Exploiting Mitochondria In Vivo as Chemical Reaction Chambers Dependent on Membrane Potential.

Recently in Cell Metabolism, Logan et al. (2016) exploit membrane potential-dependent mitochondrial accumulation of charged precursors, causing them to combine by "click" chemistry 1,000,000 times faster than without accumulation to generate an ultrasensitive indicator for membrane potentials and foreshadow targeted drug synthesis in vivo.

Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions.

Superoxide/hydrogen peroxide production by site IQ in complex I of the electron transport chain is conventionally assayed during reverse electron transport (RET) from ubiquinol to NAD. However, S1QELs (specific suppressors of superoxide/hydrogen peroxide production by site IQ) have potent effects in cells and in vivo during presumed forward electron transport (FET). Therefore, we tested whether site IQ generates S1QEL-sensitive superoxide/hydrogen peroxide during FET (site IQf), or alternatively, whether RET and associated S1QEL-sensitive superoxide/hydrogen peroxide production (site IQr) occurs in cells under normal conditions. We introduce an assay to determine if electron flow through complex I is thermodynamically forward or reverse: on blocking electron flow through complex I, the endogenous matrix NAD pool will become more reduced if flow before the challenge was forward, but more oxidised if flow was reverse. Using this assay we show in the model system of isolated rat skeletal muscle mitochondria that superoxide/hydrogen peroxide production by site IQ can be equally great whether RET or FET is running. We show that sites IQr and IQf are equally sensitive to S1QELs, and to rotenone and piericidin A, inhibitors that block the Q-site of complex I. We exclude the possibility that some sub-fraction of the mitochondrial population running site IQr during FET is responsible for S1QEL-sensitive superoxide/hydrogen peroxide production by site IQ. Finally, we show that superoxide/hydrogen peroxide production by site IQ in cells occurs during FET, and is S1QEL-sensitive.

Position statement on the definition, incidence, diagnosis and outcome of acute on chronic pancreatitis.

BACKGROUND: Acute on chronic pancreatitis (ACP) is a relatively common condition, but there are significant gaps in our knowledge on the definition, incidence, diagnosis, treatment and prognosis. METHODS: A systematic review that followed PICO (Population; Intervention; Comparator; Outcome) recommendation for quantitative questions and PICo (Population, Phenomenon of Interest, Context) for qualitative research was done to answer 10 of the most relevant questions about ACP. Quality of evidence was judged by the GRADE criteria (Grades of Recommendation, Assessment, Development and Evaluation). The manuscript was sent for review to 12 international experts from various disciplines and continents using a Delphi process. RESULTS: The quality of evidence, for most statements, was low to very low, which means that the recommendations in general are only conditional. Despite that, it was possible to reach strong levels of agreement by the expert panel for all 10 questions. A new consensus definition of ACP was reached. Although common, the real incidence of ACP is not known, with alcohol as a major risk factor. Although pain dominates, other non-specific symptoms and signs can be present. Serum levels of pancreatic enzymes may be less than 3 times the upper limit of normal and cross-sectional imaging is considered more accurate for the diagnosis in many cases. It appears that it is less severe and with a lower mortality risk than acute pancreatitis. CONCLUSIONS: Although the evidence base is poor, this position statement provides a foundation from which to advance management of ACP.

Do Antiseptic Coated Sutures Reduce Colo-Colonic Anastomotic Leaks?

BACKGROUND: Colorectal anastomotic leaks remain one of the most significant complications following colorectal surgery. Various interventions to reduce anastomotic leaks have been investigated, however few have resulted in a significant improvement. To date antiseptic coated monofilament sutures for sutured bowel anastomoses have not been assessed, hence this study was undertaken to investigate whether or not triclosan impregnated polydioxanone suture material (PDS) results in fewer anastomotic leaks. METHODS: A rabbit colo-colonic anastomotic model was developed to compare the tensile strength and local inflammatory response between triclosan coated PDS and uncoated PDS. RESULTS: Of the 42 anastomoses there were 4 (9.5%) leaks. Of the remaining 38 anastomoses neither the leak pressures, degree of bowel wall inflammation or fibrosis were statistically different (P = .11; .813 and .658 respectively) when comparing the two suture materials. CONCLUSIONS: In an animal model, triclosan coated PDS is as safe as uncoated PDS in performing colo-colonic anastomosis.

