Beyond phototoxicity: The dark side of new methylene blue on mitochondrial and cellular bioenergetics.

Molecular mechanisms and targeted intervention strategies of renal tubular epithelial cell glycolytic reprogramming in renal fibrosis.

Invited review: Fueling milk production carbon by carbon-Regulation of hepatic glucose production in dairy cattle.

Proteomic and metabolomic characterization and anti-inflammatory properties of Berberis vulgaris honey from Tibet plateau with high diastase activity.

PurposeMolecular reprogramming at the interface between malignant and non-malignant contributes to heterogeneity and metastasis across various cancers. The optic nerve metastasis serves as a significant prognostic indicator for mortality in retinoblastoma (RB). This study seeks to unveil the molecular underpinnings of this phenomenon.MethodsWe conducted a spatial proteomic analysis to delineate the heterogeneity within three RB samples exhibiting optic nerve invasion (ONI): case 1 and case 2 comparing the optic nerve invasive (ONI) group and the intraretinal group, and case 3 comparing tumor boundary and center. Data were archived in Mendeley Data [V1] (doi: 10.17632/xvtw7gmhzx.1; doi: 10.17632/mbsh6pcsnc.1). Immunofluorescence was performed to validate the expression patterns in additional RB samples of ONI versus the intraretinal group (n = 3 for each), and tumor boundary and center of neural metastasis (n = 4 for each).ResultsSpatial proteomic analysis identified significant alterations in cholesterol metabolism in optic nerve-invasive lesions compared with intraretinal lesions. The boundary of the invasive RB at the optic nerve demonstrated an upregulation of anaplerotic reaction and oxidative respiration, along with a significant enhancement of keratin-mediated cytoskeletal remodeling. Moreover, the boundary lesions exhibit epigenetic remodeling, following decreased lysine methyltransferase 5C (KMT5C) levels, an important regulator of the epithelial/mesenchymal transition. Consistently, immunofluorescence analysis of pyruvate carboxylase (PC), keratin 17 (KRT17), and KMT5C substantiated distinct expression patterns in tumor boundary and center lesions.ConclusionsThis study initially presents the spatial proteomic heterogeneity landscape in RB with ONI and uncovers its molecular reprogramming.

Succinic acid (SA)/1,4-Butanedioic acid is a key platform-chemical with broad industrial relevance, yet its biocatalytic production is constrained by redox imbalance, by-product accumulation, and limited CO2 sequestration. This study overcomes the above limitations and enhanced the SA production (0.6 g g-1; 6 g L-1) by dual-species catalysis using Citrobacter amalonaticus (NCIM 5782, CA) and Bacillus subtilis (BS, NCIM 5781), in a bioelectrocatalytic system. Comprehensive gene-expression-profiling revealed upregulation of phosphoenolpyruvate carboxylase (ppc/PPC) in CA and pyruvate carboxylase (pyc/PYC) in BS, reflecting intensified carboxylation activity via the reductive tricarboxylic acid pathway. Protein/structural modeling/docking of PPC and PYC demonstrated enhanced catalytic-site exposure under electro-fermentative co-culture conditions, correlating with greater carboxylation efficacy. Notably, beyond extracellular-CO2 fixation, intracellular-CO2 sequestration is also evident, as indicated by a marked enrichment of H2 in the biogas produced during dual-species-catalysis compared to monoculture systems. Thermodynamic and electrochemical evaluation indicated greater stability and electron flow in co-culture reactors (R9:ΔG = -42.29 kJ) compared to monocultures. Collectively, this study presents a scalable hybrid bioelectrochemical strategy leveraging species-specific metabolic roles and electron-steering to facilitate high-yield, selective SA production, offering a promising blueprint for sustainable carbon-based biomanufacturing.

Metabolic model-guided strain design for improved succinic acid production in Yarrowia lipolytica.

