Islets-on-Chip: A Tool for Real-Time Assessment of Islet Function Prior to Transplantation.

Islet transplantation improves metabolic control in patients with unstable type 1 diabetes. Clinical outcomes have been improving over the last decade, and the widely used beta-score allows the evaluation of transplantation results. However, predictive pre-transplantation criteria of islet quality for clinical outcomes are lacking. In this proof-of-concept study, we examined whether characterization of the electrical activity of donor islets could provide a criterion. Aliquots of 8 human donor islets from the STABILOT study, sampled from islet preparations before transplantation, were characterized for purity and split for glucose-induced insulin secretion and electrical activity using multi-electrode-arrays. The latter tests glucose concentration dependencies, biphasic activity, hormones, and drug effects (adrenalin, GLP-1, glibenclamide) and provides a ranking of CHIP-scores from 1 to 6 (best) based on electrical islet activity. The analysis was performed online in real time using a dedicated board or offline. Grouping of beta-scores and CHIP-scores with high, intermediate, and low values was observed. Further analysis indicated correlation between CHIP-score and beta-score, although significance was not attained (R = 0.51, p = 0.1). This novel approach is easily implantable in islet isolation units and might provide means for the prediction of clinical outcomes. We acknowledge the small cohort size as the limitation of this pilot study.

Cost-Effectiveness of Quadruple Therapy in Management of Heart Failure With Reduced Ejection Fraction in the United States.

BACKGROUND: The 2022 clinical guidelines for management of heart failure with reduced ejection fraction call for quadruple therapy. Quadruple therapy consists of an angiotensin receptor-neprilysin inhibitor (ARNi), sodium-glucose cotransporter-2 inhibitor (SGLT2i), mineralocorticoid receptor antagonist, and beta blocker. The ARNi and sodium-glucose cotransporter-2 inhibitor are newer additions to standard of care with the ARNi replacing ACE (angiotensin-converting enzyme) inhibitors and angiotensin II receptor blockers. METHODS: We investigate the cost-effectiveness of sequentially adding the SGLT2i and ARNi to form quadruple therapy as compared with the previous standard of care with ACE inhibitor/mineralocorticoid receptor antagonist/beta blocker. Using a 2-stage Markov model, we projected the expected lifetime discounted costs and quality-adjusted life years (QALYs) of a simulated cohort of US patients who underwent each treatment option and calculated incremental cost-effectiveness ratios. We assessed incremental cost-effectiveness ratios using criteria for health care value (<$50 000/quality-adjusted life year [QALY] indicating high-value, $50 000-150 000/QALY indicating intermediate value, and >$150 000/QALY indicating low-value) and a standard $100 000/QALY cost-effectiveness threshold. RESULTS: Compared with the previous standard of care, the SGLT2i addition had an incremental cost-effectiveness ratio of $73 000/QALY and weakly dominated the ARNi addition. The addition of both the ARNi and SGLT2i for quadruple therapy offered 0.68 additional discounted QALYs over the SGLT2i addition alone at a lifetime discounted cost of $66 700, resulting in an incremental cost-effectiveness ratio of $98 500/QALY. In sensitivity analysis varying drug prices, the incremental cost-effectiveness ratio for quadruple therapy ranged from $73 500/QALY using prices available to the US Department of Veterans Affairs to $110 000/QALY using drug list prices. CONCLUSIONS: While quadruple therapy offers intermediate value, it is borderline cost effective compared with adding the SGLT2i alone to previous standard of care. Thus, its cost-effectiveness is sensitive to a payer's ability to negotiate discounts off the increasing list prices for ARNI and SGLT2is. The demonstrated benefits of ARNi and SGLT2is should be weighed against their high prices in payer and policy considerations.

Systematic Review of the Economic Evaluation of Sodium-Glucose Cotransporter-2 Inhibitors Used as Treatment in Patients with Heart Failure.

