Sepsis-induced cardiomyopathy (SIC) is a severe complication of sepsis and septic shock and is characterized by cardiac dysfunction. Levosimendan (LEVO), a calcium sensitizer, has shown therapeutic potential in SIC, although its underlying mechanism remains unclear. Nrf2, a pivotal regulator of antioxidant and anti-inflammatory responses, may represent a potential target for SIC treatment. In this study, we examine the effects of LEVO on SIC and explore the mechanistic role of Nrf2 in mediating its cardioprotective effects. A murine SIC model is established via cecal ligation and puncture (CLP), and cardiomyocyte injury is induced via lipopolysaccharide (LPS) exposure in HL-1 cells. The CLP procedure significantly elevates serum cTnI and IL-6 levels and reduces the survival rates of mice. Echocardiographic analysis reveals impaired cardiac structure and function, accompanied by mitochondrial morphological and functional damage, in SIC mice. Interestingly, these pathological changes in SIC are markedly attenuated by LEVO treatment. Similarly, LEVO administration restores proliferative capacity; increases mitochondrial ATP, mitochondrial membrane potential (MMP) and NADH levels; and reduces ROS production and intracellular calcium overload. Notably, the protective effects of LEVO on cardiomyocyte viability and mitochondrial function are significantly diminished following Nrf2 inhibition or knockout (KO). Collectively, these findings demonstrate that LEVO mitigates cardiomyocyte injury and mitochondrial dysfunction in SIC through an Nrf2-dependent mechanism.

Myocyte enhancer factor 2A (MEF2A), a transcription factor implicated in coronary artery disease, remains unexplored in vascular redox regulation. To address this gap and overcome the limitations of current antioxidant therapies, we investigate the role of MEF2A in oxidative defense via human umbilical vein endothelial cells (HUVECs) and murine models. Adenoviral vectors encoding MEF2A-specific shRNAs or mRNAs are used to silence or overexpress in HUVECs. For validation, endothelial-targeted knockdown is achieved via AAV1-shRNA delivery in mice fed with a high-fat diet. Systemic redox status is assessed by measuring reactive oxygen species (ROS), glutathione homeostasis (GSH/GSSG ratio), the NADH/NAD balance, the mitochondrial membrane potential (ΔΨm), and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Mechanistic insights are derived from immunofluorescence, qPCR, western blotting, and dual-luciferase reporter assays. silencing induces redox imbalance, characterized by elevated ROS, a reduced GSH/GSSG ratio, and ΔΨm collapse. Conversely, MEF2A overexpression synergizes with SIRT1 to restore the glutathione pool, maintain NAD homeostasis, and suppress ROS under oxidative stress. Chromatin immunoprecipitation confirms that MEF2A directly binds to two cis-elements in the promoter, driving transcriptional activation. , MEF2A-deficient mice present increased vascular oxidative damage, as indicated by elevated DNA damage marker (8-OHdG) and ROS levels. The downregulation of SIRT1/PGC-1α in -silenced cells is verified . Our findings establish MEF2A as a master regulator of endothelial redox defense via the SIRT1-PGC-1α axis, providing a mechanistic foundation for the treatment of oxidative cardiovascular disorders. This work suggests that pharmacological MEF2A activation is a novel strategy for precision antioxidant therapy in vascular medicine.

Ferroptosis, an iron-dependent form of regulated cell death, is characterized by excessive reactive oxygen species (ROS) accumulation and lipid peroxidation of polyunsaturated fatty acids (PUFAs) in cellular membranes. Non-small cell lung cancer (NSCLC) harboring KRAS mutations often exhibits therapeutic resistance but may display high susceptibility to ferroptosis. Eupalinolide B (EB), a natural compound with documented anti-cancer activity, has not been thoroughly explored for its ferroptosis-inducing potential in KRAS-mutant NSCLC. In this study, we demonstrate that EB treatment significantly elevates ROS levels, intracellular iron accumulation, and lipid peroxidation in KRAS-mutant NSCLC cells, resulting in ferroptotic cell death. Molecular docking and cellular thermal shift assays reveal that EB directly binds to and activates heme oxygenase-1 (HO-1), a critical component of the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2/HO-1 oxidative stress response pathway. Genetic or pharmacological inhibition of HO-1 attenuates EB-induced ferroptosis, underscoring the pivotal role of HO-1-mediated oxidative stress in this process. Furthermore, studies using KRAS-mutant H358 xenograft models confirm the potent anti-tumor effects of EB. Collectively, our findings establish that EB triggers ferroptosis in KRAS-mutant NSCLC by activating the Keap1-Nrf2/HO-1 pathway, suggesting a promising therapeutic strategy for this challenging malignancy.

