Asia Pacific journal of clinical nutritionXiaohan Guo, Xiaomin Wang, Xiaofeng Weng, Yuanqing Fu, Jun Tang, Wensheng Hu
BACKGROUND AND OBJECTIVES: The gestational weight gain (GWG) range during mid-to-late pregnancy associated with the lowest combined risk of adverse birth outcomes in women with gestational diabetes mellitus (GDM) remains unclear. This study aims to examine the associations between GWG and adverse birth out-comes among women with GDM. METHODS AND STUDY DESIGN: This study included a cohort of 1,673 pregnant women with GDM. GWG was defined as weight gain from pre-pregnancy to a measurement between 24 and 32 gestational weeks, residualized for gestational age and standardized as z-scores. Multivariable logistic regression models were used to assess associations between GWG z-scores (per 1-SD increase and categories: <-1, -1 to 1 [reference], and ≥1) and small for gestational age (SGA), large for gestational age (LGA), and preterm birth. Restricted cubic splines were fitted to explore nonlinear associations. RESULTS: Each 1-SD higher in GWG z-score was associated with lower odds of SGA (odds ratio [OR], 0.55; 95% confidence interval [CI], 0.43-0.71) and higher odds of LGA (1.48; 1.29-1.70). Compared with the reference group, women with GWG z-scores <-1 had higher odds of SGA and lower odds of LGA, whereas those with z-scores ≥1 showed the opposite pattern. No significant association was observed with preterm birth. Spline analyses indicated that a GWG z-score of approximately -0.07 (equal to 8.2 kg at 28 weeks) was associated with the lowest combined odds of SGA and LGA. CONCLUSIONS: Among women with GDM, both insufficient and excessive mid-to-late pregnancy GWG were associated with adverse size-for-gestational-age outcomes.
Reviews of physiology, biochemistry and pharmacologyUndurti N Das
Human essential hypertension is driven by a network of interacting nutritional, metabolic, inflammatory, and endothelial mechanisms in which AA and other PUFAs play a critical role. Beyond excess sodium intake, population data supports the role of inadequate calcium, potassium, and magnesium intake and low antioxidant vitamin status in shaping blood pressure regulation. These nutrients influence vascular smooth muscle tone and act as cofactors that support Δ6 and Δ5 desaturase activities, thereby governing tissue availability of AA, EPA, and DHA and downstream formation of vasodilator and antiplatelet mediators (e.g., PGE1, prostacyclin), as well as inflammation-resolving lipid mediators (lipoxins, resolvins, protectins, maresins, and nitrolipids).Endothelial dysfunction characterized by reduced endothelial nitric oxide (eNO) bioavailability and increased oxidative stress seems to play an important role in the pathobiology of HTN. High salt intake, asymmetric dimethylarginine (ADMA), and activation of NAD(P)H oxidase and angiotensin II signaling promote superoxide generation that quenches NO and shifts the vascular balance toward vasoconstriction. Clinical and experimental observations indicate that antihypertensive therapies can partly restore NO and antioxidant defenses, while dietary patterns rich in n-3 fatty acids (particularly DHA) and balanced n-6/n-3 intake may lower blood pressure by suppressing thromboxane formation and enhancing vasoprotective, pro-resolving pathways.Preeclampsia can be considered as a model of reversible hypertension linked to oxidative stress and angiogenic imbalance (sFlt1, soluble endoglin), highlighting mechanistic overlap with essential hypertension. Hypertension is a low-grade systemic inflammatory condition whose origins may be present in the perinatal period through long-term programming of PUFA metabolism and endothelial function.
Reviews of physiology, biochemistry and pharmacologyUndurti N Das
Polyunsaturated fatty acids (PUFAs), especially gamma-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), can block HMG-CoA reductase activity and thus decrease cholesterol synthesis/formation. Cholesterol has antioxidant actions and thus reduces the formation of ROS (reactive oxygen species). In contrast, PUFAs augment the generation of ROS. PUFAs undergo peroxidation to form lipid peroxides that are toxic to tumor cells but not normal cells. GPX4 (glutathione peroxidase) reduces the formation of lipid peroxides and thus enhances tumor cell resistance to the tumoricidal actions of radiation, anticancer drugs, and immune checkpoint inhibitors (ICIs). Tumor cells have relatively high amounts of GPX4 and hence are resistant to lipid peroxide-induced ferroptosis/apoptosis. In contrast, normal cells can upregulate their GPX4 activity/content upon exposure to toxic lipid peroxides and hence are resistant to ferroptosis or apoptosis when supplemented with various PUFAs or exposed to radiation, anticancer drugs, and ICIs. Thus, PUFAs (especially GLA, AA, and DHA) are selectively toxic to tumors but not normal cells. Interferon-γ (IFN-γ) is secreted by CD8+ T cells, and AA induces ferroptosis of tumor cells in an ACSL4 (the protein encoded by this gene is an isozyme of the long-chain fatty-acid-coenzyme A ligase family)-dependent lipid peroxidation process. IL-6, TNF-α, and IFN-γ activate PLA2 to release AA from membrane lipids, which is then peroxidized to trigger apoptosis or ferroptosis of cancer cells. CD8+ T cells/macrophages/TILs/NK cells and other immunocytes downregulate the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system x c - , and impair the uptake of cystine by tumor cells, because of which accumulation of toxic lipid peroxides occurs in the tumor cells, leading to their apoptosis/ferroptosis/necrosis. In C6 glioma cells, the inhibition of the cystine/glutamate (XC) antiporter and glutamate-cysteine ligase (GCL) results in inhibition of glutathione biosynthesis that leads to ferroptosis of cancer cells because of accumulation of toxic lipid peroxides. Thus, the lipid peroxidation process is at the center of tumor cell apoptosis/ferroptosis.
Methods in molecular biology (Clifton, N.J.)Marija Djordjevič, Svetlana Dinič, Ana Sarič, Aleksandra Uskokovič, Melita Vidakovič
Epigenetic regulation provides a dynamic and reversible layer of gene control that functions independently of changes in DNA sequence, primarily mediated by DNA methylation, histone modifications, and higher-order chromatin organization. Aberrant epigenetic states contribute to a wide range of human diseases. However, conventional epigenetic therapies based on small-molecule inhibitors lack locus specificity and often cause global chromatin disturbances. The emergence of programmable epigenetic editing technologies has transformed the field by enabling targeted rewriting of chromatin states at defined genomic loci. Catalytically inactive CRISPR/Cas9 platforms fused to transcriptional activators, repressors, or chromatin-modifying enzymes now allow precise addition or removal of epigenetic marks without altering the underlying DNA sequence. This chapter provides an overview of the conceptual and technical foundations of CRISPR-based epigenetic editing, including tools for gene activation and repression, DNA methylation, histone modifications, and multiplexed systems that permit coordinated regulation of multiple genomic loci or epigenetic marks. Delivery methods for in vitro and in vivo applications are discussed, with an emphasis on viral and nonviral platforms that enable tissue-specific, durable gene regulation. Finally, recent preclinical and clinical studies highlight the potential of programmable epigenetic editing as a next-generation therapy for precise and reversible gene control.
