Non-small cell lung cancer (NSCLC) frequently evades immune surveillance through defective antigen presentation and a suppressive tumor microenvironment (TME), limiting the efficacy of immune checkpoint blockade (ICB). Conventional type 1 dendritic cells (cDC1s) are essential for initiating antitumor CD8+ T-cell responses; however, their abundance and function are often diminished in NSCLC, contributing to poor outcomes and resistance to immunotherapy. We hypothesized that in situ vaccination (ISV) using gene-modified cDC1s engineered to secrete FMS-like tyrosine kinase 3 ligand (FLT3L) would enhance cDC1 function within the TME, promote antitumor immunity, and improve responses to ICB. Syngeneic murine models of NSCLC (KrasG12D/P53-/-/Lkb1-/-; KrasG12D/P53-/- ; and KrasG12D ) with varying tumor mutational burden, along with the MC38 model, were used to assess the therapeutic efficacy of FLT3L-cDC1 ISV. Flow cytometry and multiplex immunofluorescence were used to evaluate immune mechanisms of response. To assess translational relevance, immune and tertiary lymphoid structure (TLS) signatures were analyzed in The Cancer Genome Atlas (TCGA) NSCLC datasets, with TLS signatures refined using a retrained xCell2 framework incorporating curated TLS and high endothelial venule (HEV) microdissection datasets. FLT3L-cDC1 ISV remodeled the TME across multiple NSCLC models, inducing T lymphocyte infiltration and expanding cytolytic CD8+ T cells. FLT3L-cDC1 ISV was associated with increased formation of immature TLS with primary follicle-like features within the TME. TCGA analyses revealed that FLT3L expression correlates with activated DC, T cell, and B cell signatures, as well as HEV-enriched TLS-associated programs. Combination with PD-1 blockade further enhanced the antitumor immunity of FLT3L-cDC1 ISV, resulting in robust local and systemic T-cell activation and the expansion of activated CCR7+PD-L1+ cDC1s and stem-like TCF1+PD-1+ CD8+ progenitors within the TME. In an LKB1-deficient NSCLC model, FLT3L-cDC1 ISV plus PD-1 blockade induced complete and durable regression in 85% of tumors, leading to long-lasting systemic tumor-specific immune memory, consistent with effective tumor vaccination. FLT3L-cDC1 ISV represents a rational cytokine-enhanced cellular immunotherapy designed to overcome immunosuppression and restore DC function within the TME, thereby promoting tumor-specific adaptive immune responses and enhancing responsiveness to ICB.
To observe the effects of acupuncture combined with medication pretreatment on gastric mucosal injury and serum metabolites in rats with acute gastric ulcer (GU) using ultra-high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (UHPLC-Q-TOF-MS), and to explore the protective mechanism of this combined pretreatment for GU. Forty SPF-grade male SD rats were randomly divided into a blank group, a model group, an acupuncture combined with medication group (combined group) and an omeprazole group, with 10 rats in each group. In the omeprazole group, omeprazole solution was given by gavage administration at a dosage of 20 mg/kg; in the combined group, omeprazole solution was given by gavage administration, followed by acupuncture at bilateral "Zusanli"(ST36) 30 min later, the intervention was delivered once daily for 10 days in the two groups. After the intervention, except the blank group, acute GU model was established by intragastric administration of absolute ethanol in the other three groups. The gastric mucosal injury was observed, and the gastric mucosal injury index and injury inhibition rate were calculated. The morphology of gastric tissue was observed by HE staining, and the acute gastric mucosal injury score was assessed. The distribution of mucus on the gastric mucosal surface was observed by PAS staining. The levels of hexosamine (Hex) and phospholipid (PL) in the gastric tissue, as well as the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum were detected by ELISA. The positive expression of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax),Occludin and zonula occludens-1 (ZO-1) in the gastric