Synthesis, In Silico and Kinetics Evaluation of N-(ß-d-glucopyranosyl)-2-arylimidazole-4(5)-carboxamides and N-(ß-d-glucopyranosyl)-4(5)-arylimidazole-2-carboxamides as Glycogen Phosphorylase Inhibitors.

Recently studied N-(ß-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus. Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety with imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound state DFT calculations were performed on N-(ß-d-glucopyranosyl)-arylimidazole-carboxamides, revealing their potential for strong GP inhibition. The syntheses of the target compounds involved the formation of an amide bond between per-O-acetylated ß-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GPb revealed low micromolar inhibitors, with the best inhibition constants (Kis) of ~3-4 µM obtained for 1- and 2-naphthyl-substituted N-(ß-d-glucopyranosyl)-imidazolecarboxamides, 2b-c. The predicted protein-ligand interactions responsible for the observed potencies are discussed and will facilitate the structure-based design of other inhibitors targeting this important therapeutic target. Meanwhile, the importance of the careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed.

Carnosic Acid Inhibits Herpes Simplex Virus Replication by Suppressing Cellular ATP Synthesis.

Acquiring resistance against antiviral drugs is a significant problem in antimicrobial therapy. In order to identify novel antiviral compounds, the antiviral activity of eight plants indigenous to the southern region of Hungary against herpes simplex virus-2 (HSV-2) was investigated. The plant extracts and the plant compound carnosic acid were tested for their effectiveness on both the extracellular and intracellular forms of HSV-2 on Vero and HeLa cells. HSV-2 replication was measured by a direct quantitative PCR (qPCR). Among the tested plant extracts, Salvia rosmarinus (S. rosmarinus) exhibited a 90.46% reduction in HSV-2 replication at the 0.47 µg/mL concentration. Carnosic acid, a major antimicrobial compound found in rosemary, also demonstrated a significant dose-dependent inhibition of both extracellular and intracellular forms of HSV-2. The 90% inhibitory concentration (IC90) of carnosic acid was between 25 and 6.25 µg/mL. Proteomics and high-resolution respirometry showed that carnosic acid suppressed key ATP synthesis pathways such as glycolysis, citrate cycle, and oxidative phosphorylation. Inhibition of oxidative phosphorylation also suppressed HSV-2 replication up to 39.94-fold. These results indicate that the antiviral action of carnosic acid includes the inhibition of ATP generation by suppressing key energy production pathways. Carnosic acid holds promise as a potential novel antiviral agent against HSV-2.

Microcirculation-driven mitochondrion dysfunction during the progression of experimental sepsis.

Sepsis is accompanied by a less-known mismatch between hemodynamics and mitochondrial respiration. We aimed to characterize the relationship and time dependency of microcirculatory and mitochondrial functions in a rodent model of intraabdominal sepsis. Fecal peritonitis was induced in rats, and multi-organ failure (MOF) was evaluated 12, 16, 20, 24 or 28 h later (n = 8/group, each) using rat-specific organ failure assessment (ROFA) scores. Ileal microcirculation (proportion of perfused microvessels (PPV), microvascular flow index (MFI) and heterogeneity index (HI)) was monitored by intravital video microscopy, and mitochondrial respiration (OxPhos) and outer membrane (mtOM) damage were measured with high-resolution respirometry. MOF progression was evidenced by increased ROFA scores; microcirculatory parameters followed a parallel time course from the 16th to 28th h. Mitochondrial dysfunction commenced with a 4-h time lag with signs of mtOM damage, which correlated significantly with PPV, while no correlation was found between HI and OxPhos. High diagnostic value was demonstrated for PPV, mtOM damage and lactate levels for predicting MOF. Our findings indicate insufficient splanchnic microcirculation to be a possible predictor for MOF that develops before the start of mitochondrial dysfunction. The adequate subcellular compensatory capacity suggests the presence of mitochondrial subpopulations with differing sensitivity to septic insults.

Behaviour of some 1-C-acceptor-substituted glycals under azidohydroxylation conditions.