Riding the tiger - physiological and pathological effects of superoxide and hydrogen peroxide generated in the mitochondrial matrix.

Elevated mitochondrial matrix superoxide and/or hydrogen peroxide concentrations drive a wide range of physiological responses and pathologies. Concentrations of superoxide and hydrogen peroxide in the mitochondrial matrix are set mainly by rates of production, the activities of superoxide dismutase-2 (SOD2) and peroxiredoxin-3 (PRDX3), and by diffusion of hydrogen peroxide to the cytosol. These considerations can be used to generate criteria for assessing whether changes in matrix superoxide or hydrogen peroxide are both necessary and sufficient to drive redox signaling and pathology: is a phenotype affected by suppressing superoxide and hydrogen peroxide production; by manipulating the levels of SOD2, PRDX3 or mitochondria-targeted catalase; and by adding mitochondria-targeted SOD/catalase mimetics or mitochondria-targeted antioxidants? Is the pathology associated with variants in SOD2 and PRDX3 genes? Filtering the large literature on mitochondrial redox signaling using these criteria highlights considerable evidence that mitochondrial superoxide and hydrogen peroxide drive physiological responses involved in cellular stress management, including apoptosis, autophagy, propagation of endoplasmic reticulum stress, cellular senescence, HIF1α signaling, and immune responses. They also affect cell proliferation, migration, differentiation, and the cell cycle. Filtering the huge literature on pathologies highlights strong experimental evidence that 30-40 pathologies may be driven by mitochondrial matrix superoxide or hydrogen peroxide. These can be grouped into overlapping and interacting categories: metabolic, cardiovascular, inflammatory, and neurological diseases; cancer; ischemia/reperfusion injury; aging and its diseases; external insults, and genetic diseases. Understanding the involvement of mitochondrial matrix superoxide and hydrogen peroxide concentrations in these diseases can facilitate the rational development of appropriate therapies.

Controlled power: how biology manages succinate-driven energy release.

Oxidation of succinate by mitochondria can generate a higher protonmotive force (pmf) than can oxidation of NADH-linked substrates. Fundamentally, this is because of differences in redox potentials and gearing. Biology adds kinetic constraints that tune the oxidation of NADH and succinate to ensure that the resulting mitochondrial pmf is suitable for meeting cellular needs without triggering pathology. Tuning within an optimal range is used, for example, to shift ATP consumption between different consumers. Conditions that overcome these constraints and allow succinate oxidation to drive pmf too high can cause pathological generation of reactive oxygen species. We discuss the thermodynamic properties that allow succinate oxidation to drive pmf higher than NADH oxidation, and discuss the evidence for kinetic tuning of ATP production and for pathologies resulting from substantial succinate oxidation in vivo.

Use of S1QELs and S3QELs to link mitochondrial sites of superoxide and hydrogen peroxide generation to physiological and pathological outcomes.

Changes in mitochondrial superoxide and hydrogen peroxide production may contribute to various pathologies, and even aging, given that over time and in certain conditions, they damage macromolecules and disrupt normal redox signalling. Mitochondria-targeted antioxidants such as mitoQ, mitoVitE, and mitoTEMPO have opened up the study of the importance of altered mitochondrial matrix superoxide/hydrogen peroxide in disease. However, the use of such tools has caveats and they are unable to distinguish precise sites of production within the reactions of substrate oxidation and the electron transport chain. S1QELs are specific small-molecule Suppressors of site IQElectron Leak and S3QELs are specific small-molecule Suppressors of site IIIQoElectron Leak; they prevent superoxide/hydrogen production at specific sites without affecting electron transport or oxidative phosphorylation. We discuss the benefits of using S1QELs and S3QELs as opposed to mitochondria-targeted antioxidants, mitochondrial poisons, and genetic manipulation. We summarise pathologies in which site IQ in mitochondrial complex I and site IIIQo in mitochondrial complex III have been implicated using S1QELs and S3QELs.

Prophylactic antibiotics for penetrating abdominal trauma.