tissue was detected by immunohistochemistry. The endogenous metabolic profile of serum samples was analyzed by UHPLC-Q-TOF-MS to screen differential metabolites, and metabolic pathways were enriched using the MetaboAnalyst5.0 database. The protein expression of phosphatidylinositol 3-kinase(PI3K), protein kinase B (Akt), nuclear factor-κ B (NF-κ B), phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt) and phosphorylated NF-κ B (p-NF-κ B) in the gastric tissue was detected by Western blot. Compared with the blank group, the gastric mucosal injury index and acute gastric mucosal injury score were increased in the model group (P<0.01).Compared with the model group, the gastric mucosal injury index and acute gastric mucosal injury scores were decreased(P<0.01), and the injury inhibition rate was increased in the combined group and the omeprazole group. In the model group,the mucus on the gastric mucosal surface was damaged and the mucosal structure was disrupted; in the combined group and the omeprazole group, the mucus was evenly distributed and the mucosal structure was relatively intact. Compared with the blank group, the levels of Hex and PL, the positive expression of Bcl-2 in gastric tissue, as well as the proportions of Occludin and ZO-1 positive cells in gastric mucosa were decreased (P<0.01); the serum levels of TNF-α and IL-6, the positive expression of Bax, and the protein expression of p-PI3K, p-Akt and p-NF-κ B in the gastric tissue were increased in the model group (P<0.01). Compared with the model group, the levels of Hex and PL in the gastric tissue, and the proportions of Occludin positive cells in the gastric mucosa were increased (P<0.01, P<0.05); the serum levels of TNF-α, the positive expression of Bax, and the protein expression of p-PI3K, p-Akt and p-NF-κ B in the gastric tissue were decreased (P<0.01,P<0.05) in the combined group and the omeprazole group. Compared with the model group, the positive expression of Bcl-2 in the gastric tissue, and the proportion of ZO-1 positive cells in the gastric mucosa were increased (P<0.01); the serum level of IL-6 was decreased (P<0.01) in the combined group. A total of 15 potential biomarkers were identified, mainly involved in metabolic pathways such as biosynthesis of phenylalanine, tyrosine and tryptophan, phenylalanine metabolism, alanine,aspartate and glutamate metabolism, and glutathione metabolism. Acupuncture combined with medication pretreatment can enhance gastric barrier function, inhibit inflammatory response, inhibit apoptosis, and regulate metabolic disorders, thereby improving gastric mucosal injury in acute GU rats. Its mechanism may be associated with inhibiting the abnormal activation of the PI3K/Akt/NF-κ B pathway. 目的：基于超高效液相色谱-四极杆-飞行时间串联质谱（UHPLC-Q-TOF-MS）技术观察针药结合预处理对急性胃溃疡（GU）模型大鼠胃黏膜损伤及血清代谢物的影响，探索针药结合预处理对GU的保护作用机制。 方法：将40只SPF级雄性SD大鼠随机分为空白组、模型组、针药结合组和奥美拉唑组，每组10只。奥美拉唑组灌胃20 mg/kg奥美拉唑溶液；针药结合组先灌胃奥美拉唑溶液，30 min后针刺双侧“足三里”穴，均每日1次，共干预10 d。干预结束后，除空白组外，其余3组采用无水乙醇灌胃法制备大鼠急性GU模型。观察大鼠胃黏膜损伤情况，计算胃黏膜损伤指数和损伤抑制率；HE染色观察大鼠胃组织形态，计算急性胃黏膜损伤评分；PAS染色观察大鼠胃黏膜表面黏液分布；ELISA法检测大鼠胃组织氨基己糖（Hex）、磷脂（PL）含量和血清肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）含量；免疫组化法检测大鼠胃组织B细胞淋巴瘤-2基因（Bcl-2）、Bcl-2相关X蛋白（Bax）、闭合蛋白（Occludin）、闭锁连接蛋白-1（ZO-1）阳性表达；利用UHPLC-Q-TOF-MS技术检测大鼠血清样本内源性代谢轮廓谱，筛选差异代谢物，并借助MetaboAnalyst5.0数据库富集代谢通路；Western blot法检测大鼠胃组织磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）、核因子-κ B（NF-κ B）、磷酸化PI3K（p-PI3K）、磷酸化Akt（p-Akt）、磷酸化NF-κ B（p-NF-κ B）蛋白表达。 结果：与空白组比较，模型组大鼠胃黏膜损伤指数、急性胃黏膜损伤评分升高（P<0.01）；与模型组比较，针药结合组和奥美拉唑组大鼠胃黏膜损伤指数、急性胃黏膜损伤评分降低（P<0.01），损伤抑制率升高。模型组大鼠胃黏膜表面黏液受损，黏膜结构被破坏；针药结合组和奥美拉唑组大鼠胃黏膜表面黏液分布均匀，黏膜结构相对完整。与空白组比较，模型组大鼠胃组织Hex和PL含量、Bcl-2阳性表达及胃黏膜Occludin、ZO-1阳性细胞比例降低（P<0.01），血清TNF-α和IL-6含量、胃组织Bax阳性表达及胃组织p-PI3K、p-Akt、p-NF-κ B蛋白表达升高（P<0.01）。与模型组比较，针药结合组和奥美拉唑组大鼠胃组织Hex、PL含量和胃黏膜Occludin阳性细胞比例升高（P<0.01，P<0.05），血清TNF-α含量、胃组织Bax阳性表达及胃组织p-PI3K、p-Akt、p-NF-κ B蛋白表达降低（P<0.01，P<0.05）；针药结合组大鼠胃组织Bcl-2阳性表达及胃黏膜ZO-1阳性细胞比例升高（P<0.01），血清IL-6含量降低（P<0.01）。共筛选出15种潜在生物标志物，主要涉及苯丙氨酸、酪氨酸和色氨酸的生物合成，苯丙氨酸代谢，丙氨酸、天冬氨酸和谷氨酸代谢，谷胱甘肽代谢等代谢通路。 结论：针药结合预处理能够增强大鼠胃屏障功能，抑制炎症反应，抗凋亡，调节机体代谢紊乱，从而改善急性GU模型大鼠胃黏膜损伤，其作用机制与抑制PI3K/Akt/NF-κB通路的异常激活有关。.