Azidohydroxylation of 1-carbamoyl, 1-methoxycarbonyl and 1-cyano substituted d-lyxo and d-arabino configured O-peracylated glycals was studied and the reaction conditions were optimized. Under these conditions (3 equiv. NaN3/2 equiv. PIFA/0.3 equiv. TEMPO/50 equiv. H2O/dry DCM/0 °C/Ar) the expected 3-azido-3-deoxy ulopyranosonic acid derivatives were isolated in good yield with α-d-galacto configuration exclusively from the reaction of the 1-carbamoyl and 1-methoxycarbonyl substituted d-lyxo configured O-peracetylated glycals, while the transformation of the 1-cyano derivative gave a 2,3-vicinal diazide in low yield. The 1-carbamoyl d-arabino configured O-perbenzoylated glycal gave a mixture of α-d-gluco and α-d-manno configured azidohydroxylated products with d-gluco preference. The analogous 1-methoxycarbonyl derivative gave an inseparable product mixture and no transformation was detected with the respective 1-cyano glycal.

Tilorone increases glucose uptake in vivo and in skeletal muscle cells by enhancing Akt2/AS160 signaling and glucose transporter levels.

Skeletal muscle plays a major role in whole-body glucose metabolism. Insulin resistance in skeletal muscle is characterized by decreased insulin-stimulated glucose uptake resulting from impaired intracellular trafficking and decreased glucose transporter 4 (GLUT4) expression. In this study, we illustrated that tilorone, a low-molecular-weight antiviral agent, improves glucose uptake in vitro and in vivo. Tilorone increased bone morphogenetic protein (BMP) signaling in C2C12 myoblasts, the transcription of multiple BMPs (BMP2, BMP4, BMP7, and BMP14), Smad4 expression, and the phosphorylation of BMP-mediated Smad1/5/8. The activation of Akt2/AS160 (TBC1D4) signaling, the critical regulator of GLUT4 translocation, was also increased, as well as the levels of GLUT4 and GLUT1, leading to enhanced uptake of the radioactively labeled glucose analog 18 F-fluoro-2-deoxyglucose (18 FDG). However, this excess glucose content did not result in increased ATP formation by mitochondrial respiration; both basal and ATP-linked respiration were diminished, thereby contributing to the induction of AMPK. In differentiated myotubes, AS160 phosphorylation and 18 FDG uptake also increased. Moreover, tilorone administration further increased insulin-stimulated phosphorylation of Akt2 and glucose uptake of myotubes indicating an insulin-sensitizing effect. Importantly, during in vivo experiments, the systemic administration of tilorone resulted in increased 18 FDG uptake of skeletal muscle, liver, and adipose tissue in C57BL/6 mice. Our results provide new perspectives for the treatment of type 2 diabetes, which has a limited number of treatments that regulate protein expression or translocation.

Reactions of 1-C-acceptor-substituted glycals with nucleophiles under acid promoted (Ferrier-rearrangement) conditions.

The reactivity of O-peracetylated and O-perbenzoylated 1-COOMe, 1-CONH2 and 1-CN-substituted glycals was studied against O-, S-, N- and C-nucleophiles in the presence of Lewis acids. Allylic substituted products with exclusive axial stereoselectivity were formed with simple alcohols, N3-, and Cl- ions, but with benzyl thiol the Ferrier rearrangement took place and thioglycosides were obtained. The use of a sugar derived thiol resulted in the formation of both the allylic substituted and the rearranged products.

Veno-Venous Extracorporeal Membrane Oxygenation in Minipigs as a Robust Tool to Model Acute Kidney Injury: Technical Notes and Characteristics.

