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Find video protocols related to scientific articles indexed in Pubmed.
A Price to Pay for Relaxed Substrate Specificity: A Comparative Kinetic Analysis of the Class II Lanthipeptide Synthetases ProcM and HalM2.
J. Am. Chem. Soc.
PUBLISHED: 11-20-2014
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Lanthipeptides are a class of ribosomally synthesized and ¬posttranslationally modified ¬peptide natural products (RiPPs) that typically harbor multiple intramolecular thioether linkages. For class II lanthipeptides, these crosslinks are installed in a multistep reaction pathway by a single enzyme (LanM). The multi-functional nature of LanMs and the manipulability of their genetically encoded peptide substrates (LanAs) make LanM/LanA systems promising targets for the engineering of new antibacterial compounds. Here, we report the development of a semi-quantitative mass spectrometry-based assay for kinetic characterization of LanM-catalyzed reactions. The assay was used to conduct a comparative kinetic analysis of two LanM enzymes (HalM2 and ProcM) that exhibit drastically different substrate selectivity. Numerical simulation of the kinetic data was used to develop models for the multistep HalM2- and ProcM-catalyzed reactions. These models illustrate that HalM2 and ProcM have markedly different catalytic efficiencies for the various reactions they catalyze. HalM2, which is responsible for the biosynthesis of a single compound (the Hal? subunit of the lantibiotic haloduracin), catalyzes reactions with higher catalytic efficiency than ProcM, which modifies 29 different ProcA precursor peptides during prochlorosin biosynthesis. In particular, the rates of thioether ring formation are drastically reduced in ProcM, likely because this enzyme is charged with installing a variety of lanthipeptide ring architectures in its prochlorosin products. Thus, ProcM appears to pay a kinetic price for its relaxed substrate specificity. In addition, our kinetic models suggest that conformational sampling of the LanM:LanA Michaelis complex could play an important role in the kinetics of LanA maturation.
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Effects of TiO2 and Ag Nanoparticles on Polyhydroxybutyrate Biosynthesis By Activated Sludge Bacteria.
Environ. Sci. Technol.
PUBLISHED: 11-20-2014
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Manufactured nanomaterials (MNMs) are increasingly incorporated into consumer products that are disposed into sewage. In wastewater treatment, MNMs adsorb to activated sludge biomass where they may impact biological wastewater treatment performance, including nutrient removal. Here, we studied MNM effects on bacterial polyhydroxyalkanoate (PHA), specifically polyhydroxybutyrate (PHB), biosynthesis because of its importance to enhanced biological phosphorus (P) removal (EBPR). Activated sludge was sampled from an anoxic selector of a municipal wastewater treatment plant (WWTP), and PHB-containing bacteria were concentrated by density gradient centrifugation. After starvation to decrease intracellular PHB stores, bacteria were nutritionally augmented to promote PHB biosynthesis while being exposed to either MNMs (TiO2 or Ag) or to Ag salts (each at a concentration of 5 mg L-1). Cellular PHB concentration and PhyloChip community composition were analyzed. The final bacterial community composition differed from activated sludge, demonstrating that laboratory enrichment was selective. Still, PHB was synthesized to near-activated sludge levels. Ag salts altered final bacterial communities, although MNMs did not. PHB biosynthesis was diminished with Ag (salt or MNMs), indicating the potential for Ag-MNMs to physiologically impact EBPR through the effects of dissolved Ag ions on PHB producers.
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Early versus On-Demand Nasoenteric Tube Feeding in Acute Pancreatitis.
N. Engl. J. Med.
PUBLISHED: 11-20-2014
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Background Early enteral feeding through a nasoenteric feeding tube is often used in patients with severe acute pancreatitis to prevent gut-derived infections, but evidence to support this strategy is limited. We conducted a multicenter, randomized trial comparing early nasoenteric tube feeding with an oral diet at 72 hours after presentation to the emergency department in patients with acute pancreatitis. Methods We enrolled patients with acute pancreatitis who were at high risk for complications on the basis of an Acute Physiology and Chronic Health Evaluation II score of 8 or higher (on a scale of 0 to 71, with higher scores indicating more severe disease), an Imrie or modified Glasgow score of 3 or higher (on a scale of 0 to 8, with higher scores indicating more severe disease), or a serum C-reactive protein level of more than 150 mg per liter. Patients were randomly assigned to nasoenteric tube feeding within 24 hours after randomization (early group) or to an oral diet initiated 72 hours after presentation (on-demand group), with tube feeding provided if the oral diet was not tolerated. The primary end point was a composite of major infection (infected pancreatic necrosis, bacteremia, or pneumonia) or death during 6 months of follow-up. Results A total of 208 patients were enrolled at 19 Dutch hospitals. The primary end point occurred in 30 of 101 patients (30%) in the early group and in 28 of 104 (27%) in the on-demand group (risk ratio, 1.07; 95% confidence interval, 0.79 to 1.44; P=0.76). There were no significant differences between the early group and the on-demand group in the rate of major infection (25% and 26%, respectively; P=0.87) or death (11% and 7%, respectively; P=0.33). In the on-demand group, 72 patients (69%) tolerated an oral diet and did not require tube feeding. Conclusions This trial did not show the superiority of early nasoenteric tube feeding, as compared with an oral diet after 72 hours, in reducing the rate of infection or death in patients with acute pancreatitis at high risk for complications. (Funded by the Netherlands Organization for Health Research and Development and others; PYTHON Current Controlled Trials number, ISRCTN18170985 .).
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Soybean plants modify metal oxide nanoparticle effects on soil bacterial communities.
Environ. Sci. Technol.
PUBLISHED: 11-10-2014
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Engineered nanoparticles (ENPs) are entering agricultural soils through land application of nanocontaining biosolids and agrochemicals. The potential adverse effects of ENPs have been studied on food crops and soil bacterial communities separately; however, how ENPs will affect the interacting plant-soil system remains unknown. To address this, we assessed ENP effects on soil microbial communities in soybean-planted, versus unplanted, mesocosms exposed to different doses of nano-CeO2 (0-1.0 g kg(-1)) or nano-ZnO (0-0.5 g kg(-1)). Nano-CeO2 did not affect soil bacterial communities in unplanted soils, but 0.1 g kg(-1) nano-CeO2 altered soil bacterial communities in planted soils, indicating that plants interactively promote nano-CeO2 effects in soil, possibly due to belowground C shifts since plant growth was impacted. Nano-ZnO at 0.5 g kg(-1) significantly altered soil bacterial communities, increasing some (e.g., Rhizobium and Sphingomonas) but decreasing other (e.g., Ensifer, Rhodospirillaceae, Clostridium, and Azotobacter) operational taxonomic units (OTUs). Fewer OTUs decreased from nano-ZnO exposure in planted (41) versus unplanted (85) soils, suggesting that plants ameliorate nano-ZnO effects. Taken together, plants-potentially through their effects on belowground biogeochemistry-could either promote (i.e., for the 0.1 g kg(-1) nano-CeO2 treatment) or limit (i.e., for the 0.5 g kg(-1) nano-ZnO treatment) ENP effects on soil bacterial communities.
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Lantibiotic Cyclase-like protein 2 (LanCL2) is a novel regulator of Akt.
