Performance Evaluation of Selenite (SeO32−) Reduction by Enterococcus spp.

Lactic acid bacteria (LAB) such as Enterococcus spp. have an advantage over several bacteria because of their ability to easily adapt to extreme conditions which include high temperatures, highly acidic or alkaline conditions and toxic metals. Although many microorganisms have been shown to reduce selenite (SeO32−) to elemental selenium (Se0), not much work has been done on the combined effect of Enterococcus spp. In this study, aerobic batch reduction of different selenite concentrations (1, 3 and 5 mM) was conducted using Enterococcus hermanniensis sp. and Enterococcus gallinarum sp. (3.5 h, 35 ± 2 °C, starting pH > 8.5). Results from the experiments showed that the average reductions rates were 0.608, 1.921 and 3.238 mmol·(L·h)−1, for the 1, 3 and 5 mM SeO32− concentrations respectively. In addition, more selenite was reduced for the 5 mM concentration compared to the 1 and 3 mM concentrations albeit constant biomass being used for all experiments. Other parameters which were monitored were the glucose consumption rate, protein variation, pH and ORP (oxidation reduction potential). TEM analysis was also conducted and it showed the location of electron-dense selenium nanoparticles (SeNPs). From the results obtained in this study, the authors concluded that Enterococcus species’s high adaptability makes it suitable for rapid selenium reduction and biosynthesis of elemental selenium.

Liver-Specific Kisspeptin Deletion Impairs Energy Metabolism in Mice

Kisspeptin, a neuroendocrine protein critical for the control of pubertal development and fertility has been shown to be modulated by nutritional signals. While the secretion of kisspeptin from specific hypothalamic nuclei is well-known to regulate GnRH-mediated pubertal maturation and reproduction, it remains unclear what role peripheral kisspeptin, specifically of hepatic origin, plays in regulating metabolism and glucose homeostasis. To define the role of kisspeptin in the liver, we developed a novel Kiss1f/f mouse line and targeted liver-specific Kiss1 ablation by injecting a AAV8-TBG-iCre virus via the tail vein (LKiss1KO). Control mice included Kiss1f/f male and female mice injected with AAV-GFP (LKiss1WT). We previously showed that deletion of hepatic kisspeptin did not affect body weight, but resulted in decreased insulin secretion and glucose intolerance in both sexes. To clarify the effects of liver-specific Kiss1 knockout on insulin action and glucose homeostasis in vivo, we conducted hyperinsulinemic-euglycemic clamp studies three weeks after tail injections. We noted a sexual dimorphism in the glucose infusion rate (GIR), female mice have a higher GIR to maintain euglycemia associated with an elevated glucose consumption rate, suggesting that female mice are more insulin sensitive than male mice. However, the deletion of liver kisspeptin had no effect on the glucose production rate in either sex. Indirect calorimetry assessment was conducted 4 weeks post-injection. Both male and female LKiss1KO mice showed significantly higher oxygen consumption, carbon dioxide production, and increased energy expenditure as compared to the LKiss1WT groups. However, there were no differences in either the respiratory exchange ratio or total ambulatory activity among treatments. These findings clearly define a pivotal role for hepatic Kiss1 in the modulation of insulin secretion to maintain glucose homeostasis without modulating glucose production as well as in maintaining energy homeostasis in both male or female mice.

Inactivation of the Pta-AckA Pathway Impairs Fitness of Bacillus anthracis during Overflow Metabolism

ABSTRACT Under conditions of glucose excess, aerobically growing bacteria predominantly direct carbon flux toward acetate fermentation, a phenomenon known as overflow metabolism or the bacterial “Crabtree effect.” Numerous studies of the major acetate-generating pathway, the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway, have revealed its important role in bacterial fitness through the control of central metabolism to sustain balanced growth and cellular homeostasis. In this work, we highlight the contribution of the Pta-AckA pathway to the fitness of the spore-forming bacterium Bacillus anthracis. We demonstrate that disruption of the Pta-AckA pathway causes drastic growth reduction in the mutants and alters the metabolic and energy status of the cells. Our results revealed that inactivation of the Pta-AckA pathway increases the glucose consumption rate, affects intracellular ATP, NAD+, and NADH levels, and leads to a metabolic block at the pyruvate and acetyl coenzyme A (acetyl-CoA) nodes. Consequently, accumulation of intracellular acetyl-CoA and pyruvate forces bacteria to direct carbon into the tricarboxylic acid and/or glyoxylate cycles, as well as fatty acid and poly(3‐hydroxybutyrate) biosynthesis pathways. Notably, the presence of phosphotransbutyrylase (Ptb) in B. anthracis partially compensates for the loss of Pta activity. Furthermore, overexpression of the ptb gene not only eliminates the negative impact of the pta mutation on B. anthracis fitness but also restores normal growth in the pta mutant of the non-butyrate-producing bacterium Staphylococcus aureus. Taken together, the results of this study demonstrate the importance of the Pta-AckA pathway for B. anthracis fitness by revealing its critical contribution to the maintenance of metabolic homeostasis during aerobic growth under conditions of carbon overflow. IMPORTANCE B. anthracis, the etiological agent of anthrax, is a highly pathogenic, spore-forming bacterium that causes acute, life-threatening disease in both humans and livestock. A greater understanding of the metabolic determinants governing the fitness of B. anthracis is essential for the development of successful therapeutic and vaccination strategies aimed at lessening the potential impact of this important biodefense pathogen. This study is the first to demonstrate the vital role of the Pta-AckA pathway in preserving energy and metabolic homeostasis in B. anthracis under conditions of carbon overflow, thus highlighting this pathway as a potential therapeutic target for drug discovery. Overall, the results of this study provide important insights into the metabolic processes and requirements driving rapid B. anthracis proliferation during vegetative growth.