Backgrounds: Tertiary lymphoid structures (TLSs) are increasingly recognized as modulators of anti-tumor immunity, yet their clinical relevance in bladder cancer remains incompletely understood, partly owing to heterogeneity in their maturation states. Here, we demonstrate that germinal center (GC)-like TLS maturity, rather than TLS presence alone, is closely associated with immune activation and therapeutic response to Programmed Death-Ligand 1 (PD-L1) blockade in bladder cancer. The objective of this study was to systematically investigate the clinical significance, biological function, and therapeutic potential of tertiary lymphoid structure (TLS) maturation in bladder cancer. Specifically, we aimed to determine whether GC-like TLS maturity provides prognostic and predictive value beyond TLS presence alone, to elucidate the immune programs and tumor microenvironment remodeling associated with TLS maturation, and to explore whether TLS maturation can be therapeutically induced to enhance responsiveness to PD-L1 blockade. Methods: We performed an integrative analysis combining multi-cohort transcriptomics, spatially resolved histopathology, single-cell RNA sequencing, and functional murine experiments. TLS maturation states were defined using gene-expression-based GC-like TLS signatures and validated through multiplex immunohistochemistry. Clinical relevance was assessed in public immunotherapy cohorts and an independent neoadjuvant PD-L1-treated muscle-invasive bladder cancer (MIBC) cohort. Tumor immune microenvironment remodeling and chemokine-mediated cellular crosstalk were analyzed using deconvolution, Weighted Gene Co-expression Network Analysis (WGCNA), and CellChat. The therapeutic inducibility of TLS maturation was examined using a lymphotoxin-β receptor (LTβR) agonist in combination with PD-L1 blockade in a syngeneic bladder cancer model. Results: Across multiple transcriptomic cohorts, tumors enriched for GC-like TLS signatures exhibited significantly prolonged survival and higher objective response rates to anti-PD-L1 therapy, whereas less mature TLS phenotypes showed no consistent association with clinical association. These observations were independently validated in a neoadjuvant PD-L1-treated muscle-invasive bladder cancer cohort, in which high mature TLS density was associated with major pathological response and prolonged event-free survival, outperforming PD-L1 expression. Integrative histopathological and transcriptomic analyses indicated that GC formation marks a functional transition linking humoral immune programs with cytotoxic effector activity and shaping a memory-prone, pro-inflammatory tumor immune microenvironment. Chemokine signaling via the CC chemokine ligand 21 (CCL21)-C-C chemokine receptor type 7 (CCR7) and C-X-C motif chemokine ligand 12 (CXCL12)-C-X-C chemokine receptor type 4 (CXCR4) axes was strongly associated with TLS maturation and spatial organization. Finally, in a syngeneic bladder cancer model, pharmacological activation of lymphotoxin-β receptor signaling promoted TLS maturation and enhanced the antitumor efficacy of PD-L1 blockade. Conclusions: Together, these findings suggest that GC-like TLS maturity represents a clinically relevant biomarker and a potential therapeutic entry point for precision immunotherapy in bladder cancer. Therapeutic strategies that promote TLS maturation may convert immune-cold tumors into checkpoint-responsive states, providing a mechanistically grounded precision immunotherapy approach.
Xylem is essential for water and nutrient transport, mechanical support, and carbohydrate storage. Identification and quantification of vascular cell types remain manual, time-consuming, and prone to observer bias, limiting throughput and reproducibility. Automated, integrated tools are critical for scaling wood anatomical studies and enabling comparative analyses across taxa. We assembled a poplar xylem dataset of 1,790 microscopy images with 173,434 annotated instances. Using this dataset, we evaluated seven semantic segmentation models and five YOLOv8 detection models across section types for xylem cell recognition and morphometric attribute extraction and adopted a "Segmentation-then-Detection" pipeline to reduce misidentifications in complex backgrounds. Mask2Former achieved the best segmentation performance, covering transverse sections (whole xylem, vessels, fibers, rays) and tangential sections (rays and four ray cell image types). YOLOv8x and YOLOv8m performed consistently for object detection and morphometrics, and the PLXY-AI toolkit was accordingly developed based on YOLOv8 architecture. The combined pipeline markedly improved fiber identification in challenging images. In a generalization test, 34 of 42 woody angiosperms (81.0%) met >90% accuracy for identifying all cell types. The workflow and PLXY-AI toolkit enable automated identification and quantification of vessels, fibers, and rays, extracting size and area while substantially reducing manual workload and observer bias. Per-image processing time averages <1 s. Designed for batch analysis, the pipeline minimizes operational complexity and integrates easily into existing laboratory and computational environments. With a user-friendly graphical interface, this framework supports high-throughput analysis of vascular tissue structure and function across multiple tree species.
Osimertinib (OSM), a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, causes skin disorders, such as hand-foot syndrome (HFS), which impairs skin barrier function. Stratum corneum (SC) lipids play an important role in skin barrier function. This study aimed to compare the SC lipid composition and structure of healthy participants with those of patients with OSM-induced HFS. Overall, 47 patients with non-small cell lung cancer were enrolled in this study. All patients received 80 mg/day OSM orally and completed the 56-day prospective observation period. SC samples were obtained from seven patients with OSM-induced HFS (OSC). SC lipid composition (ceramide [CER] and free fatty acids [FFAs]) and structure were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and synchrotron X-ray diffraction, respectively. In healthy SC (HSC), the lipids formed two lamellar phases. Hexagonal and orthorhombic hydrocarbon chain packing was observed in the lateral lipid organization. However, in the OSC, these structures almost disappeared. UPLC-MS/MS analysis revealed that the composition of CER and FFA differed between OSC and HSC and that the carbon chain length of SC lipid components in OSCs was lower compared with that in HSCs. However, no significant differences were observed in the CER levels of the esterified ω-hydroxy fatty acid class, which is involved in the formation of long-period lamellar structures, between the two groups. OSM induces changes in SC lipid structure due to changes in SC lipid composition and a decrease in carbon chain length in OSCs, resulting in impaired barrier function.