Lung squamous cell carcinoma (LUSC) remains a major therapeutic challenge because of its pronounced resistance to chemotherapy, particularly carboplatin. In this study, we investigate the role of SOX2, a lineage-survival oncogene, in mediating carboplatin resistance in LUSC. We demonstrate that SOX2 is highly expressed in LUSC and is significantly associated with poor prognosis. Our results show that SOX2 directly transactivates the expression of NRF2, a master regulator of cellular redox homeostasis, thereby increasing glutathione (GSH) synthesis and protecting cells from carboplatin-induced oxidative stress. Pharmacological or genetic inhibition of NRF2 effectively abrogates SOX2-mediated carboplatin resistance both and , resensitizing LUSC cells to chemotherapy. These findings highlight SOX2 as a critical redox regulator that modulates NRF2 signaling to promote carboplatin resistance in LUSC. The identification of the SOX2-NRF2 axis as a potential therapeutic target suggests that NRF2 inhibition may represent a promising strategy to overcome chemoresistance in LUSC.

Spatial biology aims to elucidate cellular organization, function, and interactions within native tissue contexts, offering key insights into both normal physiology and disease. Spatial proteomics complements this by enabling high-resolution mapping of protein localization and abundance, directly reflecting functional cellular states. Unlike transcriptomics, which infers potential activity, proteomics captures actual molecular functions, including post-translational modifications and dynamic interactions. However, protein profiling poses significant challenges, as proteins cannot be directly sequenced or easily targeted via nucleic acid hybridization. Antibody-oligonucleotide conjugates (AOCs) address this limitation by converting protein recognition into a DNA-based readout, thereby enabling sensitive and scalable detection. In this review, we outline the core principles of AOC-based spatial proteomic technologies, including multiplexed protein analysis, protein-protein interactions, and integration with other biomolecular data. We highlight their applications in decoding tissue complexity and disease pathology and examine key technical challenges that remain. Overall, AOCs offer distinct advantages, including DNA-mediated signal amplification, spatially resolved proteomic profiling, and compatibility with multi-omics approaches, positioning them as powerful platforms in the advancement of spatial biology.

Postoperative cognitive dysfunction (POCD) is a serious complication in patients undergoing colorectal cancer (CRC) surgery. It is characterized by significant impairments in memory, information processing and attention, and may also result in mood and personality changes, thereby increasing the risk of postoperative mortality. Currently, there are no effective interventions available, highlighting the need for further investigation into its pathogenesis. While the current literature has identified an association between gut microbiota dysregulation and cognitive deficits, the precise mechanisms involved remain insufficiently understood. This study hypothesizes that exosome-like (Exos-like) nanoparticles derived from the gut microbiota contribute to POCD by modulating autophagy-dependent ferroptosis in hippocampal neurons. In a rat model of CRC, significant alterations in the gut microbiota composition, including reduced microbial diversity and changes in the abundance of key taxa, are observed. Exosomes derived from these microbiota enhance neuronal uptake and trigger markers of ferroptosis, as evidenced by increased expressions of ATG5 and COX2, along with decreased levels of GPX4 and FTH1. These findings establish a mechanistic link between microbial dysbiosis, ferroptosis, and cognitive decline in POCD, providing new insights into potential therapeutic targets for CRC-associated POCD.

Emerging studies have revealed that disruptions in circadian crosstalk between the gut microbiota and the host play an essential role in the pathogenesis of metabolic disorders. Under physiological conditions, host circadian clocks regulate microbial diurnal oscillations through rhythmic behaviors, including feeding patterns and sleep-wake cycles. This temporal regulation manifests as robust 24-hour oscillations in microbial community composition, spatial organization, and metabolic activity. These rhythmic microbial signals and their metabolic outputs are subsequently translated into host immune modulation, establishing a bidirectional temporal dialogue between the host and microbiota. Modern lifestyle disruptions, including erratic eating patterns and shift work, desynchronize this temporal dialogue, leading to the loss of microbial rhythms, impaired intestinal barrier function, maladaptive immune responses, chronic inflammation, and systemic metabolic dysregulation. This review delineates the mechanisms through which host-microbiota circadian crosstalk governs immunometabolic homeostasis, provides a mechanistic framework for understanding immunometabolic diseases, and highlights therapeutic strategies that target microbial rhythms to reset host immunity and metabolism.