An Investigation into the Metabolic Profiles of Human Tconvs and Tregs Identifies Divergent Metabolic Characteristics in Effector Memory Subsets

Hisashi Hashimoto, Fadi Issa, Joanna Hester Introduction Conventional T cells (Tconvs) are known to switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis upon activation. In contrast, studies of mouse in vitro-induced regulatory T cells (Tregs) have revealed an overriding preference for OXPHOS. However, the metabolic preferences of human Tregs remain unclear and warrant further investigation in order to help identify therapeutic opportunities. Methods Human CD4+ Tconvs and CD4+CD25+CD127lo Tregs were flow sorted from healthy human donors and stimulated with anti-CD3/anti-CD28 beads or left unstimulated. In some experiments, Tconvs and Tregs were sorted into naive, central memory (CM) and effector memory (EM) subsets. Aerobic glycolysis and oxygen consumption rates were investigated using Seahorse XF. Cells were also assessed for their glucose consumption rate using the fluorescent glucose analogue 2-NBDG and Glut1 surface staining. Mitochondrial membrane potential and mass were assessed using mitotrackers and Mito-ID by flow cytometry. Results In contrast to mouse Tregs, human Tregs switch to glycolysis upon activation. While Tregs appeared to mirror the metabolic profile of Tconvs, there were differences with specific subsets. Activated EM Tregs displayed the lowest glucose consumption rate, whereas their counterpart subset in Tconvs had the highest glucose consumption rate among all subsets. Assessment of mitochondrial function revealed that all subsets of Tregs maintain a high number of polarized mitochondria, whereas memory subsets of Tconvs had a comparatively lower number of mitochondria, particularly in the EM population. Conclusions This study provides an in depth characterisation of the metabolic profile of human Tregs. These data shed light on potential differences in EM metabolic activity between Tregs and Tconvs that could be exploited for therapeutic targeting.

Choline-Based Ionic Liquids as Media for the Growth of Saccharomyces cerevisiae

Ionic liquids (ILs) have garnered great attention as alternative solvents in many biological reactions and applications. However, its unknown toxicity is in line with the challenges to use it for biological applications. In this study, three choline based Ionic Liquids—choline saccharinate (CS), choline dihydrogen phosphate (CDHP), and choline tryptophanate (CT) were assessed for their suitability on the growth of Saccharomyces cerevisiae. The ILs were incorporated into the growth media of S. cerevisiae (defined as synthetic media) to access its potential as a substitute to conventional media. The compatibility of the synthetic media was evaluated based on the toxicity (EC50), growth curve, and glucose profile. The results showed that the incorporation of CDHP and CS did promote the growth of S. cerevisiae with a rapid glucose consumption rate. The growth of S. cerevisiae with the media composition of yeast extract, peptone, and CS showed improvement of 13%. We believe that these observations have implications in the biocompatibility studies of ILs to microorganisms.