Lignin, an abundant byproduct of the pulp, paper, and biorefinery industries, has a complex chemical structure containing phenolic, carboxylic, and methoxyl functional groups that confer tunable reactivity and functional properties. These characteristics enable lignin to act as an antioxidant, antimicrobial, UV-shielding agent, emulsifier, Pickering particle, and reinforcing component in films and hydrogels. This review examines the growing role of lignin as a sustainable biopolymer for food and food-related applications. It integrates current knowledge on lignin extraction, structure-function relationships, emulsion applications, bioactive delivery systems, antioxidant and antimicrobial applications, and cytotoxicity. Opportunities and limitations are discussed in the context of food-grade processing and safety requirements. Future research directions are outlined to guide the safe and effective use of lignin in food systems. Over the past five years, advances in extraction technologies, fractionation, and chemical modification have reshaped the potential of lignin for food packaging, emulsion stabilization, and bioactive delivery systems. However, challenges remain. Plant source, extraction method, and molecular heterogeneity strongly influence lignin solubility, cytotoxicity, and interfacial behavior, creating variability that complicates standardization for food use. Emerging findings on gastrointestinal interactions and cell-line cytotoxicity highlight the need for systematic toxicological assessment and clearer regulatory pathways, as the lack of standardized safety evaluation remains a major barrier to the direct application of lignin in food.
Enhancing the biomass of industrial microorganisms, such as Saccharomyces cerevisiae, under osmotic stress is of great significance for modern industrial fermentation as it directly affects growth-coupled product yield and fermentation efficiency in common hyperosmotic production environments, such as high-sugar and deep-tank fermentation conditions. Moreover, the high-efficiency osmotolerance mechanisms in microorganisms provide a theoretical basis for the directional optimization of related strains. In this study, high-efficiency osmotolerant S. cerevisiae mutants (four strains) were developed by combining heavy ion beam irradiation mutagenesis and adaptive laboratory evolution. Biomass accumulation and cellular physiological and biochemical characteristics of these mutants under hyperosmotic stress were systematically characterized. After confirming their genetic stability, the molecular mechanisms underlying their phenotypes were elucidated at multiple levels, including gene mutations, transcriptional regulation, and metabolic remodeling. Under high osmotic stress induced by 2.4 mol/L KCl, the mutant biomass was more than twice the wild-type strain biomass, without an increase in glucose consumption. This finding suggests that carbon sources are preferentially redirected toward growth rather than toward stress responses, thereby achieving high-efficiency osmotolerance. At the cellular level, the mutants exhibited efficient redox homeostasis while maintaining normal membrane function and cell morphology. Functional gene analysis revealed that mutations in genes such as mth1, hxt1, flo9, sgd1, dan4, puf3, and rph1 were associated with carbon uptake and allocation, cell adhesion, glycerol synthesis, ribosome assembly, cell wall structure, antioxidant capacity, and global transcriptional regulation. Mutations in two or more of these genes synergistically enhance osmotic tolerance. The patterns of whole-genome variation among different mutants provide new insights into gene functions. For example, mutations in genes related to carbon metabolism (such as hxt1 or mth1) were present in all four mutants, highlighting their critical role in promoting biomass accumulation-associated anabolism. Mutations in other functional modules were dispersed among different mutants. Metabolic analysis further confirmed adaptive remodeling in carbon metabolic flux distribution, membrane phospholipid composition, and antioxidant and osmoprotectant accumulation. The overexpression and knockout of hxt1 involved in carbon metabolic remodeling confirmed that functional adjustments could further optimize the high-efficiency osmotolerant phenotype. In this study, S. cerevisiae mutants with significantly enhanced biomass under hyperosmotic stress were used to preliminarily elucidate the molecular basis of their stress-growth balance regulation from the genetic to metabolic levels, providing important insights for the directional selection of robust industrial fermentation strains.