BACKGROUND: Sodium-glucose cotransporter-2 (SGLT2) inhibitors have been recently used as therapeutic agents for type 2 diabetes mellitus. Recent clinical trials have shown that they are beneficial for reducing the risk of cardiovascular mortality and hospitalization in patients with heart failure (HF). A comprehensive review regarding the cost-effectiveness of different SGLT2 inhibitors for HF treatment may be necessary to help clinicians and decision-makers select the most cost-effective HF treatment option. OBJECTIVE: This study conducted a systematic review of economic evaluation studies of SGLT2 inhibitors for the treatment of patients with reduced ejection fraction (HFrEF) and preserved ejection fraction (HFpEF). METHOD: We searched PubMed, Cochrane, Embase, and EBSCOhost to identify published economic evaluation studies on SGLT2 inhibitors for HF treatment until May 2023. Studies on the economic evaluation of SGLT2 inhibitors in the treatment of HF were included. We extracted information such as country, population, intervention, type of model, health status, and conclusion of cost-effectiveness. RESULT: Of the 410 studies, 27 were finally selected. All economic evaluation studies used the Markov model, and commonly included health status as stable HF, hospitalization due to HF, and death. All dapagliflozin studies focused on patients with HFrEF (n = 13), and dapagliflozin was cost-effective in 14 countries, but not in the Philippines. All empagliflozin studies focused on the patients with HFrEF also showed the cost-effectiveness of empagliflozin (n = 11). However, empagliflozin use in patients with HFpEF was determined to be cost-effective in studies in Finland, China, and Australia studies but not in studies in Thailand and the USA. CONCLUSIONS: Most of the studies reported the cost-effectiveness of dapagliflozin and empagliflozin in patients with HFrEF. However, the cost-effectiveness of empagliflozin differed from country to country regarding patients with HFpEF. We suggest that further economic evaluation of SGLT2 inhibitors should focus on patients with HFpEF in more countries.

Design and Synthesis of 3-(ß-d-Glucopyranosyl)-4-amino/4-guanidino Pyrazole Derivatives and Analysis of Their Glycogen Phosphorylase Inhibitory Potential.

Glycogen phosphorylase (GP) is a key regulator of glucose levels and, with that, an important target for the discovery of novel treatments against type 2 diabetes. ß-d-Glucopyranosyl derivatives have provided some of the most potent GP inhibitors discovered to date. In this regard, C-ß-d-glucopyranosyl azole type inhibitors proved to be particularly effective, with 2- and 4-ß-d-glucopyranosyl imidazoles among the most potent designed to date. His377 backbone C=O hydrogen bonding and ion-ion interactions of the protonated imidazole with Asp283 from the 280s loop, stabilizing the inactive state, were proposed as crucial to the observed potencies. Towards further exploring these features, 4-amino-3-(ß-d-glucopyranosyl)-5-phenyl-1H-pyrazole (3) and 3-(ß-d-glucopyranosyl)-4-guanidino-5-phenyl-1H-pyrazole (4) were designed and synthesized with the potential to exploit similar interactions. Binding assay experiments against rabbit muscle GPb revealed 3 as a moderate inhibitor (IC50 = 565 µM), but 4 displayed no inhibition at 625 µM concentration. Towards understanding the observed inhibitions, docking and post-docking molecular mechanics-generalized Born surface area (MM-GBSA) binding free energy calculations were performed, together with Monte Carlo and density functional theory (DFT) calculations on the free unbound ligands. The computations revealed that while 3 was predicted to hydrogen bond with His377 C=O in its favoured tautomeric state, the interactions with Asp283 were not direct and there were no ion-ion interactions; for 4, the most stable tautomer did not have the His377 backbone C=O interaction and while ion-ion interactions and direct hydrogen bonding with Asp283 were predicted, the conformational strain and entropy loss of the ligand in the bound state was significant. The importance of consideration of tautomeric states and ligand strain for glucose analogues in the confined space of the catalytic site with the 280s loop in the closed position was highlighted.

Sodium butyrate potentiates insulin secretion from rat islets at the expense of compromised expression of ß cell identity genes.

