Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, with chronic hepatitis B virus (HBV) infection representing its foremost risk factor. Although programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors (ICIs) have entered clinical practice, response rates in HBV-related HCC remain limited, underscoring the urgent need for mechanism-based strategies to overcome intrinsic resistance and improve immune checkpoint blockade (ICB) efficacy by exploiting noncanonical PD-L1 functions. Here, we identify a non-canonical pathway in which the HBV-encoded X protein (HBx) drives O-GlcNAcylation (O-GlcNAc) of PD-L1 at S283 and T285, thereby promoting PD-L1 mitochondrial translocation via the GOLPH3/Drp1 axis. Mitochondrial PD-L1 (mtPD-L1) hijacks Golgi-mitochondria communication to activate the mTOR/PGC-1α axis, enhance mitochondrial biogenesis and translation, and reprogram cellular energy metabolism, ultimately conferring resistance to anti-PD-L1 antibody (αPD-L1) therapy in HBV-related HCC. Pharmacological inhibition of O-GlcNAcylation with OSMI-1 disrupts this mtPD-L1 regulatory axis, restores mitochondrial homeostasis, and sensitizes HBV-related HCC to αPD-L1 therapy. Collectively, these findings identify O-GlcNAcylated mtPD-L1 as a previously unrecognized immunometabolic checkpoint and establish the mtPD-L1-mTOR/PGC-1α axis as a key mechanism linking mitochondrial biogenesis to immunotherapy resistance. This study provides a rationale for combining αPD-L1 with OSMI-1-mediated O-GlcNAcylation inhibition as a therapeutic strategy to improve immunotherapy sensitivity in HBV-related HCC.

Hepatitis B virus (HBV) infection remains a leading etiological driver of hepatocellular carcinoma (HCC). Cuproptosis is a recently defined copper-dependent form of regulated cell death that selectively eliminates mitochondria-dependent cells; whether HBV rewires this vulnerability remains unknown. Here we unveil a novel HBV X protein (HBx)-driven mechanism of cuproptosis evasion. Integrative analysis of clinical specimens, HBx-transgenic (HBx-Tg) mice, and multi-omics datasets revealed marked downregulation of STEAP4 (six-transmembrane epithelial antigen of prostate 4), a metalloreductase essential for cuproptosis sensitivity, in HBV-positive HCC. Mechanistically, HBx attenuates sirtuin 3 (SIRT3), impairing deacetylation of STEAP4 at lysine 404 and abolishing its mitochondrial targeting. Consequently, cells switch from the tricarboxylic acid (TCA) cycle respiration to glycolysis, reducing sensitivity to the copper ionophore elesclomol (ES). Restoring STEAP4 expression or pharmacological activation of SIRT3 with honokiol (HKL) re-instated mitochondrial STEAP4 localization and re-sensitized HBV-related HCC cells to cuproptosis; combination with ES produced synergistic tumor suppression in vitro and in orthotopic models. Collectively, our findings establish the SIRT3-STEAP4 axis as a novel regulator of cuproptosis resistance in HBV-related HCC. HBx-mediated repression of SIRT3 disrupts STEAP4 deacetylation and mitochondrial targeting, fostering metabolic reprogramming and evasion of copper-induced cell death. The results provide a pre-clinical rationale for copper-directed combination strategies in HBV-associated HCC.