O-GlcNAcylation, a prevalent reversible post-translational modification, intricately alters non-histone proteins, influencing the organization of gene transcriptional regulation within the accessible chromatin environment. This nucleoplasmic landscape, characterized by histone-free regions, fundamentally enables O-GlcNAc-mediated modulation through dynamic accessibility. However, unraveling the O-GlcNAc-open chromatin interplay that governs sophisticated transcriptional regulatory networks remains constrained by current techniques, which lack the resolution to probe this spatiotemporal crosstalk. Here, we report a general strategy to systematically and chemoselectively profile O-GlcNAc-associated chromatin accessibility on a genome-wide scale (COCA-seq). Through comprehensive validation across low- and high-throughput levels, we demonstrate COCA-seq's dual fidelity in both O-GlcNAc chemoselectivity and open chromatin specificity. We employ it to delve into doxorubicin resistance for breast cancer, scrutinizing pivotal regulatory genes and transcription factors implicated in this complex biological event. By integrating bulk RNA-seq with COCA-seq, we offer a multiomics perspective, shedding light on related biological processes and pathways like drug efflux and stress homeostasis, thereby uncovering potential mechanisms by which O-GlcNAc-associated open chromatin orchestrates tumor drug resistance. COCA-seq emerges as a general and versatile tool across various biological contexts, poised to reveal the landscape of O-GlcNAc-associated open chromatin regions across the genome and decipher the significance of glycosylation behind it.

Glycolysis provides the main energy source for the rapid proliferation and migration of colorectal cancer (CRC) cells. In our previous studies, we reported that SUN5, a nuclear membrane protein, promotes proliferation and migration. However, whether SUN5 is involved in the process of glycolysis is unclear. Here, we demonstrate that overexpression of SUN5 enhances glucose uptake and lactate production in CRC cells, whereas the opposite results are observed in -knockdown cells. Mechanistically, SUN5 activates the NF-κB signaling pathway, which can be inhibited by the IKK inhibitor BAY11-7082. Further studies reveal that SUN5 interacts with TRIM28 to increase IκB ubiquitination, leading to the nuclear translocation of phosphorylated P65 (phos-P65) and subsequent increases in the transcription of and , accelerating glycolysis. Moreover, xenograft transplantation experiments reveal that the knockdown of inhibits glycolysis and tumorigenesis . Taken together, these findings indicate that SUN5 enhances the glycolysis and tumorigenesis of CRC cells via interaction with TRIM28, which provides a potential target for the diagnosis and treatment of CRC.

As a critical component of amino acid metabolic reprogramming, serine metabolism has been demonstrated to be enhanced in a variety of cancer types, thereby supporting tumor progression. This enhancement is primarily driven by increased expression levels and augmented enzymatic activity of serine metabolic enzymes (phosphoglycerate dehydrogenase, phosphoserine aminotransferase 1, phosphoserine phosphatase and serine hydroxymethyltransferase). However, there is still lack of comprehensive summary on the regulation of serine metabolism in cancer. In this review, we provide a systematic overview of the currently discovered and proven regulatory mechanisms of serine metabolic enzymes in cancer, focusing on three levels: transcriptional, post-transcriptional, and post-translational regulation. Specifically, transcriptional regulation encompasses three major mechanisms: (1) transcription factor-mediated gene expression control, (2) histone modifications, and (3) DNA methylation. At the post-transcriptional level, regulation is primarily achieved through (1) non-coding RNAs, (2) RNA-binding proteins, and (3) RNA modifications. Post-translational regulation is predominantly mediated through diverse protein post-translational modifications. The transcriptional and post-transcriptional mechanisms primarily modulate the expression levels of serine metabolic enzymes, while post-translational modifications exert more diverse effects by altering the activity, protein stability or cellular localization of these enzymes. These regulations collectively modulate serine metabolism to influence tumor progression, offering promising targets for tumor-specific therapeutic interventions.

