Lactic acid production by Lachancea thermotolerans offers a promising biological strategy to restore wine acidity often compromised by climate change, yet this metabolic trait remains highly variable and poorly controlled. This study systematically evaluated the impact of controlled oxygenation regimes on fermentation performance and primary metabolism in three genetically distinct L. thermotolerans strains. Oxygen availability drastically altered metabolic fluxes. Under continuous aerobic conditions, carbon was redirected toward biomass formation (9- to 13-fold increase), with reduced ethanol and lactic acid yields, as well as increased succinic acid, acetoin, and 2,3-butanediol, whereas fully anaerobic conditions lowered fermentation performance but substantially increased lactic acid. These results confirmed that L. thermotolerans is a weakly Crabtree-positive yeast with mixed respiratory-fermentative metabolism. Carbon redistribution under aerobic conditions was not fully compensated by measured metabolites, resulting in substantially decreased carbon recovery (~70%) and suggesting undetermined respiratory CO2 production alongside diversion of carbon toward undetermined intracellular components. This imbalance was supported by a decline in cell density following peak growth, consistent with potential redox imbalance or oxidative stress. Lactic acid production occurred primarily under oxygen limitation, independent of ethanol formation, indicating a regulated, facultative adaptive response rather than a constitutive fermentation product. Strain-dependent differences further revealed distinct carbon allocation strategies. While oxygen strongly influenced metabolic behavior, pulse strategies showed limited, condition-dependent effects, highlighting the importance of timing and intensity for modulating lactic acid production. Overall, oxygen is a powerful lever to modulate metabolic fluxes in L. thermotolerans, but its application for predictable bio-acidification requires further optimization.IMPORTANCEWine acidity is increasingly difficult to maintain under climate change, creating a need for reliable biological solutions. Some strains of Lachancea thermotolerans can produce lactic acid during fermentation, but this metabolic trait is inconsistent and difficult to control. This study shows that oxygen availability is a key factor shaping how this yeast uses carbon, shifting it between growth and lactic acid production. Under the continuous aerobic condition, carbon flux was redirected toward biomass, whereas the fully anaerobic condition favored lactic acid production but reduced fermentation performance. Importantly, pulsed oxygen additions did not provide consistent control of lactic acid production, but they revealed trends that highlight the importance of timing and intensity. These findings show that lactic acid production is a flexible response to environmental conditions rather than a constitutive trait. This work provides a foundation for developing strategies to better control lactic acid production through timed oxygen addition during fermentation.

Protein posttranslational modifications (PTMs) are crucial and dynamic modulators of protein functions, interactions, and localizations, as well as biological pathways and cellular signaling. Lysine succinylation analysis remains challenging, but sophisticated workflows combining succinylated peptide enrichments and quantitative mass spectrometry approaches have revolutionized proteome-wide succinylome analysis. The implementation of data-independent acquisition (DIA)-mass spectrometry has greatly advanced the detection of low-abundance succinylated peptides, succinylome coverage, identification reproducibility, and quantification accuracy. However, the complexity of DIA data requires dedicated data processing algorithms and tools, which typically rely on spectral libraries. These reference libraries can be generated from experimental data-dependent acquisition (DDA) acquisitions of representative study samples submitted to DDA database search engines for confident succinylated peptide identification and precise PTM site localization. Here, we describe how to build DDA PTM spectral libraries using various software tools, specifically Spectronaut, SpectroMine, and MSFragger. The generated libraries were imported into Skyline for the analysis of previously published DIA succinylome data of Sirtuin-5 knocked-out vs wild-type mouse brains in order to accurately quantify and visualize PTM-containing peptides.