Mol. Biol. Cell
PUBLISHED: 10-03-2014
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The Serine/Threonine protein kinase Akt controls a wide range of biochemical and cellular processes under the modulation of a variety of regulators. In this study we identify the lanthionine synthetase C-like 2 (LanCL2) protein as a positive regulator of Akt activation in human liver cells. LanCL2 knockdown dampens serum- and insulin-stimulated Akt phosphorylation, whereas LanCL2 overexpression enhances it. Neither insulin receptor phosphorylation nor the interaction between insulin receptor substrate and phosphatidylinositide 3-kinase is affected by LanCL2 knockdown. LanCL2 also does not function through PP2A, a phosphatase of Akt. Instead, LanCL2 directly interacts with Akt, with a preference for inactive Akt. Moreover, we show that LanCL2 also binds to the Akt kinase mTORC2, but not PDK1. Whereas LanCL2 is not required for the Akt-mTORC2 interaction, recombinant LanCL2 enhances Akt phosphorylation by mTORC2 in vitro. Finally, consistent with a function of Akt in regulating cell survival, LanCL2 knockdown increases the rate of apoptosis, which is reversed by the expression of a constitutively active Akt. Taken together, our findings reveal LanCL2 as a novel regulator of Akt, and suggest that LanCL2 facilitates optimal phosphorylation of Akt by mTORC2 via direct physical interactions with both the kinase and the substrate.
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High Divergence of the Precursor Peptides in Combinatorial Lanthipeptide Biosynthesis.
ACS Chem. Biol.
PUBLISHED: 10-02-2014
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Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and post-translationally modified peptides (RiPPs). These compounds are widely distributed in taxonomically distant species, and their biosynthetic systems and biological activities are diverse. A unique example of lanthipeptide biosynthesis is the prochlorosin synthetase ProcM from the marine cyanobacterium Prochlorococcus MIT9313, which transforms up to 29 different precursor peptides (ProcAs) into a library of lanthipeptides called prochlorosins (Pcns) with highly diverse sequences and ring topologies. Here, we show that many ProcM-like enzymes from a variety of bacteria have the capacity to carry out post-translational modifications on highly diverse precursor peptides, providing new examples of natural combinatorial biosynthesis. We also demonstrate that the leader peptides come from different evolutionary origins, suggesting that the combinatorial biosynthesis is tied to the enzyme and not a specific type of leader peptide. For some precursor peptides encoded in the genomes, the leader peptides apparently have been truncated at the N-termini, and we show that these N-terminally truncated peptides are still substrates of the enzymes. Consistent with this hypothesis, we demonstrate that about two-thirds of the ProcA N-terminal sequence is not essential for ProcM activity. Our results also highlight the potential of exploring this class of natural products by genome mining and bioengineering.
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Lymphangiogenesis in renal diseases: passive bystander or active participant?
Expert Rev Mol Med
PUBLISHED: 09-26-2014
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Lymphatic vessels (LVs) are involved in a number of physiological and pathophysiological processes such as fluid homoeostasis, immune surveillance, and resolution of inflammation and wound healing. Lymphangiogenesis, the outgrowth of existing LVs and the formation of new ones, has received increasing attention over the past decade on account of its prominence in organ physiology and pathology, which has been enabled by the development of specific tools to study lymph vessel functions. Several studies have been devoted to renal lymphatic vasculature and lymphangiogenesis in kidney diseases, such as chronic renal transplant dysfunction, primary renal fibrotic disorders, proteinuria, diabetic nephropathy and renal inflammation. This review describes the most recent findings on lymphangiogenesis, with a specific focus on renal lymphangiogenesis and its impact on renal diseases. We suggest renal lymphatics as a possible target for therapeutic interventions in renal medicine to dampen tubulointerstitial tissue remodelling and improve renal functioning.
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Proceedings of the first international summit on intestinal anastomotic leak, chicago, illinois, october 4-5, 2012.
Surg Infect (Larchmt)
PUBLISHED: 09-12-2014
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Abstract Objective: The first international summit on anastomotic leak was held in Chicago in October, 2012 to assess current knowledge in the field and develop novel lines of inquiry. The following report is a summary of the proceedings with commentaries and future prospects for clinical trials and laboratory investigations.
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Ultrapure alginate gel reduces adhesion reformation after adhesiolysis.
Int J Colorectal Dis
PUBLISHED: 09-03-2014
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Adhesiolysis at repeat surgery induces adhesion reformation which seems more virulent than development of de novo adhesions. We studied the effect of a new ultrapure alginate gel on adhesion reformation.
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Antioxidants for pain in chronic pancreatitis.
Cochrane Database Syst Rev
PUBLISHED: 08-21-2014
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Reduced intake and absorption of antioxidants due to pain and malabsorption are probable causes of the lower levels of antioxidants observed in patients with chronic pancreatitis (CP). Improving the status of antioxidants might be effective in slowing the disease process and reducing pain in CP.
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Minimally invasive intervention for infected necrosis in acute pancreatitis.
Expert Rev Med Devices
PUBLISHED: 08-14-2014
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Infected necrosis is the main indication for invasive intervention in acute necrotizing pancreatitis. The 2013 IAP/APA guidelines state that percutaneous catheter drainage should be the first step in the treatment of infected necrosis. In 50-65% of patients, additional necrosectomy is required after catheter drainage, which was traditionally done by open necrosectomy. Driven by the perceived lower complication rate, there is an increasing trend toward minimally invasive percutaneous and endoscopic transluminal necrosectomy. The authors present an overview of current minimally invasive treatment options for necrotizing pancreatitis and review recent developments in clinical studies.
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Canine scent detection and microbial source tracking of human waste contamination in storm drains.
Water Environ. Res.
PUBLISHED: 08-12-2014
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Human fecal contamination of surface waters and drains is difficult to diagnose. DNA-based and chemical analyses of water samples can be used to specifically quantify human waste contamination, but their expense precludes routine use. We evaluated canine scent tracking, using two dogs trained to respond to the scent of municipal wastewater, as a field approach for surveying human fecal contamination. Fecal indicator bacteria, as well as DNA-based and chemical markers of human waste, were analyzed in waters sampled from canine scent-evaluated sites (urban storm drains and creeks). In the field, the dogs responded positively (70% and 100%) at sites for which sampled waters were then confirmed as contaminated with human waste. When both dogs indicated a negative response, human waste markers were absent. Overall, canine scent tracking appears useful for prioritizing sampling sites for which DNA-based and similarly expensive assays can confirm and quantify human waste contamination.
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Microbial source tracking in a coastal California watershed reveals canines as controllable sources of fecal contamination.
Environ. Sci. Technol.
PUBLISHED: 08-05-2014
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Elevated levels of fecal indicator bacteria (FIB), including Escherichia coli and enterococci, trigger coastal beach advisories and signal public health risks. Solving FIB pollution in suburban coastal watersheds is challenging, as there are many potential sources. The Arroyo Burro watershed in Santa Barbara, CA is an example, with its popular, but chronically FIB-contaminated beach. To address, a microbial source tracking study was performed. Surface waters were sampled over 2 years, FIB were quantified, and DNA was analyzed for host-associated fecal markers. Surf zone FIB were only elevated when the coastal lagoon was discharging. Among the fecal sources into the lagoon, including upstream human sources and coastal birds, canines were the most important. Canine sources included input via upstream creek water, which decreased after creek-side residences were educated about proper pet waste disposal, and direct inputs to the lagoon and surf zone, where dog waste could have been tidally exchanged with the lagoon. Based on this study, canine waste can be an influential, yet controllable, fecal source to suburban coastal beaches.
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Structural investigation of ribosomally synthesized natural products by hypothetical structure enumeration and evaluation using tandem MS.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-04-2014
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Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a growing class of natural products that are found in all domains of life. These compounds possess vast structural diversity and have a wide range of biological activities, promising a fertile ground for exploring novel natural products. One challenging aspect of RiPP research is the difficulty of structure determination due to their architectural complexity. We here describe a method for automated structural characterization of RiPPs by tandem mass spectrometry. This method is based on the combined analysis of multiple mass spectra and evaluation of a collection of hypothetical structures predicted based on the biosynthetic gene cluster and molecular weight. We show that this method is effective in structural characterization of complex RiPPs, including lanthipeptides, glycopeptides, and azole-containing peptides. Using this method, we have determined the structure of a previously structurally uncharacterized lanthipeptide, prochlorosin 1.2, and investigated the order of the posttranslational modifications in three biosynthetic systems.
