Evidences on the role of the lid loop of γ-glutamyltransferases (GGT) in substrate selection

\u3b3-Glutamyltransferase (GGT) catalyzes the transfer of the \u3b3-glutamyl moiety from a donor substrate such as glutathione to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a \u3b3-glutamyl enzyme intermediate. The vast majority of the known GGTs has a short sequence covering the glutamate binding site, called lid-loop. Although being conserved enzymes, both B. subtilis GGT and the related enzyme CapD from B. anthracis lack the lid loop and, differently from other GGTs, both accept poly-\u3b3-glutamic acid (\u3b3-PGA) as a substrate. Starting from this observation, in this work the activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from E. coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT. Results indicate that the absence of the lid loop seems not to be the sole structural feature responsible for the recognition of a polymeric substrate by GGTs. Nevertheless, time course of hydrolysis reactions carried out using oligo-\u3b3-glutamylglutamines as substrates showed that the lid loop acts as a gating structure, allowing the preferential selection of the small glutamine with respect to the oligomeric substrates. In this respect, the mutant B. subtilis GGT revealed to be more similar to E. coli GGT than to its wild-type counterpart. In addition, the transpeptidase activity of the newly produced mutant enzyme revealed to be higher with respect to that of both E. coli and wild-type B. subtilis GGT. These findings can be helpful in selecting GGTs intended as biocatalysts for preparative purposes as well as in designing mutant enzymes with improved transpeptidase activity

Omnipolarity applied to equi-spaced electrode array for ventricular tachycardia substrate mapping

Aims : Bipolar electrogram (BiEGM)-based substrate maps are heavily influenced by direction of a wavefront to the mapping bipole. In this study, we evaluate high-resolution, orientation-independent peak-to-peak voltage (Vpp) maps obtained with an equi-spaced electrode array and omnipolar EGMs (OTEGMs), measure its beat-to-beat consistency, and assess its ability to delineate diseased areas within the myocardium compared against traditional BiEGMs on two orientations: along (AL) and across (AC) array splines. Methods and results: The endocardium of the left ventricle of 10 pigs (three healthy and seven infarcted) were each mapped using an Advisor™ HD grid with a research EnSite Precision™ system. Cardiac magnetic resonance images with late gadolinium enhancement were registered with electroanatomical maps and were used for gross scar delineation. Over healthy areas, OTEGM Vpp values are larger than AL bipoles by 27% and AC bipoles by 26%, and over infarcted areas OTEGM Vpp values are 23% larger than AL bipoles and 27% larger than AC bipoles (P < 0.05). Omnipolar EGM voltage maps were 37% denser than BiEGM maps. In addition, OTEGM Vpp values are more consistent than bipolar Vpps showing less beat-by-beat variation than BiEGM by 39% and 47% over both infarcted and healthy areas, respectively (P < 0.01). Omnipolar EGM better delineate infarcted areas than traditional BiEGMs from both orientations. Conclusion: An equi-spaced electrode grid when combined with omnipolar methodology yielded the largest detectable bipolar-like voltage and is void of directional influences, providing reliable voltage assessment within infarcted and non-infarcted regions of the heart.This work was funded by Abbott Laboratories, St. Paul, MN, USA.S

Use and perceived effectiveness of non-analgesic medical therapies for chronic pancreatitis in the United States

Aliment Pharmacol Ther 2011; 33: 149–159Effectiveness of medical therapies in chronic pancreatitis has been described in small studies of selected patients.To describe frequency and perceived effectiveness of non-analgesic medical therapies in chronic pancreatitis patients evaluated at US referral centres.Using data on 516 chronic pancreatitis patients enrolled prospectively in the NAPS2 Study, we evaluated how often medical therapies [pancreatic enzyme replacement therapy (PERT), vitamins/antioxidants (AO), octreotide, coeliac plexus block (CPB)] were utilized and considered useful by physicians.Oral PERT was commonly used (70%), more frequently in the presence of exocrine insufficiency (EI) (88% vs. 61%, P  < 0.001) and pain (74% vs. 59%, P  < 0.002). On multivariable analyses, predictors of PERT usage were EI (OR 5.14, 95% CI 2.87–9.18), constant (OR 3.42, 95% CI 1.93–6.04) or intermittent pain (OR 1.98, 95% CI 1.14–3.45). Efficacy of PERT was predicted only by EI (OR 2.16, 95% CI 1.36–3.42). AO were tried less often (14%) and were more effective in idiopathic and obstructive vs. alcoholic chronic pancreatitis (25% vs. 4%, P  = 0.03). Other therapies were infrequently used (CPB – 5%, octreotide – 7%) with efficacy generally <50%.Pancreatic enzyme replacement therapy is commonly utilized, but is considered useful in only subsets of chronic pancreatitis patients. Other medical therapies are used infrequently and have limited efficacy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79114/1/j.1365-2036.2010.04491.x.pd