Lipase immobilization strategies are key to the potential use of biological catalysts in reactions of interest. The bioinspired synthesis of silica nanoparticles uses biological or environmentally friendly processes to create highly functionalized supports with desirable properties such as increased surface area and stability. Here, we describe the preparation of two immobilization methods for Thermomyces lanuginosus lipase on biomimetic silica supports: in situ entrapment and covalent attachment to the hetero-functionalized surface of the nanoparticles, and their application in the synthesis of fatty acid methyl esters.
Methods in molecular biology (Clifton, N.J.)Lorenzo Fontanelli, Carolina Ferri, Adrian Florentin Suman, Elisa Ceccherini, Giovanni Signore, Fabio Anastasio Recchia, Silvia Rocchiccioli
Cardiac organoids are increasingly used to model human cardiac development and disease, but their small size often limits molecular characterization, especially if different approaches and protocols are necessary to extract and quantify metabolites and lipids. Here, we present a multistep workflow for combined targeted free amino-acid (FAA) based metabolomic and lipidomic profiling from a single pooled cardiac organoid sample. The protocol covers organoid harvesting, detergent-assisted lysis, and a modified Folch extraction that generates an organic phase for lipid analysis and an aqueous phase for FAA metabolite analysis. Lipids are quantified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS) in Multiple Reaction Monitoring (MRM) mode using class-matched external standards and an internal standard to support calibration and reduce technical variability. Free amino acids and derivatives are analyzed from the same sample after filtration, drying, and AccQ-Tag derivatization, with norvaline as an internal standard. Together, this approach maximizes information yield from limited material and enables integrated analysis of metabolic and lipid pathways within the same biological specimen, facilitating organoid-based studies of cardiac maturation, disease modeling, and pharmacological responses.
Methods in molecular biology (Clifton, N.J.)Amin Alvani, Negar Mottaghi-Dastjerd, Abozar Ghorbani, Ahmad Gholami, Zeinab Pazhoohesh
Conventional treatments often face challenges such as the limited ability to penetrate the blood-brain barrier (BBB). The Doxorubicin-loaded graphene oxide/magnetite (DOX/GO/Fe3O4) nanocomplex offers a promising platform due to GO's high surface area and pH-sensitive release, and Fe3O4's magnetic properties. This protocol describes the methodology for evaluating the cytotoxicity of free DOX versus the DOX/GO/Fe3O4 nanocomplex in the A-172 glioblastoma cell line, followed by advanced bioinformatics analysis to identify gene networks and indirect pathways that enhance the nanomaterial's biocompatibility. The methodology integrates the MTT assay, real-time PCR for apoptosis genes (Casp3, Bax, and Bcl-2), and advanced analysis, including protein-protein interaction (PPI) networking, clustering, and promoter motif analysis. The analysis indicated that miR-92a-2-5p is a potential therapeutic target for preventing myocardial damage and enhancing biocompatibility. The findings highlight key regulatory pathways that indirectly boost nanodrug biocompatibility through the modulation of secondary components like miRNAs and cellular stress mechanisms.
Methods in molecular biology (Clifton, N.J.)Zhenlan Yao, Melody Li
Affinity purification-mass spectrometry (AP-MS) is a powerful proteomic approach for dissecting the interaction network between virus and host. Traditional AP-MS employs overexpression of viral proteins as baits to enrich host interactors. However, overexpressed viral proteins may mislocalize to inappropriate cellular compartments and trigger endoplasmic reticulum stress by overwhelming the protein-folding machinery, which leads to false identification of host factors. To overcome these limitations, we introduce an AP-MS strategy based on direct infection with an epitope-tagged chikungunya virus (CHIKV/myc-E2), which we used to successfully uncover two new antiviral factors in CHIKV cellular reservoirs-macrophages. In this protocol, we will describe this technique step by step: (1) design and construction of myc-tagged virus by advanced multi-fragment assembly, (2) in vitro transcription and preparation of infectious myc-tagged virus stocks, and (3) immunoprecipitation of myc-tagged viral protein and its interactome for mass spectrometry analysis. This strategy enables accurate identification of viral interactors in a physiologically relevant context, providing a framework for future proteomic studies using tagged viruses.
Methods in molecular biology (Clifton, N.J.)Elisa Gutierrez-Navarro, José Daniel Padilla-de la Rosa, Georgina Sandoval, Josué Solís
Flavonoids, valued for their health benefits, face challenges due to low solubility. Enzymatic acylation offers promise in enhancing properties, with the use of lipase B sourced from Candida antarctica (N435). This study has the aim to improve naringin solubility via esterification using as acyl donors short- and medium-chain fatty acids (vinyl acetate, propionate, and laurate) catalyzed by N435. Lipases play a crucial role as catalysts, facilitating the acylation process. Our study encompasses executing acylation and characterizing products. This process significantly enhances aqueous and organic solvent solubility of naringin esters. Additionally, acylating flavonoids with these fatty acids has the potential to broaden their range of applications in understanding and modulating gut health.
Methods in molecular biology (Clifton, N.J.)Syed M A Rizvi, Owais R Hakiem, Ming Tan
Chromatin immunoprecipitation (ChIP) measures binding between a specific protein and its in vivo DNA-binding sites. It is challenging to perform ChIP for bacteria that reside inside a eukaryotic cell, but we have optimized this method for Chlamydia-infected cells and used it to identify genome-wide binding sites of chlamydial transcription factors and sigma factors. The ability to measure protein-DNA interactions in intracellular chlamydiae without the need to isolate bacteria from the host cell allows changes in DNA binding to be measured during the Chlamydia developmental cycle and under different growth conditions. In this chapter, we provide a detailed protocol for Chlamydia ChIP by describing how to cross-link proteins to DNA, shear chlamydial chromatin into DNA fragments, and immunoprecipitate protein-DNA complexes with specific antibodies against the protein of interest. After a decrosslinking step, DNA fragments are analyzed by qPCR to measure binding to individual DNA-binding sites, or with deep sequencing for unbiased identification of genome-wide binding sites.