Objective: Veno-venous extracorporeal membrane oxygenation (vv-ECMO) can save lives in severe respiratory distress, but this innovative approach has serious side-effects and is accompanied by higher rates of iatrogenic morbidity. Our aims were, first, to establish a large animal model of vv-ECMO to study the pathomechanism of complications within a clinically relevant time frame and, second, to investigate renal reactions to increase the likelihood of identifying novel targets and to improve clinical outcomes of vv-ECMO-induced acute kidney injury (AKI). Methods: Anesthetized Vietnamese miniature pigs were used. After cannulation of the right jugular and femoral veins, vv-ECMO was started and maintained for 24 hrs. In Group 1 (n = 6) ECMO was followed by a further 6-hr post-ECMO period, while (n = 6) cannulation was performed without ECMO in the control group, with observation maintained for 30 h. Systemic hemodynamics, blood gas values and hour diuresis were monitored. Renal artery flow (RAF) was measured in the post-ECMO period with an ultrasonic flowmeter. At the end of the experiments, renal tissue samples were taken for histology to measure myeloperoxidase (MPO) and xanthine oxidoreductase (XOR) activity and to examine mitochondrial function with high-resolution respirometry (HRR, Oroboros, Austria). Plasma and urine samples were collected every 6 hrs to determine neutrophil gelatinase-associated lipocalin (NGAL) concentrations. Results: During the post-ECMO period, RAF dropped (96.3 ± 21 vs. 223.6 ± 32 ml/min) and, similarly, hour diuresis was significantly lower as compared to the control group (3.25 ± 0.4 ml/h/kg vs. 4.83 ± 0.6 ml/h/kg). Renal histology demonstrated significant structural damage characteristic of ischemic injury in the tubular system. In the vv-ECMO group NGAL levels, rose significantly in both urine (4.24 ± 0.25 vs. 2.57 ± 0.26 ng/ml) and plasma samples (4.67 ± 0.1 vs. 3.22 ± 0.2 ng/ml), while tissue XOR (5.88 ± 0.8 vs. 2.57 ± 0.2 pmol/min/mg protein) and MPO (11.93 ± 2.5 vs. 4.34 ± 0.6 mU/mg protein) activity was elevated. HRR showed renal mitochondrial dysfunction, including a significant drop in complex-I-dependent oxidative capacity (174.93 ± 12.7 vs. 249 ± 30.07 pmol/s/ml). Conclusion: Significantly decreased renal function with signs of structural damage and impaired mitochondrial function developed in the vv-ECMO group. The vv-ECMO-induced acute renal impairment in this 30-hr research protocol provides a good basis to study the pathomechanism, biomarker combinations or possible therapeutic possibilities for AKI.

A Porcine Sepsis Model With Numerical Scoring for Early Prediction of Severity.

Introduction: Sepsis can lead to organ dysfunctions with disturbed oxygen dynamics and life-threatening consequences. Since the results of organ-protective treatments cannot always be transferred from laboratory models into human therapies, increasing the translational potential of preclinical settings is an important goal. Our aim was to develop a standardized research protocol, where the progression of sepsis-related events can be characterized reproducibly in model experiments within clinically-relevant time frames. Methods: Peritonitis was induced in anesthetized minipigs injected intraperitoneally with autofeces inoculum (n = 27) or with saline (sham operation; n = 9). The microbial colony-forming units (CFUs) in the inoculum were retrospectively determined. After awakening, clinically relevant supportive therapies were conducted. Nineteen inoculated animals developed sepsis without a fulminant reaction. Sixteen hours later, these animals were re-anesthetized for invasive monitoring. Blood samples were taken to detect plasma TNF-α, IL-10, big endothelin (bET), high mobility group box protein1 (HMGB1) levels and blood gases, and sublingual microcirculatory measurements were conducted. Hemodynamic, respiratory, coagulation, liver and kidney dysfunctions were detected to characterize the septic status with a pig-specific Sequential Organ Failure Assessment (pSOFA) score and its simplified version (respiratory, cardiovascular and renal failure) between 16 and 24 h of the experiments. Results: Despite the standardized sepsis induction, the animals could be clustered into two distinct levels of severity: a sepsis (n = 10; median pSOFA score = 2) and a septic shock (n = 9; median pSOFA score = 8) subgroup at 18 h of the experiments, when the decreased systemic vascular resistance, increased DO2 and VO2, and markedly increased ExO2 demonstrated a compensated hyperdynamic state. Septic animals showed severity-dependent scores for organ failure with reduced microcirculation despite the adequate oxygen dynamics. Sepsis severity characterized later with pSOFA scores was in correlation with the germ count in the induction inoculum (r = 0.664) and CFUs in hemocultures (r = 0.876). Early changes in plasma levels of TNF-α, bET and HMGB1 were all related to the late-onset organ dysfunctions characterized by pSOFA scores. Conclusions: This microbiologically-monitored, large animal model of intraabdominal sepsis is suitable for clinically-relevant investigations. The methodology combines the advantages of conscious and anesthetized studies, and mimics human sepsis and septic shock closely with the possibility of numerical quantification of host responses.