The iron regulatory protein IREB2 (Iron Responsive Element Binding Protein 2) plays a crucial role in maintaining cellular iron homeostasis through the posttranscriptional regulation of genes involved in iron metabolism. Mutations in the gene have been linked to NDCAMA (OMIM#618451), a rare genetic neurological disorder characterized by early-onset neurodegeneration, choreoathetoid movements, and microcytic anemia. However, the absence of an -mutated animal model has left the underlying pathogenic mechanisms poorly understood. To investigate this, we establish a CRISPR-Cas9-mediated mouse model, which carries the c.2477A>T (p.D826V) pathogenic variant in identified in a Chinese pedigree with NDCAMA. Behavioral studies, including the Morris water maze (MWM), open field test (OFT), and Y-maze, reveal significant neurobehavioral deficits, such as impaired spatial learning and memory and reduced motor activity, in mice. Furthermore, we observe increased microglial activation and decreased dendritic spine density in the hippocampus, along with impaired long-term potentiation (LTP) and elevated paired-pulse facilitation (PPF), indicating synaptic dysfunction. Mechanistically, mice present reduced Ireb2 protein levels, dysregulated iron metabolism, and an altered expression profile associated with neurological function. This study elucidates the molecular mechanisms underlying NDCAMA and establishes mice as a model for iron metabolism-driven neurodegeneration. This finding links the instability of IREB2 to synaptic failure and neuroinflammation, highlighting potential therapeutic implications for neurodegenerative diseases.

This review synthesizes how neurotransmitters-including glutamate, acetylcholine (ACh), γ-aminobutyric acid (GABA), serotonin (5-HT), and catecholamines-modulate T-cell immunity in the tumor microenvironment through activation, differentiation, trafficking, and checkpoint dependence. Glutamate amplifies T-cell receptor signaling but is counterbalanced by tumor-derived glutamate export. Cholinergic pathways exert dual effects through nicotinic and muscarinic receptors, whereas GABA generally imposes metabolic and signaling brakes that favor regulatory programs. Serotonin shows spatial divergence-suppressing peripheral responses but enhancing intratumoral cytotoxicity-and chronic β-adrenergic stress dampens effector function and limits immunotherapy efficacy. Advances in spatial multi-omics, single-cell profiling, and neuromodulation will help discover new targets across these axes. This review provides mechanistic insights and translational implications, highlighting emerging strategies such as glutamate receptor, metabotropic glutamate receptor 4 (mGluR4) or xCT (SLC7A11) inhibition, receptor subtype modulation, and β-blockade. Integrating neurotransmitter-receptor targeting with checkpoint inhibitors or cell therapies may improve the depth and durability of cancer immunotherapy.

Myocardial infarction (MI) causes irreversible cardiomyocyte loss, creating a need for cardiac repair therapies. The role of cell division cycle 5-like (CDC5L), a cell cycle regulator, in cardiac repair is unknown. This study aims to define the role of CDC5L in mitigating ischemia-reperfusion (I/R) injury by assessing its impact on cardiomyocyte proliferation and apoptosis and to determine the mechanism involving the FGF10-YAP axis. We model cardiac injury using oxygen-glucose deprivation/reoxygenation (OGD/R) in neonatal mouse cardiomyocytes and I/R in adult mice. To investigate CDC5L function, we modulate its expression via adenoviral or AAV9-mediated overexpression or knockdown. Proliferation markers (EdU , Ki67 , pH3 ), apoptosis (TUNEL staining, Bax/Bcl-2 ratio), and cardiac function (echocardiography) are assessed. Through transcriptomic screening, we identify CDC5L downstream targets and validate their functional roles using knockdown rescue assays. We find that CDC5L is upregulated in the post-I/R murine myocardium. Its overexpression enhances cardiomyocyte proliferation, preserves cardiac function, reduces apoptosis, and diminishes infarct size. Transcriptomic analysis identifies FGF10 as a key downstream effector, and we confirm that CDC5L upregulates FGF10 expression. Notably, knockdown reverses the proliferative and anti-apoptotic effects of CDC5L. Moreover, the CDC5L-mediated reduction in YAP phosphorylation is also dependent on FGF10, as this effect is abolished upon knockdown. In conclusion, CDC5L attenuates cardiac I/R injury by promoting cardiomyocyte proliferation and inhibiting apoptosis through the FGF10-YAP pathway. This CDC5L-FGF10-YAP axis represents a promising therapeutic target to improve myocardial regeneration and recovery after myocardial infarction.