Methods in molecular biology (Clifton, N.J.)Leandro Battini
Structure-based virtual screening is an attractive strategy in drug design. It enables the selection of potential binders to target biomolecules with known molecular structures, providing a rational framework for studying their mechanisms of action and guiding further optimization. However, accurately predicting the free energy of binding remains an extremely challenging task, and the available tools only provide approximate estimates. In this context, one strategy to improve the predictive power of virtual screening and increase the probability of identifying true binders is to employ multiple docking algorithms and combine their results using a consensus method. In this chapter, we will describe a step-by-step methodology to perform this task. We will conduct a virtual screening using AutoDock Vina, AutoDock4, and LeDock, and then combine their results employing the Exponential Consensus Ranking algorithm. Finally, we will discuss possible extensions of this strategy, such as incorporating Molecular Dynamics simulations as an additional filter in the virtual screening or integrating the novel Artificial Intelligence-based methodologies into the consensus framework.
Methods in molecular biology (Clifton, N.J.)Romina Edith Avanzo, Orlando Elso, Fabricio Freije García, Guadalupe García Liñares
In this chapter, we describe the application of lipases as catalysts in the synthesis of structurally different amides with pharmacological activities. Numerous products, many of them new compounds, were obtained in very good to excellent yield and a highly regioselective manner. The influence of various reaction parameters in the enzymatic reactions, such as enzyme source, nucleophile/substrate ratio, enzyme/substrate ratio, solvent, and temperature, was examined for each case.
Methods in molecular biology (Clifton, N.J.)Suzana Ferreira-Dias, Natália M Osório, Carla Tecelão
In this chapter, some examples of laboratory protocols to produce nutritional structured lipids, namely human milk fat substitutes, dietetic triacylglycerols, and interesterified fat blends with improved functional and rheological properties, catalyzed by either immobilized commercial or noncommercial lipase preparations, are presented. The use of crude oils instead of refined counterparts to reduce oil purification costs is also addressed. In addition to batch synthesis, the continuous production in packed- or fluidized-bed bioreactors is addressed, as well as the evaluation of operational stability of the biocatalysts used (either in batch reuses or in continuous mode).
Methods in molecular biology (Clifton, N.J.)Rachel Weild, Richard D Hayward
This chapter describes methodological approaches for studying intracellular ion dynamics during Chlamydia trachomatis infection. It emphasizes the requirement for live-cell imaging to capture active and compartmentalized ion fluxes in infected cells precluded by fixed-cell techniques. Specifically, we present step-by-step protocols for labeling Chlamydia-infected cells with a fluorescent potassium ion (K+)-sensitive probe for performing live imaging with confocal microscopy or spinning disc confocal microscopy for higher resolution and procedures for image processing and quantification. We also describe time-lapse microscopy to monitor K+ dynamics over the extended time course of the infection. These advanced microscopy methods have the potential to visualize ion transport in real time at the host-pathogen interface.
Methods in molecular biology (Clifton, N.J.)Carine Alcon, Tou Cheu Xiong
Iron (Fe) is involved in numerous key physiological processes due to its transfer or gain electron capacity. However, in excess, Fe is toxic to cells. The understanding of mechanisms governing its homeostasis requires imaging techniques capable of localizing and quantifying Fe and its redox state in situ. The Perls-DAB histochemical staining is a widely used method for visualizing Fe at a cellular level. Other advanced approaches, such as micro X-ray fluorescence and X-ray absorption near-edge structure spectroscopy, also provide information on the spatial distribution and oxidation state of Fe. However, all of these methods require sample fixation before observing Fe localization, which can introduce biases in the results. Recent development in synthetic fluorescent probes now enables the real-time visualization of ferrous (Fe2+) and ferric (Fe3+) forms in living roots, offering a robust and user-friendly approach to study Fe redox distribution in planta. This live imaging approach provides access to Fe dynamics and enables semi-quantitative analyses, thereby opening new avenues for Fe homeostasis research. This chapter provides detailed protocols for applying these methods in Arabidopsis roots.
Methods in molecular biology (Clifton, N.J.)Michele Cervellera, Hang Zhou, Bram van den Broek, Kees Jalink, Ruslan I Dmitriev
Intracellular calcium signaling plays a crucial role in intestinal epithelial function. In this chapter, we describe a method to monitor calcium dynamics in live pig small intestinal organoids using stably expressed genetically encoded FRET-based biosensor in combination with intensity-based ratiometric and fluorescence lifetime imaging (FLIM) microscopy readouts. The calcium biosensor Twitch-2B was introduced into organoids by electroporation. Despite the modest lifetime contrast, the biosensor provides a highly reliable ratiometric signal and is compatible with both conventional fluorescence and FLIM-based imaging platform. Following electroporation, organoids are allowed to form and subsequently were selected to establish stable biosensor-expressing lines. This is followed by the live microscopy of heterogeneous and cell-specific calcium responses upon pharmacological stimulation. The presented protocol offers a versatile approach for studies of calcium signaling in relevant 3D cell models, such as organoids and tissues-on-a-chip.
Methods in molecular biology (Clifton, N.J.)Mónica R Montes, Santiago E Faraj
In this protocol, we describe a PAGE-based assay to evaluate the steady-state kinetics of the nsP2 protease. Using a recombinant GFP-TRX fusion substrate containing the nsP3/4 cleavage site, proteolytic activity is monitored through the appearance of cleavage products resolved by SDS-PAGE. Detection is achieved by Coomassie blue staining or GFP fluorescence emission, followed by densitometric analysis to quantify product formation and calculate initial reaction rates. The protocol provides a robust and accessible method for comparing enzyme activity under varying conditions, and is suitable for studies of substrate specificity, inhibitor screening, and the mechanistic characterization of protease function.
Reviews of physiology, biochemistry and pharmacologyUndurti N Das
AA has several physiological functions. Its various metabolites also have many significant actions and thus participate in innumerable physiological and pathological processes. There is strong evidence to suggest that AA and its metabolites play a significant role in the pathophysiology of diabetes mellitus (DM), hypertension (HTN), and coronary heart disease. In addition, high-fat diet (HFD), high-cholesterol diet (HCD), calorie-dense foods (CDF), high-salt and low-potassium diet, low magnesium, lack of exercise, sedentary lifestyle, and aging alter the generation of AA from its dietary precursor linoleic acid (LA) by altering the activities of desaturases. This may enhance the occurrence of a pro-inflammatory milieu in the cells/tissues/organs that can be ameliorated to a certain extent by AA.