BackgroundCancer cells undergo profound metabolic reprogramming to sustain proliferation, redox homeostasis, and epigenetic remodeling. While the Warburg effect and glutaminolysis have long been recognized as central paradigms, the anabolic and regulatory role of lactate under normoxic conditions remains poorly defined.HypothesisThe Cancer-Induced Lactate Load and Oncologic Remodeling (CILLO) hypothesis proposes that lactate, either imported through MCT1 or produced endogenously, is oxidized to pyruvate by LDHB and subsequently carboxylated to oxaloacetate (OAA) by pyruvate carboxylase. OAA then acts as a metabolic hub driving malate-dependent NADPH production, aspartate synthesis for nucleotide metabolism, activation of the serine/glycine/folate cycle, lipogenesis, and S-adenosylmethionine–mediated epigenetic modifications. In this framework, lactate is no longer a mere by-product of glycolysis but a central integrator of anabolic flux, redox balance, and chromatin dynamics.ConclusionThe CILLO hypothesis unifies previously fragmented mechanisms into a coherent paradigm, emphasizing lactate-derived carbon skeletons as active drivers of tumor growth and metabolic plasticity. Key rate-limiting steps—MCT1-mediated uptake, LDHB-dependent oxidation, PC-driven anaplerosis, and PEPCK-M–mediated cataplerosis—emerge as therapeutic nodes for intervention. This model not only advances our understanding of cancer metabolism but also suggests novel strategies for biomarker development, metabolic imaging, and targeted therapies. By reframing lactate as a central determinant of oncologic remodeling, the CILLO hypothesis provides a foundation for translational advances in oncology and personalized medicine.

BTD encodes the biotinidase enzyme, responsible for recycling and maintaining biotin homeostasis in the human body. Biotin is a water-soluble micronutrient essential for various metabolic processes, with most being recycled by the biotinidase enzyme under normal physiological conditions. The process involves Holocarboxylase synthetase covalently attaching free biotin to Apocarboxylases, such as pyruvate carboxylase, 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and acetyl-CoA carboxylase, forming active Holocarboxylases. These active forms are then proteolyzed into biocytin and/or biotin peptides, which are subsequently cleaved by biotinidase enzyme, thus completing the biotin recycling loop. Variants within BTD disrupt the catalytic activity of biotinidase, leading to an inability to recycle biotin. Biotinidase deficiency, an autosomal recessive inherited metabolic disorder, can result from this disruption, causing the accumulation of biotin metabolites and subsequent damage to the peripheral and central nervous systems. The objective of this study was to analyze BTD variants and assess their structural, functional, and clinical significance in biotinidase deficiency. This study presents a comprehensive analysis of BTD variants, identifying a total of 740 reported variants, with exon 4 being a significant hotspot with 452 variants, indicating its potential importance for future genetic screening and diagnostic strategies. The research further provides an in-silico analysis of the BTD proteins, detailing their pathogenicity, domain structure, conserved regions, and key amino acids involved in interaction and structural integrity. Functional studies utilizing animal models demonstrate that BTD knockout adversely affects physiological features and metabolic pathways, with these effects being reversible upon biotin supplementation.

Triacetic acid lactone (TAL) is a promising platform chemical to produce valuable compounds. The development of engineered microbial hosts to efficiently produce TAL from lipid-containing waste streams could be a cost-effective, sustainable and environmentally friendly approach to meet the industrial demand. In this study, we engineered the yeast Candida viswanathii, possessing robust fatty acid conversion capabilities, to develop an alternative route for TAL production from fatty acids that aims to maximize conversion of the acetyl-CoA pool generated by β-oxidation in the peroxisome. To do so, we inactivated the carnitine acetyltransferase gene to block the transport of acetyl-CoA out of the peroxisome and overexpressed the enzymes methylmalonyl-CoA carboxyltransferase, 2-pyrone synthase and pyruvate carboxylase in the peroxisome to convert acetyl-CoA into TAL. We also performed an adaptive laboratory evolution experiment to obtain mutants with higher growth rate in medium with oleic acid and observed marked differences in central carbon metabolism and organic acid production pathways between the evolved and parental strains. These strains were further engineered by integrating additional copies of TAL biosynthetic genes while reducing competing reactions like ω-oxidation and Lipid biosynthesis, resulting in up to 50-fold increase in titers relative to the initial strain, reaching 280 mg/L. This study contributes to the development of bioprocesses that valorize fatty acids as microbial conversion substrates for the production of valuable compounds.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-025-02841-7.

