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Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. Despite advances in diagnosis and treatment, the lack of effective therapeutic targets continues to limit improvements in patient outcomes. ASAP3 has been implicated in tumor progression; however, its role and underlying mechanisms in ESCC remain unclear. Paraffin-embedded ESCC tissues were analyzed by immunohistochemistry to evaluate the expression of ASAP3, NEMO, p65, phosphorylated p65 (p-p65), and MMP9 and their associations with clinicopathological parameters. In vitro, ASAP3 was silenced to assess its effects on ESCC cell proliferation, migration, and invasion and to explore the underlying mechanisms. In vivo, the role of ASAP3 in ESCC growth was validated using a mouse xenograft model, with tumor size and volume measured and changes in ASAP3, NEMO, p65, and p-p65 expression further analyzed. Clinicopathological analysis showed that elevated ASAP3, NEMO, p65, p-p65, and MMP9 expression in ESCC tissues was associated with poor prognosis. In vitro, ASAP3 knockdown inhibited the proliferation, migration, and invasion of KYSE450 cells. In vivo, ASAP3 silencing suppressed xenograft tumor growth and reduced tumor proliferative activity. Mechanistically, ASAP3 downregulation decreased NEMO expression and p65 phosphorylation, thereby suppressing ESCC cell proliferation, migration, and invasion. Collectively, ASAP3 may promote ESCC progression by interacting with NEMO and activating NF-κB signaling. These findings suggest that ASAP3 functions as an oncogenic driver and may serve as a potential biomarker in ESCC.

Understanding bacterial gene expression in natural environments remains challenging because most regulatory networks are inferred from in vitro models that poorly capture in situ environmental conditions. Here, we present NEMO (network transcriptomics of microbes in native environments), a generalizable pangenome-based metatranscriptomic framework for extracting microbial transcriptional programs from metatranscriptomes generated from natural samples. We applied NEMO to Staphylococcus aureus metatranscriptomes from human chronic wounds and cystic fibrosis sputum, revealing infection-associated transcriptional states that diverged markedly from in vitro growth conditions. Notably, small RNAs (sRNAs) were key drivers of the transcriptional divergence between human infection-derived and in vitro transcriptomes. Among these, we identify rsaX20 as a previously uncharacterized, zinc-responsive sRNA that also encodes a small peptide. Meta-analysis of more than 2,000 publicly available S. aureus RNA-seq data sets showed that rsaX20 is induced under zinc limitation and host-associated stress and is co-regulated with known metal-responsive sRNAs. Genetic, transcriptomic, and proteomic analyses demonstrated that rsaX20 is repressed by the zinc regulator Zur and functions as a dual-purpose sRNA/small open reading frame, with the RNA and peptide exerting distinct, sometimes opposing, effects on target proteins. Together, these findings establish NEMO as a broadly applicable framework for interrogating microbial gene regulation directly from native metatranscriptomes and identify rsaX20 as a key regulator of zinc homeostasis during chronic human S. aureus infection. Most knowledge of bacterial gene regulation comes from laboratory cultures, yet pathogens behave very differently inside the human body. We introduce NEMO, a pangenome-based framework that enables direct analysis of microbial gene expression from natural infection samples. Applying NEMO to human Staphylococcus aureus infections revealed transcriptional programs that differ substantially from standard in vitro models and highlighted small regulatory RNAs as major drivers of this divergence. Using this approach, we discovered rsaX20, a previously uncharacterized zinc-responsive regulatory RNA that also encodes a small peptide with distinct biological functions. These findings demonstrate the power of studying microbes in their native environments and uncover a key regulator of zinc homeostasis during chronic human infection.

Procaspase-8 activation at the death-inducing signalling complex (DISC) is largely blocked by short c-FLIP isoforms (c-FLIPS). The interaction between c-FLIPS and procaspase-8 is mediated by their tandem death effector domains (tDED). The canonical model of tDED interaction suggests that c-FLIPS uses H1a/H4a α helices of DED1 to interact with the H2b/H5b α helices of procaspase-8 DED2. Here, based on the NMR chemical shift perturbation of c-FLIPS (F114G) resonances induced by titration with peptides derived from the helical regions of procaspase-8, a model structure of the c-FLIPS:procaspase-8 complex was constructed using HADDOK. The complex revealed that their interactions are mediated by the two binding clefts on DED1 of c-FLIPS and helices H1a/H4a of procaspase-8, the two clefts are formed by the α helices H1a, H5a, and H7a. The binding clefts on DED1 of c-FLIPS were also suggested to bind nuclear factor kappa B (NF-κB) essential modulator (NEMO) and initiate NF-κB pathway activation. To further investigate the interaction between c-FLIPS and NEMO, a c-FLIPS:NEMO complex model was also constructed. Two complex structural models revealed that c-FLIPS may interact with procaspase-8 and NEMO via a common binding surface. Furthermore, we hypothesized that c-FLIPS uses a similar binding surface to interact with both procaspase-8 and NEMO, thereby promoting cell survival by inhibiting caspase-8 activation and inducing NF-κB activity.