Brown adipose tissue (BAT) activity in humans is linked to better glucose metabolism and cardiometabolic health. In mice, cold exposure increases circulating succinate and enhances BAT thermogenesis, but whether similar succinate responses occur in humans remains undetermined. We analysed data from 33 young (18-25 years), sedentary adults (20 women) from the ACTIBATE cohort (NCT02365129). Plasma succinate was measured enzymatically at baseline and 60 and 120 min during a personalized cooling protocol. BAT volume, activity, and radiodensity were assessed via static [18]F-fluorodeoxyglucose positron emission tomography-computed tomography ([18]F-FDG PET-CT) at the end of the cold exposure. Circulating succinate changed over time during cold exposure, with no significant change at 60 min and a significant increase at 120 min versus baseline (+ 23.8%, P = 0.017), with substantial interindividual variability. Exploratory stratification by median baseline succinate revealed divergent temporal patterns: the low-baseline group showed increases at 60 min (+ 46%) and 120 min (+ 80.5%; both P ≤ 0.021), while the high-baseline group showed reductions at both timepoints (-31.4% and - 2.4%; both P ≤ 0.003). These profiles were not explained by phenotypic or BAT-related parameters. In the full cohort, changes in circulating succinate were not associated with BAT outcomes. Overall, our findings indicate that circulating succinate responses during acute cold exposure are highly heterogeneous and no associated with BAT PET/CT-derived parameters in humans.

Rheumatoid Arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation. Recent studies have revealed that immune metabolites, particularly succinate, play a critical role in its pathogenesis. In this review, we systematically analyze the molecular mechanisms underlying succinate accumulation and elucidate how succinate drives inflammatory and immune dysregulation in RA. Hypoxia and inflammation microenvironment drive metabolic alterations, leading to succinate accumulation via three primary mechanisms: 1) succinate retention caused by impaired mitochondrial oxidation, 2) enhanced succinate synthesis through alternative biosynthetic pathways, and 3) increased succinate export into the extracellular space. Accumulated succinate exerts its pathogenic effects through three distinct pathways: (i) binding to its receptor Succinate Receptor 1 (SUCNR1) as an extracellular signaling molecule; (ii) stabilizing Hypoxia-Inducible Factor-1α (HIF-1α) in the intracellular space; and (iii) acting as a key epigenetic modulator. These insights into succinate-mediated pathogenesis provide a rationale for the development of targeted RA therapies, including strategies aimed at SUCNR1, Succinate Dehydrogenase (SDH), and the combination of these metabolic interventions with existing anti-inflammatory treatments.

To develop a green enzymatic method for the remediation of parabens (PBs) contaminants, a ternary composite system comprising laccase, 2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO) mediator, and cutinase SE50 (LCTM) was constructed. Using methylparaben (MP) as a model substrate, the degradation performance, kinetic characteristics, and underlying mechanisms of the system were systematically investigated. The results demonstrated that TEMPO significantly enhanced the catalytic conversion of MP by laccase, and the subsequent introduction of cutinase SE50 further boosted the overall degradation efficiency. Under the optimized conditions (0.5 U/mL cutinase SE50, 1.2 U/mL laccase, 25 mg/L TEMPO, temperature 50 °C, and pH 6.0), the degradation rate of MP reached 96.8% within 24 h. The degradation process well conformed to a pseudo-first-order kinetic model with an apparent reaction rate constant of 191.84 × 10[-3] h[-1]. Subsequently, identification of the degradation products revealed that MP was initially hydrolyzed into p-hydroxybenzoic acid, and then underwent decarboxylation and pivotal hydroxylation to generate the core intermediate catechol. Catechol then underwent oxidative ring-cleavage into small-molecule organic acids, including succinic, pyruvic, and glycolic acids, which were ultimately mineralized. Furthermore, the effects of different PBs structures on degradation efficiency were investigated, with higher degradation efficiency observed for short-chain substrates possessing lower steric hindrance. This study validates the potential application of the LCTM system in the remediation of ester-based pollutants and provides a theoretical basis for composite enzymatic degradation mechanisms.