BACKGROUND: Penetrating abdominal trauma occurs when the peritoneal cavity is breached. Routine laparotomy for penetrating abdominal injuries began in the 1800s, with antibiotics first being used in World War II to combat septic complications associated with these injuries. This practice was marked with a reduction in sepsis-related mortality and morbidity. Whether prophylactic antibiotics are required in the prevention of infective complications following penetrating abdominal trauma is controversial, however, as no randomised placebo controlled trials have been published to date. There has also been debate about the timing of antibiotic prophylaxis. In 1972 Fullen noted a 7% to 11% post-surgical infection rate with pre-operative antibiotics, a 33% to 57% infection rate with intra-operative antibiotic administration and 30% to 70% infection rate with only post-operative antibiotic administration. Current guidelines state there is sufficient class I evidence to support the use of a single pre-operative broad spectrum antibiotic dose, with aerobic and anaerobic cover, and continuation (up to 24 hours) only in the event of a hollow viscus perforation found at exploratory laparotomy. OBJECTIVES: To assess the benefits and harms of prophylactic antibiotics administered for penetrating abdominal injuries for the reduction of the incidence of septic complications, such as septicaemia, intra-abdominal abscesses and wound infections. SEARCH METHODS: Searches were not restricted by date, language or publication status. We searched the following electronic databases: the Cochrane Injuries Group Specialised Register, CENTRAL (The Cochrane Library 2019, issue 7 of 12), MEDLINE (OvidSP), Embase (OvidSP), ISI Web of Science: Science Citation Index Expanded (SCI-EXPANDED), ISI Web of Science: Conference Proceedings Citation Index- Science (CPCI-S) and PubMed. Searches were last conducted on 23 July 2019. SELECTION CRITERIA: All randomised controlled trials of antibiotic prophylaxis in patients with penetrating abdominal trauma versus no antibiotics or placebo. DATA COLLECTION AND ANALYSIS: Two authors screened the literature search results independently. MAIN RESULTS: We identified no trials meeting the inclusion criteria. AUTHORS' CONCLUSIONS: There is currently no information from randomised controlled trials to support or refute the use of antibiotics for patients with penetrating abdominal trauma.

Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions.

Mitochondrial production of superoxide and hydrogen peroxide is potentially important in cell signaling and disease. Eleven distinct mitochondrial sites that differ markedly in capacity are known to leak electrons to oxygen to produce O2̇̄ and/or H2O2 We discuss their contributions to O2̇̄/H2O2 production under native conditions in mitochondria oxidizing different substrates and in conditions mimicking physical exercise and the changes in their capacities after caloric restriction. We review the use of S1QELs and S3QELs, suppressors of mitochondrial O2̇̄/H2O2 generation that do not inhibit oxidative phosphorylation, as tools to characterize the contributions of specific sites in situ and in vivo.

Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements.

Partitioning of ATP generation between glycolysis and oxidative phosphorylation is central to cellular bioenergetics but cumbersome to measure. We describe here how rates of ATP generation by each pathway can be calculated from simultaneous measurements of extracellular acidification and oxygen consumption. We update theoretical maximum ATP yields by mitochondria and cells catabolizing different substrates. Mitochondrial P/O ratios (mol of ATP generated per mol of [O] consumed) are 2.73 for oxidation of pyruvate plus malate and 1.64 for oxidation of succinate. Complete oxidation of glucose by cells yields up to 33.45 ATP/glucose with a maximum P/O of 2.79. We introduce novel indices to quantify bioenergetic phenotypes. The glycolytic index reports the proportion of ATP production from glycolysis and identifies cells as primarily glycolytic (glycolytic index > 50%) or primarily oxidative. The Warburg effect is a chronic increase in glycolytic index, quantified by the Warburg index. Additional indices quantify the acute flexibility of ATP supply. The Crabtree index and Pasteur index quantify the responses of oxidative and glycolytic ATP production to alterations in glycolysis and oxidative reactions, respectively; the supply flexibility index quantifies overall flexibility of ATP supply; and the bioenergetic capacity quantifies the maximum rate of total ATP production. We illustrate the determination of these indices using C2C12 myoblasts. Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how extracellular fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics. We provide a simple spreadsheet to calculate glycolytic and oxidative ATP production rates from raw extracellular acidification and respiration data.