The development of biomimetic scaffolds capable of promoting both cartilage and subchondral bone regeneration remains a major challenge in osteochondral tissue engineering. In this study, type I acid-soluble collagen (ASC) was successfully extracted from black flounder (Paralichthys olivaceus) skin and systematically characterized. The purified ASC retained its native triple-helical structure, as confirmed by SDS-PAGE, FTIR, CD, and XRD analyses, and exhibited favorable self-assembly behavior near physiological pH. Based on this natural matrix, photocrosslinkable methacrylated chondroitin sulfate (CSMA) was synthesized and combined with ASC or mineralized collagen (MC) to fabricate injectable composite hydrogels via UV-initiated polymerization. The resulting CSMA/COL and CSMA/MC hydrogels demonstrated tunable gelation times (90-120 s), high porosity, excellent swelling capacity, and superior mechanical strength (compressive modulus up to ∼40 kPa). Rheological analysis revealed stable viscoelastic properties with G' consistently exceeding G″. The composites also exhibited remarkable self-healing ability. In vitro, all hydrogel extracts displayed outstanding cytocompatibility, promoting primary chondrocyte adhesion, proliferation, and migration. Hydrogels containing collagen and MC (especially CS5M1) significantly enhanced the alkaline phosphatase (ALP) activity and upregulated chondrogenic gene expression (COL II, Acan, and Sox9). In vivo implantation in a rat full-thickness cartilage defect model demonstrated that CSMA-based composite hydrogels facilitated seamless defect filling, enhanced proteoglycan and glycosaminoglycan deposition, and promoted subchondral bone remodeling. Among all formulations, CS5M1 achieved the most complete repair, regenerating hyaline-like cartilage integrated with surrounding tissue after 12 weeks. Collectively, these results demonstrate that the composite hydrogels provide a biomimetic, injectable, and photo-curable platform with excellent osteochondral regenerative potential.
Astrocyte dysfunction plays a pivotal role in the pathogenesis of POLG-related mitochondrial diseases, yet the underlying mechanisms remain poorly understood. Here, we employed human iPSC-derived astrocytes, cortical organoids and astrocyte-neuron co-culture systems to model POLG mutations and investigate astrocyte-mediated neurotoxicity. Single-cell transcriptomic profiling revealed a marked expansion of A1 neurotoxic astrocytes, depletion of A2 neuroprotective astrocytes, and reduction of neuronal populations in POLG organoids. A1 astrocytes exhibited transcriptional signatures of mitochondrial dysfunction, inflammatory signaling (TGF-β, JAK-STAT), impaired neuro-supportive functions, and activation of senescence, autophagy, and proteostasis stress pathways. Co-cultured dopaminergic neurons displayed impaired morphology and widespread transcriptional downregulation of mitotic, cytoskeletal, and synaptic genes, along with activation of inflammatory and ion transport pathways. Treatment with the NAD⁺ precursor nicotinamide riboside (NR) attenuated astrocyte reactivity, reduced IL-6 and CXCL1 secretion, improved neuronal structure and synaptic marker expression, and increased mtDNA copy number and ATP production in POLG astrocytes. Our study identifies NAD⁺ augmentation as a promising strategy to mitigate astrocyte-driven pathology in mitochondrial encephalopathies.
Hashimoto's thyroiditis (HT) is a chronic autoimmune disorder in which persistent immune dysregulation gradually impairs thyroid structure and endocrine function. Although levothyroxine replacement can correct hypothyroidism, it has little effect on the underlying autoimmune activity that drives the progression of the disease. Tetrahedral framework nucleic acids (tFNAs) are self-assembled DNA nanostructures that have demonstrated immunomodulatory capabilities in several inflammatory conditions. In this study, we investigated whether tFNAs could exert therapeutic effects in experimental models of HT and explored the involved mechanisms. In vivo administration of tFNAs alleviated multiple disease-associated symptoms, including thyroid enlargement, lymphocytic infiltration, elevated autoantibodies levels, hormonal dysfunction, and follicular cell apoptosis. These changes were accompanied by a shift in CD4+ T-cell differentiation, characterized by decreased T helper 1 (Th1), T helper 17 (Th17), and follicular helper T (Tfh) responses and a relative increase in T helper 2 (Th2) cells and regulatory T cells (Tregs). In vitro, tFNAs were efficiently internalized by thyroid follicular cells, where they mitigated the inflammatory environment induced by interferon-gamma (IFN-γ) by reducing cytokine release, preserving mitochondrial membrane potential, and limiting cell death. Further analyses indicated that these effects were closely associated with the suppression of NOTCH1 pathway activation. No obvious systemic toxicity was observed. Collectively, the data suggest that tFNAs may provide a strategy to modulate autoimmune activity in HT beyond hormone replacement therapy alone.
The research study is dedicated towards synthesis of novel spiro[indoline-3,2'-pyrrolidines] incorporating a urea functionality. Crucially, the study seeks to elucidate their anticipated multi-targeted mechanism of action, moving towards the final objective of producing multi-functional antineoplastic agents. 1,3-Dipolar cycloaddition was used to construct the final compounds, the spiro-analogs 15a-m. 2-Propen-1-ones 12a-j provided a crucial part of the structure (the urea group). Isatins 13a,b and sarcosine 14, reacted together during the process to form in situ the necessary dipole, azomethine ylide. This synthetic protocol produced the targeted agents 15a-m with high yields. Biological evaluation identified compound 15l as a highly potent agent against the HCT116 colon cancer line, outperforming sunitinib and 5-fluorouracil by 3.7-fold and 7.9-fold, respectively. It is also identified with efficacy against MCF7 (breast cancer) close to that of 5-fluorouracil. Additionally, compound 15h exhibited exceptional potency against A431 skin squamous carcinoma surpassing 5-fluorouracil by 9.1-fold. Mechanistic studies confirmed the dual-action of rational design. Compound 15h demonstrated VEGFR-2 inhibition comparable to sorafenib, while 15g emerged as a dual MDM2 inhibitor and p53 activator, significantly outperforming doxorubicin. The correlation between MDM2 inhibition and p53 activation suggests that the disruption of the MDM2-p53 protein-protein interaction, alongside VEGFR-2 inhibition, drives the antiproliferative effects. Furthermore, compounds 15g, 15h, 15j, and 15k induced apoptosis through the upregulation of caspase-3 and BAX and the downregulation of Bcl-2. Cell cycle (flow cytometry) studies of 15h and 15j confirmed G0/G1 phase arrest. Moreover, the Annexin V-FITC/PI dual staining assay evidenced the apoptotic potential of compounds 15h and 15j. CAM assays supported the anti-angiogenic potential of 15g, 15h, 15j, and 15k. Finally, molecular docking (PDB ID: 5LAW) and dynamic simulations validated the binding stability and mechanism within the MDM2 pocket.