PURPOSE:The transcription factor Ets-1 is widely implicated in hepatic carcinogenesis, yet its precise role in balancing fibrogenesis and regeneration remains poorly defined. This study elucidated the clinical relevance, upstream regulatory mechanisms, and dual cytoprotective/antifibrotic functions of Ets-1 during chronic liver injury and repair. MATERIALS AND METHODS:Ets-1 networks were evaluated using human cirrhotic tissues adjacent to hepatocellular carcinoma, a rat carbon tetrachloride fibrosis/recovery model, and Ets-1 knockout mice. Upstream regulation and epigenetic gating were characterized in vitro across human hepatocellular carcinoma lines via Western blotting, real-time reverse-transcription PCR, methylation-specific PCR, chromatin immunoprecipitation, and enzyme-linked immunosorbent assay. RESULTS:Hepatic Ets-1 expression was profoundly depleted in human and rodent fibrosis, inversely correlating with disease severity, but rebounded robustly during regeneration, colocalizing with hepatocellular heme oxygenase-1 (HO-1). Ets-1 knockout mice exhibited exacerbated fibrosis, blunted HO-1 induction, and heightened injury markers under toxic stress. Mechanistically, in vitro analyses identified Ets-1 as a downstream effector of the proregenerative epidermal growth factor/extracellular signal-regulated kinase axis. Crucially, Ets-1 responsiveness to mitogens was restricted by promoter DNA hypermethylation, acting as an epigenetic gatekeeper in specific cell lines. Functionally, Ets-1 overexpression suppressed profibrogenic transforming growth factor beta 1 secretion and mitigated oxidative cytotoxicity via redox-sensitive recruitment to the HO-1 promoter. This significantly reduced macromolecular damage, as evidenced by attenuated 4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine adduct formation. CONCLUSIONS:Ets-1 acts as a pivotal molecular switch that integrates growth factor signaling with antioxidant defenses to facilitate hepatic recovery. Restoring Ets-1-mediated cytoprotection offers a potential therapeutic strategy for chronic liver disease, provided that tumorigenic risks are carefully managed via targeted hepatocyte-specific delivery systems.

Nanoplastics (NPs) exhibit neurotoxicity, yet the precise molecular mechanisms remain elusive. In this study, we established a human-relevant polystyrene nanoplastics (PS-NPs, 50 mg kg-1) oral exposure model in C57BL/6 mice in vivo and a neuro-immune microglial-neuron co-culture system (HMC-3/SH-SY5Y cells) in vitro to dissect these mechanisms. We demonstrate that PS-NPs exposure triggers microglial M1 activation and drives neuronal senescence. Mechanistically, PS-NPs activate the protein phosphatase 2A (PP2A)-B56γ subunit, which selectively dephosphorylates the ribosome biogenesis regulator ErbB3-binding protein 1 (Ebp1) at Ser335. This post-translational modification reduces Ebp1 nucleolar localization, suppresses 47S pre-ribosomal RNA transcription, and induces nucleolar stress. Consequently, the p53/p21 pathway is engaged, promoting neuronal senescence. Pharmacological inhibition of PP2A with LB-100 restored ribosome biogenesis, prevented neuronal senescence, and rescued cognitive deficits and neurodegenerative phenotypes in PS-NP-exposed mice. This is the first study to identify the PP2A-B56γ-p-Ebp1Ser335-ribosome biogenesis axis as a novel cascade mechanism driving PS-NP-induced neuronal senescence. Our findings offer a targetable strategy to mitigate nanoplastics-associated neurodegeneration.

Background/Objectives: To develop and validate a model system using deep learning algorithms for the automatic detection of type A aortic dissection (AD), and differentiate it from normal and type B AD patients. Methods: In this retrospective study, a deep learning model is developed, based on aortic computed tomography angiography (CTA) scans of 498 patients using training, validation and test sets of 398, 50 and 50 patients, respectively. An independent test set of 316 patients is used to validate and evaluate its performance. Results: Our model comprises two components. The first one is an objection detection model, which can identify the aorta from CTA. The second one is a dissection classification model, which can automatically detect the presence of aortic dissection and determine its type based on Stanford classification. Overall, the sensitivity and specificity for Type A AD were 0.969 and 0.982, for Type B AD were 0.946 and 0.996 and for normal cases were 0.988 and 1.000, respectively. The average processing time per CTA scan was 7.9 ± 2.8 s. (mean ± standard deviation). Conclusions: This deep learning automatic model can accurately and quickly detect type A AD patients, and could serve as an imaging triage in an emergency setting and facilitate early intervention and surgery to decrease the mortality rates of type A AD patients.