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Hydrogen sulfide - physiological properties and therapeutic potential in ischaemia.
Br. J. Pharmacol.
PUBLISHED: 07-27-2014
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Hydrogen sulfide (H2 S) has become a molecule of high interest in recent years, and it is now recognized as the third gasotransmitter in addition to nitric oxide and carbon monoxide. In this review, we discuss the recent literature on the physiology of endogenous and exogenous H2 S, focusing on the protective effects of hydrogen sulfide in models of hypoxia and ischaemia.
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Timing of enteral nutrition in acute pancreatitis: meta-analysis of individuals using a single-arm of randomised trials.
Pancreatology
PUBLISHED: 07-23-2014
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In acute pancreatitis, enteral nutrition (EN) reduces the rate of complications, such as infected pancreatic necrosis, organ failure, and mortality, as compared to parenteral nutrition (PN). Starting EN within 24 h of admission might further reduce complications.
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Mechanistic studies on the substrate-tolerant lanthipeptide synthetase ProcM.
J. Am. Chem. Soc.
PUBLISHED: 07-08-2014
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Lanthipeptides are a class of post-translationally modified peptide natural products. They contain lanthionine (Lan) and methyllanthionine (MeLan) residues, which generate cross-links and endow the peptides with various biological activities. The mechanism of a highly substrate-tolerant lanthipeptide synthetase, ProcM, was investigated herein. We report a hybrid ligation strategy to prepare a series of substrate analogues designed to address a number of mechanistic questions regarding catalysis by ProcM. The method utilizes expressed protein ligation to generate a C-terminal thioester of the leader peptide of ProcA, the substrate of ProcM. This thioester was ligated with a cysteine derivative that resulted in an alkyne at the C-terminus of the leader peptide. This alkyne in turn was used to conjugate the leader peptides to a variety of synthetic peptides by copper-catalyzed azide-alkyne cycloaddition. Using deuterium-labeled Ser and Thr in the substrate analogues thus prepared, dehydration by ProcM was established to occur from C-to-N-terminus for two different substrates. Cyclization also occurred with a specific order, which depended on the sequence of the substrate peptides. Furthermore, using orthogonal cysteine side-chain protection in the two semisynthetic peptide substrates, we were able to rule out spontaneous non-enzymatic cyclization events to explain the very high substrate tolerance of ProcM. Finally, the enzyme was capable of exchanging protons at the ?-carbon of MeLan, suggesting that ring formation could be reversible. These findings are discussed in the context of the mechanism of the substrate-tolerant ProcM, which may aid future efforts in lanthipeptide engineering.
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Abdominal compartment syndrome in acute pancreatitis: a systematic review.
Pancreas
PUBLISHED: 06-13-2014
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Abdominal compartment syndrome (ACS) is a lethal complication of acute pancreatitis. We performed a systematic review to assess the treatment and outcome of these patients.A systematic literature search for cohorts of patients with acute pancreatitis and ACS was performed. The main outcomes were number of patients with ACS, radiologic and surgical interventions, morbidity, mortality, and methodological quality.After screening 169 articles, 7 studies were included. Three studies were prospective and 4 studies were retrospective. The overall methodological quality of the studies was moderate to low. The pooled data consisted of 271 patients, of whom 103 (38%) developed ACS. Percutaneous drainage of intraabdominal fluid was reported as first intervention in 11 (11%) patients. Additional decompressive laparotomy was performed in 8 patients. Decompressive laparotomy was performed in a total of 76 (74%) patients. The median decrease in intraabdominal pressure was 15 mm Hg (range, 33-18 mm Hg). Mortality in acute pancreatitis patients with ACS was 49% versus 11% without ACS. Morbidity ranged from 17% to 90%.Abdominal compartment syndrome during acute pancreatitis is associated with high mortality and morbidity. Studies are relatively small and have methodological shortcomings. The optimal timing and method of invasive interventions, as well as their effect on clinical outcomes, should be further evaluated.
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Treatment options for chronic pancreatitis.
Nat Rev Gastroenterol Hepatol
PUBLISHED: 06-10-2014
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This Review covers the latest developments in the treatment options for chronic pancreatitis. Pain is the most frequent and dominant symptom in patients with chronic pancreatitis, which ranges from severe disabling continuous pain to mild pain attacks and pain-free periods. Conventional treatment strategies and recent changes in the treatment of pain in patients with chronic pancreatitis are outlined. The different treatment options for pain consist of medical therapy, endoscopy or surgery. Their related merits and drawbacks are discussed. Finally, novel insights in the field of genetics and microbiota are summarized, and future perspectives are discussed.
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Substrate specificity of the lanthipeptide peptidase ElxP and the oxidoreductase ElxO.
ACS Chem. Biol.
PUBLISHED: 06-06-2014
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The final step in lanthipeptide biosynthesis involves the proteolytic removal of an N-terminal leader peptide. In the class I lanthipeptide epilancin 15X, this step is performed by the subtilisin-like serine peptidase ElxP. Bioinformatic, kinetic, and mass spectrometric analysis revealed that ElxP recognizes the stretch of amino acids DLNPQS located near the proteolytic cleavage site of its substrate, ElxA. When the ElxP recognition motif was inserted into the noncognate lanthipeptide precursor NisA, ElxP was able to proteolytically remove the leader peptide from NisA. Proteolytic removal of the leader peptide by ElxP during the biosynthesis of epilancin 15X exposes an N-terminal dehydroalanine on the core peptide of ElxA that hydrolyzes to a pyruvyl group. The short-chain dehydrogenase ElxO reduces the pyruvyl group to a lactyl moiety in the final step of epilancin 15X maturation. Using synthetic peptides, we also investigated the substrate specificity of ElxO and determined the 1.85 Å resolution X-ray crystal structure of the enzyme.
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Efficacy and safety of the C-Qur™ Film Adhesion Barrier for the prevention of surgical adhesions (CLIPEUS Trial): study protocol for a randomized controlled trial.
Trials
PUBLISHED: 06-05-2014
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Adhesions develop in over 90% of patients after intra-abdominal surgery. Adhesion barriers are rarely used despite the high morbidity caused by intra-abdominal adhesions. Only one of the currently available adhesion barriers has demonstrated consistent evidence for reducing adhesions in visceral surgery. This agent has limitations through poor handling characteristics because it is sticky on both sides. C-Qur™ Film is a novel thin film adhesion barrier and it is sticky on only one side, resulting in better handling characteristics. The objective of this study is to assess efficacy and safety of C-Qur™ Film to decrease the incidence of adhesions after colorectal surgery.
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Conjugation to albumin-binding molecule tags as a strategy to improve both efficacy and pharmacokinetic properties of the complement inhibitor compstatin.
ChemMedChem
PUBLISHED: 05-22-2014
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The compstatin family of complement inhibitors has shown promise in various immuno-inflammatory disorders. Although recent analogues show beneficial pharmacokinetics, further extension of the plasma half-life is expected to benefit systemic application of these peptidic inhibitors. We therefore synthesized conjugates of compstatin analogues and albumin-binding molecules (ABM) to increase circulatory residence. Equilibrium dialysis in complement-depleted serum showed a marked increase in plasma protein binding from <8?% to >99?% for a resulting chimera (ABM2-Cp20). Further analysis confirmed interaction with albumin from different species, primarily via site?II. Importantly, ABM2-Cp20 bound 20-fold stronger to its target protein C3b (KD =150?pM) than the parent peptide. Kinetic and in silico analysis suggested that ABM2 occupies a secondary site on C3b and improves the dissociation rate via additional contacts. Addition of an ABM modifier thereby not only improved plasma protein binding but also produced the most potent compstatin analogue to date with potential implications for the treatment of systemic complement-related diseases.
