Staphylococcus aureus is a facultative anaerobe that can generate energy through oxidative phosphorylation or solely glycolysis, and inhibiting oxidative phosphorylation results in the formation of small colony variants (SCVs). SCVs lack a proton motive force (PMF), increasing antibiotic tolerance and contributing to persistent infection in the host. Bicarbonate is an abundant antimicrobial compound in the human host that S. aureus encounters during infection. Bicarbonate alters the PMF, enhancing antibiotic susceptibility in S. aureus, but its impact on S. aureus SCVs remains unexplored. We report that bicarbonate inhibits the growth of S. aureus SCVs at concentrations that do not affect S. aureus wild type, due to defective bicarbonate anaplerotic metabolism, resulting in increased cytoplasmic pH and alkaline toxicity. Inactivation of pyruvate carboxylase (Pyc), a critical enzyme in bicarbonate anaplerotic metabolism that combines bicarbonate and pyruvate to form oxaloacetate, increases bicarbonate sensitivity in S. aureus, indicating that bicarbonate anaplerotic metabolism plays a vital role in bicarbonate detoxification. While SCVs upregulate Pyc in response to bicarbonate, cellular pyruvate levels are insufficient to sustain bicarbonate anaplerotic metabolism. Exogenous pyruvate restores bicarbonate anaplerotic metabolism and lowers the cytoplasmic pH, protecting SCVs from bicarbonate toxicity. Cytoplasmic pH alterations by bicarbonate also resensitize SCVs to aminoglycosides. S. aureus treated with bicarbonate is more susceptible to neutrophil killing, indicating that bicarbonate decreases the virulence of S. aureus. This study identifies bicarbonate anaplerotic metabolism as a S. aureus detoxification mechanism for bicarbonate toxicity and demonstrates that modulating anaplerotic metabolism may be an effective treatment for S. aureus infections. Staphylococcus aureus is one of the major bacterial contributors to human deaths around the world. Metabolic flexibility allows S. aureus to alter energy generation and resist oxidative and antibiotic killing, facilitating persistence in the host. Bicarbonate has been used for over a century for cleaning and hygiene without completely understanding its antimicrobial properties. We report that small colony variants (SCVs) are defective for bicarbonate anaplerotic metabolism, which is required to detoxify bicarbonate. As a result, bicarbonate inhibits the growth of SCVs by alkalinizing the cytoplasm. Cytoplasmic alkalinization also resensitizes SCVs to aminoglycoside killing, implicating bicarbonate as an effective antimicrobial adjuvant for treating glycolytic S. aureus. Our study defines the impacts of bicarbonate on the growth of SCVs and the metabolic pathways involved in detoxification, indicating that bicarbonate could be effective at controlling chronic S. aureus infections.

The alpha-ketoglutarate dehydrogenase complex (KGDHc), also known as the 2-oxoglutarate dehydrogenase complex, plays a crucial role in oxidative metabolism. It catalyzes a key step in the tricarboxylic acid (TCA) cycle, producing NADH (primarily for oxidative phosphorylation) and succinyl-CoA (for substrate-level phosphorylation, among others). Additionally, KGDHc is also capable of generating reactive oxygen species, which contribute to mitochondrial oxidative stress. Hence, the KGDHc and its dysfunction are implicated in various pathological conditions, including selected neurodegenerative diseases. The pathological roles of KGDHc in these diseases are generally still obscure. The aim of this study was to assess whether the mitochondrial malfunctions observed in the dihydrolipoamide succinyltransferase (DLST) and dihydrolipoamide dehydrogenase (DLD) double-heterozygous knockout (DLST+/-DLD+/-, DKO) mice are associated with neuronal and/or metabolic abnormalities. In the DKO animals, the mitochondrial O2 consumption and ATP production rates both decreased in a substrate-specific manner. Reduced H2O2 production was also observed, either due to Complex I inhibition with α-ketoglutarate or reverse electron transfer with succinate, which is significant in ischaemia-reperfusion injury. Middle-aged DKO mice exhibited minor cognitive decline, associated with microgliosis in the cerebral cortex and neuronal death in the Cornu Ammonis subfield 1 (CA1) of the hippocampus, indicating neuroinflammation. This was supported by increased levels of dynamin-related protein 1 (Drp1) and reduced levels of mitofusin 2 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in DKO mice. Observations on activity, food and oxygen consumption, and blood amino acid and acylcarnitine profiles revealed no significant differences. However, middle-aged DKO animals showed decreased performance in the treadmill fatigue-endurance test as compared to wild-type animals, accompanied by subtle resting cardiac impairment, but not skeletal muscle fibrosis. In conclusion, DKO animals compensate well the double-heterozygous knockout condition at the whole-body level with no major phenotypic changes under resting physiological conditions. However, under high energy demand, middle-aged DKO mice exhibited reduced performance, suggesting a decline in metabolic compensation. Additionally, microgliosis, neuronal death, decreased mitochondrial biogenesis, and altered mitochondrial dynamics were observed in DKO animals, resulting in minor cognitive decline. This is the first study to highlight the in vivo changes of this combined genetic modification. It demonstrates that unlike single knockout rodents, double knockout mice exhibit phenotypical alterations that worsen under stress situations.