Methods in molecular biology (Clifton, N.J.)Almudena Escobar-Niño, Olivier Coste, Maria Victoria Calcis-Marzán, Francisco Javier Fernandez-Acero
Proteomics has been revealed as a key set of technologies that provide a detailed description of the molecular processes involved in the development of a specific phenotype. "Omics" technologies can collect an incredible amount of information. Among them, proteomics is an invaluable tool for defining specific biological information by studying the complete set of proteins under specific conditions, the proteome; or specific subsets of proteins, the subproteome. It is a crucial instrument for describing protein post-translational modifications, the functional annotation of the genome, and the detection of orphan genes. Protein extraction procedures are necessary to obtain B. cinerea protein extracts of sufficient quality to be analyzed by LC-MS/MS, avoiding contaminants that interfere with the identification process. After experimental design, collect the samples and replicates as defined in each experimental approach; we will describe protocols and procedures for the next steps of proteome and subproteome extraction and LC-MS analysis.
Methods in molecular biology (Clifton, N.J.)Martina Špundová, Jakub Hajný
Water potential is the driving force behind the movement of water within the soil-plant-atmosphere continuum. Measuring water potential and its components provides valuable information about plant- and root water relations, including osmoregulation. Here, we describe a straightforward method for determining water and osmotic potential, as well as cell turgor in roots, using dew point hygrometry. The chapter begins with a brief overview of issues related to plant and root water potential, and then proceeds to describe the principles and measurement procedures of dew point hygrometry in more detail, including calibration, equilibration, and sample preparation. Practical recommendations are provided to minimize common error sources. This methodology may be useful for researchers studying plant-water balance, root water uptake, plant stress physiology, and osmoregulation.
Methods in molecular biology (Clifton, N.J.)Yulin Gan, Yakun Peng, Shutang Tan
The polar transport of the phytohormone auxin regulates plant organogenesis, morphogenesis, and gravitropism, which are coordinately regulated by auxin influx and efflux carriers. To measure the basipetal (shootward) transport of auxin in roots, we applied radiolabeled IAA to the root tips of 7-day-old seedlings in the model plant Arabidopsis thaliana. A scintillation counter was then used to measure the radioactivity of the samples. Here, we describe a method for measuring auxin transport in A. thaliana roots via 3H-indole-3-acetic acid (3H-IAA).
Chikungunya virus (CHIKV) is an emerging arbovirus lacking widely effective antiviral therapies, highlighting the need to characterize molecular interactions essential for its replication and pathogenesis. Surface plasmon resonance (SPR) is a sensitive, label-free technique that enables real-time analysis of biomolecular interactions by detecting refractive index changes at a sensor surface. SPR provides precise kinetic and affinity measurements for protein-protein, protein-RNA, and protein-ligand interactions using minimal sample quantities and supporting high-throughput screening. In CHIKV research, it has been applied to evaluate antiviral compounds, antibodies, receptor binding, and viral protein complexes. This chapter outlines key methodological considerations, including ligand preparation, immobilization strategies, assay optimization, and kinetic analysis.
Methods in molecular biology (Clifton, N.J.)Luis Felipe Chávez-Flores, Dolores Reyes-Duarte
Sugar fatty acid esters have a wide range of applications in food, cosmetic, detergent, and pharmaceutical industries due to their excellent surface and antimicrobial properties. Additionally, some sugar esters are insecticides and acaricides. In this work, we described the enzymatic synthesis of lactulose monoesters, their purification, and qualitative and quantitative chromatographic analysis.
Methods in molecular biology (Clifton, N.J.)Nadia Guajardo
This chapter of the book details the experimental methodology for obtaining a 5-hydroxymethyl furfural (HMF) ester through enzymatic catalysis. The esterification reaction consists of obtaining 5-acetyl-hydroxymethylfurfural from HMF and ethyl acetate catalyzed by the lipase CALB from immobilized Candida antarctica (Novozym 435®). Esterification is carried out in a batch and continuously packed-bed reactor. The operating conditions in batch mode are 35°C, 100 mM HMF, 0.02 g of biocatalyst, and 5 mL of reaction volume. In continuous mode, the reaction was carried out using a stainless-steel column of 0.4 cm internal diameter and 12.6 cm long, packed with 0.5 g of biocatalyst. The operating conditions are 35°C, 100 mM HMF, and flows of 0.02, 0.05, and 0.1 mL/min. Continuous esterification stands out over the batch mode for its simplicity of operation and high productivity.
Methods in molecular biology (Clifton, N.J.)Jose L Gonzalez-Alfonso, David Rodriguez-Garcia, Carlos Uceda-Dominguez, Antonio O Ballesteros, Francisco J Plou
We describe an efficient method for acylating non-glycosylated polyphenols, which involves an initial α-glucosylation step catalyzed by sucrose phosphorylase, followed by acylation using an immobilized lipase. This method is applied to phloretin. It is first glucosylated at the 4'-OH position, followed by quantitative acylation at the 6-OH of glucose with C8, C12, and C16 acyl chains using the lipase from Thermomyces lanuginosus. The aqueous solubility of the acyl glucosides is similar to that of the aglycone. The radical scavenging capacity of the new phloretin derivatives measured with ABTS·+ shows a slight decrease. This methodology offers a means to adjust the physicochemical and biological properties of polyphenols by varying the length and nature of the acyl donor.