Generation of superoxide and hydrogen peroxide by side reactions of mitochondrial 2-oxoacid dehydrogenase complexes in isolation and in cells.

Mitochondrial 2-oxoacid dehydrogenase complexes oxidize 2-oxoglutarate, pyruvate, branched-chain 2-oxoacids and 2-oxoadipate to the corresponding acyl-CoAs and reduce NAD+ to NADH. The isolated enzyme complexes generate superoxide anion radical or hydrogen peroxide in defined reactions by leaking electrons to oxygen. Studies using isolated mitochondria in media mimicking cytosol suggest that the 2-oxoacid dehydrogenase complexes contribute little to the production of superoxide or hydrogen peroxide relative to other mitochondrial sites at physiological steady states. However, the contributions may increase under pathological conditions, in accordance with the high maximum capacities of superoxide or hydrogen peroxide-generating reactions of the complexes, established in isolated mitochondria. We assess available data on the use of modulations of enzyme activity to infer superoxide or hydrogen peroxide production from particular 2-oxoacid dehydrogenase complexes in cells, and limitations of such methods to discriminate specific superoxide or hydrogen peroxide sources in vivo.

The immune imbalance in the second hit of pancreatitis is independent of IL-17A.

BACKGROUND: Severe acute pancreatitis (SAP) is characterised by two distinct clinical phases. Organ dysfunction and death is initially as a result of a systemic inflammatory response syndrome (SIRS). Systemic sepsis from infected pancreatic necrosis characterises the second phase, the so called 'second hit' of acute pancreatitis (AP). An immune imbalance during the second hit is postulated to contribute to the formation of the septic complications that occur in these patients. The pro-inflammatory T-helper (Th) 17 pathway has been shown to be an initiator of early SIRS in AP, however to date its role has not been established in the second hit in AP. METHODS: Thirty-six patients with mild (n = 16), moderate (n = 10) and severe (n = 10) acute pancreatitis were enrolled. Peripheral blood samples were drawn on days 7, 9, 11 and 13 of illness for analysis of routine clinical markers as well as cytokine analysis. Flow cytometry and a IL-17A ELISA was performed to determine cytokine concentrations. RESULTS: There were no significant differences between days 7, 9, 11 and 13 for either the mild/moderate or SAP groups for IL-17A (CBA assay or ELISA), IFN-γ, TNF-α, IL-2 or IL-4. For each of the study days, the mean IL-6 and IL-10 concentrations were significantly higher in the SAP group compared to the mild/moderate group. WCC, CRP and PCT were all significantly higher in severe acute pancreatitis over the study days. CONCLUSIONS: An immune imbalance exists in patients with SAP, however secreted IL-17A is not responsible for the second hit in AP.

Surgical portosystemic shunts versus transjugular intrahepatic portosystemic shunt for variceal haemorrhage in people with cirrhosis.