Serum deprivation is a well-established inducer of apoptosis, yet the molecular mechanisms governing this process remain incompletely understood. Here, we show that serum starvation selectively triggers intrinsic apoptosis in high-density murine embryonic fibroblasts (MEFs) through coordinated HIF-1α activation and JNK signaling suppression. Knockdown of abolishes caspase-3 activation and apoptosis induced by serum deprivation, whereas upregulation of HIF-1α in low-density cells recapitulates the apoptotic response observed in high-density cultures. Simultaneously, serum deprivation leads to the suppression of the JNK pathway, which contributes to apoptosis. Notably, combined HIF-1α activation and JNK inhibition in low-density cells fully mimics the apoptotic phenotype of high-density conditions, underscoring the interplay between these pathways. Together, these findings define a density-dependent apoptotic switch in which HIF-1α drives metabolic stress adaptation, whereas JNK suppression removes a critical survival signal, converging to promote mitochondrial-mediated cell death. This work provides a mechanistic framework for understanding nutrient stress-induced apoptosis and suggests potential therapeutic targets for diseases characterized by aberrant cell survival.

Triple-negative breast cancer (TNBC), a highly aggressive molecular subtype characterized by a lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression, remains a major clinical challenge, with a median overall survival of approximately 12-15, months in advanced-stage patients. Andrographolide (AG), a diterpenoid phytochemical derived from , has demonstrated promising anti-inflammatory, antioxidant, and antitumor activities. However, its precise molecular and therapeutic mechanisms in TNBC remain poorly understood. AG inhibits the proliferation and metastasis of TNBC. Through an integrative approach combining network pharmacology and machine learning algorithms, including least absolute shrinkage and selection operator (LASSO) and random forest, cyclin-dependent kinase 1 (CDK1) has been identified as a critical molecular gene of AG. Experimental validation via western blot and immunofluorescence analyses reveal that AG treatment significantly downregulates CDK1 expression while concurrently upregulating the expression of the tumor suppressor p53, suggesting a functional interplay between these pathways. These mechanistic insights indicate that AG potentially exerts antiproliferative effects on TNBC cells through modulation of the p53/CDK1 signaling axis, thereby establishing a robust preclinical basis for its potential clinical translation in this challenging malignancy.

Taste Receptor Type 2 (TAS2R) has an obvious function of sensing bitterness and transmitting neural signals. Our study aims to elucidate the upstream regulatory pathways of TAS2R14 in thyroid tumors, revealing new facets of TAS2R investigation. Bioinformatics analyses are conducted utilizing UALCAN, TargetScan, ENCORI, and GEPIA. Gene expression is assessed via microarray, qPCR, and western blot analysis. Molecular interactions are probed through dual-luciferase reporter assay, biotin-coupled pull-down, and chromatin immunoprecipitation (ChIP) assay. Ferroptosis assessment is performed using incorporated iron-centric techniques. Cellular phenotypes are determined by cell invasion, viability, apoptosis, and wound healing assays. studies employ a nude mouse xenograft model. We discover that TAS2R14 can inhibit thyroid cancer cell proliferation, invasion, and migration, concurrently inducing ferroptosis. The expression of TAS2R14 is governed by miR-450b-5p, which is sponged by the lncRNA HCG11 via a STAT3-dependent pathway. TAS2R14 promotes cell ferroptosis and arrests thyroid cancer progression through an upstream regulatory cascade that involves STAT3, the lncRNA HCG11, and miR-450b-5p. Our research reveals an unsuspected regulatory paradigm of the TAS2R family, shedding new light on its function and extending our understanding beyond its established role in bitter taste sensation.

Immunotherapy, including cellular therapy, has emerged as a crucial pillar in cancer treatment, complementing established modalities such as surgery, chemotherapy and radiotherapy. The clinical observation that immunotherapy is effective in only a limited proportion of patients inspires mechanistic research on the complicated regulatory network within the tumor microenvironment (TME). Circadian regulation significantly affects immune cell behavior, including the activity of immune cells and cytokine production, and emerging evidence suggests the key role of circadian regulation in the TME, which subsequently affects the effectiveness of immunotherapy. Results from preclinical and clinical studies indicate that appropriate timing of adoptive cellular therapy and immune checkpoint blockade therapy improves their efficacy. Therefore, understanding the molecular mechanism of the circadian rhythm together with its role in immunotherapy is essential for optimizing cellular function, proliferation and persistence in the TME. Here, we review how circadian rhythms influence immunotherapy and the TME across different stages of tumor progression. Future clinical protocols may integrate concepts of circadian rhythm and immunotherapy to enhance treatment response.