Coupling Reactions of Anhydro-Aldose Tosylhydrazones with Boronic Acids.

A catalyst-free coupling reaction between O-peracetylated, O-perbenzoylated, O-permethylated, and O-permethoxymethylated 2,6-anhydro-aldose tosylhydrazones (C-(ß-d-glycopyranosyl)formaldehyde tosylhydrazones) and aromatic boronic acids is reported. The base-promoted reaction is operationally simple and exhibits a broad substrate scope. The main products in most of the transformations were open-chain 1-C-aryl-hept-1-enitol type compounds while the expected ß-d-glycopyranosylmethyl arenes (benzyl C-glycosides) were formed in subordinate yields only. A mechanistic rationale is provided to explain how a complex substrate may change the well-established course of the reaction.

Characterization of technical skill progress in a standardized rabbit model for training in laparoscopic duodenal atresia repair.

BACKGROUND: Laboratory skills training is an essential step before conducting minimally invasive surgery in clinical practice. Our main aim was to develop an animal model for training in clinically highly challenging laparoscopic duodenal atresia repair that could be useful in establishing a minimum number of repetitions to indicate safe performance of similar interventions on humans. MATERIALS AND METHODS: A rabbit model of laparoscopic duodenum atresia surgery involving a diamond-shaped duodeno-duodenostomy was designed. This approach was tested in two groups of surgeons: in a beginner group without any previous clinical laparoscopic experience (but having undergone previous standardized dry-lab training, n = 8) and in an advanced group comprising pediatric surgery fellows with previous clinical experience of laparoscopy (n = 7). Each participant performed eight interventions. Surgical time, expert assessment using the Global Operative Assessment of Laparoscopic Skills (GOALS) score, anastomosis quality (leakage) and results from participant feedback questionnaires were analyzed. RESULTS: Participants in both groups successfully completed all eight surgeries. The surgical time gradually improved in both groups, but it was typically shorter in the advanced group than in the beginner group. The leakage rate was significantly lower in the advanced group in the first two interventions, and it reached its optimal level after five operations in both groups. The GOALS and participant feedback scores showed gradual increases, evident even after the fifth surgery. CONCLUSIONS: Our data confirm the feasibility of this advanced pediatric laparoscopic model. Surgical time, anastomosis quality, GOALS score and self-assessment parameters adequately quantify technical improvement among the participants. Anastomosis quality reaches its optimal value after the fifth operation even in novice, but uniformly trained surgeons. A minimum number of wet-lab operations can be determined before surgery can be safely conducted in a clinical setting, where the development of further non-technical skills is also required.

The microbial composition of the initial insult can predict the prognosis of experimental sepsis.

We hypothesized that the composition of sepsis-inducing bacterial flora influences the course of fecal peritonitis in rodents. Saline or fecal suspensions with a standardized dose range of bacterial colony-forming units (CFUs) were injected intraperitoneally into Sprague-Dawley rats. The qualitative composition of the initial inoculum and the ascites was analyzed separately by MALDI-TOF mass spectrometry. Invasive monitoring was conducted in separate anesthetized groups (n = 12-13/group) after 12, 24, 48 and 72 h to determine rat-specific organ failure assessment (ROFA) scores. Death and ROFA scores peaked at 24 h. At this time, 20% mortality occurred in animals receiving a monomicrobial E. coli suspension, and ROFA scores were significantly higher in the monomicrobial subgroup than in the polymicrobial one (median 6.5; 5.0-7.0 and 5.0; 4.75-5.0, respectively). ROFA scores dropped after 48 h, accompanied by a steady decrease in ascites CFUs and a shift towards intra-abdominal monomicrobial E. coli cultures. Furthermore, we found a relationship between ascites CFUs and the evolving change in ROFA scores throughout the study. Hence, quantitatively identical bacterial loads with mono- or polymicrobial dominance lead to a different degree of sepsis severity and divergent outcomes. Initial and intraperitoneal microbiological testing should be used to improve translational research success.