Journal of the International Society of Sports NutritionCui Xingchen, Liu Meng, Cao Yinhang, Zhao Can
BACKGROUND: Weight reduction before competition has long been a major challenge for combat-sports athletes. Unscientific weight-loss practices not only increase the risk of hormonal imbalance, electrolyte disturbance, and impaired immune function but also compromise athletic capacities such as aerobic and anaerobic performance and explosive power. This study investigated the potential benefits of tangeretin, a natural citrus extract, on athletic performance. These benefits include the regulation of hormone secretion, the optimization of carbohydrate and lipid metabolism, and the promotion of muscle synthesis and lipid utilization, which may help athletes reach their target weight. OBJECTIVE: To examine the effects of tangeretin supplementation on body-composition and sports-performance indicators in female combat-sports athletes, and to evaluate its efficacy for simulated precompetition weight loss. METHODS: Thirty female combat-sports athletes (4 wrestlers, 3 boxers, 9 sanda athletes, 3 judokas, and 11 taekwondo athletes) were recruited. A paired, randomized, double-blind design was used. All 30 participants were first paired according to sport and competitive level, then randomly allocated to either a tangeretin group or a placebo group (15 per group). The 30-day intervention comprised a double-blind nutritional supplement, a dietary questionnaire, blood sampling and analysis, body-composition assessment, isokinetic muscle-strength testing, and aerobic-capacity testing, to evaluate the effects of tangeretin (400 mg/day) on performance and health-related outcomes. These outcomes included body-composition indicators (e.g. body-fat percentage, fat mass, lean-body mass), fat-distribution indicators (e.g. limb and regional fat mass, gynoid fat mass), glucose and lipid-metabolism indicators (e.g. LDL-C, total cholesterol, free fatty acids), hormone indicators (e.g. cortisol, testosterone), muscle-strength indicators (e.g. peak torque of hip, knee, and ankle flexors and extensors), and maximal-oxygen-uptake-derived indicators (e.g. VO2max heart rate, time to exhaustion). Statistical significance was assessed using two-way repeated-measures analysis of variance (ANOVA). RESULTS: (1) Body weight: 80.0% of athletes in the tangeretin group lost weight to varying degrees, and 33.3% (5 athletes) reached their competition weight class. (2) Body fat: body-fat percentage in the tangeretin group decreased from 23.92 ± 0.87% to 22.92 ± 0.89%; trunk, upper-limb, lower-limb, and android-region fat mass decreased by 6.77%, 8.77%, 4.43%, and 8.09%, respectively. (3) Glucose and lipid metabolism: HDL-C increased by 8.86% (P = 0.002), whereas triglycerides decreased by 25.00% (P = 0.048). (4) Sports performance: peak torque during hip, knee, and ankle flexion and extension increased significantly in the tangeretin group. VO2max also improved from 42.80 ± 1.13 to 43.46 ± 1.32 ml/kg/min. CONCLUSION: Tangeretin supplementation effectively reduced body weight and fat mass (both overall and regionally) in female combat-sports athletes. Among the participants, 80.0% lost weight to varying degrees, and 33.3% reached their competition weight class, indicating a strong effect on precompetition weight loss. Moreover, tangeretin improved aerobic capacity and lower-limb joint muscle strength, thereby enhancing competitive performance.
Plant signaling & behaviorSusmita Das, Yamshi Arif, Hesam Mousavi, Henrik Aronsson, Nadia Bazihizina, Mohammad Sarraf
Salinity is a major abiotic constraint on plant growth, productivity, and global food security. It disrupts cellular protein homeostasis (proteostasis) at every step, from synthesis to degradation. Plant survival under salt stress therefore depends on the coordinated regulation of protein synthesis, folding, post-translational modification, trafficking, quality control, and turnover. The individual contributions of the nucleus, ribosomes, endoplasmic reticulum (ER), and Golgi apparatus to salinity responses are increasingly well described, but how these compartments communicate remains far less understood. This review synthesizes current evidence on this inter-organellar signaling network, addressing four questions: how salinity-induced osmotic, ionic, and oxidative stress reprograms nuclear transcription and chromatin state; how ribosomes sustain selective translation of protective proteins while clearing stalled or damaged translational products; how the ER expands its folding capacity through the unfolded protein response and removes terminally misfolded proteins by ER-associated degradation; and how the Golgi sustains glycosylation, sorting, and secretion of the transporters needed for ion and osmotic homeostasis. Beyond these organelle-level responses, the review discusses two signaling layers that couple them into a single circuit: post-translational modifications (phosphorylation, ubiquitination, SUMOylation, and redox- and nitrosative/persulfidation-based marks) and phytohormone/gasotransmitter signals, including abscisic acid, nitric oxide, hydrogen sulfide, carbon monoxide, and melatonin. By framing proteostasis as an emergent property of nucleus-ribosome-ER-Golgi coordination rather than of any single compartment, this review identifies specific inter-organellar signaling nodes as candidate targets for breeding or engineering salinity-resilient crops.
The global prevalence of Metabolic dysfunction-associated steatotic liver disease (MASLD) among persons with HIV (PWHIV) is estimated at 34%. This study examined the influence of gut microbial and metabolic alterations in PWHIV with MASLD. We conducted 16S rRNA sequencing on 60 fecal samples (24 from HIV group and 36 from HIV-MASLD group) to assess microbial composition, and employed LC/MS-based metabolomics on both fecal and plasma samples to identify variations in metabolites. An exploratory analysis was performed to compare plasma cytokines and oxidative stress markers between the two groups. Sequencing in the HIV-MASLD group showed lower Faecalibacterium and Anaerobutyricum, and higher Klebsiella, Escherichia, and Enterococcus. Metabolomic analysis revealed a reduction in anti-inflammatory metabolites (Dodecanedioic acid, 5-Phenylvaleric acid, Kynurenic acid) and antioxidant metabolites, coupled with an increase in the pro-inflammatory metabolite Gamma-Glutamyl-L-Putrescine in the HIV-MASLD group. And plasma analysis indicated a decline in 20-carboxy arachidonic acid. Additionally, Faecalibacterium and Anaerobutyricum correlated positively with anti-inflammatory metabolites, while Klebsiella, Enterobacter, and Citrobacter correlated negatively. Furthermore, Faecalibacterium and Anaerobutyricum also showed a moderate negative correlation with MDA levels. A random forest model using plasma metabolites showed the optimal discriminative ability for HIV-MASLD (AUC = 0.819 ± 0.0059). Differences in gut microbiota and metabolites in PWHIV may affect MASLD progression by influencing inflammation and oxidative stress. Modulating gut microbiota, such as adding Faecalibacterium and Anaerobutyricum, could be a treatment strategy for HIV-MASLD. Plasma metabolites have potential as noninvasive markers.