BACKGROUND:Nasopharyngeal carcinoma (NPC) is a prevalent malignant tumor in East Asia, particularly impacting China. The association between multiple constituents of fine particulate matter (PM2.5) and the survival time of NPC patients remains unclear, which poses a challenge for targeted public health interventions. METHODS:An accelerated failure-time model with a 12-year cohort design was used to analyze the impact of long-term PM2.5 and its constituents on the survival time of 1492 NPC patients. Restricted cubic splines (RCS) functions and stratification analyses were conducted to identify the exposure-response curve and vulnerable subgroups, respectively. RESULTS:PM2.5 and its constituents were significantly associated with reduced survival time in NPC patients. For per interquartile range (IQR) increase in concentrations, the time ratio changing percentage (TRCP) ranged from -28.8 % to -33.6 % for PM2.5, -34.7 % to -39.6 % for black carbon (BC), -13.6 % to -17.4 % for nitrate (NO3-), -21.9 % to -26.6 % for ammonium (NH4+), -29.5 % to -35.5 % for organic matter (OM), and -31.5 % to -36.2 % for sulfate (SO42-). The exposure-response relationship exhibited a nonlinear trend, with a steep slope at lower concentrations. Furthermore, females, patients with lower monocyte levels, and those with a drinking history faced a higher risk of reduced survival time. CONCLUSIONS:The study reveals the urgent need for environmental regulations to mitigate PM2.5 and its constituents, particularly BC. The evidence of accelerated loss of survivorship is crucial for establishing air quality guidelines concerning PM constituents and formulating public health interventions and protective measures for high-risk NPC patients.

Lung cancer is a leading cause of cancer-related mortality worldwide, largely due to its heterogeneity and intrinsic drug resistance. Malignant pleural effusions (MPEs) provide diverse tumor cell populations ideal for studying these complexities. Although chemotherapy and targeted therapies can be initially effective, subpopulations of cancer cells with phenotypic plasticity often survive treatment, eventually developing resistance. Here, we integrated single-cell isolation and three-dimensional (3D) spheroid culture to dissect subclonal heterogeneity and drug responses, aiming to inform precision medicine approaches. Using A549 lung cancer cells, we established a cisplatin-resistant line and isolated three resistant subclones (Holoclone, Meroclone, Paraclone) via single-cell sorting. In 3D spheroids, Docetaxel and Alimta displayed higher IC50 values than in 2D cultures, suggesting that 3D models better reflect clinical dosing. Additionally, MPE-derived Holoclone and Paraclone subclones exhibited distinct sensitivities to Giotrif and Capmatinib, revealing their heterogeneous drug responses. Molecular analyses confirmed elevated ABCB1, ABCG2, cancer stem cell (CSC) markers (OCT4, SOX2, CD44, CD133), and epithelial-mesenchymal transition (EMT) markers (E-cadherin downregulation, increased Vimentin, N-cadherin, Twist) in resistant subclones, correlating with enhanced migration and invasion. This integrated approach clarifies the interplay between heterogeneity, CSC/EMT phenotypes, and drug resistance, providing a valuable tool for predicting therapeutic responses and guiding personalized, combination-based lung cancer treatments.

Chronic diabetic skin wounds are among the most common complications of diabetes mellitus. Persistent cellular senescence, primarily driven by hyperglycemia and oxidative stress, impairs all phases of the wound healing cascade, posing a major therapeutic challenge. To address this, we engineered an innovative glucose- and reactive oxygen species (ROS)-dual-responsive hydrogel (HG) based on marine polysaccharides. This advanced platform, termed CCM@CS-HG, combines carboxymethyl chitosan (CMCS) and oxidized sodium alginate (OSA), and is embedded with functionalized fullerenol [C60(OH)n] nanoparticles (NPs) that co-deliver the mitochondria-targeting agents carnosine (Car) and elamipretide (MTP-131). Within the CCM@CS-HG system, Car and MTP-131 act synergistically to scavenge excessive ROS in the wound microenvironment, while C60(OH)n NPs confer intrinsic antioxidant and cytoprotective effects and simultaneously enhance intracellular delivery of Car and MTP-131. In vitro, under a diabetic wound-mimicking microenvironment (high glucose and hydrogen peroxide), CCM@CS-HG effectively reversed fibroblast senescence, promoting proliferation and migration, while also demonstrating potent antibacterial activity. In vivo, in a streptozotocin-induced diabetic mouse model with full-thickness skin wounds, CCM@CS-HG markedly accelerated wound closure, with enhanced collagen deposition and angiogenesis. Collectively, these findings highlight the potential of CCM@CS-HG as a promising and multifunctional therapeutic strategy for the repair of chronic diabetic skin wounds.

