, and could help to identify key areas for TB programme improvement.

Protein-protein interactions: PDZ domain networks.

PDZ domains can dimerize or bind to the carboxyl termini of unrelated proteins. Crystallographic studies demonstrate the structural basis for these interactions, which contribute to the ability of PDZ domains to create networks associated with the plasma membrane.

Tobacco and tuberculosis: a qualitative systematic review and meta-analysis.

OBJECTIVES: To assess the strength of evidence in published articles for an association between smoking and passive exposure to tobacco smoke and various manifestations and outcomes of tuberculosis (TB). Clinicians and public health workers working to fight TB may not see a role for themselves in tobacco control because the association between tobacco and TB has not been widely accepted. A qualitative review and meta-analysis was therefore undertaken. METHODS: Reference lists, PubMed, the database of the International Union Against Tuberculosis and Lung Disease and Google Scholar were searched for a final inclusion of 42 articles in English containing 53 outcomes for data extraction. A quality score was attributed to each study to classify the strength of evidence according to each TB outcome. A meta-analysis was then performed on results from included studies. RESULTS: Despite the limitations in the data available, the evidence was rated as strong for an association between smoking and TB disease, moderate for the association between second-hand smoke exposure and TB disease and between smoking and retreatment TB disease, and limited for the association between smoking and tuberculous infection and between smoking and TB mortality. There was insufficient evidence to support an association of smoking and delay, default, slower smear conversion, greater severity of disease or drug-resistant TB or of second-hand tobacco smoke exposure and infection. CONCLUSIONS: The association between smoking and TB disease appears to be causal. Smoking can have an important impact on many aspects of TB. Clinicians can confidently advise patients that quitting smoking and avoiding exposure to others' tobacco smoke are important measures in TB control.

Tuberculosis among nomads in Adamawa, Nigeria: outcomes from two years of active case finding.

BACKGROUND: Nomadic populations are often isolated and have difficulty accessing health care, leading to increased morbidity and mortality. Although Nigeria has one of the highest tuberculosis (TB) burdens in Africa, case detection rates remain relatively low. METHODS: Active case finding for TB among nomadic populations was implemented over a 2-year period in Adamawa State. A total of 378 community screening days were organised with local leaders; community volunteers provided treatment support. Xpert(®) MTB/RIF was available for nomads with negative smear results. RESULTS: Through active case finding, 96 376 nomads were verbally screened, yielding 1310 bacteriologically positive patients. The number of patients submitting sputum for smear microscopy statewide increased by 112% compared with the 2 years before the intervention. New smear-positive notifications increased by 49.5%, while notifications of all forms of TB increased by 24.5% compared with expected notifications based on historical trends. Nomads accounted for respectively 31.4% and 26.0% of all smear-positive and all forms TB notifications. Pre-treatment loss to follow-up and treatment outcomes were similar among nomads and non-nomads. DISCUSSION: Nomads in Nigeria have high TB rates, and active case-finding approaches may be useful in identifying and successfully treating them. Large-scale interventions in vulnerable populations can improve TB case detection.

Fish oils in hypertriglyceridemia: a dose-response study.

The effects of three supplemental doses of fish oil on plasma lipids, lipoproteins, and bleeding times were studied in ten hypertriglyceridemic patients. After a 3-wk baseline period each patient was given 15, 25, or 40 mL fish oil/d (containing 4.5, 7.5, and 12 g n-3 fatty acids) for three successive 6-wk periods, each separated by a 4-wk period of no supplementation. Plasma cholesterol concentrations decreased from 7.40 mmol/L to 6.35, 6.55, and 6.40 mmol/L with increasing doses of fish oil (p less than 0.01 vs baseline for each). Plasma triglyceride concentrations decreased from 6.10 mmol/L to 2.90, 2.80, and 2.35 mmol/L (p less than 0.01 vs baseline for each). Low-density-lipoprotein cholesterol concentrations increased significantly (by 23% and 28%) with the two higher doses, respectively. Bleeding times increased only with the largest dose. The lowest dose was the most hypolipidemic per gram n-3 fatty acids.

Current and potential treatment of tuberculosis.

A recent resurgence of interest in tuberculosis as a global health problem has accompanied the resurgence of tuberculosis in both industrialised and developing countries. It has also been demonstrated recently that tuberculosis treatment and control is one of the most cost effective of all medical interventions. The human immunodeficiency virus (HIV) epidemic and increasing resistance to antituberculous drugs complicate our response to the problem of tuberculosis. Chemotherapy with currently available agents is highly effective, not only in pulmonary tuberculosis in adults, but also in extrapulmonary disease, and in disease in children and even patients with concomitant HIV infection. Short course chemotherapy and intermittent therapy are as effective as older regimens. Measures, including directly observed therapy, to maximise compliance with therapy, are of utmost importance. An efficient programme which assures compliance with effective antituberculosis chemotherapy should be a priority for health spending even in those countries with fewest resources.

The molecular epidemiology of tuberculosis in western Canada.