The inhibitor of κB kinase (IKK) complex integrates diverse cellular inflammatory responses, and induces transcription factor NF-κB. The molecular mechanism by which IKK becomes catalytically active in response to signaling remains unclear despite structural knowledge of the individual IKK1/α, IKK2/β, and NEMO/IKKγ protein components within its hetero-oligomeric assembly. Cryo-EM of the IKK2/β homodimer bound to an associating NEMO/IKKγ protein fragment, reveals multiple conformers. Mutual exclusivity of dimeric conformers, canonical versus alternate, is reflected in and dependent upon order-to-disorder transition of the canonical 6-helical bundle dimerization interface. Correlation of this unusual structural plasticity of IKK2/β with its biochemical and cellular activities suggests mechanistic possibilities for how association with its partner scaffold protein NEMO/IKKγ and polyubiquitin chains might dictate catalytic activation of IKK through distinct IKK2/β conformers. The inhibitor of κB kinase (IKK) complex is central to inflammatory signaling via the NF-κB family transcription factors. Its activation mechanism has remained unclear. Cryo-EM analysis reveals that the constituent kinase IKK2/β adopts structurally distinct, mutually exclusive dimeric conformations controlled by an ordered-to-disordered transition at its canonical dimerization interface. Stabilization of select IKK2/β conformers by the scaffold protein NEMO in association with poly-ubiquitin chains is regulated through modular architecture and structural plasticity of distinctive kinase-associated domains, present only in kinases of this family. This unique regulatory mechanism governing catalytic activation of IKK2/β provides a conceptual framework for targeting dysregulated NF-κB signaling in human diseases.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATLL) and the neuroinflammatory disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax regulatory protein plays a critical role in HTLV-1 persistence and pathogenesis; however, the underlying mechanisms are poorly understood. Here we show that Tax dynamically regulates mitochondrial reactive oxygen species (ROS) and membrane potential to trigger mitochondrial dysfunction. Tax is recruited to damaged mitochondria through its interaction with the IKK regulatory subunit IKBKG/NEMO and directly engages the ubiquitin-dependent PINK1-PRKN/parkin pathway to induce mitophagy. Tax also recruits autophagy receptors CALCOCO2/NDP52 and SQSTM1/p62 to damaged mitochondria to induce mitophagy. Furthermore, Tax requires PRKN to limit the extent of CGAS-STING1 activation and suppress type I interferon (IFN) induction. HTLV-1-transformed T-cell lines and PBMCs from HAM/TSP patients exhibit hallmarks of chronic mitophagy, and inhibition of PRKN in HTLV-1-transformed cell lines downregulates p19 Gag expression and induces cell death. Collectively, our findings suggest that Tax manipulation of the PINK1-PRKN mitophagy pathway represents a new HTLV-1 immune evasion strategy important for maintaining viral gene expression and cell survival.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATLL: adult T-cell leukemia/lymphoma; BafA1: bafilomycin A1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CGAS: cyclic GMP-AMP synthase; co-IP: co-immunoprecipitation; DOX: doxycycline; GFP: green fluorescent protein; DNM1L/DRP1: dynamin 1 like; HAM/TSP: HTLV-1-associated myelopathy/tropical spastic paraparesis; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; HTLV-1: Human T-cell leukemia virus type 1; IFN: interferon; IkB: inhibitor of nuclear factor kappa B; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; IKK: IkB kinase; IRF3: interferon regulatory factor 3; KO: knockout; LAMP2: lysosome associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mtDNA: mitochondrial DNA; mtROS: mitochondrial reactive oxygen species; NAC: N-acetylcysteine; NBR1: NBR1 autophagy cargo receptor; NFKB: nuclear factor kappa B; OPTN: optineurin; PBMCs: peripheral blood mononuclear cells; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; qRT-PCR: quantitative reverse transcription polymerase chain reaction; RFP: red fluorescence protein; ROS: reactive oxygen species; SAR: selective autophagy receptor; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; Ub: ubiquitin; VCL: vinculin; WT: wild-type.