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Serosal abrasion of bowel ends does not enhance anastomotic healing.
J. Surg. Res.
PUBLISHED: 04-28-2014
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Anastomotic leakage rates remain unacceptably high, warranting reconsideration of current anastomotic technique. Anastomotic healing may improve by abrading the serosal surface of bowel ends that are invertedly anastomosed, based on the concept that serosal damage evokes inflammatory adherent processes. It is studied if local abrasion leads to stronger anastomoses and reduces leakage.
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The Effect of Renin Angiotensin System Genetic Variants in Acute Pancreatitis.
Ann. Surg.
PUBLISHED: 04-19-2014
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We sought association of genetic variants in the renin-angiotensin system (RAS) and vitamin D system with acute pancreatitis (AP) development and severity.
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Modulating the Copper-Sulfur Interaction in Type 1 Blue Copper Azurin by Replacing Cys112 with Nonproteinogenic Homocysteine.
Inorg Chem Front
PUBLISHED: 04-08-2014
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The Cu-SCys interaction is known to play a dominant role in defining the type 1 (T1) blue copper center with respect to both its electronic structure and electron transfer function. Despite this importance, its role has yet to be probed by mutagenesis studies without dramatic change of its T1 copper character. We herein report replacement of the conserved Cys112 in azurin with the nonproteinogenic amino acid homocysteine. Based on electronic absorption, electron paramagnetic resonance, and extended x-ray absorption fine structural spectroscopic studies, this variant displays typical type 1 copper site features. Surprisingly, instead of increasing the strength of the Cu-sulfur interaction by the introduction of the extra methylene group, the Cys112Hcy azurin showed a decrease in the covalent interaction between SHcy and Cu(II) when compared with the WT SCys-Cu(II) interaction. This is likely due to geometric adjustment of the center that resulted in the copper ion moving out of the trigonal plane defined by two histidines and one Hcy and closer to Met121. These structural changes resulted in an increase of reduction potential by 35 mV, consistent with lower Cu-S covalency. These results suggest that the Cu-SCys interaction is close to being optimal in native blue copper protein. It also demonstrates the power of using nonproteinogenic amino acids in addressing important issues in bioinorganic chemistry.
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Treatment options for acute pancreatitis.
Nat Rev Gastroenterol Hepatol
PUBLISHED: 03-25-2014
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This Review covers the latest developments in the treatment of acute pancreatitis. The Atlanta Classification of acute pancreatitis has been revised, proposing several new terms and abandoning some of the old and confusing terminology. The 2012 Revised Atlanta Classification and the determinant-based classification aim to universally define the different local and systemic complications and predict outcome. The most important differences between these classifications are discussed. Several promising treatment options for the early management of acute pancreatitis have been tested, including the use of enteral nutrition and antibiotics as well as novel therapies such as haemofiltration and protease inhibitors. The results are summarized and the quality of evidence is discussed. Finally, new developments in the management of patients with infected pancreatic necrosis are addressed, including the use of the 'step-up approach' and results of minimally invasive necrosectomy.
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Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB.
Nature
PUBLISHED: 03-14-2014
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Lantibiotics are a class of peptide antibiotics that contain one or more thioether bonds. The lantibiotic nisin is an antimicrobial peptide that is widely used as a food preservative to combat food-borne pathogens. Nisin contains dehydroalanine and dehydrobutyrine residues that are formed by the dehydration of Ser/Thr by the lantibiotic dehydratase NisB (ref. 2). Recent biochemical studies revealed that NisB glutamylates Ser/Thr side chains as part of the dehydration process. However, the molecular mechanism by which NisB uses glutamate to catalyse dehydration remains unresolved. Here we show that this process involves glutamyl-tRNA(Glu) to activate Ser/Thr residues. In addition, the 2.9-Å crystal structure of NisB in complex with its substrate peptide NisA reveals the presence of two separate domains that catalyse the Ser/Thr glutamylation and glutamate elimination steps. The co-crystal structure also provides insights into substrate recognition by lantibiotic dehydratases. Our findings demonstrate an unexpected role for aminoacyl-tRNA in the formation of dehydroamino acids in lantibiotics, and serve as a basis for the functional characterization of the many lantibiotic-like dehydratases involved in the biosynthesis of other classes of natural products.
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Preoperative characteristics of patients with presumed pancreatic cancer but ultimately benign disease: a multicenter series of 344 pancreatoduodenectomies.
Ann. Surg. Oncol.
PUBLISHED: 02-28-2014
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Preoperative differentiation between malignant and benign pancreatic tumors can be difficult. Consequently, a proportion of patients undergoing pancreatoduodenectomy for suspected malignancy will ultimately have benign disease. The aim of this study was to compare preoperative clinical and diagnostic characteristics of patients with unexpected benign disease after pancreatoduodenectomy with those of patients with confirmed (pre)malignant disease.
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MRI-based tumor motion characterization and gating schemes for radiation therapy of pancreatic cancer.
Radiother Oncol
PUBLISHED: 01-28-2014
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To characterize pancreatic tumor motion and to develop a gating scheme for radiotherapy in pancreatic cancer.
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Coaching during a trauma surgery team training: perceptions versus structured observations.
Am. J. Surg.
PUBLISHED: 01-22-2014
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Using the concept perception to quantify coaching skills during surgical training is questionable. This study compared the perceptions reported by the trainees and the faculty members following an emergency surgery team training with structured observations made on the basis of video registrations.
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NMR structure of the S-linked glycopeptide sublancin 168.
ACS Chem. Biol.
PUBLISHED: 01-17-2014
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Sublancin 168 is a member of a small group of glycosylated antimicrobial peptides known as glycocins. The solution structure of sublancin 168, a 37-amino-acid peptide produced by Bacillus subtilis 168, has been solved by nuclear magnetic resonance (NMR) spectroscopy. Sublancin comprises two ?-helices and a well-defined interhelical loop. The two helices span residues 6-16 and 26-35, and the loop region encompasses residues 17-25. The 9-amino-acid loop region contains a ?-S-linked glucose moiety attached to Cys22. Hydrophobic interactions as well as hydrogen bonding are responsible for the well-structured loop region. The three-dimensional structure provides an explanation for the previously reported extraordinary high stability of sublancin 168.
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Endoscopic transluminal necrosectomy in necrotising pancreatitis: a systematic review.
Surg Endosc
PUBLISHED: 01-08-2014
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We performed a systematic review to assess the outcome of endoscopic transluminal necrosectomy in necrotising pancreatitis with additional focus on indication, disease severity, and methodological quality of studies.
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Retention of first aid and basic life support skills in undergraduate medical students.
Med Educ Online
PUBLISHED: 01-01-2014
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Undergraduate medical students follow a compulsory first aid (FA) and basic life support (BLS) course. Retention of BLS seems poor and only little information is provided on the retention of FA skills. This study aims at evaluating 1- and 2-year retention of FA and BLS training in undergraduate medical students.
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Genetic analysis of intracapillary glomerular lipoprotein deposits in aging mice.
PLoS ONE
PUBLISHED: 01-01-2014
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Renal aging is characterized by functional and structural changes like decreased glomerular filtration rate, and glomerular, tubular and interstitial damage. To gain insight in pathways involved in renal aging, we studied aged mouse strains and used genetic analysis to identify genes associated with aging phenotypes.