Abstract The use of minerals complexed with organic molecules has been used to enhance their bioavailability and intestinal absorption. The increased nutrient intake provided by finishing diets promotes muscle growth and weight gain; however, it also intensifies the oxidative challenge to which cells are exposed. We aimed to evaluate the proteomic alterations in the Longissimus thoracis muscle of feedlot cattle supplemented with carbo-amino-phospho-chelated minerals compared with inorganic minerals. Sixteen crossbred Nellore × Angus bulls (iBW: 472 ± 4.9 kg, 18 mo old) were randomly assigned to two treatments: (1) INORG - supplementation with sulfate-based mineral sources; (2) TM - supplementation with minerals (Cr, Zn, Mn, Se, S, Cu, and Co; Tortuga® Minerals, dsm-firmenich) in the form of carbo-amino-phospho-chelates. The bulls were fed a high-concentrate diet for 89 days. At the end of the finishing period, animals were slaughtered, and Longissimus thoracis muscle samples were collected and stored at -80ºC. Proteomic analysis was performed using a Label-Free approach on the high-resolution ACQUITY IClass Xevo -G2Xs mass spectrometer system (Waters, Manchester, UK). Data were processed using the Progenesis QI for Proteomics software. A total of 1,165 proteins were identified, of which 134 showed differential abundance (DA, p< 0.05). The TM treatment increased the abundance of 61 proteins and decreased the abundance of 73. Functional analysis of the DA protein list using WebGestalt identified that TM increased the abundance of proteins that utilize Zn2+ as a cofactor, such as CA2 and CA3 (p=0.001), which are responsible for CO2 removal from the cellular environment. Additionally, an increase was observed in ALDOC and pyruvate carboxylase (p=0.019), involved in both glycolysis and amino acid biosynthesis, utilizing Mg2+ and Cr indirectly. NADK2 and GPHN proteins (p=0.069), involved in cofactor biosynthesis, also demonstrated increased abundance. Proteins CALR and HSPA2, associated with inflammatory processes, were also upregulated (p=0.024). Supplementation with INORG increased the abundance of proteins related to pyruvate and carbon metabolism (MDH2 and ME2, p=0.037), as well as proteins involved in identifying energy status (PRKAB2) and enhancing fatty acid uptake (FATP1; p=0.036). However, a higher abundance of proteins associated with cellular stress (HSPD1, p=0.011), regulation of inflammatory response (DUSP7, FGF8, and MK2; p=0.048), cellular senescence (MK2 and SQSTM1; p=0.072), and mitophagy (EIF2AK3 and SQSTM1; p=0.036), was also observed phenomena interconnected and linked to oxidative stress. Supplementation with carbo-amino-phospho-chelated minerals enhances the abundance of proteins related to cofactor renewal and glycolytic energy metabolism, improves the cellular response to oxidative stress, and mitigates mechanisms associated with natural killer cell activation in the muscle tissue of feedlot cattle fed high-concentrate diets. These findings highlight the potential of carbo-amino-phospho-chelated mineral supplementation in optimizing muscle metabolism and oxidative stress, which are crucial factors for promoting performance, health and meat quality in feedlot cattle.

Mid-life obesity is a major risk factor for neurodegenerative diseases, with mitochondrial and cerebrovascular dysfunction considered key mediators. Lysine acetylation is a reversible post-translational modification that regulates several mitochondrial metabolic and biochemical processes. The present study investigatedthe sex-dependent effects of brain lysine acetylation and cerebrovascular and cognitive health in a high fat diet (HFD)-induced obesity mouse model. We hypothesize that a HFD will cause an increase in acetylation, dysregulating mitochondrial respiration, potentially due to the decline in overall cerebrovascular health. Six-month-old C57/Bl6 mice (M/F) were placed on a 60% HFD or normal chow (CON) for 4months. Changes in cerebral blood flux (CBF), behavioural testing, glucose tolerance testing and body composition were tested. Brain lysates were probed for various substrate utilizations, bioenergetics proteins and lysine acetylation. A HFD resulted in global metabolic dysregulation, with a substantial increase in weight and fat mass, with a greater increase in female mice; however, no cognitive changes were noted. Additionally, unlike female mice, males demonstrated a decrease in CBF after a HFD. Brain lysine acetylation was decreased in male HFD mice but increased in female HFD mice. Similarly, acetylation levels of fatty acid oxidation protein (long-chain acyl-CoA dehydrogenase), glucose oxidation proteins (pyruvate dehydrogenase, pyruvate carboxylase) and electron transport chain complex I (NDUFB8) and IV (MTCO1) proteins were decreased in male and increased in female brains after a HFD. In summary, our findings propose lysine acetylation as a novel and potential regulatory mechanism that impacts vascular and metabolic function in the brain mitochondria in a sex-dependent manner.