Short-chain fatty acids (SCFAs) produced by the gut microbiota have been well demonstrated to improve metabolic homeostasis. However, the role of SCFAs in islet function remains controversial. In the present study, none of the sodium acetate, sodium propionate, and sodium butyrate (SB) displayed acute impacts on insulin secretion from rat islets, whereas long-term incubation of the three SCFAs significantly potentiated pancreatic ß cell function. RNA sequencing (RNA-seq) revealed an unusual transcriptome change in SB-treated rat islets, with the downregulation of insulin secretion pathway and ß cell identity genes, including Pdx1, MafA, NeuroD1, Gck, and Slc2a2. But these ß cell identity genes were not governed by the pan-HDAC inhibitor trichostatin A. Overlapping analysis of H3K27Ac ChIP-seq and RNA-seq showed that the inhibitory effect of SB on the expression of multiple ß cell identity genes was independent of H3K27Ac. SB treatment increased basal oxygen consumption rate (OCR), but attenuated glucose-stimulated OCR in rat islets, without altering the expressions of genes involved in glycolysis and tricarboxylic acid cycle. SB reduced the expression of Kcnj11 (encoding KATP channel) and elevated basal intracellular calcium concentration. On the other hand, SB elicited insulin gene expression in rat islets through increasing H3K18bu occupation in its promoter, without stimulating CREB phosphorylation. These findings indicate that SB potentiates islet function as a lipid molecule at the expense of compromised expression of islet ß cell identity genes.

Assessment of individual and standardized glucagon kinetics in healthy humans.

Impaired glucose tolerance arises out of impaired postprandial insulin secretion and delayed suppression of glucagon. These defects occur early and independently in the pathogenesis of prediabetes. Quantification of the contribution of α-cell dysfunction to glucose tolerance has been lacking because knowledge of glucagon kinetics in humans is limited. Therefore, in a series of experiments examining the interaction of glucagon suppression with insulin secretion we studied 51 nondiabetic subjects (age = 54 ± 13 yr, BMI = 28 ± 4 kg/m2). Glucose was infused to mimic the systemic appearance of an oral challenge. Somatostatin was used to inhibit endogenous hormone secretion. 120 min after the start of the experiment, glucagon was infused at 0.65 ng/kg/min. The rise in glucagon concentrations was used to estimate its kinetic parameters [volume of distribution (Vd), half-life (t1/2), and clearance rate (CL)]. A single-exponential model provided the best fit for the data, and individualized kinetic parameters were estimated: Vd = 8.2 ± 2.7 L, t1/2 = 4 ± 1.1 min, CL = 1.4 ± 0.33 L/min. Stepwise linear regression was used to correlate Vd with BMI and sex (R2adj = 0.44), whereas CL similarly correlated with lean body mass or BSA (both R2 = 0.28). This enabled the development of a population-based model using anthropometric characteristics to predict Vd and CL. These data demonstrate that it is feasible to derive glucagon kinetic parameters from anthropometric characteristics, thereby enabling quantitation of the rate of glucagon appearance in the systemic circulation in large populations.NEW & NOTEWORTHY State-of-the-art measurement of insulin secretion in humans is accomplished by deconvolution of peripheral C-peptide concentrations using population-derived parameters of C-peptide kinetics. In contrast, knowledge of the kinetic parameters of glucagon in humans is lacking so that measurement of glucagon secretion to date is largely qualitative. This series of experiments enabled measurement of glucagon kinetics in 51 subjects, and subsequently, stepwise linear regression was used to correlate these parameters with anthropometric characteristics. This enabled the development of a population-based model using anthropometric characteristics to predict the volume of distribution and the rate of clearance. This is a necessary first step in the development of a model to quantitate of glucagon secretion and action (and its contribution to glucose tolerance) in large populations.

Noninvasive and in vivo assessment of upper and lower limb skeletal muscle oxidative metabolism activity and microvascular responses to glucose ingestion in humans.