The oral performance of Biopharmaceutics Classification System (BCS) Class II and IV drugs is primarily limited by poor aqueous solubility and intestinal efflux, resulting in low, variable absorption. Saponins, amphiphilic natural glycosides, have gained recognition as biogenic multifunctional excipients for integrated absorption enhancement strategies. This review synthesizes a comprehensive structure-function-bioavailability (SFB) knowledge base, relating individual saponins and derived nanocarriers' molecular architecture to micelle formation, self-assembly interactions, nanostructure formation, and quantifiable pharmacokinetic (PK) endpoints. Signalling factors including aglycone formulation type, log P, sugar chain length and branching, and HLB govern critical micellar concentration, nanostructure persistence, drug loading, cellular membrane dynamics, and modulation of intestinal efflux. Selected drug-saponin systems are used to compare diverse nanocarrier formats, for example, micelles, nano emulsions, liposomes, solid lipid nanoparticles, and silk-like nanostructures, with individual BCS Class II drugs, using both experimental and theoretical PK data. These nanocarriers achieve nanoemulsion stabilization, mucus penetration, cell membrane disruption, tight junction opening, and efflux pump inhibition, leading to significantly enhanced oral absorption (2-7 × increases in bioavailability, with dose reduction). This work defines the visualization and design rules essential to guide future nanocarrier optimization and clinical translation.
The stability of liver function in pregnant hepatitis B virus (HBV) carriers is closely related to both maternal and infant outcomes. However, the role of gut microbiota in HBV infection during pregnancy remains unclear. This study aimed to analyze the compositional characteristics of gut microbiota in pregnant HBV carriers and to explore their associations with liver biochemical indicators. This study enrolled 20 pregnant women with hepatitis B virus infection (HBV group) and 13 healthy pregnant women (control group, CON). Clinical data were collected from all subjects. Fresh fecal samples were obtained and subjected to 16S rRNA gene high-throughput sequencing to characterize gut microbial alpha diversity, beta diversity, and between-group differences in community composition. Spearman correlation analysis was employed to assess associations between gut microbiota and liver biochemical indicators as well as HBV DNA load. Beta diversity analysis indicated a significant separation of overall microbiota structure between the HBV and CON groups. At the phylum level, the gut microbiota in both groups was predominantly composed of Bacteroidota, Firmicutes, and Proteobacteria. At the genus level, the dominant genera were Bacteroides, Prevotella, and Faecalibacterium. Compared with the CON group, the HBV group showed significantly decreased abundances of Synergistota and Desulfobacterota at the phylum level, Synergistia and Desulfovibrionia at the class level, and NK4A214_group and Desulfovibrio at the genus level (all p < 0.05), whereas Acidaminococcus was significantly enriched (p < 0.05). Spearman analysis showed that Bacteroides, Bifidobacterium, Lachnospira, Muribaculum, Parabacteroides, Parasutterella, [Eubacterium] eligens group, and Prevotella were negatively correlated with liver function-related indicators (p < 0.05). In contrast, Coprococcus, Prevotella, and Ruminococcus were positively correlated with liver function-related indicators (p < 0.05). Notably, Escherichia-Shigella demonstrated positive correlations with both liver function indicators and HBV-DNA levels (p < 0.05). Pregnant HBV carriers exhibit distinct gut microbiota compositional features compared to healthy pregnant controls. Specific bacterial genera show correlations with HBV DNA levels and liver biochemical parameters.