Hepatitis B virus (HBV) X protein (HBx) plays a critical role in the progression of HBV-related hepatocellular carcinoma (HCC). Long non-coding RNAs (lncRNAs) regulate various biological processes and contribute to HCC development, with their therapeutic potential in disease progression recently gaining significant attention. However, the involvement of lncRNAs in HBx-related hepatocarcinogenesis and the underlying regulatory mechanisms remain unclear. In this study, we conducted a comprehensive analysis of multi-database sequencing data to identify metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as an HBx-associated lncRNA and observed its upregulation in HBV-related HCC tissues and cells upon HBx expression. Additionally, high MALAT1 expression was correlated with poor prognosis and advanced HCC progression. MALAT1 overexpression significantly promoted the proliferation, migration, and invasion of HCC cells. Mechanistic investigations revealed that MALAT1 was transported to the cytoplasm and enhanced RNA stability in a N6-methyladenosine (m6A)-dependent manner through direct interaction with and recruitment of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). Targeting MALAT1 in vivo with antisense oligonucleotides (ASO)-MALAT1 treatment effectively suppressed the progression of xenograft tumors and orthotopic liver tumors in HBx-related HCC. Moreover, hydrodynamic-based gene delivery (HGD) was utilized to introduce anti-HBx transposon plasmids into murine hepatocytes, thereby suppressing MALAT1-m6A-mediated HBV-related hepatocarcinogenesis in HBx transgenic (HBx-Tg) mice. Overall, our findings shed novel light on the regulatory role of IGF2BP3-mediated MALAT1 nuclear-cytoplasmic shuttling and RNA stabilization via m6A modification during HCC progression. These results suggest that m6A-based MALAT1 expression serves as a novel diagnostic and prognostic biomarker for targeted epigenetic intervention in HBV-related HCC.

BACKGROUND/PURPOSE:Early detection of severe dengue (SD) and appropriate management are crucial in reducing the case fatality rate. The objective of this study was to investigate the clinical characteristics of SD and identify independent risk factors associated with mortality among SD patients. METHODS:A retrospective study was conducted at two medical center hospitals between 2002 and 2019, involving patients aged ≧18 years with laboratory-confirmed SD. RESULTS:This study included 294 patients with SD, of whom 203 (69%) survived and 91 (31%) died. Among the 294 SD patients, 103 (35%) experienced acute kidney injury, 54 (18.4%) had pneumonia, and 19 (6.5%) had bacteremia. Among the 286 patients with available alanine aminotransferase (ALT) data, 41 (14.3%) experienced severe hepatitis (ALT>1000U/L). The median time from illness onset to death among the 91 SD patients who died was 5 days. Multivariable regression analysis revealed increasing odds of death associated with older age (odds ratio [OR], 1.037; 95% confidence interval [CI], 1.009-1.066), altered consciousness (OR, 8.591; 95% CI, 2.914-25.330), gastrointestinal bleeding (OR, 1.939; 95% CI, 1.037-3.626), and leukocytosis (OR, 2.504; 95% CI, 1.124-5.578) upon arrival, as well as organ impairment during hospitalization, including acute kidney injury (OR, 2.627; 95% CI, 1.373-5.028), severe hepatitis (OR, 5.324; 95% CI, 2.199-12.889), and pneumonia (OR, 2.250; 95% CI, 1.054-4.802). CONCLUSIONS:Our findings underscore the importance of early recognition and intervention by frontline physicians in identifying SD patients at high risk of mortality. This information can significantly contribute to reducing fatalities and improving the overall management of SD cases.

This study identifies glutathione (GSH) as an endogenous A-A type allosteric activator of pyruvate kinase M2 (PKM2), stabilizing it in its active tetrameric form through binding at the A-A interface. This PKM2-GSH interaction links GSH metabolism to ferroptosis regulation. Transcriptomic analyses across cancers demonstrate strong correlations between GSH, SLC7A11, PKM2, glycolysis, and ferroptosis pathways. By depleting GSH and activating PKM2, ferroptosis is enhanced in PKM2-dependent cancer models. This approach leads to significant changes in central carbon and lipid metabolism, disrupts mitochondrial function, and drives ferroptotic cell death. The combined treatment markedly suppresses tumor growth in a xenograft model. Elevated PKM2 and SLC7A11 expression levels correlate with poorer survival outcomes, indicating their potential as biomarkers for ferroptosis-based therapy. The findings demonstrate a dual role for GSH in cellular homeostasis and identify the PKM2-GSH-SLC7A11 axis as a therapeutic target for aggressive cancers.