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The role of routine preoperative EUS when performed after contrast enhanced CT in the diagnostic work-up in patients suspected of pancreatic or periampullary cancer.
Pancreatology
PUBLISHED: 01-01-2014
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In patients suspected of pancreatic or periampullary cancer, abdominal contrast-enhanced computed tomography (CT) is the standard diagnostic modality. A supplementary endoscopic ultrasonography (EUS) is often performed, although there is only limited evidence of its additional diagnostic value. The aim of the study is to evaluate the additional diagnostic value of EUS over CT in deciding on exploratory laparotomy in patients suspected of pancreatic or periampullary cancer.
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Chemical rescue and inhibition studies to determine the role of Arg301 in phosphite dehydrogenase.
PLoS ONE
PUBLISHED: 01-01-2014
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Phosphite dehydrogenase (PTDH) catalyzes the NAD(+)-dependent oxidation of phosphite to phosphate. This reaction requires the deprotonation of a water nucleophile for attack on phosphite. A crystal structure was recently solved that identified Arg301 as a potential base given its proximity and orientation to the substrates and a water molecule within the active site. Mutants of this residue showed its importance for efficient catalysis, with about a 100-fold loss in k cat and substantially increased K m,phosphite for the Ala mutant (R301A). The 2.35 Å resolution crystal structure of the R301A mutant with NAD(+) bound shows that removal of the guanidine group renders the active site solvent exposed, suggesting the possibility of chemical rescue of activity. We show that the catalytic activity of this mutant is restored to near wild-type levels by the addition of exogenous guanidinium analogues; Brønsted analysis of the rates of chemical rescue suggests that protonation of the rescue reagent is complete in the transition state of the rate-limiting step. Kinetic isotope effects on the reaction in the presence of rescue agents show that hydride transfer remains at least partially rate-limiting, and inhibition experiments show that K i of sulfite with R301A is ?400-fold increased compared to the parent enzyme, similar to the increase in K m for phosphite in this mutant. The results of our experiments indicate that Arg301 plays an important role in phosphite binding as well as catalysis, but that it is not likely to act as an active site base.
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The Glycosyltransferase Involved in Thurandacin Biosynthesis Catalyzes Both O- and S-Glycosylation.
J. Am. Chem. Soc.
PUBLISHED: 12-16-2013
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The S-glycosyltransferase SunS is a recently discovered enzyme that selectively catalyzes the conjugation of carbohydrates to the cysteine thiol of proteins. This study reports the discovery of a second S-glycosyltransferase, ThuS, and shows that ThuS catalyzes both S-glycosylation of the thiol of cysteine and O-glycosylation of the hydroxyl group of serine in peptide substrates. ThuS-catalyzed S-glycosylation is more efficient than O-glycosylation, and the enzyme demonstrates high tolerance with respect to both nucleotide sugars and peptide substrates. The biosynthesis of the putative products of the thuS gene cluster was reconstituted in vitro, and the resulting S-glycosylated peptides thurandacin A and B exhibit highly selective antimicrobial activity toward Bacillus thuringiensis.
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Hydrogen Sulfide Attenuates sFlt1-Induced Hypertension and Renal Damage by Upregulating Vascular Endothelial Growth Factor.
J. Am. Soc. Nephrol.
PUBLISHED: 12-12-2013
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Soluble fms-like tyrosine kinase 1 (sFlt1), a circulating antiangiogenic protein, is elevated in kidney diseases and contributes to the development of preeclampsia. Hydrogen sulfide is a vasorelaxant and proangiogenic gas with therapeutic potential in several diseases. Therefore, we evaluated the potential therapeutic effect and mechanisms of action of hydrogen sulfide in an animal model of sFlt1-induced hypertension, proteinuria, and glomerular endotheliosis created by adenovirus-mediated overexpression of sFlt1 in Sprague-Dawley rats. We injected sFlt1-overexpressing animals intraperitoneally with the hydrogen sulfide-donor sodium hydrosulfide (NaHS) (50 µmol/kg, twice daily) or vehicle (n=7 per group). Treatment with NaHS for 8 days significantly reduced sFlt1-induced hypertension, proteinuria, and glomerular endotheliosis. Measurement of plasma protein concentrations with ELISA revealed a reduction of free plasma sFlt1 and an increase of free plasma vascular endothelial growth factor (VEGF) after treatment with NaHS. Renal VEGF-A mRNA expression increased significantly with NaHS treatment. In vitro, NaHS was proangiogenic in an endothelial tube assay and attenuated the antiangiogenic effects of sFlt1. Stimulation of podocytes with NaHS resulted in both short-term VEGF release (120 minutes) and upregulation of VEGF-A mRNA levels (24 hours). Furthermore, pretreatment of mesenteric vessels with a VEGF receptor 2-neutralizing antibody significantly attenuated NaHS-induced vasodilation. These results suggest that hydrogen sulfide ameliorates sFlt1-induced hypertension, proteinuria, and glomerular endotheliosis in rats by increasing VEGF expression. Further studies are warranted to evaluate the role of hydrogen sulfide as a novel therapeutic agent for vascular disorders such as preeclampsia.
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Potential Mechanisms and Environmental Controls of TiO2 Nanoparticle Effects on Soil Bacterial Communities.
Environ. Sci. Technol.
PUBLISHED: 11-27-2013
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It has been reported that engineered nanoparticles (ENPs) alter soil bacterial communities, but the underlying mechanisms and environmental controls of such effects remain unknown. Besides direct toxicity, ENPs may indirectly affect soil bacteria by changing soil water availability or other properties. Alternatively, soil water or other environmental factors may mediate ENP effects on soil bacterial communities. To test, we incubated nano-TiO2-amended soils across a range of water potentials for 288 days. Following incubation, the soil water characteristics, organic matter, total carbon, total nitrogen, and respiration upon rewetting (an indicator of bioavailable organic carbon) were measured. Bacterial community shifts were characterized by terminal restriction fragment length polymorphism (T-RFLP). The endpoint soil water holding had been reported previously as not changing with this nano-TiO2 amendment; herein, we also found that some selected soil properties were unaffected by the treatments. However, we found that nano-TiO2 altered the bacterial community composition and reduced diversity. Nano-TiO2-induced community dissimilarities increased but tended to approach a plateau when soils became drier. Taken together, nano-TiO2 effects on soil bacteria appear to be a result of direct toxicity rather than indirectly through nano-TiO2 affecting soil water and organic matter pools. However, such directs effects of nano-TiO2 on soil bacterial communities are mediated by soil water.
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Transluminal endoscopic step-up approach versus minimally invasive surgical step-up approach in patients with infected necrotising pancreatitis (TENSION trial): design and rationale of a randomised controlled multicenter trial [ISRCTN09186711].
BMC Gastroenterol
PUBLISHED: 10-08-2013
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Infected necrotising pancreatitis is a potentially lethal disease that nearly always requires intervention. Traditionally, primary open necrosectomy has been the treatment of choice. In recent years, the surgical step-up approach, consisting of percutaneous catheter drainage followed, if necessary, by (minimally invasive) surgical necrosectomy has become the standard of care. A promising minimally invasive alternative is the endoscopic transluminal step-up approach. This approach consists of endoscopic transluminal drainage followed, if necessary, by endoscopic transluminal necrosectomy. We hypothesise that the less invasive endoscopic step-up approach is superior to the surgical step-up approach in terms of clinical and economic outcomes.
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Use of a Phosphonate Methyltransferase in the Identification of the Fosfazinomycin Biosynthetic Gene Cluster.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 09-24-2013
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Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for "stable isotope labeling of phosphonates in extract" (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S-adenosyl methionine. Mass-guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.
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Early oral feeding after pancreatoduodenectomy enhances recovery without increasing morbidity.