BACKGROUND: Variceal haemorrhage that is refractory or recurs after pharmacologic and endoscopic therapy requires a portal decompression shunt (either surgical shunts or radiologic shunt, transjugular intrahepatic portosystemic shunt (TIPS)). TIPS has become the shunt of choice; however, is it the preferred option? This review assesses evidence for the comparisons of surgical portosystemic shunts versus TIPS for variceal haemorrhage in people with cirrhotic portal hypertension. OBJECTIVES: To assess the benefits and harms of surgical portosystemic shunts versus transjugular intrahepatic portosystemic shunt (TIPS) for treatment of refractory or recurrent variceal haemorrhage in people with cirrhotic portal hypertension. SEARCH METHODS: We searched the Cochrane Hepato-Biliary Group Controlled Trials Register, CENTRAL, MEDLINE, Embase, LILACS, Science Citation Index Expanded, and Conference Proceedings Citation Index - Science. We also searched on-line trial registries, reference lists of relevant articles, and proceedings of relevant associations for trials that met the inclusion criteria for this review (date of search 8 March 2018). SELECTION CRITERIA: Randomised clinical trials comparing surgical portosystemic shunts versus TIPS for the treatment of refractory or recurrent variceal haemorrhage in people with cirrhotic portal hypertension. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trials and extracted data using methodological standards expected by Cochrane. We assessed risk of bias according to domains and risk of random errors with Trial Sequential Analysis (TSA). We assessed the certainty of the evidence using the GRADE approach. MAIN RESULTS: We found four randomised clinical trials including 496 adult participants diagnosed with variceal haemorrhage due to cirrhotic portal hypertension. The overall risk of bias in all the trials was judged at high risk. All the trials were conducted in the United States of America (USA). Two of the trials randomised participants to selective surgical shunts versus TIPS. The other two trials randomised participants to non-selective surgical shunts versus TIPS. The diagnosis of liver cirrhosis was by clinical and laboratory findings. We are uncertain whether there is a difference in all-cause mortality at 30 days between surgical portosystemic shunts compared with TIPS (risk ratio (RR) 0.94, 95% confidence interval (CI) 0.44 to 1.99; participants = 496; studies = 4). We are uncertain whether there is a difference in encephalopathy between surgical shunts compared with TIPS (RR 0.56, 95% CI 0.27 to 1.16; participants = 496; studies = 4). We found evidence suggesting an increase in the occurrence of the following harms in the TIPS group compared with surgical shunts: all-cause mortality at five years (RR 0.61, 95% CI 0.42 to 0.90; participants = 496; studies = 4); variceal rebleeding (RR 0.18, 95% CI 0.07 to 0.49; participants = 496; studies = 4); reinterventions (RR 0.13, 95% CI 0.06 to 0.28; participants = 496; studies = 4); and shunt occlusion (RR 0.14, 95% CI 0.04 to 0.51; participants = 496; studies = 4). We could not perform an analysis of health-related quality of life but available evidence appear to suggest improved health-related quality of life in people who received surgical shunt compared with TIPS. We downgraded the certainty of the evidence for all-cause mortality at 30 days and five years, irreversible shunt occlusion, and encephalopathy to very low because of high risk of bias (due to lack of blinding); inconsistency (due to heterogeneity); imprecision (due to small sample sizes of the individual trials and few events); and publication bias (few trials reporting outcomes). We downgraded the certainty of the evidence for variceal rebleeding and reintervention to very low because of high risk of bias (due to lack of blinding); imprecision (due to small sample sizes of the individual trials and few events); and publication bias (few trials reporting outcomes). The small sample sizes and few events did not allow us to produce meaningful trial sequential monitoring boundaries, suggesting plausible random errors in our estimates. AUTHORS' CONCLUSIONS: We found evidence suggesting that surgical portosystemic shunts may have benefit over TIPS for treatment of refractory or recurrent variceal haemorrhage in people with cirrhotic portal hypertension. Given the very low-certainty of the available evidence and risks of random errors in our analyses, we have very little confidence in our review findings.

Surgical portosystemic shunts versus devascularisation procedures for prevention of variceal rebleeding in people with hepatosplenic schistosomiasis.