Kynurenic Acid and Its Synthetic Derivatives Protect Against Sepsis-Associated Neutrophil Activation and Brain Mitochondrial Dysfunction in Rats.

Background and Aims: The systemic host response in sepsis is frequently accompanied by central nervous system (CNS) dysfunction. Evidence suggests that excessive formation of neutrophil extracellular traps (NETs) can increase the permeability of the blood-brain barrier (BBB) and that the evolving mitochondrial damage may contribute to the pathogenesis of sepsis-associated encephalopathy. Kynurenic acid (KYNA), a metabolite of tryptophan catabolism, exerts pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenous KYNA or its synthetic analogues SZR-72 and SZR-104 affect BBB permeability secondary to NET formation and influence cerebral mitochondrial disturbances in a clinically relevant rodent model of intraabdominal sepsis. Methods: Sprague-Dawley rats were subjected to fecal peritonitis (0.6 g kg-1 ip) or a sham operation. Septic animals were treated with saline or KYNA, SZR-72 or SZR-104 (160 µmol kg-1 each ip) 16h and 22h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic parameters to calculate rat organ failure assessment (ROFA) scores. NET components (citrullinated histone H3 (CitH3); myeloperoxidase (MPO)) and the NET inducer IL-1ß, as well as IL-6 and a brain injury marker (S100B) were detected from plasma samples. After 24h, leukocyte infiltration (tissue MPO) and mitochondrial complex I- and II-linked (CI-CII) oxidative phosphorylation (OXPHOS) were evaluated. In a separate series, Evans Blue extravasation and the edema index were used to assess BBB permeability in the same regions. Results: Sepsis was characterized by significantly elevated ROFA scores, while the increased BBB permeability and plasma S100B levels demonstrated brain damage. Plasma levels of CitH3, MPO and IL-1ß were elevated in sepsis but were ameliorated by KYNA and its synthetic analogues. The sepsis-induced deterioration in tissue CI-CII-linked OXPHOS and BBB parameters as well as the increase in tissue MPO content were positively affected by KYNA/KYNA analogues. Conclusion: This study is the first to report that KYNA and KYNA analogues are potential neuroprotective agents in experimental sepsis. The proposed mechanistic steps involve reduced peripheral NET formation, lowered BBB permeability changes and alleviation of mitochondrial dysfunction in the CNS.

Halogen addition to some 1-C-substituted pyranoid glycals.

Addition of bromine and chlorine to O-peracylated 1-CN-, COOMe- and CONH2-substituted glycals was studied under ionic and radical conditions. The main or exclusive products were the corresponding 2,3-trans-diaxial (3-bromo-3-deoxy-α-d-heptopyranosylbromide)onic acid derivatives. Bromination of the O-peracetylated d-lyxo-hept-2-enopyranosononitrile and all chlorinations proved selective towards the 2-axial-3-equatorial (3-halogeno-3-deoxy-α-d-heptopyranosylhalide)onic acid derivatives. Silver triflate promoted glycosylation of methanol was successful with each 2,3-trans-diaxial (3-bromo-3-deoxy-α-d-heptopyranosylbromide)onic acid derivative, however, several attempted nucleophilic substitution and elimination reactions gave the parent glycal only.

Bioactivity of Inhaled Methane and Interactions With Other Biological Gases.

A number of studies have demonstrated explicit bioactivity for exogenous methane (CH4), even though it is conventionally considered as physiologically inert. Other reports cited in this review have demonstrated that inhaled, normoxic air-CH4 mixtures can modulate the in vivo pathways involved in oxidative and nitrosative stress responses and key events of mitochondrial respiration and apoptosis. The overview is divided into two parts, the first being devoted to a brief review of the effects of biologically important gases in the context of hypoxia, while the second part deals with CH4 bioactivity. Finally, the consequence of exogenous, normoxic CH4 administration is discussed under experimental hypoxia- or ischaemia-linked conditions and in interactions between CH4 and other biological gases, with a special emphasis on its versatile effects demonstrated in pulmonary pathologies.