MRCP compared to diagnostic ERCP for diagnosis when biliary obstruction is suspected: a systematic review

BACKGROUND: Magnetic resonance cholangiopancreatography (MRCP) is an alternative to diagnostic endoscopic retrograde cholangiopancreatography (ERCP) for investigating biliary obstruction. The use of MRCP, a non-invasive procedure, may prevent the use of unnecessary invasive procedures. The aim of the study was to compare the findings of MRCP with those of ERCP by the computation of accuracy statistics. METHODS: Thirteen electronic bibliographic databases, covering biomedical, science, health economics and grey literature were searched. A systematic review of studies comparing MRCP to diagnostic ERCP in patients with suspected biliary obstruction was conducted. Sensitivity, specificity, likelihood ratios, acceptability and adverse events were reported. RESULTS: 25 studies were identified reporting several conditions including choledocholithiasis (18 studies), malignancy (four studies), obstruction (three studies), stricture (two studies) and dilatation (five studies). Three of the 18 studies reporting choledocholithiasis were excluded from the analysis due to lack of data, or differences in study design. The sensitivity for the 15 studies of choledocholithiasis ranged from 0.50 to 1.00 while specificity ranged from 0.83 to 1.00. The positive likelihood ratio ranged: from 5.44–47.72 and the negative likelihood ratio for the 15 studies ranged from 0.00–0.51. Significant heterogeneity was found across the 15 studies so the sensitivities and specificities were summarised by a Receiver Operating Characteristic (ROC) curve. For malignancy, sensitivity ranged from 0.81 to 0.94 and specificity from 0.92 to 1.00. Positive likelihood ratios ranged from 10.12 to 43 and negative likelihood ratios ranged from 0.15 to 0.21, although these estimates were less reliable. CONCLUSION: MRCP is a comparable diagnostic investigation in comparison to ERCP for diagnosing biliary obstruction

Chronic pancreatitis: Pediatric and adult cohorts show similarities in disease progress despite different risk factors

Objectives: To investigate the natural history of chronic pancreatitis (CP), patients in the North American Pancreatitis Study2 (NAPS2, adults) and INternational Study group of Pediatric Pancreatitis: In search for a cuRE (INSPPIRE, pediatric) were compared. Methods: Demographics, risk factors, disease duration, management and outcomes of 224 children and 1,063 adults were compared using appropriate statistical tests for categorical and continuous variables. Results: Alcohol was a risk in 53% of adults and 1% of children (p<0.0001); tobacco in 50% of adults and 7% of children (p<0.0001). Obstructive factors were more common in children (29% vs 19% in adults, p=0.001). Genetic risk factors were found more often in children. Exocrine pancreatic insufficiency was similar (children 26% vs adult 33%, p=0.107). Diabetes was more common in adults than children (36% vs 4% respectively, p<0.0001). Median emergency room visits, hospitalizations, and missed days of work/school were similar across the cohorts. As a secondary analysis, NAPS2 subjects with childhood onset (NAPS2-CO) were compared to INSPPIRE subjects. These two cohorts were more similar than the total INSPPIRE and NAPS2 cohorts, including for genetic risk factors. The only risk factor significantly more common in the NAPS2-CO cohort compared with the INSPPIRE cohort was alcohol (9% NAPS2-CO vs 1% INSPPIRE cohorts, p=0.011). Conclusions: Despite disparity in age of onset, children and adults with CP exhibit similarity in demographics, CP treatment, and pain. Differences between groups in radiographic findings and diabetes prevalence may be related to differences in risk factors associated with disease and length of time of CP

Rectal indomethacin alone versus indomethacin and prophylactic pancreatic stent placement for preventing pancreatitis after ERCP: study protocol for a randomized controlled trial