HPB (Oxford)
PUBLISHED: 08-20-2013
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The aim of this study was to evaluate whether a change in the routine feeding strategy applied after pancreatoduodenectomy (PD) from nasojejunal tube (NJT) feeding to early oral feeding improved clinical outcomes.
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Structure and Function of Phosphonoacetaldehyde Dehydrogenase: The Missing Link in Phosphonoacetate Formation.
Chem. Biol.
PUBLISHED: 08-05-2013
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Phosphonates (C-PO3(2-)) have applications as antibiotics, herbicides, and detergents. In some environments, these molecules represent the predominant source of phosphorus, and several microbes have evolved dedicated enzymatic machineries for phosphonate degradation. For example, most common naturally occurring phosphonates can be catabolized to either phosphonoacetaldehyde or phosphonoacetate, which can then be hydrolyzed to generate inorganic phosphate and acetaldehyde or acetate, respectively. The phosphonoacetaldehyde oxidase gene (phnY) links these two hydrolytic processes and provides a previously unknown catabolic mechanism for phosphonoacetate production in the microbial metabolome. Here, we present biochemical characterization of PhnY and high-resolution crystal structures of the apo state, as well as complexes with substrate, cofactor, and product. Kinetic analysis of active site mutants demonstrates how a highly conserved aldehyde dehydrogenase active site has been modified in nature to generate activity with a phosphonate substrate.
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Insights into the evolution of lanthipeptide biosynthesis.
Protein Sci.
PUBLISHED: 07-31-2013
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Lanthipeptides are a group of posttranslationally modified peptide natural products that contain multiple thioether crosslinks. These crosslinks are formed by dehydration of Ser/Thr residues followed by addition of the thiols of Cys residues to the resulting dehydroamino acids. At least four different pathways to these polycyclic natural products have evolved, reflecting the high efficiency and evolvability of a posttranslational modification route to generate conformationally constrained peptides. The wealth of genomic information that has been made available in recent years has started to provide insights into how these remarkable pathways and their posttranslational modification machineries may have evolved. In this review, we discuss a model for the evolution of the lanthipeptide biosynthetic enzymes that has recently been developed based on the currently available data.
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A general method for fluorescent labeling of the N-termini of lanthipeptides and its application to visualize their cellular localization.
J. Am. Chem. Soc.
PUBLISHED: 07-08-2013
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Labeling of natural products with biophysical probes has greatly contributed to investigations of their modes of action and has provided tools for visualization of their targets. A general challenge is the availability of a suitable functional group for chemoselective modification. We demonstrate here that an N-terminal ketone is readily introduced into various lanthipeptides by the generation of a cryptic N-terminal dehydro amino acid by the cognate biosynthetic enzymes. Spontaneous hydrolysis of the N-terminal enamines results in ?-ketoamides that site-specifically react with an aminooxy-derivatized alkyne or fluorophore. The methodology was successfully applied to prochlorosins 1.7 and 2.8, as well as the lantibiotics lacticin 481, haloduracin ?, and haloduracin ?. The fluorescently modified lantibiotics were added to bacteria, and their cellular localization was visualized by confocal fluorescence microscopy. Lacticin 481 and haloduracin ? localized predominantly at sites of new and old cell division as well as in punctate patterns along the long axis of rod-shaped bacilli, similar to the localization of lipid II. On the other hand, haloduracin ? was localized nonspecifically in the absence of haloduracin ?, but formed specific patterns when coadministered with haloduracin ?. Using two-color labeling, colocalization of both components of the two-component lantibiotic haloduracin was demonstrated. These data with living cells supports a model in which the ? component recognizes lipid II and then recruits the ?-component.
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Performance of forty-one microbial source tracking methods: a twenty-seven lab evaluation study.
Water Res.
PUBLISHED: 07-05-2013
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The last decade has seen development of numerous new microbial source tracking (MST) methodologies, but many of these have been tested in just a few laboratories with a limited number of fecal samples. This method evaluation study examined the specificity and sensitivity of 41 MST methodologies by analyzing data generated in 27 laboratories. MST methodologies that targeted human, cow, ruminant, dog, gull, pig, horse, and sheep were tested against sewage, septage, human, cow, dog, deer, pig, chicken, pigeon, gull, horse, and goose fecal samples. Each laboratory received 64 blind samples containing a single source (singletons) or two sources (doubletons), as well as diluted singleton samples to assess method sensitivity. Laboratories utilized their own protocols when performing the methods and data were deposited in a central database before samples were unblinded. Between one and seven laboratories tested each method. The most sensitive and specific assays, based on an analysis of presence/absence of each marker in target and non-target fecal samples, were HF183 endpoint and HF183SYBR (human), CF193 and Rum2Bac (ruminant), CowM2 and CowM3 (cow), BacCan (dog), Gull2SYBR and LeeSeaGull (gull), PF163 and pigmtDNA (pig), HoF597 (horse), PhyloChip (pig, horse, chicken, deer), Universal 16S TRFLP (deer), and Bacteroidales 16S TRFLP (pig, horse, chicken, deer); all had sensitivity and specificity higher than 80% in all or the majority of laboratories. When the abundance of MST markers in target and non-target fecal samples was examined, some assays that performed well in the binary analysis were found to not be sensitive enough as median concentrations fell below a minimum abundance criterion (set at 50 copies per colony forming units of enterococci) in target fecal samples. Similarly, some assays that cross-reacted with non-target fecal sources in the binary analysis were found to perform well in a quantitative analysis because the cross-reaction occurred at very low levels. Based on a quantitative analysis, the best performing methods were HF183Taqman and BacH (human), Rum2Bac and BacR (ruminant), LeeSeaGull (gull), and Pig2Bac (pig); no cow or dog-specific assay met the quantitative specificity and sensitivity criteria. Some of the best performing assays in the study were run by just one laboratory so further testing of assay portability is needed. While this study evaluated the marker performance in defined samples, further field testing as well as development of frameworks for fecal source allocation and risk assessment are needed.
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Early angiopoietin-2 levels after onset predict the advent of severe pancreatitis, multiple organ failure, and infectious complications in patients with acute pancreatitis.
J. Am. Coll. Surg.
PUBLISHED: 07-03-2013
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Acute pancreatitis is a severe condition that requires early identification of patients at risk of developing potentially lethal complications. Current clinical scoring systems and biochemical parameters are insufficient. In this study, we aimed to assess whether early plasma Angiopoietin-2 (Ang-2) is associated with adverse outcomes in patients with predicted severe acute pancreatitis (SAP).
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Early management of acute pancreatitis.
Best Pract Res Clin Gastroenterol
PUBLISHED: 06-27-2013
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Acute pancreatitis is the most common gastro-intestinal indication for acute hospitalization and its incidence continues to rise. In severe pancreatitis, morbidity and mortality remains high and is mainly driven by organ failure and infectious complications. Early management strategies should aim to prevent or treat organ failure and to reduce infectious complications. This review addresses the management of acute pancreatitis in the first hours to days after onset of symptoms, including fluid therapy, nutrition and endoscopic retrograde cholangiography. This review also discusses the recently revised Atlanta classification which provides new uniform terminology, thereby facilitating communication regarding severity and complications of pancreatitis.
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Lack of consensus on the role of endoscopic retrograde cholangiography in acute biliary pancreatitis in published meta-analyses and guidelines: a systematic review.
Pancreas
PUBLISHED: 06-19-2013
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Several randomized controlled trials studied the role of endoscopic retrograde cholangiopancreaticography (ERCP) and endoscopic sphincterotomy (ES) in acute biliary pancreatitis (ABP). No study assessed whether these trials resulted in international consensus in published meta-analyses and treatment guidelines.