Background/Objectives: The clinical application of the potent antitumor alkaloid camptothecin is severely limited by its high systemic toxicity, poor solubility, and rapid inactivation. This study aimed to design, synthesize, and evaluate a novel series of camptothecin-lipid conjugates where natural steroids and triterpenoids serve as lipophilic carriers to enhance cellular penetration and mitigate cardiotoxicity. Herein, we implemented an endogenous targeting strategy by conjugating camptothecin with natural steroids and triterpenoids, which serve as biomimetic lipophilic vectors to achieve tumor-selective intracellular delivery. Methods: Eleven novel camptothecin-based hybrids linked via succinic acid or ethylene glycol spacers were synthesized and characterized. Their in vitro cytotoxicity was evaluated against eight cancer cell lines of varied embryologic origin (Jurkat, HCT 116, A549, HL60, K562, HeLa, HEK293, and Fibroblasts). Cell cycle distribution, apoptosis induction, and topoisomerase I inhibitory activity were assessed using flow cytometry and enzyme assays. In silico pharmacokinetic profiling, including P-glycoprotein interaction and cardiotoxicity mitigation, was substantiated via ADME algorithms. Results: Among the series, compounds 12 (cholestanol conjugate) and 26 (betulin derivative) emerged as the primary lead candidates based on an optimal composite of submicromolar cytotoxicity, exceptional tumor selectivity indices (up to 7.0 for 12), and active topoisomerase I inhibition. Compounds 11-14 generally shared prominent S-phase cell cycle arrest, while the lead hybrids 12 and 26 successfully combined high apoptotic triggering with favorable in silico ADME safety profiling, including eliminated cardiotoxicity for the structural analogs. Conclusions: The integration of steroidal and triterpenoid pharmacophores via strategic spacers provides a reliable foundation for developing targeted, low-toxicity camptothecin-lipid conjugates with optimized safety profiles for cancer therapy.

Kiwifruit wine is valued for its nutrients and health benefits, but conventional brewing often results in a weak aroma due to precursor insufficiency and oxidative losses during fermentation. Therefore, adding natural polysaccharides with antioxidant and metabolic regulatory properties prior to fermentation may be a promising quality improvement strategy. Bletilla striata polysaccharide (BSP), a plant polysaccharide with multiple bioactivities, may have the potential to regulate kiwifruit wine quality. This study explored the regulatory effects of 100-400 mg/L BSP on the physicochemical properties, antioxidant activity, and aroma compounds of kiwifruit wine, which suggested its possible mechanism of action at the metabolite level. The results showed that the 300 mg/L BSP treatment achieved the best overall quality, maximizing the DPPH and ABTS radical scavenging rates and increasing the total relative abundance of volatile compound concentration by 28.69% compared with the control. Electronic nose testing, odor activity value analysis, and sensory evaluation all indicated that the 300 mg/L BSP group exhibited the best overall aroma profile, and non-targeted metabolomics further suggested that the 300 mg/L BSP addition was associated with the depletion of quinic acid, the accumulation of aromatic amino and succinic acids, and changes in lipid-related metabolites, which were correlated with enhanced aroma production. In conclusion, exogenous BSP addition appears to improve kiwifruit wine quality, and this study provides a theoretical basis for its application in fruit wine brewing.

Opuntia ficus-indica (L.) Mill., an edible cactus, has gained increasing attention as a potential food resource due to its adaptability and nutritional value. This study aimed to characterize the bioactive compound profiles and structural characteristics of the epidermis and cortex of cladodes across different maturity stages. Numerically higher levels of bioactive compounds were generally observed in the epidermis than in the cortex, particularly gentisic acid (384.58-668.23 µg/g DW in the epidermis vs. 191.11-426.69 µg/g DW in the cortex) and protocatechuic acid (154.04-234.45 µg/g DW in the epidermis vs. 40.78-118.44 µg/g DW in the cortex). The epidermis and cortex also exhibited distinct organic acid profiles, with oxalic and malic acids being the predominant organic acids detected in both tissues. FTIR analysis further revealed a more pronounced O-H absorption band associated with water molecules in the cortex than in the epidermis. Furthermore, stage-dependent variations were observed in both bioactive composition and structural characteristics. Organic acids exhibited stage-dependent variations in both tissues. Oxalic, malic, and succinic acids generally occurred at higher levels during the later maturity stages, whereas fumaric acid was more abundant at the earliest maturity stages. Their concentrations ranged from 13.61-47.20, 10.74-16.27, 7.56-14.47, and 0.51-20.46 mg/g DW in the epidermis, and 8.19-46.82, 9.67-52.26, 2.76-14.72, and 0.27-34.41 mg/g DW in the cortex, respectively. These findings provide baseline information on the compositional and structural characteristics of edible cactus cladodes and may support future studies on their potential food-related applications.