This study investigated changes in muscle oxidative metabolism and microvascular responsiveness induced by glucose ingestion in the upper and lower limbs using near-infrared spectroscopy (NIRS). Fourteen individuals (aged 27 ± 1.4 years) underwent 5 vascular occlusion tests (VOT) (pre-intervention (Pre), 30 min, 60 min, 90 min, and 120 min after glucose challenge). NIRS-derived oxygen saturation (StO2) was measured on the forearm and leg muscle at each VOT. Muscle oxidative metabolism was determined by the StO2 downslope during cuff inflation (deoxygenation slope); microvascular responsiveness was estimated by the StO2 upslope (reperfusion slope) following cuff deflation. There was a significant increase in arm (p < 0.05; 1-ß = 0.860) and leg (p < 0.05; 1-ß = 1.000) oxidative metabolism activity as represented by the faster deoxygenation slope at 60, 90, and 120 min (0.08 ± 0.03, 0.08 ± 0.03, 0.08 ± 0.02%·s-1, respectively) (leg) and at 90 min (0.16 ± 0.08%·s-1) (arm) observed after glucose ingestion when compared with their respective Pre values (leg = 0.06 ± 0.02; arm = 0.11 ± 0.04%·s-1). There was a significant increase in arm (p < 0.05; 1-ß = 0.880) and leg (p < 0.05; 1-ß = 0.983) reperfusion slope at 60 min (arm = 3.63 ± 2.1%·s-1; leg = 1.56 ± 0.6%·s-1), 90 min (arm = 3.91 ± 2.1%·s-1; leg = 1.60 ± 0.6%·s-1), and 120 min (arm = 3.91 ± 1.6%·s-1; leg = 1.54 ± 0.6%·s-1) when compared with their Pre values (arm = 2.79 ± 1.7%·s-1; leg = 1.26 ± 0.5%·s-1). Our findings showed that NIRS-VOT technique is capable of detecting postprandial changes in muscle oxidative metabolism activity and microvascular reactivity in the upper and lower limb. Novelty NIRS-VOT is a promising noninvasive clinical approach that may help in the early, limb-specific detection of impairments in glucose oxidation and microvascular function.

Continuous Succinic Acid Fermentation by Actinobacillus Succinogenes: Assessment of Growth and Succinic Acid Production Kinetics.

Succinic acid is one of the most interesting platform chemicals that can be produced in a biorefinery approach. The paper reports the characterization of the growth kinetics of Actinobacillus succinogenes DSM 22257 using glucose as carbon source. Tests were carried out in a continuous bioreactor operated under controlled pH. Under steady-state conditions, the conversion process was characterized in terms of concentration of glucose, cells, acids, and pH. The effects of acid-succinic, acetic, and formic-concentration in the medium on fermentation performance were investigated. The fermentation was interpreted according to several models characterized by substrate and product inhibition. The selected kinetic model of biomass growth and of metabolite production described the microorganism growth rate under a broad interval of operating conditions. Under the investigated operating conditions, results pointed out that: no substrate inhibition was observed; acetic acid did not inhibit the cell growth and succinic acid production.

Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti-diabetic agents.

Temporin A (FLPLIGRVLSGIL-NH2 ), temporin F (FLPLIGKVLSGIL-NH2 ), and temporin G (FFPVIGRILNGIL-NH2 ), first identified in skin secretions of the frog Rana temporaria, produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal ß-cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 µM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca2+ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration-dependent stimulation of insulin release from 1.1B4 human-derived pancreatic ß-cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 µM), but not temporin G, protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon-like peptide-1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.

Effects of glucose, lactate and basic FGF as limiting factors on the expansion of human induced pluripotent stem cells.

Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses showed that reducing medium change frequency increased differentiation marker expressions but high glucose concentration downregulated these expressions. In contrast, heparan sulfate did not prevent differentiation marker expressions. According to analyses of growth rate, cell growth with extended medium change interval was decreased in later stage of log growth phase despite the existence of high glucose concentration and heparan sulfate. This result and culturing iPSCs with lactate showed that the accumulation of excreted lactate decreased the growth rate regardless of pH control. Conclusively, these experiments show that adding glucose and removing lactate are important to expand iPSCs with reduced culture medium usage. This knowledge should be useful to design economical iPSC mass production and differentiation system.