Divergent Effects of the N-Methyl-D-Aspartate Receptor Antagonist Kynurenic Acid and the Synthetic Analog SZR-72 on Microcirculatory and Mitochondrial Dysfunction in Experimental Sepsis.

Introduction: Sepsis is a dysregulated host response to infection with macro- and microhemodynamic deterioration. Kynurenic acid (KYNA) is a metabolite of the kynurenine pathway of tryptophan catabolism with pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenously administered KYNA or the synthetic analog SZR-72 affects the microcirculation and mitochondrial function in a clinically relevant rodent model of intraabdominal sepsis. Methods: Male Sprague-Dawley rats (n = 8/group) were subjected to fecal peritonitis (0.6 g kg-1 feces ip) or a sham operation. Septic animals were treated with sterile saline or received ip KYNA or SZR-72 (160 µmol kg-1 each) 16 and 22 h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic dysfunctions (PaO2/FiO2 ratio, mean arterial pressure, urea, AST/ALT ratio and lactate levels, respectively) based on the Rat Organ Failure Assessment (ROFA) score. The ratio of perfused vessels (PPV) of the ileal serosa was quantified with the intravital imaging technique. Complex I- and II-linked (CI; CII) oxidative phosphorylation capacities (OXPHOS) and mitochondrial membrane potential (ΔΨmt) were evaluated by High-Resolution FluoRespirometry (O2k, Oroboros, Austria) in liver biopsies. Plasma endothelin-1 (ET-1), IL-6, intestinal nitrotyrosine (NT) and xanthine oxidoreductase (XOR) activities were measured as inflammatory markers. Results: Sepsis was characterized by an increased ROFA score (5.3 ± 1.3 vs. 1.3 ± 0.7), increased ET-1, IL-6, NT and XOR levels, and decreased serosal PPV (65 ± 12% vs. 87 ± 7%), ΔΨmt and CI-CII-linked OXPHOS (73 ± 16 vs. 158 ± 14, and 189 ± 67 vs. 328 ± 81, respectively) as compared to controls. Both KYNA and SZR-72 reduced systemic inflammatory activation; KYNA treatment decreased serosal perfusion heterogeneity, restored PPV (85 ± 11%) and complex II-linked OXPHOS (307 ± 38), whereas SZR-72 improved both CI- and CII-linked OXPHOS (CI: 117 ± 18; CII: 445 ± 107) without effects on PPV 24 h after sepsis induction. Conclusion: Treatment with SZR-72 directly modulates mitochondrial respiration, leading to improved conversion of ADP to ATP, while administration of KYNA restores microcirculatory dysfunction. The results suggest that microcirculatory and mitochondrial resuscitation with KYNA or the synthetic analog SZR-72 might be an appropriate supportive tool in sepsis therapy.

Glycogen phosphorylase inhibitor, 2,3-bis[(2E)-3-(4-hydroxyphenyl)prop-2-enamido] butanedioic acid (BF142), improves baseline insulin secretion of MIN6 insulinoma cells.

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of ß cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of ß cells. The purpose of this study was to assess whether an analogue of FR258900 can influence ß cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence ß cell function and can support the insulin producing ability of ß cells.

Synthesis and photoinitiated thiol-ene reactions of exo-mannals - a new route to C-ß-d-mannosyl derivatives.

Syntheses of acyl protected exo-mannal derivatives were developed starting from O-peracylated mannopyranoses via the corresponding anhydro-aldose tosylhydrazones under modified Bamford-Stevens conditions. The synthesis of analogous O-peralkylated (benzylated and isopropylenated) derivatives was carried out from pyranoid and furanoid mannonolactones using methylene transfer reagents. Photoinitiated thiol-ene additions of these exo-mannals resulted in the corresponding C-(mannopyranosyl/mannofuranosyl)methyl sulfides in medium to good yields with exclusive regio- and ß(d) stereoselectivities.

Endothelin A and B Receptors: Potential Targets for Microcirculatory-Mitochondrial Therapy in Experimental Sepsis.