The identification of microbial strains harbouring bioactive genes is important for the discovery of novel therapeutic agents. The study objective was to identify the genes in Kurthia gibsonii VITAM20 that are associated with anti-inflammatory and antioxidant properties and validate these findings with in vitro functional assays. Genes associated with anti-inflammatory and antioxidant activities were identified by BLAST analysis against curated databases. The anti-inflammatory, antioxidant, and antibacterial potential of Kurthia gibsonii VITAM20 were subsequently evaluated using in vitro assays. BLAST analysis identified several genes associated with anti-inflammatory properties, including eps (95.455%), NEMO (100%), spa (100%), and spaB (90.625%). Multiple genes associated with antioxidant potential were also detected, including CAT (92.308%), dps (95.238%), gshR3 (91.304%), gshR4 (100%), KATA (100%), oxyR (100%), KatA (88.462%), perR (74.041%, bit score 126), sodA (100%), sodM (76.471%), and trxA (77.778%). The genomic findings were supported by in vitro analyses, which demonstrated notable anti-inflammatory activity. Kurthia gibsonii VITAM20 exhibited human red blood cell membrane stabilization rates of 90.78% ± 1.03% and 69.92% under non-heated and heated conditions, respectively, compared with diclofenac (83.01% ± 0.72% and 83.83% ± 1.23%). In the egg albumin denaturation assay, Kuthia gibsonii VITAM20 demonstrated a concentration-dependent increase in inhibition from 80.94% ± 0.57% to 84.47% ± 0.39%, comparable to that of diclofenac. Furthermore, Kurthia gibsonii VITAM20 exhibited 64.27% ± 0.46% antioxidant activity compared with ascorbic acid (91.40% ± 0.46%), with an IC50 value of 286.5 µg/mL. The crude enzyme extract demonstrated antibacterial activity against Listeria monocytogenes and Staphylococcus aureus, producing inhibition zones of 13 mm. The MIC was determined to be 250 µg/mL for both pathogens. At this concentration, the crude enzyme extract inhibited the growth of L. monocytogenes and S. aureus by 81.46% and 77.82%, respectively. The combined genomic and functional analyses confirmed the presence of conserved genes associated with anti-inflammatory and antioxidant activities in Kurthia gibsonii VITAM20. These findings suggest that Kurthia gibsonii VITAM20 represents a promising source of bioactive molecules with potential therapeutic applications.

Small ubiquitin-like modifiers (SUMO) are reversible post-translational modifiers of intracellular proteins. SUMOylation plays an important regulatory role in both innate and adaptive immunity. The natural compound anacardic acid (AA) has been shown to bind and inhibit the SUMO-activating enzyme E1, the first enzyme in the SUMOylation pathway. Here, we examined the consequences of AA treatment on the development of innate and adaptive immune responses in vitro and in vivo. We examined the inhibitory effects of anacardic acid on SUMOylation and de-SUMOylation both in vitro and in vivo. The in vitro studies on the effect of anacardic acid on the development of an immune response were conducted in RAW264.7 cells and naïve or antigen-driven splenocytes. AA inhibited activation of NF-κB by preventing SUMOylation of NEMO, a key requirement for activation of the canonical NF-κB pathway. Stimulation of splenocytes with LPS, a known immunostimulant, resulted in reduced production of inflammatory mediators, including IL-12, IL-23, TNF-α, and iNOS, in the presence of AA. In antigen-primed lymphocytes stimulated with MOGp₃₅-₅₅, AA reduced the induction of IL-17, IFN-γ, TNF-α, IL-6, and GM-CSF. Following transfer of MOGp₃₅-₅₅-primed lymphocytes into naïve mice, AA treatment significantly reduced clinical paralysis and pathological CNS inflammation in experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. The ability of AA to inhibit NF-κB-driven inflammatory response and CNS inflammation after autoreactive T cells are already primed and circulating suggests that AA may have therapeutic potential in established Th1/Th17-mediated inflammatory diseases.