Assessment of ß Cell Mass and Function by AIRmax and Intravenous Glucose in High-Risk Subjects for Type 1 Diabetes.

Context: There is little information regarding ß cell mass in individuals at early stages of type 1 diabetes (T1D). Objective: To investigate both acute insulin response to arginine at hyperglycemia (AIRmax), as a correlate of ß cell mass, and ß cell function by the intravenous glucose tolerance test (IVGTT) in subjects at early stages of T1D. Design/Setting/Participants: Forty subjects were enrolled: (1) low-risk group: relatives of patients with T1D with 0 to 1 antibody (n = 21) and (2) high-risk group: relatives with ≥2 antibodies (n = 19). Main Outcome Measure: Acute insulin and C-peptide responses to IVGTT and to AIRmax. Participants underwent two IVGTT and AIRmax procedures on different days. Results: AIRmax was reproducible, well tolerated, and correlated to first-phase insulin response (FPIR) from IVGTT (r = 0.779). The high-risk group had greater impaired ß cell function compared with the low-risk group, determined both by lower mean FPIR and a greater number of subjects below an established threshold for abnormal function [10 of 19 (52.6%) versus 4 of 21 (19%)]. There was a heterogeneous AIRmax response in these subjects with low FPIR, ranging from 38 to 250 µU/mL. Conclusions: There is significant variation in insulin secretory reserve as assessed by AIRmax in family members with low ß cell function assessed by FPIR. As AIRmax is a functional measure of ß cell mass, these data suggest heterogeneity in disease pathogenesis in which mass is preserved in relation to function in some individuals. The tolerability and reproducibility of AIRmax suggest it could be a useful stratification measure in clinical trials of disease-modifying therapy.

Improved yield of canine islet isolation from deceased donors.

BACKGROUND: Canine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes. RESULTS: Here, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group). CONCLUSIONS: In summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.

Proteomic Assessment of the Expression of Genes Related to Toluene Catabolism and Porin Synthesis in Pseudomonas stutzeri ST-9.

The organization and expression of Pseudomonas stutzeri ST-9 genes related to toluene catabolism and porin synthesis was investigated. Toluene-degrading genes were found to be localized in the chromosome close to a phage-type integrase. A regulatory gene and 21 genes related to an aromatics degradation pathway are organized as a putative operon. These proteins are upregulated in the presence of toluene. Fourteen outer membrane proteins were identified as porins in the ST-9 genome. The identified porins showed that the main detected porins are related to the OmpA and OprD superfamilies. The percentage of porins in the outer membrane protein fraction, as determined by mass spectrometry, was 73% and 54% when the cells were cultured with toluene and with glucose, respectively. Upregulation of OmpA and downregulation of OprD occurred in the presence of toluene. A porin fraction (90% OprD) from both cultures was isolated and examined as a toluene uptake system using the liposome-swelling assay. Liposomes were prepared with the porin fraction from a culture that was grown on toluene (T-proteoliposome) or glucose (G-proteoliposome). There was no significant difference in the permeability rate of the different solutes through the T-proteoliposome and the G-proteoliposome.

Glucose challenge test screening for prediabetes and early diabetes.