Efficient regeneration of NADPH can be a limiting factor for anabolic processes in engineered microbial cells. We tested the ability of four distinct Pyruvate-Oxaloacetate-Malate “POM” cycles composed of Saccharomyces cerevisiae pyruvate carboxylase (PYC1 or PYC2), malate dehydrogenase (‘MDH1 or ‘MDH2), and malic enzyme (sMAE1) to improve NADPH regeneration. Only the PYC1, ‘MDH2, sMAE1 combination increased the titer of fatty alcohols produced by engineered S. cerevisiae indicating that not all combinations of POM cycle enzymes could drive this pathway. Metabolomic analysis revealed that introduction of the POM cycle altered the concentration of intermediates in amino acid biosynthetic pathways and the trichloroacetic acid cycle suggesting that the POM cycle had wider effects than previously anticipated. Overexpression of the endogenous NAD+ kinases UTR1, YEF1, and a cytosolic version of POS5 were also tested. Only expression of POS5c resulted a significant increase in fatty alcohol titer. In these minimally engineered strains, combined overexpression of the PYC1, ‘MDH2, sMAE1 POM cycle and POS5c did not further increase titers. These findings indicate that more extensive metabolomic and proteomic investigations are required to identify combinations of enzymes that will yield an optimal increase in NADPH to meet anabolic demands without imposing excessive metabolic burden or disrupting pathways that might compromise bioproduct synthesis.

The invasive white-backed planthopper (WBP) poses a severe threat to global rice crop security, and most populations have developed significant resistance to neonicotinoids. Although these species remain sensitive to mesoionic triflumezopyrim (TFM), both neonicotinoids and TFM are hazardous to pollinating insects. Herein, we disclose a series of new spirocompounds designed via isosteric ring replacement of scaffold hopping. Of these, spirocyclic molecule A11 exhibits satisfactory biosafety to bees (LD50 > 11.0 μg a.i./bee) and comparable efficacy (LC50 = 11.0 μg/mL) against WBPs to that of spirotetramat (LC50 = 11.6 μg/mL). Proteomics, quantitative real-time PCR, and enzyme activity assay experiments collaboratively verify the mode of action of A11 being a pest acetyl-CoA carboxylase inhibitor. Noteworthily, the suppression of pyruvate carboxylase in WBP by spiro compounds is observed for the first time, which implies a multitarget effect of these insecticides on key enzymes involved in tricarboxylic acid cycle. This impact enables effective inhibition of lipid biosynthesis pathways in WBPs, ultimately leading to the death of the pests. We believe this study provides valuable insights into the molecular basis of spiro substances on WBPs.

Efficient trans-aconitic acid production using systematically metabolic engineered Escherichia coli

Histoplasma capsulatum is a human fungal pathogen that survives and proliferates within phagocytic immune cells. To sustain growth in the nutrient-limited phagosome environment, the pathogenic yeast scavenges available carbon sources, which must be metabolized through central carbon metabolism for respiration and biomass synthesis. However, Histoplasma carbon metabolic pathways operating in the pathogenic yeast phase have not been extensively mapped. To address this gap, we employed a fluxomic platform using stable isotope tracers to quantify the cellular reaction rates of central carbon metabolism. This approach revealed that, in Histoplasma yeasts, carbon resides within five main reservoirs: fatty acids, proteins, mannitol, nucleic acids, and cell wall components. Carbon conversion efficiency, or biomass yield, was approximately 50%, indicating substantial CO2 loss from supplemented carbon substrates, glucose, and glutamate. 13C-labeling analysis demonstrated simultaneous glycolysis and gluconeogenesis, and enriched serine labeling confirmed threonine aldolase activity in serine biosynthesis. Compartmentalization of pyruvate metabolism was evident from the labeling of amino acids derived from pyruvate, with the methylcitrate cycle identified as the primary source of labeled pyruvate. Notably, malic enzyme and pyruvate carboxylase exhibited negligible fluxes, while mitochondrial reactions, particularly CO2-producing ones, were the most active. These results offer insight into key metabolic reactions, alternative pathways, and metabolite/enzyme compartmentalization in Histoplasma yeast metabolism. This foundational framework supports future studies aimed at identifying metabolic targets for novel histoplasmosis therapeutics.IMPORTANCETo our knowledge, this study represents the first application of 13C-metabolic flux analysis to a human fungal pathogen, where we identified carbon reservoirs and quantified the metabolic fluxes of pathogenic Histoplasma yeasts. Our findings demonstrated that Histoplasma metabolizes carbon toward cellular respiration to robustly produce CO2 and energy but also uses alternative pathways within central metabolism for biosynthesis. Given the potential for other pathogenic fungi to share similar metabolic features, especially biomass, our study offers a comprehensive framework for deciphering fungal metabolism, providing insights into their infection-enabling metabolism and offering a foundation for identifying new therapeutic targets.