The identification of novel therapeutics for malignant glioblastoma multiforme (GBM) remains critical, given the tumor's highly invasive nature and poor prognosis. This study aimed to investigate the effect of Ginsenoside Rg1 (GS Rg1) on human GBM U87-MG and U251-MG cells. Cell viability and proliferation was detected by MTT and BrdU assay, respectively. The level of NF-κB phosphorylation was quantified. The activity of the cathepsin B, Caspase-3 and Caspase-9 was measured by ELISA assay. The level of transcription of all genes were quantified by QRT-PCR assay. GS Rg1 exerted potent dose-dependent anti-tumor effects on U87MG and U251MG cells. It significantly suppressed cell viability and proliferation, as determined by MTT and BrdU assays, respectively. Mechanistically, GS Rg1 induced apoptosis by activating caspase-3 and caspase-9 and shifting the Bax/Bcl-2 ratio in favor of pro-apoptotic signaling. This was accompanied by the downregulation of key anti-apoptotic (Survivin, Bfl-1) and proliferative (c-Myc, hTERT) genes. A central finding was the potent inhibition of the canonical NF-κB pathway, evidenced by reduced phosphorylation of NF-κB and decreased expression of its upstream kinase, IKK2. Consequently, GS Rg1 suppressed the transcription and activity of major NF-κB-dependent invasion mediators, including MMP-2, MMP-9, cathepsin B, uPA, and MMP-14. Furthermore, it inhibited the expression of the pH regulator CA9. Intriguingly, GS Rg1 upregulated the expression of NEMO, STAT3, and NHE1, suggesting compensatory mechanisms or alternative signaling modulation. Our findings demonstrate that GS Rg1 exerts multi-faceted anti-tumor effects in glioblastoma cells by targeting the NF-κB signaling pathway.

Phytoplankton are key contributors to global primary production and serve as sensitive indicators of changes in marine ecosystems. Changes in phytoplankton phenology, and in particular the onset, termination, and duration of their growing period, have important implications for higher trophic levels that feed on them. Several local studies have provided evidence linking phenological shifts to climate change. However, global analyses have so far relied on time-series observations of surface chlorophyll covering less than two decades, making it difficult to separate trends from variability and establish the coherence of underlying drivers at the global level. Here we use the OC-CCIv5 satellite-derived observations harmonised across satellite sensors and the NEMO-PlankTOM12 global ocean biogeochemical model to revisit recent trends in phytoplankton phenology and identify their global drivers during the 1998-2020 period. Using statistical analysis to separate and control for covariance among underlying drivers, we show that SST warming trends are clearly associated with earlier initiation, later termination, and longer duration of the phytoplankton growing season. In contrast, changes in MLD, SST variability, and chlorophyll are associated with shifts in initiation and termination in the same direction, such that both boundaries move in parallel, partly cancelling their effect on duration. The PlankTOM12 model successfully reproduces most of the observed patterns in phenology and their association with the underlying drivers, providing evidence of the mechanistic relationships, and helping to ascertain the global significance of the drivers. Our findings suggest that trends towards earlier initiation, later termination, and longer duration of phytoplankton growing periods are likely associated with climate change, and may therefore persist in the near future under continued global warming.

Large language models often mishandle psychiatric emergencies, offering harmful or inappropriate advice. This study evaluated the Verily Mental Health Guardrail (VMHG) on two clinician-labeled datasets: the Verily Mental Health Crisis Dataset v1.0, containing 1800 simulated messages and the NVIDIA Aegis AI Content Safety Dataset subsetted to 794 mental health-related messages. Performance was benchmarked against OpenAI Omni Moderation Latest and NVIDIA NeMo Guardrails. The VMHG demonstrated high sensitivity (0.990) and specificity (0.992) on the Verily dataset, with an F1-score of 0.939 and high category-level sensitivity (0.917-0.992) and specificity (&#x2265;0.978). On the NVIDIA dataset, it maintained strong sensitivity (0.982) and accuracy (0.921) with reduced specificity (0.859). Compared with NVIDIA and OpenAI guardrails, the VMHG achieved significantly higher sensitivity (all p&#x2009;<&#x2009;0.001) and comparable specificity (NVIDIA p&#x2009;<&#x2009;0.001, OpenAI p&#x2009;=&#x2009;0.094). Overall, the VMHG demonstrated robust, generalizable, and clinically oriented safety performance that prioritizes sensitivity to minimize missed mental health crises.