The mitochondrial dicarboxylate carrier SLC25A10 mediates reversible exchange among succinate, malate, and phosphate, contributing to mitochondrial metabolic regulation. Structural studies establish a ping-pong mechanism, but most mathematical models still assume sequential binding, lacking mechanistic justification and overlooking the alternation of a single binding site. Here, we present the first mechanistically derived and thermodynamically consistent model of SLC25A10 based on a ping-pong framework. The model incorporates competitive binding of succinate, malate, and phosphate, heteroexchange, reversibility, and electroneutrality, and is calibrated using experimental datasets from intact mitochondria and reconstituted proteoliposomes. To estimate kinetic parameters and quantify their uncertainty, we employed Bayesian inference, enabling statistically rigorous calibration to uptake and competition assays. The model introduces new terms that quantify which substrate and from which side of the membrane is most likely to start the transport cycle. Beyond reproducing experimentally observed exchange kinetics, the model resolves non-equilibrium transport dynamics that are difficult to access directly in classical uptake assays. In particular, the simulations reveal a two-phase response in which an initial phosphate-driven high-flux uptake regime for malate and succinate is followed by a slower redistribution phase in which the two dicarboxylates continue to readjust primarily against each other. The model also predicts that mitochondrial morphology modulates early transport behaviour, with matrix swelling increasing and matrix condensation decreasing the initial SLC25A10 flux magnitude. More broadly, the framework provides a quantitative basis for studying how substrate competition, thermodynamic driving forces, and compartment geometry shape SLC25A10-mediated exchange, and it offers a transferable modelling strategy for other carriers in the SLC25 family.

Succinic semialdehyde dehydrogenase is a key enzyme involved in the catabolism of g-aminobutyric acid. Biallelic variants in the ALDH5A1 gene cause a rare monogenic neurometabolic disorder, SSADH deficiency, characterized by a total or partial enzyme loss-of-function. Patients present symptoms from infancy on and mainly show developmental delay and intellectual disabilities, with seizures and/or autistic spectrum disorders as the main distinctive traits associated with the severe forms of the disorder. A valuable instrument to interpret variant function is provided by bioinformatic tools that are based on evolutionary and structural data. However, the results need to be complemented by other experimental approaches to dissect the molecular basis for the defect and be supportive of the clinical phenotype of patients. Here, we validate a bioinformatic prediction of loss-of-function using two recombinant enzyme variants and/or transient transfection in a ALDH5A1 knockout HEK-293T cell model. These SSADH protein variants are synthesized by two functionally hemizygous SSADH deficiency patients. The known loss-of-function p.Gly176Arg variant was predicted by bioinformatic analyses to affect oligomerization. Indeed, we demonstrate that the variant acquires a tetrameric altered structure, possibly responsible for the loss-of-function. The pathogenicity of the new p.Pro234Ser variant is mainly due to its failure to reach the mature form, possibly due to its intrinsic instability rather than a defect in the maturation apparatus, thus preventing a correct folding process. Altogether, we provide evidence that combining recombinant protein studies with structural predictions can offer valuable insights into loss-of-function, which is essential for precision therapy.