NOD-like receptor protein 3 (NLRP3) is a key driver of hepatotoxicant-induced nonalcoholic fatty liver disease (NAFLD). Phosphorylation of NLRP3 at serine 295 (p-NLRP3S295) is crucial for pyroptosis. Monoclonal antibodies (mAbs) have been designed to target extracellular molecules or cell membrane surface receptors and have achieved progress in NAFLD treatment. However, research on mAbs targeting intracellular biomarkers for NAFLD treatment remains limited. In this study, aflatoxin B1 (AFB1), lipopolysaccharide (LPS) combined with ATP, or palmitic acid (PA) were used to induce p-NLRP3S295-dependent pyroptosis and inflammation mediated by lipotoxicity in hepatocytes in vitro. We generated a specific anti-p-NLRP3S295 mAb (14C7) and internalized it into hepatocytes via an enhanced TAT-based intracellular delivery system (eTAT), which inhibited p-NLRP3S295-dependent pyroptosis and inflammation in hepatocytes subjected to simulated lipotoxic injury and in the livers of NAFLD mice. The recombinant mAb@p-NLRP3S295 expression system was constructed with 14C7. The intracellularly expressed recombinant monoclonal antibody (R-mAb) efficiently blocked p-NLRP3S295-dependent pyroptosis and inflammation in hepatocytes exposed to hepatotoxicant through the proteasome degradation pathway mediated by tripartite motif-containing 40 (TRIM40). In conclusion, this study presents a novel approach for the targeted inhibition of p-NLRP3S295 through intracellular recombinant mAbs, offering new insights into the treatment of hepatotoxicant-related NAFLD via specific intracellular targeting.

The M1/M2-macrophage imbalance that characterizes diabetic skin wounds sustains chronic inflammation, oxidative stress and bacterial colonization, thereby hindering healing. Although fullerenol [C60(OH)n]-loaded hydrogels (HGs) exert antioxidant properties that accelerate repair, a hydrogel system that specifically targets mitochondria to regulate macrophage polarization has not yet been reported. Here we synthesized a C60(OH)nquaternized chitosan-carbomer hydrogel (C60@QM-HG) utilizing quaternized chitosan modified with phenylboronic acid. C60@QM-HG exhibited excellent injectability and water absorption capacity. Controlled release of C60(OH)n was achieved through reversible borate ester bonds, enabling tailored antioxidant effects under the conditions typical of diabetic wounds. C60@QM-HG modulated mitochondrial redox responsive via the mitochondrial HSPA8-ROMO1 signaling axis to regulate M1/M2 polarization, resulting in significant antioxidant and anti-inflammatory effects. In summary, C60@QM-HG improved the inflammatory microenvironment, accelerated re-epithelialization and collagen deposition, and enhanced healing of diabetic and infected wounds. To enhance mitochondrial targeting, C60(OH)n was conjugated with Apoptozole, an HSPA8 inhibitor, yielding AP&C60@QMHG, which further suppressed M1 polarization and promoted diabetic wound healing. This study provides new insights into anti-inflammatory and antimicrobial strategies for diabetic skin wounds through mitochondriatargeted regulation and controlled release of C60(OH)n.