OBJECTIVE: To define the molecular epidemiology of TB in western Canada, and in particular the risk factors for clustering. MEASUREMENTS: We prospectively identified all positive cultures from newly diagnosed cases of TB diagnosed between February 1995 and January 1997 and carried out restriction fragment length polymorphism (RFLP) testing on all isolates. RESULTS: Of 956 cases identified, 944 fulfilled the entry criteria. The mean age was 49.65 years (+/- 22.33), and 508 (53.6%) were males. Three hundred and three (32.1%) subjects were clustered; this varied from 20.2% of the foreign born, 48.4% of Canadian non-Aboriginal and 61.1% of all Aboriginal persons. Younger persons (P = 0.0001), males (P = 0.015), those with pulmonary disease (P < 0.001), living in a shelter in the past year (P < 0.001), drug-susceptible disease (P < 0.036), predisposing factors (P < 0.001), prior contact (P < 0.001), and prior skin test (P < 0.002) were more likely to cluster. Among specific risk factors, HIV infection, injection drug use, alcohol excess, and weight loss were all significant. CONCLUSIONS: In this description of the molecular epidemiology of TB in Western Canada, previous results have been confirmed and extended. These results highlight the importance of identifying specific high risk groups, especially in the context of renewed efforts to target persons for treatment of latent TB infection.

Stop TB-Halte à la Tuberculose-Canada: engaging industrialised nations in the challenge to meet global targets.

The Stop TB Partnership has engaged the 22 high-burden countries in a drive toward the goal of finding 70% of cases and curing 85% by 2005. Traditional partners, aid agencies and governments of industrialised nations have joined the Partnership, but the broader range of civil society remains outside the discourse, risking disinterest on the part of the donor community. Stop TB-Halte à la Tuberculose-Canada was organised to engage new partners to support the Canadian government's commitment to the goal of reducing poverty and diseases of poverty, including tuberculosis, by 50% by 2010. The successes and challenges are explored, and the possibility raised that having a Stop TB movement in every country will ensure that support is sustained and goals of global tuberculosis control reached.

Transmission characteristics of tuberculosis in the foreign-born and the Canadian-born populations of Alberta, Canada.

SETTING: All notified cases of tuberculosis in the province of Alberta, Canada, 1994-1998. OBJECTIVE: To compare the transmission characteristics of tuberculosis among foreign-born and Canadian-born cases. DESIGN: Retrospective analysis using DNA fingerprinting (IS6110 restriction fragment length polymorphism and spoligotyping) and patient information from the Alberta Tuberculosis Registry. Transmission indexes were determined by calculating the average number of culture-positive pulmonary cases generated by a single source case. RESULTS: Of the 750 cases of active tuberculosis, 437 (58.3%) were in the foreign-born. DNA fingerprinting of Mycobacterium tuberculosis isolates from all 573 culture-positive cases over the 5 years from 1994 to 1998 showed that there was significantly less clustering among foreign-born isolates (9.8%) compared to Canadian-born non-Aboriginal (28.8%) and Aboriginal (44.7%) isolates. The transmission index was significantly higher for males, lower for those > or =65 years of age, and higher for Aboriginals. CONCLUSION: Although cases of tuberculosis in the foreign-born constitute the majority in Alberta, there is little transmission to other foreign-born or to Canadian-born individuals. Transmission of tuberculosis among the Aboriginal population remains a significant problem in Alberta.

Availability of quality fixed-dose combinations for the treatment of tuberculosis: what can we learn from studying the World Health Organization's vaccine model?

SETTING: In efforts to promote the use of fixed-dose combinations (FDCs) for the treatment of tuberculosis (TB), the World Health Organization (WHO) and partners address the issue of quality assurance. OBJECTIVE: To provide guidance for the development of strategies for quality assurance of FDCs. DESIGN: This review examines the WHO strategies for and experience with quality assurance and supply of vaccines. RESULTS: Several elements in the strategies for quality assurance and supply of vaccines may be applicable for FDCs. At national level, the important strategies are to strengthen National Regulatory Authorities (NRA) and procurement systems and develop planning activities. Stressing quality assurance of FDCs in training activities for regulatory personnel and recommending that aid agencies require adherence to quality assurance policies as conditions for support would promote the implementation of quality assurance of FDCs at country level. At the global level, pre-qualification of manufacturers of FDCs should be explored as a mechanism to assure quality. The pre-qualification process should include evaluation of product files, initial testing for compliance and consistency of specifications, and site visits to producers and NRAs. The vaccine model defines criteria for reassessment that can be used for FDCs.

The economic impact of flexible endoscopy in a large academic surgical department.

BACKGROUND: There has been considerable controversy concerning the value added to a general surgery practice when flexible gastrointestinal endoscopy is incorporated. The purpose of this study was to assess the economic impact of flexible endoscopy performed by general surgeons in a large academic practice. METHODS: A retrospective review of gross billing charges for a group practice of 11 surgeons over the fiscal year 1999 was performed at the Cleveland Clinic Foundation. The total billing for clinic visits and outpatient and inpatient surgical procedures was compiled, and the percentage attributable to flexible endoscopy was determined. Of the 11 surgeons, three had incorporated flexible endoscopy into their practice. RESULTS: The three endosurgeons generated 33% of the total gross billing for the Department of General Surgery. Flexible endoscopy alone accounted for 12.2% of the total percentage of gross billings for the department. Meanwhile, this revenue was generated from only 8% of the workweek when performing flexible endoscopic procedures were performed. CONCLUSION: Flexible endoscopy can contribute significantly to the financial productivity of the general surgeon.