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Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-17-2013
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Bioactive natural products containing a C-P bond act as mimics of phosphate esters and carboxylic acids, thereby competing with these compounds for active sites of enzymes. Dehydrophos (DHP), a broad-spectrum antibiotic, is a phosphonotripeptide produced by Streptomyces luridus, in which glycine and leucine are linked to an aminophosphonate analog of dehydroalanine, ?Ala(P). This unique feature, in combination with the monomethylation of the phosphonic acid, renders DHP a Trojan horse type antibiotic because peptidase-mediated hydrolysis will release methyl acetylphosphonate, a potent inhibitor of pyruvate dehydrogenase. Bioinformatic analysis of the biosynthetic gene cluster suggested that ?Ala(P) would be generated from Ser(P), the phosphonate analog of Ser, by phosphorylation and subsequent elimination, and that ?Ala(P) would be condensed with Leu-tRNA(Leu). DhpH was anticipated to carry out this elimination/ligation cascade. DhpH is a multidomain protein, in which a pyridoxal phosphate binding domain is fused to an N-acetyltransferase domain related to the general control nonderepressible-5 (GCN5) family. In this work, the activity of DhpH was reconstituted in vitro. The enzyme was able to catalyze the ?-elimination reaction of pSer(P) to generate ?Ala(P), but it was unable to condense ?Ala(P) with Leu. Instead, ?Ala(P) is hydrolyzed to acetyl phosphonate, which is converted to Ala(P) by a second pyridoxal phosphate-dependent enzyme, DhpD. Ala(P) is the substrate for the condensation with Leu-tRNA(Leu) catalyzed by the C-terminal domain of DhpH. DhpJ, a 2-oxoglutarate/Fe(II)-dependent enzyme, introduces the vinyl functionality into Leu-Ala(P) acting as a desaturase, and addition of Gly by DhpK in a Gly-tRNA(Gly)-dependent manner completes the in vitro biosynthesis of dehydrophos.
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The role of routine fine-needle aspiration in the diagnosis of infected necrotizing pancreatitis.
Surgery
PUBLISHED: 05-23-2013
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Diagnosing infected necrotizing pancreatitis (INP) may be challenging. The aim of this study was to determine the added value of routine fine-needle aspiration (FNA) in addition to clinical and imaging signs of infection in patients who underwent intervention for suspected INP.
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Preoperative opioid use and the outcome of thoracoscopic splanchnicectomy in chronic pancreatitis: a systematic review.
Surg Endosc
PUBLISHED: 05-15-2013
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Thoracoscopic splanchnicectomy (TS) is a minimally invasive intervention to relieve pain in patients with chronic pancreatitis (CP) with equivocal results. Preoperative opioid use seems to impair TS outcome but this has not been investigated in a systematic matter.
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Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.
Water Res.
PUBLISHED: 05-07-2013
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A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study.
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Chemical synthesis of the lantibiotic lacticin 481 reveals the importance of lanthionine stereochemistry.
J. Am. Chem. Soc.
PUBLISHED: 05-03-2013
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Lantibiotics are a family of antibacterial peptide natural products characterized by the post-translational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single naturally occurring stereochemical configuration for each of these cross-links was known. The discovery of lantibiotics with alternative lanthionine and methyllanthionine stereochemistry has prompted an investigation of its importance to biological activity. Here, solid-supported chemical synthesis enabled the total synthesis of the lantibiotic lacticin 481 and analogues containing cross-links with non-native stereochemical configurations. Biological evaluation revealed that these alterations abolished the antibacterial activity in all of the analogues, revealing the critical importance of the enzymatically installed stereochemistry for the biological activity of lacticin 481.
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In vitro activity of the nisin dehydratase NisB.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-15-2013
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The biosynthesis of several classes of ribosomally synthesized and posttranslationally modified peptides involves dehydration of serine and threonine residues. For class I lantibiotics, thiopeptides, and goadsporin, this dehydration is catalyzed by lanthionine biosynthetic enzyme B (LanB) or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. We show here the in vitro activity of the dehydratase involved in the biosynthesis of the food preservative nisin (NisB). In vitro, NisB dehydrated its substrate peptide NisA eight times in the presence of glutamate, ATP, Mg(2+), and the ribosomal/membrane fraction of bacterial cell extract. Mutation of 23 highly conserved residues of NisB identified a number of amino acids that are essential for dehydration activity. In addition, these mutagenesis studies identified three mutants, R786A, R826A, and H961A, that result in multiple glutamylations of the NisA substrate. Glutamylation was observed during both Escherichia coli coexpression of NisA with these mutants and in vitro assays. Treatment of the glutamylated substrate with WT NisB results in dehydrated NisA, suggesting that the glutamylated peptide is an intermediate in dehydration. Collectively, these studies suggest that dehydration involves glutamylation of the side chains of Ser and Thr followed by elimination. The latter step has precedent in the virginiamycin resistance protein virginiamycin B lyase. These studies will facilitate investigation of other LanB proteins involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin.
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Discovery of the antibiotic phosacetamycin via a new mass spectrometry-based method for phosphonic acid detection.
ACS Chem. Biol.
PUBLISHED: 03-13-2013
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Naturally occurring phosphonates such as phosphinothricin (Glufosinate, a commercially used herbicide) and fosfomycin (Monurol, a clinically used antibiotic) have proved to be potent and useful biocides. Yet this class of natural products is still an under explored family of secondary metabolites. Discovery of the biosynthetic pathways responsible for the production of these compounds has been simplified by using gene based screening approaches, but detection and identification of the natural products the genes produce have been hampered by a lack of high-throughput methods for screening potential producers under various culture conditions. Here, we present an efficient mass-spectrometric method for the selective detection of natural products containing phosphonate and phosphinate functional groups. We have used this method to identify a new phosphonate metabolite, phosacetamycin, whose structure, biological activity, and biosynthetic gene cluster are reported.
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[Imaging techniques in acute cholecystitis].
Ned Tijdschr Geneeskd
PUBLISHED: 03-08-2013
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A recent meta-analysis of 57 studies investigated the diagnostic accuracy of imaging techniques in acute cholecystitis. The main findings were that abdominal ultrasound has a sensitivity of 81% and a specificity of 83%, that ultrasound seems comparable to magnetic resonance imaging (MRI), that cholescintigraphy has a sensitivity of 96% and a specificity of 90%, and that there are insufficient studies to evaluate the role of computed tomography (CT). In daily practice, ultrasound is the preferred modality as it is safe, widely available and cheap. MRI and cholescintigraphy are less available and have additional disadvantages. Patients with a negative or inconclusive ultrasound examination should undergo CT as this is in line with the general diagnostic strategy for acute abdominal pain that results in the highest sensitivity for urgent conditions and lowest exposure to radiation. Patients with clinical suspicion of acute cholecystitis but negative imaging findings should undergo prompt diagnostic laparoscopy and potentially cholecystectomy.
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Bacteroidales terminal restriction fragment length polymorphism (TRFLP) for fecal source differentiation in comparison to and in combination with universal bacteria TRFLP.
Water Res.