Mammalian orthoreoviruses (MRVs), commonly known as reoviruses, are an emerging zoonotic threat that are known for their broad host tropism and potential for causing severe clinical pathology in both humans and animals. Despite this epidemic risk, currently, there are no approved therapeutic agents that are able to disrupt MRV transmission. The viral attachment protein sigma1 (σ1), mediating the entry of the virus into the host cells is a critical target for antiviral intervention. This study used an in silico structure-based drug design strategy to screen for bioactive phytochemicals that are capable of inhibiting the function of σ1. We screened a library of 376 bioactive phytochemicals with known antiviral potential against the σ1 receptor binding domain using molecular docking. Among the candidates, catechin gallate was the most potent inhibitor, possessing a superior binding affinity of -8.1 kcal/mol followed by bilobetin, which also showed a favorable binding affinity of -7.8 kcal/mol. Structural interaction analysis showed that catechin gallate and bilobetin occupies the active JAM-A binding pocket, forming stable interactions with some of the residues, including Gly381, Glu384, and Arg316, which are essential for the reovirus in the cellular attachment process. Subsequent pharmacokinetic and toxicity profiling proved that catechin gallate possessed favorable safety and drug-like characteristics, whereas bilobetin exhibited an unfavorable toxicity profile. In addition, molecular dynamics (MD) simulations supported the stability of σ1-catechin gallate complex relative to the σ1-bilobetin complex. Extensive post-trajectory analyses including RMSD, RMSF, Rg, SASA, and H-bond, showed that the binding of the catechin gallate significantly increases the rigidity and compactness of the protein. PCA indicated that the first three principal components (PC1-PC3) accounted for 74.1% and 76.2% of the total variance for catechin gallate and bilobetin, respectively, with the σ1-catechin gallate complex displaying a more compact conformational cluster consistent with greater stability. MM-GBSA analysis also showed favorable binding for both complexes, with estimated binding energies of -15.6097 ± 3.21 kcal/mol and -13.7327 ± 5.44 kcal/mol for the σ1-catechin gallate and σ1-bilobetin complexes, respectively, with catechin gallate showing comparatively stronger binding. Our results reveal a precise mechanism by which the lead compound catechin gallate sterically occludes the σ1 receptor-binding pocket, thereby likely abrogating viral attachment to the host cell. This comprehensive preclinical evaluation provides supporting evidence for the further development of catechin gallate using in vivo models and clinical trials as a promising antiviral candidate against reovirus infection.
Trypanosoma brucei, a divergent eukaryote parasite, is responsible for neglected tropical diseases in humans and animals, specifically sleeping sickness or human African trypanosomiasis and nagana. Beyond its scientific significance, a comprehensive understanding of its biology has substantial medical and economical implications. Nuclear pore complexes (NPCs) are large multiprotein channels embedded in the nuclear envelope that regulate nucleocytoplasmic transport. In addition to this critical function, NPCs are involved in essential nuclear processes such as chromosome segregation, transcription, and cytokinesis. This study demonstrates that Myosin-like protein-1 (MLP1) localizes to the nuclear basket of NPCs in T. brucei. Silencing of TbMLP1 by RNA interference in T. brucei procyclic cells resulted in severe growth, significant impairment of messenger RNA export, disorganization of nuclear structure, and marked genomic instability. Flow cytometry and fluorescence in situ hybridization (FISH) analyses revealed abnormal DNA content and a reduction in disomic cells, alongside an increase in monosomic, trisomic, and polysomic cells, indicating intolerable aneuploidy detrimental to cell viability. Together, these findings demonstrate that TbMLP1 links NPC function to multiple key cellular pathways. This research provides new insights into the mechanisms that maintain nuclear architecture, preserve nuclear envelope morphology, ensure genome stability, and faithful chromosome segregation, and support appropriat kinetochore distribution and mitotic spindle organization.
Acinetobacter baumannii is a rapidly evolving opportunistic pathogen that has developed strategies to resist multiple antimicrobials and evade the host immune system. Its global emergence as a multidrug-resistant (MDR) pathogen has severely limited treatment options and increased mortality in hospital settings. Virulence factors associated with the cell envelope and outer membrane vesicles play central roles in bacterial survival and pathogenicity. In this context, this review aims to provide a concise overview of the virulence apparatus present on the surface of A. baumannii and evaluate its role in bacterial survival, pathogenicity, and host-pathogen interactions, while also exploring its potential as a therapeutic target. A scoping review of the published literature was conducted to address the structure, function, and host-interaction dynamics of surface-associated virulence factors and secreted outer membrane vesicles (OMVs) along with their translational therapeutic potential. Virulence factors associated with the bacterial cell envelope and OMVs play integral roles in the survival and pathogenicity of A. baumannii. These surface components are involved in biofilm formation, adhesion to both biotic and abiotic surfaces, and modulation of host cell responses. Their surface accessibility and functional conservation make them attractive therapeutic targets and candidate vaccine antigens. The efficacy of subunit vaccines targeting components of the virulence apparatus has also been explored in several studies. Additionally, secreted OMVs perform diverse biological functions and have demonstrated immunogenic potential. However, strategies targeting single virulence determinants have often shown limited efficacy due to functional redundancy and adaptive plasticity, whereas multi-target approaches (utilising combinations of multiple surface-associated virulence factors of A. baumannii) may represent as one of the effective anti-virulence therapy approaches to prevent disease progression caused by MDR strains, ultimately contributing to improved therapeutic outcomes and reducing the burden of hospital-acquired infections.