The hypoxia-sensitive endothelin (ET) system plays an important role in circulatory regulation through vasoconstrictor ETA and ETB2 and vasodilator ETB1 receptors. Sepsis progression is associated with microcirculatory and mitochondrial disturbances along with tissue hypoxia. Our aim was to investigate the consequences of treatments with the ETA receptor (ETA-R) antagonist, ETB1 receptor (ETB1-R) agonist, or their combination on oxygen dynamics, mesenteric microcirculation, and mitochondrial respiration in a rodent model of sepsis. Sprague Dawley rats were subjected to fecal peritonitis (0.6 g kg i.p.) or a sham operation. Septic animals were treated with saline or the ETA-R antagonist ETR-p1/fl peptide (100 nmol kg i.v.), the ETB1-R agonist IRL-1620 (0.55 nmol kg i.v.), or a combination therapy 22 h after induction. Invasive hemodynamic monitoring and blood gas analysis were performed during a 90-min observation, plasma ET-1 levels were determined, and intestinal capillary perfusion (CPR) was detected by intravital videomicroscopy. Mitochondrial Complex I (CI)- and CII-linked oxidative phosphorylation (OXPHOS) was evaluated by high-resolution respirometry in liver biopsies. Septic animals were hypotensive with elevated plasma ET-1. The ileal CPR, oxygen extraction (ExO2), and CI-CII-linked OXPHOS capacities decreased. ETR-p1/fl treatment increased ExO2 (by >45%), CPR, and CII-linked OXPHOS capacity. The administration of IRL-1620 countervailed the sepsis-induced hypotension (by >30%), normalized ExO2, and increased CPR. The combined ETA-R antagonist-ETB1-R agonist therapy reduced the plasma ET-1 level, significantly improved the intestinal microcirculation (by >41%), and reversed mitochondrial dysfunction. The additive effects of a combined ETA-R-ETB1-R-targeted therapy may offer a tool for a novel microcirculatory and mitochondrial resuscitation strategy in experimental sepsis.

Methane and Inflammation - A Review (Fight Fire with Fire).

Mammalian methanogenesis is regarded as an indicator of carbohydrate fermentation by anaerobic gastrointestinal flora. Once generated by microbes or released by a non-bacterial process, methane is generally considered to be biologically inactive. However, recent studies have provided evidence for methane bioactivity in various in vivo settings. The administration of methane either in gas form or solutions has been shown to have anti-inflammatory and neuroprotective effects in an array of experimental conditions, such as ischemia/reperfusion, endotoxemia and sepsis. It has also been demonstrated that exogenous methane influences the key regulatory mechanisms and cellular signalling pathways involved in oxidative and nitrosative stress responses. This review offers an insight into the latest findings on the multi-faceted organ protective activity of exogenous methane treatments with special emphasis on its versatile effects demonstrated in sepsis models.

Ca(2+)N It Be Measured? Detection of Extramitochondrial Calcium Movement With High-Resolution FluoRespirometry.

Our aim was to develop a method to detect extramitochondrial Ca2+ movement and O2 fluxes simultaneously. Using High-Resolution FluoRespirometry, we also tested whether mitochondrial permeability transition pore (mPTP) inhibition or anoxia affects the mitochondrial Ca2+ flux. Ca2+ movement evoked by CaCl2 or anoxia was assessed with CaGreen-5N dye using Blue-Fluorescence-Sensor in isolated liver mitochondria, liver homogenates and duodenal biopsies. Exogenous CaCl2 (50 µM) resulted in an abrupt elevation in CaGreen-5N fluorescence followed by a decrease (Ca2+ uptake) with simultaneous elevation in O2 consumption in liver preparations. This was followed by a rapid increase in the fluorescence signal, reaching a higher intensity (Ca2+ efflux) than that of the initial CaCl2-induced elevation. Chelation of Ca2+ with EGTA completely abolished the fluorescence of the indicator. After pre-incubation with cyclosporin A, a marked delay in Ca2+ movement was observed, not only in isolated liver mitochondria, but also in tissue homogenates. In all samples, the transition to anoxia resulted in immediate increase in the level of extramitochondrial Ca2+. The results demonstrate that the CaGreen-5N method is suitable to monitor simultaneous O2 and Ca2+ fluxes, and the opening of mPTP in various biological samples. In this system the duration of stimulated Ca2+ fluxes may provide a novel parameter to evaluate the efficacy of mPTP blocker compounds.