PUBLISHED: 03-04-2013
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Terminal restriction fragment length polymorphism (TRFLP) is an attractive community analysis method for microbial source tracking (MST) because it is accessible, relatively inexpensive, and can discern multiple fecal sources simultaneously. A new Bacteroidales TRFLP (Bac-TRFLP) method was developed and its source identification performance was evaluated by itself, in comparison to, and in combination with an existing universal bacterial TRFLP method in two laboratories. Sixty-four blind samples from 12 fecal sources (sewage, septage, human, dog, horse, cow, deer, pig, chicken, goose, pigeon, and gull) were used for evaluation. Bac- and Univ-TRFLP exhibited similarly high overall correct identification (>88% and >89%, respectively), excellent specificity regardless of fecal sources, variable sensitivity depending on the source, and stable performance across two laboratories. Compared to Univ-TRFLP, Bac-TRFLP had better sensitivity and specificity with horse, cow, and pig fecal sources but was not suited for certain avian sources such as goose, gull, and pigeon. Combining the general and more targeted TRFLP methods (Univ&Bac-TRFLP) achieved higher overall correct identification (>92%), higher sensitivity and specificity metrics, and higher reproducibility between laboratories. Our results suggest that the Bac-TRFLP and Univ&Bac-TRFLP methods are promising additions to the MST toolbox and warrant further evaluation and utilization in field MST applications.
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Phosphonate biosynthesis and catabolism: a treasure trove of unusual enzymology.
Curr Opin Chem Biol
PUBLISHED: 02-27-2013
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Natural product biosynthesis has proven a fertile ground for the discovery of novel chemistry. Herein we review the progress made in elucidating the biosynthetic pathways of phosphonate and phosphinate natural products such as the antibacterial compounds dehydrophos and fosfomycin, the herbicidal phosphinothricin-containing peptides, and the antimalarial compound FR-900098. In each case, investigation of the pathway has yielded unusual, and often unprecedented, biochemistry. Likewise, recent investigations have uncovered novel ways to cleave the CP bond to yield phosphate under phosphorus starvation conditions. These include the discovery of novel oxidative cleavage of the CP bond catalyzed by PhnY and PhnZ as well as phosphonohydrolases that liberate phosphate from phosphonoacetate. Perhaps the crown jewel of phosphonate catabolism has been the recent resolution of the longstanding problem of the C-P lyase responsible for reductively cleaving the CP bond of a number of different phosphonates to release phosphate. Taken together, the strides made on both metabolic and catabolic fronts illustrate an array of fascinating biochemistry.
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Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.
Nat Prod Rep
PUBLISHED: 02-09-2013
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This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
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Radiofrequency ablation for unresectable locally advanced pancreatic cancer: a systematic review.
HPB (Oxford)
PUBLISHED: 02-07-2013
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BACKGROUND: Median survival in patients with unresectable locally advanced pancreatic cancer lies in the range of 9-15?months. Radiofrequency ablation (RFA) may prolong survival, but data on its safety and efficacy are scarce. METHODS: A systematic literature search was performed in PubMed, EMBASE and the Cochrane Library with the syntax (radiofrequency OR RFA) AND (pancreas OR pancreatic) for studies published until 1 January 2012. In addition, a search of the proceedings of conferences on pancreatic disease that took place during 2009-2011 was performed. Studies with fewer than five patients were excluded as they were considered to be case reports. The primary endpoint was survival. Secondary endpoints included morbidity and mortality. RESULTS: Five studies involving a total of 158 patients with pancreatic cancer treated with RFA fulfilled the eligibility criteria. These studies reported median survival after RFA of 3-33?months, morbidity related to RFA of 4-37%, mortality of 0-19% and overall morbidity of 10-43%. Pooling of data was not appropriate as the study populations and reported outcomes were heterogeneous. Crucial safety aspects included ensuring a maximum RFA tip temperature of
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Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources.
Water Res.
PUBLISHED: 02-04-2013
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Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR(®) Green qPCR method, and two TaqMan(®) qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.
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Association of Sex with Long-Term Outcomes after Popliteal Artery Aneurysm Repair.
Ann Vasc Surg
PUBLISHED: 02-03-2013
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We compared initial and long-term outcomes between men and women after endovascular and open repair of popliteal artery aneurysms (PAAs).
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Evaluation of molecular community analysis methods for discerning fecal sources and human waste.
Water Res.
PUBLISHED: 02-02-2013
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Molecular microbial community analyses provide information on thousands of microorganisms simultaneously, and integrate biotic and abiotic perturbations caused by fecal contamination entering water bodies. A few studies have explored community methods as emerging approaches for microbial source tracking (MST), however, an evaluation of the current state of this approach is lacking. Here, we utilized three types of community-based methods with 64 blind, single- or dual-source, challenge samples generated from 12 sources, including: humans (feces), sewage, septage, dogs, pigs, deer, horses, cows, chickens, gulls, pigeons, and geese. Each source was a composite from multiple donors from four representative geographical regions in California. Methods evaluated included terminal restriction fragment polymorphism (TRFLP), phylogenetic microarray (PhyloChip), and next generation (Illumina) sequencing. These methods correctly identified dominant (or sole) sources in over 90% of the challenge samples, and exhibited excellent specificity regardless of source, rarely detecting a source that was not present in the challenge sample. Sensitivity, however, varied with source and community analysis method. All three methods distinguished septage from human feces and sewage, and identified deer and horse with 100% sensitivity and 100% specificity. Method performance improved if the composition of blind dual-source reference samples were defined by DNA contribution of each single source within the mixture, instead of by Enterococcus colony forming units. Data analysis approach also influenced method performance, indicating the need to standardize data interpretation. Overall, results of this study indicate that community analysis methods hold great promise as they may be used to identify any source, and they are particularly useful for sources that currently do not have, and may never have, a source-specific single marker gene.
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Ribosomally synthesized and post-translationally modified peptide natural products: new insights into the role of leader and core peptides during biosynthesis.
Chemistry
PUBLISHED: 01-31-2013
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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with a high degree of structural diversity and a wide variety of bioactivities. Understanding the biosynthetic machinery of these RiPPs will benefit the discovery and development of new molecules with potential pharmaceutical applications. In this Concept article, we discuss the features of the biosynthetic pathways to different RiPP classes, and propose mechanisms regarding recognition of the precursor peptide by the post-translational modification enzymes. We propose that the leader peptides function as allosteric regulators that bind the active form of the biosynthetic enzymes in a conformational selection process. We also speculate how enzymes that generate polycyclic products of defined topologies may have been selected for during evolution.
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Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.
Water Res.
PUBLISHED: 01-17-2013
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Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.
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The sequence of the enterococcal cytolysin imparts unusual lanthionine stereochemistry.
Nat. Chem. Biol.
PUBLISHED: 01-13-2013
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The enterococcal cytolysin is a two-component lantibiotic of unknown structure with hemolytic activity that is important for virulence. We prepared cytolysin by coexpression of each precursor peptide with the synthetase CylM in Escherichia coli and characterized its structure. Unexpectedly, cytolysin is to our knowledge the first example of a lantibiotic containing lanthionine and methyllanthionine structures with different stereochemistries in the same peptide. The stereochemistry is determined by the sequence of the substrate peptide.
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Association Analysis of Genetic Variants in the Myosin IXB Gene in Acute Pancreatitis.
PLoS ONE
PUBLISHED: 01-01-2013
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Impairment of the mucosal barrier plays an important role in the pathophysiology of acute pancreatitis. The myosin IXB (MYO9B) gene and the two tight-junction adaptor genes, PARD3 and MAGI2, have been linked to gastrointestinal permeability. Common variants of these genes are associated with celiac disease and inflammatory bowel disease, two other conditions in which intestinal permeability plays a role. We investigated genetic variation in MYO9B, PARD3 and MAGI2 for association with acute pancreatitis.
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Altered resting state EEG in chronic pancreatitis patients: toward a marker for chronic pain.
J Pain Res
PUBLISHED: 01-01-2013
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Electroencephalography (EEG) may be a promising source of physiological biomarkers accompanying chronic pain. Several studies in patients with chronic neuropathic pain have reported alterations in central pain processing, manifested as slowed EEG rhythmicity and increased EEG power in the brains resting state. We aimed to investigate novel potential markers of chronic pain in the resting state EEG of patients with chronic pancreatitis.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.