Gynecological endocrinology : the official journal of the International Society of Gynecological EndocrinologyXiangyan Ruan, Juan Du, Muqing Gu, Fengyu Jin, Jing Jin, Yanglu Li, Jiaojiao Cheng, Yanqiu Li, Weimin Kong, Qi Zhang, Zecheng Wang, Mingzhen Zhang, Anming Liu,…
OBJECTIVE: This article reports on the world's first live birth after ovarian tissue cryopreservation and transplantation (OTCT) in a patient with colon cancer. METHODS: A 29-year-old unmarried nulliparous patient underwent ovarian tissue cryopreservation before chemotherapy. After chemotherapy-induced premature ovarian insufficiency, she received the first transplantation of six thawed ovarian cortex strips. Despite normal ovarian function being maintained for over 2 years, she requested and underwent a second transplantation with another six strips to maximize her chances of pregnancy. RESULTS: After the first transplantation, she resumed spontaneous menstruation with restored ovarian function, which was remained functional for 28 months until the second transplantation. Following the second transplantation, she conceived spontaneously without assisted reproductive technology two months after grafting. Routine prenatal examinations showed normal fetal development without abnormalities. She delivered a healthy baby girl weighing 2785 g with Apgar scores of 8/9 at 1 and 5 min. CONCLUSIONS: This case demonstrates the feasibility and safety of OTCT for fertility and ovarian function preservation in patients with colon cancer.
Journal of the International Society of Sports NutritionDamoon Ashtary-Larky, Darren G Candow, Jose Antonio, Christian Quiceno, Fabrizio Angelini, Scott C Forbes, Sajjad Moradi, Fatemeh Kazeminasab, Reza Bagheri
AIM: Soccer places repeated demands on short-duration, high-intensity performance, including sprints, accelerations, jumps, and technical actions performed over the course of a match. Because these efforts depend heavily on rapid adenosine triphosphate (ATP) resynthesis, creatine is a plausible ergogenic aid for soccer players. However, findings in soccer-specific studies have been inconsistent, and previous reviews have addressed narrower questions using smaller evidence sets. This systematic review therefore examined the effects of creatine supplementation on performance, body composition, and recovery/fatigue-related outcomes in soccer players. METHODS: PubMed, Scopus, and Web of Science were searched from inception to 15 May 2025 in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement. We included controlled clinical trials of creatine supplementation in male or female soccer players of any age or competitive level when an eligible placebo, control, or comparison condition was available. Outcomes were grouped into performance, body composition, and recovery/fatigue domains. Domains from the revised Cochrane risk-of-bias tool for randomised trials (RoB 2) and the Risk Of Bias In Non-randomised Studies - of Interventions (ROBINS-I) tool were used to produce study-level methodological summaries rather than formal result-specific risk-of-bias assessments. Because the studies differed substantially in design, supplementation protocols, populations, and outcomes, findings were synthesised qualitatively rather than pooled in a meta-analysis. RESULTS: Source-level verification left 45 eligible reports representing 41 unique studies: 28 randomised parallel-group studies, 2 randomised crossover/repeated-condition studies, and 11 non-randomised or allocation-unverifiable controlled studies. Some trials reported favourable findings for repeated-sprint performance, anaerobic power, and muscular strength, but the comparative evidence was inconsistent and the certainty of evidence was very low. Findings for vertical jump performance and technical skills were mixed, while change-of-direction and endurance outcomes showed no consistent benefit. Recovery- and fatigue-related findings were also largely null or inconsistent. Body mass often increased, particularly after loading protocols, whereas evidence for gains in lean mass was limited and inconsistent. CONCLUSION: The soccer-specific evidence for creatine supplementation remains suggestive rather than conclusive. Favourable findings have been reported for some repeated-sprint, anaerobic-power, and strength outcomes, but the comparative evidence is heterogeneous, often limited by unclear or nonsignificant between-group effects, and of very low certainty. Firm conclusions about efficacy are therefore not possible. Body mass commonly increased, especially after loading protocols, while effects on lean mass, endurance, change-of-direction ability, and recovery/fatigue-related outcomes remain uncertain.
Clinical and experimental hypertension (New York, N.Y. : 1993)Zhongbo Chen, Wang Chunbin, Hanmei Zhang, Ting Luo, Jinhua Yan
Hypertension remains one of the leading modifiable risk factors for cardiovascular disease and premature mortality worldwide. Blood pressure (BP) management is particularly challenging in patients with coexisting type 2 diabetes (T2D), chronic kidney disease (CKD), or heart failure (HF). Sodium-glucose cotransporter 2 (SGLT2) inhibitors have become an integral component of contemporary cardiorenal therapy and have consistently demonstrated additional BP-lowering effects across diverse clinical settings. This narrative review synthesizes the current clinical evidence regarding the antihypertensive effects of dapagliflozin and summarizes the biological mechanisms that may contribute to these effects, including osmotic diuresis and natriuresis, weight reduction, modulation of the sympathetic nervous system, enhanced uric acid excretion, and attenuation of oxidative stress, inflammation, and vascular dysfunction. Across clinical studies, reductions in systolic BP (SBP) generally exceed those in diastolic BP (DBP), although the physiological basis underlying this differential response remains incompletely understood. Dapagliflozin has demonstrated good tolerability and antihypertensive effects in clinical applications, its long-term use may be limited by the risk of genital infections. Collectively, the available evidence supports dapagliflozin as a cardiorenal therapeutic agent that may provide additional BP-lowering benefits in patients with hypertension and concomitant T2D, CKD, or HF, rather than as a substitute for conventional antihypertensive therapy. Future hypertension-focused clinical studies are warranted to further define its role in hypertension management.
Drug deliveryYoungbeom Kim, Chae-Ok Yun, A-Rum Yoon
Nose-to-brain (N2B) delivery is a promising noninvasive strategy to circumvent the blood‒brain barrier (BBB). However, the clinical translation of N2B platforms remains hindered by a conceptual oversimplification that equates crude central nervous system (CNS) exposure with effective therapeutic delivery. This review reconceptualizes the N2B pathway as a stage-resolved sequential transport cascade extending from nasal entry to meaningful parenchymal and cellular access. We identify a critical imbalance in current formulation strategies, which have advanced two extreme poles, namely initial mucosal retention and epithelial permeation at one end and final target-cell engagement at the other, while leaving the intermediate post-epithelial gateways, including cerebrospinal fluid (CSF) and perivascular space (PVS)-mediated redistribution and deep intraparenchymal diffusion, as structural and interpretive blind spots. To bridge this gap, we propose an integrated engineering paradigm with three core design principles. First, multistage-aware design harmonizes sequential spatiotemporal functionalities within a single vehicle architecture. Second, disease-informed design integrates pathologically remodeled barrier states and glymphatic hydrodynamics into the formulation rationale. Third, cargo-informed design establishes the distinct biophysical identity and inherent liabilities of each payload, from small molecules to viral vectors and CRISPR machinery, as the primary engineering starting point. Finally, we advocate for a methodological standard demanding stage-specific validation of vehicle integrity, cross-boundary penetration, and functional target engagement, beyond compartment-blind whole-brain readouts. We further underscore the biofate and clearance of delivery systems as an emerging axis of safety and regulatory evaluation. Collectively, this framework provides a systematic basis for transitioning N2B engineering into a viable clinical modality.