BACKGROUND AND PURPOSE:Obstructive sleep apnoea (OSA) worsens asthma control. Oxygen desaturation increases oxidative stress, contributing to corticosteroid insensitivity, a hallmark of severe asthma. This study investigated the impact of hypoxaemia and reactive oxygen species on corticosteroid responsiveness in asthma with OSA. EXPERIMENTAL APPROACH:Asthmatic patients with apnoea-hypopnoea index (AHI) ≥ 5 h-1 were classified as OSA. Interleukin (IL)-8 and IL-6 production by peripheral blood mononuclear cells (PBMCs), serum cytokines, oxidative stress markers and nuclear histone deacetylase 2 (HDAC2) were quantified by enzyme-linked immunosorbent assay. HDAC2 and hypoxia-inducible factor-1α (HIF-1α) expression were evaluated by Western blotting and flow cytometry. KEY RESULTS:Compared with non-OSA asthmatics, OSA patients used higher inhaled corticosteroid doses and had increased serum thiobarbituric acid-reactive substances and 8-hydroxy-2-deoxyguanosine, but lower superoxide dismutase and total antioxidant capacity. HDAC2 was lower in OSA PBMCs and in non-OSA PBMCs exposed to 5% O₂ than in normoxia. HDAC2 was correlated inversely with AHI, corticosteroid dose, serum IL-8, oxidative stress, baseline production of IL-8/IL-6 and dexamethasone-induced IL-8 suppression. Dexamethasone inhibited TNF-α-induced IL-8 and lipopolysaccharide (LPS)-induced IL-6 in non-OSA PBMCs, but not in OSA PBMCs. HDAC2 inhibitor CAY10683 impaired corticosteroid action, while N-acetylcysteine and inhibitors of HIF-1α (CAY10585) or phosphoinositide 3-kinase (LY294002) restored HDAC2 and corticosteroid sensitivity. CONCLUSIONS AND IMPLICATIONS:OSA is associated with oxidative stress, reduced HDAC2, and corticosteroid insensitivity in asthma. Antioxidants may help restore corticosteroid efficacy.

Metabolic dysfunction-associated steatohepatitis (MASH) is one of the fastest-growing chronic liver diseases and is characterized by excessive steatosis, inflammation, and progressive liver injury. The hepatitis B virus (HBV) X protein (HBx) is a major viral factor that contributes to the onset and progression of MASH. Emerging evidence highlights the role of epigenetic modifications, particularly N6-methyladenosine (m6A), as prevalent modifications of mRNAs that play crucial roles in MASH pathogenesis by regulating mRNA stability, translation, processing, and nuclear export. However, the epigenetic mechanisms by which m6A modification contributes to HBx-related MASH remain poorly defined. In this study, we observed that NOD-like receptor protein 3 (NLRP3)-dependent pyroptosis and intracellular lipid accumulation are markedly elevated in the livers of HBx-transgenic (HBx-Tg) mice in vivo and in HBx-expressing hepatocytes in vitro, exacerbating liver injury and driving MASH progression. Integrated metabolomic and transcriptomic analyses of HBx-Tg mice revealed distinct gene expression alterations, suggesting a key role for m6A modification in mediating hepatic inflammation and lipotoxicity. Mechanistically, we identified methyltransferase-like 3 (METTL3) as a critical positive regulator of this process. HBx upregulated METTL3 expression and the m6A level of NLRP3 mRNA in HBx-expressing hepatocytes, whereas METTL3 knockdown or catalytic inactivation suppressed NLRP3-dependent pyroptosis. Further investigation revealed that METTL3 enhances NLRP3 mRNA stability via m6A modification at A2748 site in the coding sequence. Moreover, the protein phosphatase 2A (PP2A) B56α subunit was found to interact with the METTL3 methyltransferase domain (MTD), facilitating its enzymatic activity and further increasing NLRP3 m6A methylation, thereby promoting pyroptosis and lipid accumulation in HBx-expressing hepatocytes. Importantly, treatment with STM2457, a selective inhibitor targeting the METTL3 MTD, significantly attenuated hepatic inflammation, steatohepatitis, and lipotoxicity. Taken together, our findings advance the understanding of HBx-induced hepatic lipid accumulation, steatosis, inflammasome formation, and pyroptosis, and indicate that targeting METTL3 with STM2457 intervention is a promising approach for MASH treatment.