Abstract Background The combination of prophylactic pancreatic stent placement (PSP) – a temporary plastic stent placed in the pancreatic duct – and rectal non-steroidal anti-inflammatory drugs (NSAIDs) is recommended for preventing post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) in high-risk cases. Preliminary data, however, suggest that PSP may be unnecessary if rectal NSAIDs are administered. Given the costs and potential risks of PSP, we aim to determine whether rectal indomethacin obviates the need for pancreatic stent placement in patients undergoing high-risk ERCP. Methods/Design The SVI (Stent vs. Indomethacin) trial is a comparative effectiveness, multicenter, randomized, double-blind, non-inferiority study of rectal indomethacin alone versus the combination of rectal indomethacin and PSP for preventing PEP in high-risk cases. One thousand four hundred and thirty subjects undergoing high-risk ERCP, in whom PSP is planned solely for PEP prevention, will be randomized to indomethacin alone or combination therapy. Those who are aware of study group assignment, including the endoscopist, will not be involved in the post-procedure care of the patient for at least 48 hours. Subjects will be assessed for PEP and its severity by a panel of independent and blinded adjudicators. Indomethacin alone will be declared non-inferior to combination therapy if the two-sided 95 % upper confidence bound of the treatment difference is less than 5 % between the two groups. Biological specimens will be obtained from trial participants and centrally banked. Discussion The SVI trial is designed to determine whether PSP remains necessary in the era of NSAIDs pharmacoprevention. The associated bio-repository will establish the groundwork for important scientific breakthrough. Trial registration NCT02476279, registered June 2015

Rectal indomethacin alone versus indomethacin and prophylactic pancreatic stent placement for preventing pancreatitis after ERCP: Study protocol for a randomized controlled trial

Background: The combination of prophylactic pancreatic stent placement (PSP) - a temporary plastic stent placed in the pancreatic duct - and rectal non-steroidal anti-inflammatory drugs (NSAIDs) is recommended for preventing post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) in high-risk cases. Preliminary data, however, suggest that PSP may be unnecessary if rectal NSAIDs are administered. Given the costs and potential risks of PSP, we aim to determine whether rectal indomethacin obviates the need for pancreatic stent placement in patients undergoing high-risk ERCP. Methods/Design: The SVI (Stent vs. Indomethacin) trial is a comparative effectiveness, multicenter, randomized, double-blind, non-inferiority study of rectal indomethacin alone versus the combination of rectal indomethacin and PSP for preventing PEP in high-risk cases. One thousand four hundred and thirty subjects undergoing high-risk ERCP, in whom PSP is planned solely for PEP prevention, will be randomized to indomethacin alone or combination therapy. Those who are aware of study group assignment, including the endoscopist, will not be involved in the post-procedure care of the patient for at least 48 hours. Subjects will be assessed for PEP and its severity by a panel of independent and blinded adjudicators. Indomethacin alone will be declared non-inferior to combination therapy if the two-sided 95 % upper confidence bound of the treatment difference is less than 5 % between the two groups. Biological specimens will be obtained from trial participants and centrally banked. Discussion: The SVI trial is designed to determine whether PSP remains necessary in the era of NSAIDs pharmacoprevention. The associated bio-repository will establish the groundwork for important scientific breakthrough

Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR, and SPINK1 variants were associated with pancreatitis risk. We now report two significant genome-wide associations identified and replicated at PRSS1-PRSS2 (1×10-12) and x-linked CLDN2 (p < 1×10-21) through a two-stage genome-wide study (Stage 1, 676 cases and 4507 controls; Stage 2, 910 cases and 4170 controls). The PRSS1 variant affects susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous male) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men – male hemizygous frequency is 0.26, female homozygote is 0.07

Flowcharts for the management of biliary tract and ampullary carcinomas

No strategies for the diagnosis and treatment of biliary tract carcinoma have been clearly described. We developed flowcharts for the diagnosis and treatment of biliary tract carcinoma on the basis of the best clinical evidence. Risk factors for bile duct carcinoma are a dilated type of pancreaticobiliary maljunction (PBM) and primary sclerosing cholangitis. A nondilated type of PBM is a risk factor for gallbladder carcinoma. Symptoms that may indicate biliary tract carcinoma are jaundice and pain in the upper right area of the abdomen. The first step of diagnosis is to carry out blood biochemistry tests and ultrasonography (US) of the abdomen. The second step of diagnosis is to find the local extension of the carcinoma by means of computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance cholangiopancreatography (MRCP), percutaneous transhepatic cholangiography (PTC), and endoscopic retrograde cholangiopancreatography (ERCP). Because resection is the only way to completely cure biliary tract carcinoma, the indications for resection are determined first. In patients with resectable disease, the indications for biliary drainage or portal vein embolization (PVE) are checked. In those with nonresectable disease, biliary stenting, chemotherapy, radiotherapy, and/or best supportive care is selected