Fusobacterium nucleatum (F. nucleatum) has been increasingly implicated in the pathogenesis of inflammatory bowel disease (IBD), yet the mechanisms underlying its effects remain incompletely defined. In this study, we integrated human fecal and mucosal samples, comparative metabolomics, multiple experimental colitis models, bacterial genetic manipulation, macrophage functional assays, and host signaling analyses to identify a macrophage-centered mechanism through which F. nucleatum exacerbates colitis. We show that F. nucleatum colonization increases intestinal and systemic levels of its metabolite succinic acid, upregulates the expression of its cognate receptor SUCNR1 on intestinal macrophages, activates NF-κB signaling, and promotes pro-inflammatory macrophage activation. This macrophage inflammatory response is associated with epithelial barrier disruption, increased epithelial apoptosis, and aggravated mucosal and systemic inflammation. A fumarate reductase-deficient (frdA-KO) F. nucleatum strain with impaired succinic acid production showed a markedly reduced capacity to activate macrophage NF-κB signaling, induce macrophage inflammatory activation, and aggravate colitis, whereas exogenous succinic acid restored these effects in the frdA-KO setting. Moreover, siSUCNR1 and pharmacological NF-κB inhibition substantially attenuated succinic acid-induced macrophage inflammatory activation, supporting the involvement of a SUCNR1-NF-κB signaling cascade. Collectively, these findings demonstrate that F. nucleatum exacerbates colitis by producing succinic acid and engaging SUCNR1-NF-κB-dependent inflammatory activation of macrophages, highlighting the F. nucleatum-succinic acid-SUCNR1-NF-κB axis as a potential therapeutic target in IBD.

Native starch is difficult to use directly for microcapsule preparation due to its strong hydrophilicity, poor emulsification capacity, and large particle size. This study provides a novel strategy for developing essential oil-loaded microcapsules using Pickering emulsions stabilized by octenyl succinic anhydride (OSA) modified starch nanocrystals (SNCs). The SNCs were prepared via acid hydrolysis of corn starch and further modified with different amounts (3%, 6%, and 12% w/w) of OSA. The SNCs and OSA-modified SNCs (OSNCs) were used to encapsulate cinnamon essential oil (CEO) via interfacial crosslinking in Pickering emulsions, where SNCs/OSNCs (aqueous phase) served as the wall material and isophorone diisocyanate (oil phase) as the cross-linker. The resulting microcapsules (CEO@SNCs, CEO@3%OSNCs, CEO@6%OSNCs, CEO@12%OSNCs) were characterized on physicochemical properties. All OSNC-based microcapsules exhibited superior performance, featuring smaller particle sizes (around 600 nm vs. 2960 nm for CEO@SNCs), higher CEO loading capacity (about 90% compared to 72% for CEO@SNCs), and enhanced long-term stability across a broad temperature range (4-54 °C). Microencapsulation notably improved the functional performance of CEO. In particular, the CEO@6%OSNCs microcapsules demonstrated a release profile consistent with the first-order kinetic model, indicating sustained release behavior, along with effective antioxidant activity and enhanced antifungal efficacy (against B. cinerea and R. stolonifer).

This study investigated the effects of traditional frying (TF, 0-9 min), air frying (AF, 0-17 min), and microwave frying (MF, 0-12 min) on non-volatile flavor compounds in grass carp fillets. Prolonged frying time promoted the accumulation of total free amino acids and reducing sugars, with thermal degradation acting as the primary driver of this increase. Histidine, 5'-guanosine monophosphate, and tartaric acid were identified as key flavor contributors, while succinic acid also showed significant contribution in TF and the early AF groups. The equivalent umami concentration exhibited a continuous increase with frying time. Electronic tongue revealed general enhancements in bitterness, saltiness, and sweetness attributes after frying. Statistical modeling identified 13 key flavor differentiation compounds. Both frying time and method significantly influenced non-volatile flavor compounds, with AF exhibiting the greatest efficacy in promoting umami compound accumulation. These findings provide theoretical guidance for taste quality regulation in fried fish fillet production.