Enhancer Reprogramming Confers Dependence on Glycolysis and IGF signaling in KMT2D Mutant Melanoma

SUMMARYEpigenetic modifiers have emerged as important regulators of tumor progression. We identified histone methyltransferase KMT2D as a potent tumor-suppressor through an in vivo epigenome-focused pooled RNAi screen in melanoma. KMT2D harbors frequent somatic point mutations in multiple tumor types. How these events contribute to tumorigenesis and whether they impart therapeutic vulnerability are poorly understood. To address these questions, we generated a genetically engineered mouse model of melanoma based on conditional and melanocyte-specific deletion of KMT2D. We demonstrate KMT2D as a bona fide tumor suppressor which cooperates with activated BRAF. KMT2D-deficient tumors showed substantial reprogramming of key metabolic pathways including glycolysis. Glycolysis enzymes, intermediate metabolites and glucose consumption rate were aberrantly upregulated in KMT2D mutant cells. The pharmacological inhibition of glycolysis reduced proliferation and tumorigenesis preferentially in KMT2D mutant cells. Mechanistically, KMT2D loss caused drastic reduction of H3K4me1-marked active enhancer states. Loss of distal enhancer and subsequent reduction in expression of IGFBP5 activated IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating increased usage of glycolysis pathway for enhanced biomass needs via enhancer reprogramming. Our data imply that inhibition of glycolysis or IGFR pathway could be a potential therapeutic strategy in KMT2D mutant tumors.

Synthesis of a thin-layer gelatin nanofiber mat for cultivating retinal cell

Thin-layer gelatin nanofiber mats were fabricated as a biodegradable scaffold for proliferating human retinal pigment epithelium. Together with MTT assay, the glucose consumption rate, lactate formation, and lactate dehydrogenase activity of the human retinal pigment epithelium cells—on the gelatin nanofibers—were analyzed as indicators for cell growth and viability. The results showed that gelatin nanofiber did not make any toxic effect on the cells and the growth rate was comparable to the tissue culture plates. Using the fabricated thin-layer nanofibers let the by-product to leave which in turn cause less adverse effect on the cells. The biodegradability and stability of the gelatin nanofibers were optimized as a function of reaction time.

Enhanced Chondrocyte Proliferation in a Prototyped Culture System with Wave-Induced Agitation

One of the actual challenges in tissue engineering applications is to efficiently produce as high of number of cells as it is only possible, in the shortest time. In static cultures, the production of animal cell biomass in integrated forms (i.e. aggregates, inoculated scaffolds) is limited due to inefficient diffusion of culture medium components observed in such non-mixed culture systems, especially in the case of cell-inoculated fiber-based dense 3D scaffolds, inside which the intensification of mass transfer is particularly important. The applicability of a prototyped, small-scale, continuously wave-induced agitated system for intensification of anchorage-dependent CP5 chondrocytes proliferation outside and inside three-dimensional poly(lactic acid) (PLA) scaffolds has been discussed. Fibrous PLA-based constructs have been inoculated with CP5 cells and then maintained in two independent incubation systems: (i) non-agitated conditions and (ii) culture with wave-induced agitation. Significantly higher values of the volumetric glucose consumption rate have been noted for the system with the wave-induced agitation. The advantage of the presented wave-induced agitation culture system has been confirmed by lower activity of lactate dehydrogenase (LDH) released from the cells in the samples of culture medium harvested from the agitated cultures, in contrast to rather high values of LDH activity measured for static conditions. Results of the proceeded experiments and their analysis clearly exhibited the feasibility of the culture system supported with continuously wave-induced agitation for robust proliferation of the CP5 chondrocytes on PLA-based structures. Aside from the practicability of the prototyped system, we believe that it could also be applied as a standard method offering advantages for all types of the daily routine laboratory-scale animal cell cultures utilizing various fiber-based biomaterials, with the use of only regular laboratory devices.

Quantitative assessment of brain glucose metabolic rates using in vivo deuterium magnetic resonance spectroscopy

Quantitative assessment of cerebral glucose consumption rate (CMRglc) and tricarboxylic acid cycle flux (VTCA) is crucial for understanding neuroenergetics under physiopathological conditions. In this study, we report a novel in vivo Deuterium (2H) MRS (DMRS) approach for simultaneously measuring and quantifying CMRglc and VTCA in rat brains at 16.4 Tesla. Following a brief infusion of deuterated glucose, dynamic changes of isotope-labeled glucose, glutamate/glutamine (Glx) and water contents in the brain can be robustly monitored from their well-resolved 2H resonances. Dynamic DMRS glucose and Glx data were employed to determine CMRglc and VTCA concurrently. To test the sensitivity of this method in response to altered glucose metabolism, two brain conditions with different anesthetics were investigated. Increased CMRglc (0.46 vs. 0.28 µmol/g/min) and VTCA (0.96 vs. 0.6 µmol/g/min) were found in rats under morphine as compared to deeper anesthesia using 2% isoflurane. This study demonstrates the feasibility and new utility of the in vivo DMRS approach to assess cerebral glucose metabolic rates at high/ultrahigh field. It provides an alternative MRS tool for in vivo study of metabolic coupling relationship between aerobic and anaerobic glucose metabolisms in brain under physiopathological states.