BACKGROUND: Hepatosplenic schistosomiasis is an important cause of variceal bleeding in low-income countries. Randomised clinical trials have evaluated the outcomes of two categories of surgical interventions, shunts and devascularisation procedures, for the prevention of variceal rebleeding in people with hepatosplenic schistosomiasis. The comparative overall benefits and harms of these two interventions are unclear. OBJECTIVES: To assess the benefits and harms of surgical portosystemic shunts versus oesophagogastric devascularisation procedures for the prevention of variceal rebleeding in people with hepatosplenic schistosomiasis. SEARCH METHODS: We searched the Cochrane Hepato-Biliary Group Controlled Trials Register, CENTRAL, MEDLINE, Embase, Science Citation Index Expanded, LILACS, reference lists of articles, and proceedings of relevant associations for trials that met the inclusion criteria (date of search 11 January 2018). SELECTION CRITERIA: Randomised clinical trials comparing surgical portosystemic shunts versus oesophagogastric devascularisation procedures for the prevention of variceal rebleeding in people with hepatosplenic schistosomiasis. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed the trials and extracted data using methodological standards expected by Cochrane. We assessed risk of bias according to domains and risk of random errors with GRADE and Trial Sequential Analysis. We assessed the certainty of evidence using the GRADE approach. MAIN RESULTS: We found two randomised clinical trials including 154 adult participants, aged between 18 years and 65 years, diagnosed with hepatosplenic schistosomiasis. One of the trials randomised participants to proximal splenorenal shunt versus distal splenorenal shunt versus oesophagogastric devascularisation with splenectomy, and the other randomised participants to distal splenorenal shunt versus oesophagogastric devascularisation with splenectomy. In both trials the diagnosis of hepatosplenic schistosomiasis was made based on clinical and biochemical assessments. The trials were conducted in Brazil and Egypt. Both trials were at high risk of bias.We are uncertain as to whether surgical portosystemic shunts improved all-cause mortality compared with oesophagogastric devascularisation with splenectomy due to imprecision in the trials (risk ratio (RR) 2.35, 95% confidence interval (CI) 0.55 to 9.92; participants = 154; studies = 2). We are uncertain whether serious adverse events differed between surgical portosystemic shunts and oesophagogastric devascularisation with splenectomy (RR 2.26, 95% CI 0.44 to 11.70; participants = 154; studies = 2). None of the trials reported on health-related quality of life. We are uncertain whether variceal rebleeding differed between surgical portosystemic shunts and oesophagogastric devascularisation with splenectomy (RR 0.39, 95% CI 0.13 to 1.23; participants = 154; studies = 2). We found evidence suggesting an increase in encephalopathy in the shunts group versus the devascularisation with splenectomy group (RR 7.51, 95% CI 1.45 to 38.89; participants = 154; studies = 2). We are uncertain whether ascites and re-interventions differed between surgical portosystemic shunts and oesophagogastric devascularisation with splenectomy. We computed Trial Sequential Analysis for all outcomes, but the trial sequential monitoring boundaries could not be drawn because of insufficient sample size and events. We downgraded the overall certainty of the body of evidence for all outcomes to very low due to risk of bias and imprecision. AUTHORS' CONCLUSIONS: Given the very low certainty of the available body of evidence and the low number of clinical trials, we could not determine an overall benefit or harm of surgical portosystemic shunts compared with oesophagogastric devascularisation with splenectomy. Future randomised clinical trials should be designed with sufficient statistical power to assess the benefits and harms of surgical portosystemic shunts versus oesophagogastric devascularisations with or without splenectomy and with or without oesophageal transection.

Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant.

We explore the possibility of re-engineering mitochondrial genes and expressing them from the nucleus as an approach to rescue defects arising from mitochondrial DNA mutations. We have used a patient cybrid cell line with a single point mutation in the overlap region of the ATP8 and ATP6 genes of the human mitochondrial genome. These cells are null for the ATP8 protein, have significantly lowered ATP6 protein levels and no Complex V function. Nuclear expression of only the ATP8 gene with the ATP5G1 mitochondrial targeting sequence appended restored viability on Krebs cycle substrates and ATP synthesis capabilities but, failed to restore ATP hydrolysis and was insensitive to various inhibitors of oxidative phosphorylation. Co-expressing both ATP8 and ATP6 genes under similar conditions resulted in stable protein expression leading to successful integration into Complex V of the oxidative phosphorylation machinery. Tests for ATP hydrolysis / synthesis, oxygen consumption, glycolytic metabolism and viability all indicate a significant functional rescue of the mutant phenotype (including re-assembly of Complex V) following stable co-expression of ATP8 and ATP6 Thus, we report the stable allotopic expression, import and function of two mitochondria encoded genes, ATP8 and ATP6, resulting in simultaneous rescue of the loss of both mitochondrial proteins.

Managing Infected Pancreatic Necrosis.

The management of infected pancreatic necrosis has historically been based on early, open necrosectomy, associated with significant mortality. In recent years, an evidence based transformation has occurred towards the step-up approach consisting of percutaneous catheter drainage, if necessary, followed by minimally invasive necrosectomy. More recently the endoscopic step-up approach has gained popularity. This review evaluates the diagnosis, prevention and treatment of infected necrotizing pancreatitis. Key points in managing infected pancreatic necrosis: - multidisciplinary team approach in tertiary level centres; - no indication for prophylactic antibiotics or probiotics; - nasogastric, enteral nutrition indicated after 72 hours, if oral feeding is insufficient; - only intervene in infected necrosis; - delay intervention until "walled-off necrosis"; - step-up approach of percutaneous or endoscopic catheter drainage, followed by minimally invasive necrosectomy, if required; - endoscopic strategies are preferable where possible.