Epigallocatechin-3-gallate (EGCG), the main catechin in green tea, is associated with antidiabetic and anti-obesity effects, although its acute hepatic actions remain unclear. We investigated short-term effects of EGCG (10-500 μm) using isolated perfused rat livers and complementary assays in mitochondrial, microsomal, and cytosolic fractions. EGCG markedly inhibited gluconeogenesis from lactate (up to 52%), glycerol (33%), and alanine (47%), while it stimulated glycolysis, glycogenolysis, and oleic acid oxidation (+42% total ketone bodies). Oxygen uptake was stimulated under glycogenolytic and fatty acid oxidizing conditions but inhibited under gluconeogenic conditions. Mechanistic analyses revealed EGCG-induced mild mitochondrial uncoupling, inhibition of pyruvate carboxylase and glucose-6-phosphatase (with no effect on fructose-1,6-bisphosphatase) and stimulation of phosphoenolpyruvate carboxykinase. EGCG shifted cytosolic and mitochondrial NADH/NAD+ ratios toward oxidation, increased mitochondrial and plasma membrane permeability (LDH leakage from 10 μm), and altered redox-sensitive fluxes, while the total hepatic ATP content remained unchanged. In summary, EGCG's multifaceted actions suggest that suppression of gluconeogenesis may contribute to its antihyperglycemic effect and the stimulation of fatty acid oxidation to its anti-obesity action. Finally, EGCG's membrane-disruptive properties raise concerns about potential hepatotoxicity in compromised livers.

Ganoderic acid T, a novel activator of pyruvate carboxylase, exhibits potent anti-liver cancer activity.

Tailoring Escherichia coli for high-yield production of O-acetyl-L-homoserine through multi-node metabolic regulation.

Soil Pseudomonas species, which thrive on lignin derivatives, are widely explored for biotechnology applications in lignin valorization. However, how the native metabolism coordinates phenolic carbon processing with required cofactor generation remains poorly understood. Here, we achieve quantitative understanding of this metabolic balance through a detailed multi-omics investigation of Pseudomonas putida KT2440 grown on four common phenolic acid substrates: ferulate, p-coumarate, vanillate, and 4-hydroxybenzoate. Relative to succinate, proteomics reveals > 140-fold increase in transport and catabolic proteins for aromatics, but metabolomics identifies bottlenecks in initial catabolism to maintain favorable cellular energy charge, which is compromised in mutants with resolved bottlenecks. Up to 30-fold increase in pyruvate carboxylase and glyoxylate shunt proteins implies a metabolic remodeling confirmed by kinetic 13C-metabolomics. Quantitative analysis by 13C-fluxomics demonstrates coupling of this remodeling with cofactor production. Specifically, anaplerotic carbon recycling through pyruvate carboxylase promotes tricarboxylic acid cycle fluxes to generate 50-60% NADPH yield and 60-80% NADH yield, resulting in up to 6-fold greater ATP surplus than with succinate metabolism; the glyoxylate shunt sustains cataplerotic flux through malic enzyme for the remaining NADPH yield. This quantitative blueprint affords cofactor imbalance predictions in proposed engineering of key metabolic nodes in lignin valorization pathways.