AIMS: To test the hypothesis that a 50-g oral glucose challenge test with 1-h glucose measurement would have superior performance compared with other opportunistic screening methods. METHODS: In this prospective study in a Veterans Health Administration primary care clinic, the following test performances, measured by area under receiver-operating characteristic curves, were compared: 50-g oral glucose challenge test; random glucose; and HbA1c level, using a 75-g oral glucose tolerance test as the 'gold standard'. RESULTS: The study population was comprised of 1535 people (mean age 56 years, BMI 30.3 kg/m2 , 94% men, 74% black). By oral glucose tolerance test criteria, diabetes was present in 10% and high-risk prediabetes was present in 22% of participants. The plasma glucose challenge test provided area under receiver-operating characteristic curves of 0.85 (95% CI 0.78-0.91) to detect diabetes and 0.76 (95% CI 0.72-0.80) to detect high-risk dysglycaemia (diabetes or high-risk prediabetes), while area under receiver-operating characteristic curves for the capillary glucose challenge test were 0.82 (95% CI 0.75-0.89) and 0.73 (95% CI 0.69-0.77) for diabetes and high-risk dysglycaemia, respectively. Random glucose performed less well [plasma: 0.76 (95% CI 0.69-0.82) and 0.66 (95% CI 0.62-0.71), respectively; capillary: 0.72 (95% CI 0.65-0.80) and 0.64 (95% CI 0.59-0.68), respectively], and HbA1c performed even less well [0.67 (95% CI 0.57-0.76) and 0.63 (95% CI 0.58-0.68), respectively]. The cost of identifying one case of high-risk dysglycaemia with a plasma glucose challenge test would be $42 from a Veterans Health Administration perspective, and $55 from a US Medicare perspective. CONCLUSIONS: Glucose challenge test screening, followed, if abnormal, by an oral glucose tolerance test, would be convenient and more accurate than other opportunistic tests. Use of glucose challenge test screening could improve management by permitting earlier therapy.

Economic 3D-printing approach for transplantation of human stem cell-derived ß-like cells.

Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing ß cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived ß (SC-ß) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-ß cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-ß cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies.

Eight-Hour Continuous Normothermic Ex Vivo Kidney Perfusion Is a Safe Preservation Technique for Kidney Transplantation: A New Opportunity for the Storage, Assessment, and Repair of Kidney Grafts.

BACKGROUND: Hypothermic kidney storage causes preservation injury and is poorly tolerated by renal grafts. We investigated whether static cold storage (SCS) can be safely replaced with a novel technique of pressure-controlled normothermic ex vivo kidney perfusion (NEVKP) in heart-beating donor kidney transplantation. METHODS: Right kidneys were removed from 30 kg Yorkshire pigs in a model of heart-beating donation and either preserved in cold histidine-tryptophan-ketoglutarate solution for 8 hours (n = 5), or subjected to 8 hours of pressure-controlled NEVKP (n = 5) followed by renal heterotopic autotransplantation. RESULTS: During NEVKP, physiologic perfusion conditions were maintained with low intrarenal resistance and normal electrolyte and pH parameters. Aspartate aminotransferase and lactate dehydrogenase as injury markers were below the detectable analyzer range (<4 and <100 U/L, respectively). Perfusate lactate concentration decreased from baseline until the end of perfusion (10.38 ± 0.76 mmol/L vs 1.22 ± 0.26 mmol/L; P < 0.001). Posttransplantation, animals transplanted with NEVKP versus SCS grafts demonstrated similar serum creatinine peak levels (NEVKP, 2.0 ± 0.5 vs SCS 2.7 ± 0.7 mg/dL; P = 0.11) and creatinine clearance on day 10 (NEVKP, 65.9 ± 18.8 mL/min vs SCS 61.2 ± 15.6 mL/min; P = 0.74). After 10 days of follow-up, animals transplanted with NEVKP grafts had serum creatinine and blood urea nitrogen values comparable to their basal levels (P = 0.49 and P = 0.59), whereas animals transplanted with SCS grafts had persistently elevated serum creatinine and blood urea nitrogen when compared with basal levels (P = 0.01 and P = 0.03). CONCLUSIONS: Continuous pressure-controlled NEVKP is feasible and safe in good quality heart-beating donor kidney grafts. It maintains a physiologic environment and excellent graft function ex vivo during preservation without causing graft injury.

Highly efficient rice straw utilization for poly-(γ-glutamic acid) production by Bacillus subtilis NX-2.