Gut microbesXianxi Ye, Qingpeng Cai, Yulong Li, Mingjie You, Junli Chen, Yangjie Pan, Ming Zhu, Zhuofan Lu, Siyuan Chen, Yan Lin, Weiru Zhang, Xuelian Liu, Zhen Wang, Guan…
Dysregulation of the gut-brain metabolic axis has been implicated in the progression of diabetes-associated cognitive decline (DACD), yet its underlying mechanism remains largely unknown. Herein, we identified a low abundance of Akkermansia muciniphila (AKK) and high leucine levels as key driving factors of DACD in both diabetic mice and patients. Our results revealed that AKK bacteria hold the capacity to catabolize leucine, and the elevated leucine levels observed in diabetic mice and patients are partly attributed to a low abundance of AKK bacteria. Moreover, leucine supplementation and deprivation further confirmed that excessive leucine accelerated and aggravated the progression of DACD in mice. Mechanistically, leucine directly modulated the phosphorylation of TBK1 and, in turn, phosphorylated the autophagy adaptor OPTN, thereby enhancing its binding capacity to LC3 and Ub chains and triggering neuronal excessive mitophagy. Therefore, this study uncovers a new mechanism of the gut-brain metabolic axis involved in DACD progression and provides a novel perspective for gut-targeted therapy of cognitive disorders.
The Libyan journal of medicineMadjda Akacha, Abdelhalim Khenchouche, Mounira Amrane, Abbas Mehnane, Abdelmadjid Lacheheb, Nasir A Ibrahim, Nosiba S Basher, Mohammed Saad Aleissa, Assia Derg…
The COVID-19 pandemic revealed the importance of pre-existing conditions, or comorbidities, as major predictors of disease severity and mortality worldwide. In this context, the major goal of this study was to examine the impact of common comorbidities on the clinical outcomes of COVID-19 patients in Sétif, Algeria, as well as to evaluate the local epidemiological risk profile. We conducted a thorough analysis of 12,932 COVID-19 participants in the Sétif community, focusing on the prevalence and impact of many illnesses, most notably diabetes, cardiovascular disease (CVD), and hypertension. We also looked at geographical inequalities in comorbidity prevalence to help influence local patient care methods. Our data revealed a strong link between these comorbidities and poor COVID-19 outcomes. Patients with heart disease, hypertension, and diabetes had a considerably greater incidence of severe COVID-19, with computed odds ratios (ORs) of 1.50, 1.34, and 2.89, respectively, indicating that diabetes was the best predictor of severity in this cohort. This investigation demonstrated that the presence of comorbidities considerably exacerbated the severity and mortality rate of SARS-CoV-2 infection. These findings highlight the importance of establishing thorough, personalised techniques and more effective diagnostic and treatment approaches for COVID-19 patients that explicitly account for the varied prevalence of underlying health issues.
Gut microbesTim Hensen, Lora Khatib, Lucas Patel, Daniel McDonald, Antonio González, Siamak MahmoudianDehkordi, Colette Blach, Rob Knight, Rima Kaddurah-Daouk, Ines Thiele
The human gut microbiome has been linked to metabolic disturbances in Alzheimer's disease (AD). However, the mechanisms by which gut microbes might influence metabolic dysfunction in AD remain poorly understood. Previously, gut microbiome-personalized whole-body models of human metabolism have been applied to predict how altered gut microbiome compositions may influence metabolites in the blood of healthy aging individuals with increased risk of AD. However, these previous results have not been validated in AD. In this study, we aimed to test these prior predictions in a cohort of AD dementia patients and individuals with mild cognitive impairment (MCI) and a probable AD diagnosis. Therefore, we created gut microbiome-personalized whole-body metabolic models for 34 AD dementia patients, 51 MCI patients, and 298 healthy controls. These in silico models were profiled to predict the metabolic influences of gut microbiomes on blood metabolites with previously reported alterations in AD. We found increased capacities of the in silico host-microbiome co-metabolism to produce S-adenosyl-L-methionine, L-arginine, creatine, taurine, and formate in the blood of AD patients. The metabolic predictions were then linked to key microbial taxa using a novel method that combines modeling-informed prediction sensitivity to alternative microbial abundances with LASSO-based taxonomic stability selection and elastic net regressions. This method found that increased relative abundances of Bacteroides uniformis and Bacteroides thetaiotamicron in AD were major factors driving the predicted metabolic changes. Furthermore, the metabolic predictions were associated with allelic variations in the APOE risk gene in healthy individuals, confirming our previous findings. In conclusion, we identified blood metabolites with known links to AD that were differentially influenced by gut microbiota in AD, and identified possible microbial drivers of these predicted shifts in host-microbiome interactions. These findings may facilitate the development of microbiome-informed treatments of AD.
Aquaporins and nitric oxide (NO) are essential regulators of lateral root (LR) development in plants. However, whether an aquaporin could modulate NO-affected LR formation remains elusive. Here, we show that the tonoplast intrinsic aquaporin protein AtTIP5;1 negatively regulates NO-promoted LR development. AtTIP5;1 is highly expressed in root pericycle cells. AtTIP5;1 overexpression causes significant suppression of LR formation induced by sodium nitroprusside (SNP), an NO donor. Moreover, AtTIP5;1 overexpression results in a clear decrease in NO accumulation in the roots. The application of exogenous auxin influx inhibitor naphthoxyacetic acid boosts the effect of AtTIP5;1 on LR growth evoked by SNP, whereas the auxin efflux inhibitors N-1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid notably attenuate this effect. In addition, AtTIP5;1 overexpression leads to elevated hydrogen peroxide levels, decreased superoxide anion accumulation, enhanced superoxide dismutase activity, and reduced activities of catalase, ascorbate peroxidase, and peroxidase in roots under SNP treatment. These results suggest that AtTIP5;1 may inhibit NO-facilitated LR development by reducing NO accumulation, affecting auxin transport, and altering ROS homeostasis in Arabidopsis.