Early identification and multidisciplinary management of complex conditions such as COXPD-38 are crucial for optimizing outcomes in pediatric patients. Ongoing monitoring of metabolic status, developmental progress, and nutritional needs is essential for supporting growth and improving quality of life.

Congenital heart disease (CHD) is the most common type of birth defect and the main noninfectious cause of death during the neonatal stage. The non-POU domain containing, octamer-binding gene, NONO, performs a variety of roles involved in DNA repair, RNA synthesis, transcriptional and post-transcriptional regulation. Currently, hemizygous loss-of-function mutation of NONO have been described as the genetic origin of CHD. However, essential effects of NONO during cardiac development have not been fully elucidated. In this study, we aim to understand role of Nono in cardiomyocytes during development by utilizing the CRISPR/Cas9 gene editing system to deplete Nono in the rat cardiomyocytes H9c2. Functional comparison of H9c2 control and knockout cells showed that Nono deficiency suppressed cell proliferation and adhesion. Furthermore, Nono depletion significantly affected the mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, resulting in H9c2 overall metabolic deficits. Mechanistically we demonstrated that the Nono knockout impeded the cardiomyocyte function by attenuating phosphatidyl inositol 3 kinase-serine/threonine kinase (Pi3k/Akt) signaling via the assay for transposase-accessible chromatin using sequencing in combination with RNA sequencing. From these results we propose a novel molecular mechanism of Nono to influence cardiomyocytes differentiation and proliferation during the development of embryonic heart. We conclude that NONO may represent an emerging possible biomarkers and targets for the diagnosis and treatment of human cardiac development defects.

A recent epigenome-wide association study of genes associated with type 2 diabetics (T2D), used integrative cross-omics analysis to identify 22 abnormally methylated CpG sites associated with insulin and glucose metabolism. Here, in this epigenetic analysis we preliminarily determine whether the same CpG sites identified in T2D also apply to type 1 diabetes (T1D). We then determine whether BCG vaccination could correct the abnormal methylation patterns, considering that the two diseases share metabolic derangements. T1D (n = 13) and control (n = 8) subjects were studied at baseline and then T1D subjects studied yearly for 3 years after receiving BCG vaccinations in a clinical trial. In this biomarker analysis, methylation patterns were evaluated on CD4+ T-lymphocytes from baseline and yearly blood samples using the human Illumina Methylation EPIC Bead Chip. Methylation analysis combined with mRNA analysis using RNAseq. Broad but not complete overlap was observed between T1D and T2D in CpG sites with abnormal methylation. And in the three-year observation period after BCG vaccinations, the majority of the abnormal methylation sites were corrected in vivo. Genes of particular interest were related to oxidative phosphorylation (CPT1A, LETM1, ABCG1), to the histone lysine demethylase gene (KDM2B), and mTOR signaling through the DDIT4 gene. The highlighted CpG sites for both KDM2B and DDIT4 genes were hypomethylated at baseline compared to controls; BCG vaccination corrected the defect by hypermethylation. Glycolysis is regulated by methylation of genes. This study unexpectedly identified both KDM2B and DDIT4 as genes controlling BCG-driven re-methylation of histones, and the activation of the mTOR pathway for facilitated glucose transport respectively. The BCG effect at the gene level was confirmed by reciprocal mRNA changes. The DDIT4 gene with known inhibitory role of mTOR was re-methylated after BCG, a step likely to allow improved glucose transport. BCGs driven methylation of KDM2B's site should halt augmented histone activity, a step known to allow cytokine activation and increased glycolysis.