Lignocellulosic biomass has been identified as an economic and environmental feedstock for future biotechnological production. Here, for the first time, poly-(γ-glutamic acid) (PGA) production by Bacillus subtilis NX-2 using rice straw is investigated. Based on two-stage hydrolysis and characteristic consumption of xylose and glucose by B. subtilis NX-2, a co-fermentation strategy was designed to better accumulate PGA in a 7.5L fermentor by two feeding methods. The maximum cumulative respective PGA production and PGA productivity were 73.0 ± 0.5 g L(-1) and 0.81 g L(-1) h(-1) by the continuous feeding method, with carbon source cost was saved by 84.2% and 42.5% compared with glucose and cane molasse, respectively. These results suggest that rice straw, a type of abundant, low-cost, non-food lignocellulosic feedstock, may be feasibly and efficiently utilized for industrial-scale production of PGA.

Assessment of metabolic fluxes in the mouse brain in vivo using 1H-[13C] NMR spectroscopy at 14.1 Tesla.

(13)C magnetic resonance spectroscopy (MRS) combined with the administration of (13)C labeled substrates uniquely allows to measure metabolic fluxes in vivo in the brain of humans and rats. The extension to mouse models may provide exclusive prospect for the investigation of models of human diseases. In the present study, the short-echo-time (TE) full-sensitivity (1)H-[(13)C] MRS sequence combined with high magnetic field (14.1 T) and infusion of [U-(13)C6] glucose was used to enhance the experimental sensitivity in vivo in the mouse brain and the (13)C turnover curves of glutamate C4, glutamine C4, glutamate+glutamine C3, aspartate C2, lactate C3, alanine C3, γ-aminobutyric acid C2, C3 and C4 were obtained. A one-compartment model was used to fit (13)C turnover curves and resulted in values of metabolic fluxes including the tricarboxylic acid (TCA) cycle flux VTCA (1.05 ± 0.04 µmol/g per minute), the exchange flux between 2-oxoglutarate and glutamate Vx (0.48 ± 0.02 µmol/g per minute), the glutamate-glutamine exchange rate V(gln) (0.20 ± 0.02 µmol/g per minute), the pyruvate dilution factor K(dil) (0.82 ± 0.01), and the ratio for the lactate conversion rate and the alanine conversion rate V(Lac)/V(Ala) (10 ± 2). This study opens the prospect of studying transgenic mouse models of brain pathologies.

Exposure to L-cycloserine incurs survival costs and behavioral alterations in Aedes aegypti females.

BACKGROUND: It was previously demonstrated that alanine aminotransferase (ALAT, EC 2.6.1.2) participates in maintaining the alanine-proline cycle between flight muscles and fat body during Aedes aegypti flight. ALAT is also actively involved in the metabolism of ammonia in A. aegypti. Here, we investigated the survival and behavioral costs of ALAT inhibition in A. aegypti females to better understand the role of ALAT in blood-fed mosquitoes. METHODS: We analyzed how A. aegypti female mosquitoes respond to blood meals supplemented with 0, 2.5, 5 and 10 mM L-cycloserine, a well-known inhibitor of ALAT in animals. Mosquitoes were also exposed to blood meals supplemented with L-cycloserine and different concentrations of glucose (0, 10 and 100 mM). Additionally, the effects of ALAT inhibitor and glucose in mosquitoes starved for 24 or 48 h were investigated. Survival and behavioral phenotypes were analyzed during a time course (1, 2, 4, 6, 12, 24, 48 and 72 h after feeding). RESULTS: L-cycloserine at 10 mM resulted in high mortality relative to control, with an acute effect during the first 6 h after treatment. A significant decrease in the number of active mosquitoes coinciding with an increase in futile wing fanning during the first 24 h was observed at all inhibitor concentrations. A high occurrence of knockdown phenotype was also recorded at this time for both 5 and 10 mM L-cycloserine. The supplementation of glucose in the blood meal amplified the effects of the ALAT inhibitor. In particular, we observed a higher mortality rate concomitant with an increase in the knockdown phenotype. Starvation prior to blood feeding also increased the effects of L-cycloserine with a rapid increase in mortality. CONCLUSIONS: Our results provide evidence that exposure of high doses of L-cycloserine during A. aegypti blood feeding affects mosquito survival and motor activity, suggesting an interference with carbohydrate and ammonia metabolism in a time-dependent manner.