Centrosomes are tubulin-based organelles that undergo glutamylation, a post-translational modification that conjugates glutamic acid residues to tubulins. Although centrosomal glutamylation has been known for several decades, how this modification regulates centrosome structure and function remains unclear. To address this long-standing issue, we developed a method to spatiotemporally reduce centrosomal glutamylation by recruiting an engineered deglutamylase to centrosomes. We found that centrosome structure remains largely unaffected by centrosomal hypoglutamylation. Intriguingly, glutamylation physically recruits, via electrostatic forces, the NEDD1/CEP192/γ-tubulin complex to centrosomes, ensuring microtubule nucleation and proper trafficking of centriolar satellites. The consequent defect in centriolar satellite trafficking leads to reduced levels of the ciliogenesis factor Talpid3, suppressing ciliogenesis. Centrosome glutamylation also promotes proper mitotic spindle formation and mitosis. In summary, our study provides a new approach to spatiotemporally manipulate glutamylation at centrosomes, and offers novel insights into how centrosomes are organized and regulated by glutamylation.

Background: Non-pharmaceutical interventions (NPIs) were widely used during the coronavirus disease 2019 (COVID-19) pandemic, however their impact on acute asthma exacerbations (AEs) is not well studied. Methods: We had retrospectively collected patients with asthma AEs between 2019 and 2020 and retrieved data from the Chang Gung Research Database, including clinical manifestations, medications, pulmonary function, clinic and emergency department visits and hospitalizations. Results: A total of 39,108 adult patients with asthma were enrolled, of whom 1502 were eligible for analysis. The prevalence of acute AEs significantly decreased throughout 2020 compared with 2019 after implementation of the NPI policy. The patients were categorized into four groups: Group 1, acute AEs in 2019 with influenza infection (n=692); Group 2: acute AEs in 2019 without influenza infection (n=328); Group 3: acute AEs in 2020 with influenza infection (n=268); Group 4: acute AEs in 2020 without influenza infection (n=214). The patients in group 4 were significantly older (73.3 +/- 29.1 vs 65.5 +/- 29.2, 69.7 +/- 26.2 years, p< 0.01) and had significantly worse forced expiratory volume in one second/forced vital capacity ratio (70.5 +/- 13.9 vs 79.6 +/- 15.5, 72.9 +/- 18.0, p< 0.01) than those in group 1 and 2, and the highest rate of oral corticosteroid prescriptions (17%, p< 0.01). The patients in group 3 and 4 had significantly lower rates of oxygen therapy, ventilator use and mortality at 3 and 12 months of follow-up than those in group 1 and 2. Conclusion: The use of NPIs during the COVID-19 pandemic in Taiwan may reduce the frequency and severity of asthma AEs. This may provide some cost-effective strategies to attenuate acute asthma AEs.

Background/purpose: Oral squamous cell carcinoma (OSCC) often recurs and has poor clinical outcomes, partly attributable to subpopulations that develop resistance to 5 fluorouracil (5FU). Elucidating how these resistant clones emerge and drive tumour aggressiveness is essential for improving OSCC treatment approaches. Materials and methods: To establish 5FU-resistant cells, SCC25 cells were repeatedly exposed to 5FU, and single-cell clones were subsequently isolated using a microfluidic system. Three subclones-Holoclone, Meroclone, and Paraclone-were evaluated for their 5FU responses, expression of drug-efflux pumps (ABCB1, ABCG2), and resistance in three-dimensional (3D) cultures. Their levels of cancer stem cell (CSC) markers (OCT4, SOX2, CD44, CD133) and epithelial–mesenchymal transition (EMT) markers (E-cadherin, Vimentin, Twist) were also examined. In addition, Transwell assays were performed to assess migration and invasion. Results: Compared with parental SCC25 cells, the three subclones exhibited markedly higher resistance to 5FU under 3D spheroid conditions, concurrent with upregulated ABCB1 and ABCG2 expression. All three subclones showed enhanced sphere-forming capacity and increased OCT4 and SOX2 levels, consistent with higher proportions of CD44+/CD133+ cells. Moreover, Holoclone, Meroclone, and Paraclone each displayed reduced E-cadherin alongside elevated Vimentin, and Twist, characteristic of EMT. Transwell assays confirmed increased migration and invasion, with Holoclone and Paraclone exhibiting particularly pronounced effects. Conclusion: Extended 5FU treatment in OSCC selects for distinct subclones that exhibit CSC-like traits and EMT-related motility, promoting robust chemoresistance and heightened malignancy. These findings emphasise the importance of developing comprehensive therapeutic strategies that simultaneously target drug-efflux mechanisms, CSC markers, and EMT pathways to more effectively control OSCC progression.