Gut microbesFlorine H M Westerbeke, Moritz V Warmbrunn, Melany Rios-Morales, Xinmin S Li, Melissa Y Tian, Madelyn A Novak, Austin M Williams, Stanley L Hazen, Ilias Attaye…
The gut microbiota influences host metabolism through diverse metabolites, many of which have been linked to glucose homeostasis and type 2 diabetes (T2D). Understanding microbial contributions to metabolite biosynthesis is essential for developing dietary and microbiota-targeted T2D prevention and treatment strategies. We performed targeted plasma metabolomics in individuals with T2D and healthy controls, all receiving histidine supplementation, before and after gut microbiota suppression using 7-day broad-spectrum antibiotic treatment. Associations between pre-antibiotic metabolite levels and fecal metagenomics-derived gut microbiota composition were examined using co-abundance network analysis and Random Forest modeling. Indole-3-propionic acid (IPA) was the only gut-derived metabolite differing between groups before antibiotics, with lower levels in T2D and higher levels associated with reduced T2D odds. Antibiotic treatment reduced IPA to near-undetectable levels in both groups, confirming its predominantly microbial origin. Beyond established inverse associations with BMI and glycemic markers, we found a novel inverse correlation between IPA and glycemic variability, consistent with a protective association with T2D. Plasma IPA was associated with gut microbiota beta diversity. IPA-associated species clustered within a single co-abundance module, but did not include known IPA producers, suggesting plasma IPA is influenced by broader microbial community composition rather than IPA-producing capacity of individual taxa alone. This study provides direct human evidence that plasma IPA is virtually exclusively gut microbiota-derived in individuals with T2D, extending prior findings in healthy populations. It highlights IPA's relevance to metabolic health and T2D, and guides future research on dietary and microbiota-targeted strategies to modulate IPA, advancing T2D prevention and treatment.
Drug deliveryJongho Cho, Dongjun Han, Hyemi Lee, Dong-Wook Park
Polydeoxyribonucleotide (PDRN) is a bio-derived therapeutic agent known to exhibit regenerative, angiogenic, and anti-inflammatory effects. However, its relatively large molecular size limits penetration across the skin barrier, thereby reducing the feasibility of noninvasive transdermal delivery. Iontophoresis is a promising approach for enhancing the transport of charged macromolecules, yet quantitative understanding of PDRN iontophoresis remains limited. Here, we present an in silico framework for cathodal PDRN iontophoresis based on the Nernst-Planck equation, focusing on passive diffusion and electromigration. The PDRN diffusion coefficient was determined from Franz diffusion cell experiments using the lag-time method, and the effective charge was estimated from prior experimental reports on dsDNA. A three-layer skin model comprising the stratum corneum, epidermis, and dermis was employed, and PDRN transport was analyzed as a function of applied current density, initial PDRN concentration, delivery time, and PDRN diffusion coefficient. Transport was quantified using molar flux and cumulative permeation at the epidermis-dermis interface and mid-dermis. The results clarify parameter-dependent regulation of delivery and reveal a depth-dependent shift in the dominant transport mechanism, highlighting the importance of iontophoresis for macromolecular delivery. Using calibrated outputs, we assessed agreement with experimental permeation data using the root-mean-square deviation (RMSD), mean absolute percentage error (MAPE), and the coefficient of determination (R2). Overall, this work provides a computational baseline to predict PDRN iontophoresis outcomes and proposes practical iontophoresis strategies for PDRN to guide device design.
Biochimica et biophysica acta. Molecular basis of diseaseYongqing Zhao, Jian Yang, Weixiong Zhu, Guoqing Zhang, Shaozhen Rui, Teng Ma, Zhao Guo, Wence Zhou
BACKGROUND: The global burden of metabolic-associated hepatocellular carcinoma (HCC) is increasing, with obesity emerging as a key causal factor. However, the molecular mechanisms linking lipid metabolic dysregulation to HCC progression and therapeutic vulnerability remain unclear. METHODS: We analyzed Global Burden of Disease 2021 data to assess liver cancer burden attributable to metabolic risks from 1990 to 2021. Mendelian randomization was used to evaluate causal associations between metabolic traits and liver cancer risk. TCGA, GTEx, and GEO datasets were integrated to identify lipid stress-responsive regulators. Clinical relevance was assessed using public datasets and tissue microarray immunohistochemistry. Functional validation was performed in HCC cells and a high-fat diet-fed syngeneic mouse tumor model. RESULTS: Liver cancer deaths and DALYs attributable to metabolic risks increased markedly from 1990 to 2021. Mendelian randomization showed that obesity-related traits, including BMI, waist circumference, and body fat percentage, were causally associated with liver cancer risk, whereas glycemic traits were not. Bioinformatics screening identified GPAT3 as a lipid metabolism regulator upregulated in HCC, induced by palmitic acid, associated with poor prognosis, and enriched in patients with higher BMI. Tissue microarray analysis confirmed increased GPAT3 protein expression in HCC and its association with higher BMI and GPX4 expression. GPAT3 depletion sensitized HCC cells to palmitic acid-induced ferroptosis, whereas Fer-1 rescue and GPAT3 overexpression supported its protective role. In vivo, FSG67 enhanced sorafenib-associated antitumor effects and increased tumor lipid peroxidation. CONCLUSIONS: GPAT3 protects HCC cells from lipid stress-induced ferroptosis and represents a potential metabolic vulnerability in obesity-associated HCC.
Stroke remains one of the leading causes of death and long-term disability worldwide, with neuroinflammation and metabolic dysfunction playing central roles in its pathogenesis. In this study, we investigated the function of Y-box binding protein 3 (YBX3) in ischemic stroke, with particular focus on its regulation of glycolysis and neuroinflammatory responses. Using a middle cerebral artery occlusion (MCAO) model in C57BL/6 mice and an oxygen-glucose deprivation/reperfusion (OGD/R) model in BV2 microglia, we observed that YBX3 expression was markedly upregulated following ischemia-reperfusion. Overexpression of YBX3 aggravated cerebral infarct size, worsened motor and neurological outcomes, and increased the release of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) both in vivo and in vitro. Mechanistically, YBX3 enhanced glycolysis in BV2 cells by stabilizing pyruvate kinase M2 (PKM2) mRNA and promoting its expression, while simultaneously activating glycolysis through the mTOR-HIF-1α signaling pathway. Pharmacological inhibition of mTOR with rapamycin reversed YBX3-induced glycolytic activation, neuroinflammation, and neuronal injury. Collectively, these results identify YBX3 as a key regulator of ischemic brain damage through glycolysis-dependent mechanisms and underscore its potential as a therapeutic target for stroke.