Positive Feedback Amplifies the Response of Mitochondrial Membrane Potential to Glucose Concentration in Clonal Pancreatic Beta Cells.

Analysis of the cellular mechanisms of metabolic disorders, including type 2 diabetes mellitus, is complicated by the large number of reactions and interactions in metabolic networks. Metabolic control analysis with appropriate modularization is a powerful method for simplifying and analyzing these networks. To analyze control of cellular energy metabolism in adherent cell cultures of the INS-1 832/13 pancreatic ß-cell model we adapted our microscopy assay of absolute mitochondrial membrane potential (ΔψM) to a fluorescence microplate reader format, and applied it in conjunction with cell respirometry. In these cells the sensitive response of ΔψM to extracellular glucose concentration drives glucose-stimulated insulin secretion. Using metabolic control analysis we identified the control properties that generate this sensitive response. Force-flux relationships between ΔψM and respiration were used to calculate kinetic responses to ΔψM of processes both upstream (glucose oxidation) and downstream (proton leak and ATP turnover) of ΔψM. The analysis revealed that glucose-evoked ΔψM hyperpolarization is amplified by increased glucose oxidation activity caused by factors downstream of ΔψM. At high glucose, the hyperpolarized ΔψM is stabilized almost completely by the action of glucose oxidation, whereas proton leak also contributes to the homeostatic control of ΔψM at low glucose. These findings suggest a strong positive feedback loop in the regulation of ß-cell energetics, and a possible regulatory role of proton leak in the fasting state. Analysis of islet bioenergetics from published cases of type 2 diabetes suggests that disruption of this feedback can explain the damaged bioenergetic response of ß-cells to glucose. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.

Suppressors of superoxide production from mitochondrial complex III.

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

Inflammatory cytokines and angiogenic factors as potential biomarkers in South African pancreatic ductal adenocarcinoma patients: A preliminary report.

BACKGROUND/OBJECTIVES: Several studies have investigated the association of differentially expressed cytokines with pancreatic ductal adenocarcinoma (PDAC), but none in African countries. This study aimed at investigating T-helper (Th) cell and angiogenic markers as diagnostic or prognostic biomarkers for PDAC in Black South Africans. METHODS: We conducted a prospective, case-control study comprising of 34 PDAC patients and 27 control participants with either critical limb ischemia, abdominal aortic aneurysm or other abdominal pathology from causes other than pancreatic disease. Plasma levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, IL-17A, VEGF, sVEGF-R1, FGF, PIGF, PDGF and P-selectin were measured using commercially available cytometric bead array, ELISA and multi-analyte Luminex kits. RESULTS: Significantly higher levels of IFN-γ (p < 0.001), TNF (p < 0.001), IL-2 (p = 0.001), IL-4 (p < 0.01), IL-10 (p < 0.01), IL-17A (p < 0.01), PlGF (p < 0.0001) and basic FGF (p < 0.0001) were found in cases compared to control participants. PDAC patients with irresectable tumours had higher levels of VEGF (p = 0.02) and IL-6 (p = 0.01). A univariate analysis showed significant associations between IFN-γ, TNF, IL-10, -4, -2, basic FGF, PlGF and PDAC. In a multivariate logistic regression model, basic FGF (p = 0.002) and PlGF (p = 0.007) were independent risk factors for PDAC with a combined sensitivity of 71% and specificity of 100%. CONCLUSION: Our preliminary data suggests a potential role for basic FGF and PlGF as diagnostic, and VEGF and IL-6 as prognostic biomarkers of PDAC in Black South African patients.

Sites of superoxide and hydrogen peroxide production by muscle mitochondria assessed ex vivo under conditions mimicking rest and exercise.

The sites and rates of mitochondrial production of superoxide and H2O2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site IIIQo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site IF) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H2O2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site IQ) and the flavin site in complex II (site IIF) each account for about a quarter of the total measured rate of H2O2 production. Site IF, site IIIQo, and perhaps site EF in the ß-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site IF dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H2O2 in vivo.