Ca2+-dependent exocytosis initiates with the formation of fusion pores comprising the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Although cellular signalling typically occurs in transient oscillations on the order of tens of seconds, it remains unclear how such rapid SNARE phosphorylation influences fusion pore kinetics, analogous to transient regulation observed in ion channels. Here we demonstrate that protein kinase A (PKA)-mediated phosphorylation of SN25b (the neuronal isoform of synaptosome-associated protein of 25 kD) modulates secretion rate and fusion pore kinetics in PC12 cells (rat pheochromocytoma derivatives). Upon acute application of KCl and forskolin, cells overexpressing SN25b exhibited a reduced secretion rate compared to the control. This reduction was occluded by overexpressing a PKA-phosphodeficient mutant, SN25b-T138A, rather than a PKA-phosphomimetic mutant, SN25b-T138E. Notably, SN25b, SN25b-T138A or SN25b-T138E did not alter the fraction of incomplete fusion events or quantal size compared to the control. Further kinetic analysis indicated that SN25b-T138A destabilized initial fusion pores by promoting the closure and dilatation of fusion pores. Mechanistically, in situ proximity ligation assays showed that SN25b-T138A reduced its interaction with the other t-SNARE syntaxin-1 compared to the control and SN25b, correlating with destabilized fusion pores. Moreover, compared to SN25b-T138E, SN25b-T138A decreased whole-cell Ca2+ currents and weakened its interaction with synaptobrevin-2 and L-type Ca2+ channel subunits. These changes in interaction were associated with increased secretion and full-fusion rate, implying efficient disassembly after dilatation. Together, PKA-mediated phosphorylation of SN25b rapidly modulates fusion pore kinetics in response to transient signalling oscillations, thereby fine-tuning exocytotic efficiency in real time. KEY POINTS: Protein kinase A (PKA)-mediated SNAP-25 phosphorylation rapidly reduces the rate of secretion. PKA-phosphodeficiency of SNAP-25 destabilizes the kinetics of initial fusion pores, correlating with its decreased interaction with syntaxin-1. PKA-phosphodeficiency of SNAP-25 decreases the interaction with synaptobrevin-2 and the L-type calcium channel subunit, leading to efficient priming. PKA-mediated SNAP-25 phosphorylation rapidly regulates fusion pore kinetics and shapes exocytotic kinetics on the order of tens of seconds.

BACKGROUND:Taiwan experienced a major dengue outbreak in 2023 following the relaxation of COVID-19 border controls. The contributing factors remained unclear. This study investigated potential virological, immunological, and clinical drivers. METHODS:We retrospectively analyzed laboratory-confirmed dengue virus (DENV) infections at a tertiary care hospital in southern Taiwan. Serotypes were identified by qRT-PCR. Viral origins were assessed through phylogenetic and envelope (E) gene amino acid analyses. Clinical features of DENV-1 and DENV-2 cases were compared. Neutralization and antibody-dependent enhancement (ADE) were evaluated using PRNT and ADE assays. RESULTS:DENV-1 and DENV-2 were identified as the predominant circulating serotypes. Clinical analysis revealed that DENV-2 infection was significantly associated with older age, diabetes mellitus, hypertension, and elevated hematocrit levels (p < 0.05), and these associations remained statistically significant in multivariate analysis. Phylogenetic analysis showed that DENV-1 isolates belonged to genotypes I and IV, while DENV-2 strains were of the cosmopolitan genotype. These viruses clustered closely with strains from Southeast Asia. Amino acid analysis indicated that DENV-1 strains exhibited 2-10 substitutions relative to 2014 isolates, while DENV-2 strains closely matched those from 2015. Sera from the 2014-2015 outbreaks demonstrated potent homotypic but limited heterotypic neutralization. ADE was observed in heterotypic infection contexts. CONCLUSIONS:The 2023 dengue outbreak in Taiwan was driven by co-circulation of DENV-1 and DENV-2, limited heterotypic immunity, and ADE. These findings highlight the importance of integrated virological surveillance, genotype monitoring, and immunological assessment to inform dengue control strategies in non-endemic regions experiencing imported viral threats.