Cell Structure and Function (CSF), the official journal of the Japan Society for Cell Biology (JSCB), celebrates its 50th anniversary in 2025. This essay traces the scientific evolution of CSF from its founding in 1975 to the present, drawing on bibliometric data retrieved from OpenAlex at ten-year intervals. Over five decades, CSF published 1,737 articles, with the Field-Weighted Citation Impact (FWCI) showing a consistent upward trend, even as total output declined following the journal's shift to electronic publication in 2005. A decade-by-decade analysis of the five most-cited articles reveals a clear evolution in research themes: early issues were dominated by plant cell biology and methodological papers in microscopy and biochemistry, while subsequent decades saw increasing focus on autophagy, the unfolded protein response, and intracellular membrane trafficking-fields in which Japanese researchers have played globally recognized pioneering roles. The turn of the millennium marked a peak in absolute citations, with landmark papers on bafilomycin A1, SNARE proteins, and a review of autophagy co-authored by Nobel Prize laureate Yoshinori Ohsumi. Two major milestones-electronic publication in 2005 and gold open-access adoption in 2016-fundamentally transformed the journal's publishing model. Looking ahead, the essay considers the role of artificial intelligence in peer review, arguing that while AI can assist in assessing novelty and reproducibility, the judgment of a manuscript's scientific significance must remain a human responsibility. CSF remains committed to disseminating reliable, foundational cell biology to the international community.Key words: Cell Structure and Function (CSF), bibliometrics, open access, artificial intelligence in peer review.
Type 2 diabetes mellitus (T2DM) is a major global health burden characterized by insulin resistance, progressive pancreatic β-cell dysfunction, and chronic metabolic dysregulation. Marine seaweeds have emerged as a valuable source of bioactive natural products, particularly polyphenols and polysaccharides, with promising potential for diabetes management. This review focuses on three major contributions: first, the structural diversity of seaweed-derived polyphenols and polysaccharides; second, their multi-target mechanisms of glucose regulation; and third, the structure-activity relationships governing their bioactivities. Current evidence shows that these compounds may help manage type 2 diabetes in several ways, including inhibition of α-amylase and α-glucosidase, attenuation of oxidative stress and chronic inflammation, enhancement of insulin secretion and insulin sensitivity, regulation of lipid metabolism, and modulation of gut microbiota. Key structural determinants such as degree of polymerization, hydroxyl group density, sulfation level, molecular weight, and chemical modifications are discussed in relation to their functional properties. By linking chemical structure with biological function, these findings highlight marine seaweeds as a rich reservoir of multi-target therapeutic candidates for T2DM management and provide a scientific basis for their development as functional food ingredients or lead compounds for novel diabetes management drugs.
A possible method for improving anti-cancer efficacy is the combination of the inhibition of epidermal growth factor receptor (EGFR) and poly (ADP-ribose) polymerase-1 (PARP-1). This work describes the rational design, synthesis, and complete characterization of a novel class of boomerang-shaped dual PARP-1/EGFR inhibitors (3a-o). HepG-2 and MDA-MB-231 cancer cell lines were used to test the synthesized compounds' antiproliferative potential; MDA-MB-231 cells showed greater sensitivity. Compounds 3h, 3i, and 3j had the strongest cytotoxic effects among the series, with IC50 values of 0.23, 0.90, and 1.40 µM, respectively, against MDA-MB-231 cells. Compared with the reference medications erlotinib and olaparib, compound 3h showed the highest dual inhibition (EGFR IC50 = 1.62 µM and PARP-1 IC50 = 0.36 µM) in enzymatic experiments, demonstrating that these compounds effectively inhibited both EGFR and PARP-1. Compound 3h strongly promoted apoptosis in MDA-MB-231 cells, increasing the total apoptotic population to 20.04% and causing G1-phase cell-cycle arrest, as determined by mechanistic studies. In vivo tumor growth inhibition trials showed a tumor inhibition rate (TIR%) of 41.4% for compound 3h compared to 48.8% for doxorubicin (DOX). Liver function biomarkers and hematological parameters remained within the acceptable levels following compound 3h treatment. The dual-target activity of compound 3h was further validated by molecular docking and molecular dynamics simulations, which demonstrated persistent binding contacts within the active sites of PARP-1 and EGFR.
DCTPP1 is a nucleotide pyrophosphatase that helps preserve genomic stability and epigenetic programming by hydrolyzing and preventing the misincorporation of methylated base-modified deoxycytosine triphosphates into DNA. Through this role, DCTPP1 can degrade the efficacy of nucleotide analog-based DNA methyltransferase inhibitors and thus represents a compelling therapeutic target in cancer treatment. To identify prospective antagonists of DCTPP1, we conducted a high-throughput chemical screen against the enzyme, identifying both existing and previously unreported inhibitor classes with potent submicromolar activity. Structural characterization using X-ray crystallography revealed that the inhibitors all occupy DCTPP1's nucleotide-binding pocket, associating primarily with a pair of tryptophans and two critical histidine residues that mimic interactions observed with natural substrates. Biochemical assays using modified chemical scaffolds confirmed the relevancy of the observed DCTPP1-antagonist interactions, while cell-based experiments demonstrated significant synergy between the lead inhibitors and the nucleoside analog decitabine in blocking the growth of prostate cancer cells. The specificity and efficacy of the compounds were further validated through loss- and gain-of-function studies, confirming the dependence of their therapeutic synergy on DCTPP1 activity. These findings advance our understanding of DCTPP1 as a therapeutic target while uncovering chemical scaffolds that can potentiate the action of existing nucleotide-based cancer therapies.