Pregnancy induces profound physiological adaptations to meet the dynamic nutritional demands of fetal development, including a deliberate reduction in maternal insulin sensitivity to ensure fetal glucose availability. However, excessive insulin resistance may precipitate gestational diabetes mellitus (GDM), increasing the risk of both obstetric complications and long-term metabolic disorders in mothers and offspring. Although the role of adipose tissue in pregnancy-associated metabolic adaptation has been extensively studied, the contribution of skeletal muscle remains poorly understood. Here, we systematically characterize pregnancy-induced molecular and metabolic changes in maternal skeletal muscle through multi-omics profiling. We use transcriptomic, metabolomic, computational single-cell deconvolution, and qPCR validation in an established C57BL/6J mouse pregnancy model (8-week-old females). Pregnancy triggers remarkable skeletal muscle remodelling, featuring histological reorganization with myofiber depletion and expanded endothelial compartments. Concurrent metabolic disturbances include insulin resistance, dysregulated TCA cycle activity, and impaired ubiquinone biosynthesis. This study represents a multi-omics-based systematic elucidation of pregnancy-induced maternal skeletal muscle adaptations. Our findings demonstrate that pregnancy induces profound structural reorganization and metabolic reprogramming in maternal skeletal muscle, characterized by prioritized fetal nutrient provision at the expense of maternal tissue utilization. These observations not only reveal previously unrecognized mechanisms of pregnancy-specific metabolic regulation but also, more importantly, establish a critical theoretical foundation for developing skeletal muscle-targeted intervention strategies to prevent gestational diabetes mellitus.

Hypertension is commonly accompanied by endothelial dysfunction, characterized by an imbalance between vasodilatation and constriction, increased levels of the proinflammatory factors interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1), and decreased nitric oxide (NO) bioavailability. Using an angiotensin II (Ang II)-induced endothelial dysfunction model, we show that treatment with the hydrogen sulfide (H₂S) donor GYY4137 significantly reverses Ang II-induced damage. GYY4137 restores sirtuin 6 (SIRT6) expression, suppresses inflammation, and improves vasodilatory function. Furthermore, endothelial-specific cystathionine-γ-lyase (CSE)-deficient mice exhibit inflammation and endothelial dysfunction in blood vessels, which is reversed by H₂S supplementation. Critically, SIRT6 inhibitors block the protective effects of H₂S in the endothelium. This study demonstrates that H₂S protects vascular endothelial function by activating the SIRT6 anti-inflammatory pathway.

Tuberculosis (TB), caused by (MTB), remains a significant global health threat. However, the licensed Bacille Calmette-Guérin- (BCG) vaccine provides only limited protection in adults, underscoring the urgent need for more effective preventive strategies. Recent studies have shown that multi-epitope DNA vaccines are superior to traditional vaccines in terms of immunogenicity, safety and stability. In this study, we develop a multi-epitope DNA vaccine that contains CD8 T-cell epitopes, CD4 T-cell epitopes, and B-cell epitopes using bioinformatics tools. These epitopes are derived from three genome-encoded proteins, ESAT-6, Rv2660c, and RpfB, which exhibit stage-specific immunodominance in the early, resting, and convalescent stages of MTB infection. Using reverse vaccinology and computational immunomodulation, we demonstrate that the multiepitope vaccine increases antigen-specific antibody titres, activates CD8 T and CD4 T cells, and enhances IFN-γ secretion. validation studies in HEK293T cells confirm high-yield expression of multi-epitope-encoded antigens, whereas immunization experiments reveal significant expansion of NK cells and Th1-polarized lymphocytes, with concomitant upregulation of pro-inflammatory mediators. Collectively, these results highlight the potent activation of adaptive immunity through Th1-driven mechanisms and IFN-γ-mediated mycobacterial clearance, which are crucial for defending against MTB.