Allergic asthma is promoted by type 2 inflammation involving cytokines such as IL-4, IL-5, and IL-13, with group 2 innate lymphoid cells (ILC2s) playing a key pathogenic role. Here, we identify T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) as a negative regulator of ILC2 function. Tim-3 expression is upregulated in activated pulmonary ILC2s, and engagement with Tim-3 agonists inhibits ILC2 activation, proliferation, and type 2 cytokine production via the Nemo Like Kinase (NLK) signaling pathway and suppression of mitochondrial metabolism. In vivo, Tim-3 agonists alleviate airway hyperreactivity (AHR) and inflammation in both IL-33- and Alternaria alternata-induced AHR models, while ILC2-specific Tim-3 deletion exacerbates AHR. These results are confirmed in human ILC2s and humanized mice, supporting the translational relevance. Our findings establish Tim-3 as an inhibitory checkpoint for ILC2s and suggest its potential as a therapeutic target in allergic asthma and other ILC2-mediated diseases.

Radiation and chemotherapy rely on an intact DNA damage response (DDR) to halt cell-cycle progression and eliminate damaged cells, yet many tumors evade these outcomes and develop resistance. Adenoviruses remodel host signaling networks in ways that mirror tumor evolution, providing a powerful system to dissect how DDR pathways are subverted. Here, we identify two scenarios in which the central DDR kinases ATM and ATR are reprogrammed from enforcing CHK1/CHK2-dependent checkpoint arrest to activating a NEMO-NF-&#x3ba;B survival pathway. This rewiring induces transcriptional programs associated with stress tolerance, anti-apoptotic signaling, and chemoresistance, and promotes the accumulation of cells with abnormal DNA content. These findings reveal a previously unrecognized mode of DDR plasticity that generates a pro-survival state reminiscent of early tumor evolution and suggest how ATM- and ATR-dependent pathways can be co-opted to promote therapeutic resistance.

Osteophytes are a characteristic feature of osteoarthritis (OA), and clarifying their molecular regulation may contribute to preventive and therapeutic strategies. Uhrf1 (ubiquitin-like containing PHD and RING finger domains 1), a regulator of DNA methylation maintenance, is indispensable for chondrocyte proliferation and differentiation in the growth plate. Because osteophytes develop via endochondral ossification, we hypothesized that Uhrf1 is involved in osteophyte formation. platelet-derived growth factor receptor &#x3b1; (PDGFR&#x3b1;)-lineage cell-specific Uhrf1-knockout mice showed that Uhrf1 regulates the proliferation and chondrogenic potential of synovial PDGFR-&#x3b1;-lineage cells during osteophyte development, influencing osteophyte formation. Furthermore, experiments using human synovial cells revealed that UHRF1 maintains DNA methylation and identified NLK (nemo-like kinase) as a candidate gene that may be regulated by UHRF1-mediated DNA methylation. These findings suggest that UHRF1 may modulate Wnt signaling and chondrogenic differentiation through the regulation of NLK. Together, these results highlight the role of Uhrf1 in osteophyte formation and provide insight into the mechanisms underlying OA progression, suggesting Uhrf1-mediated pathways as targets for OA.

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) remains incompletely understood. This study aimed to identify potential protein biomarkers for the accurate diagnosis and phenotyping of HFpEF and to construct a machine learning-based diagnostic model incorporating these biomarkers and key clinical features. In a cross-sectional study of 249 cardiac patients, HFpEF-associated plasma proteins were identified using Olink PEA and validated by ELISA. A machine learning nomogram was developed and its diagnostic performance was evaluated. Analysis identified 92 plasma proteins,among which Serine protease 27(PRSS27), P-selectin glycoprotein ligand 1 (PSGL-1), Biregional Cell Adhesion Molecule-related (BOC), NF-&#x3ba;B essential modulator (NEMO), Glyoxalase 1(GLO1))) were specifically expressed in HFpEF group. Enrichment analysis indicated these differential proteins were primarily involved in inflammatory response, immune response, and the Phosphatidylinositol 3-kinase-AKT serine/threonine kinase (PI3K-AKT) signaling pathway. A diagnostic model integrating three proteins with clinical features (LDL-C, ALB) demonstrated excellent performance (AUC: 0.895), showing strong discriminatory power, good calibration, and potential clinical applicability. This study identifies potential protein biomarkers for HFpEF diagnosis, provides new insights into its pathophysiology, and offers a practical diagnostic tool for clinical use.