Small open reading frames (sORFs, encoding 30–100 amino acid peptides) are systematically excluded by conventional gene-prediction pipelines, yet emerging evidence indicates that their products participate in development, signalling and stress adaptation across plants. The recent release of the gap-free telomere-to-telomere assembly of Citrullus lanatus (97103 v3) provides an unprecedented opportunity to interrogate previously inaccessible intergenic territory in watermelon. Here, we coupled six-frame open reading frame scanning (EMBOSS getorf) with a two-stage purification pipeline to construct the first genome-wide catalogue of novel intergenic sORFs in watermelon, recovering 143,288 non-redundant entries distributed near-symmetrically across both strands. Codon adaptation index (CAI) profiling against a reference panel of 362 ribosomal-related genes retained 134,887 nsORFs (94.14%) at CAI ≥0.7, and relative synonymous codon usage analysis identified 24 codons jointly over-represented in the nsORF pool and the ribosomal reference, indicating substantial translational compatibility with the host machinery. Infernal–Rfam scanning revealed that only 1.22% of CAI-passing nsORFs overlap annotated non-coding RNA families, with miRNA-associated hits dominated by developmentally and stress-relevant families (miR156/166/169/171/393/399/408). Subcellular trafficking inference uncovered a heterogeneous architecture (NES, n = 39,216 ≫ NLS, n = 8,855 ≫ SP, n = 429), together with a rare “tri-signal” sextet harbouring SP, NLS and NES simultaneously. Re-analysis of 134 RNA-seq libraries across six biotic and abiotic stress regimes (drought, low-light, salinity, root-knot nematode, CGMMV–boron and powdery mildew) identified 18,625 transcriptionally engaged nsORFs (TPM ≥1) and 30 DESeq2 contrasts revealed stress-specific differential programmes with the most pronounced reprogramming under drought and nematode infection. PredHPI screening against four major watermelon pathogens, together with FFPred4 and MULocDeep analyses, prioritized five high-priority host–pathogen-interacting candidate sequences (wlsORF1–wlsORF5; 53–86 aa) for further investigation. These candidates were predicted to possess transmembrane features and were assigned putative functional annotations spanning transporter, kinase, transcription factor, cytokine-like, and ATP-binding categories, pointing to possible involvement in organellar membrane-associated and electron-transport processes. Collectively, this work delivers the first T2T-anchored nsORF resource for watermelon and provides a tiered evidence framework spanning sequence composition, transcriptional engagement and predicted interactomes, laying the groundwork for micropeptide-targeted breeding strategies in cucurbit crops.

We investigate six singly Cabibbo-suppressed decay channels in D^0→ VP ( V and P stand for the ground state vector and pseudoscalar mesons, respectively), i.e. D^0→ ρ^+π^-, ρ^-π^+, K^*+K^-, K^*-K^+, K^*0K̅^0, and K̅^*0K^0. These decay channels share the similar transition mechanisms involving only the direct emission (DE) and internal conversion (IC) processes. We show that a combined analysis of these channels can explicitly highlight the role played by the IC processes which contribute to the amplitudes at the same order of magnitude as the DE processes.

Background: Haploinsufficiency of A20 (HA20) is an immune dysregulation disorder caused by loss-of-function TNFAIP3 mutations. This international multicenter study aimed to delineate its clinical spectrum, genetic basis, and natural history. Methods: A cross-sectional, retrospective analysis was conducted in HA20 patients with pathogenic or likely pathogenic TNFAIP3 variants. Clinical, laboratory and treatment data were assessed. Clustering analysis was applied to evaluate clinical features and disease phenotypes. Patients were stratified by age (< 16 years vs ≥ 16 years), country of origin (China vs U.S.), and gender. Results: A total of 185 patients from 41 clinics across 7 countries were included (median onset 3.3 years). Common clinical features were mucocutaneous involvement (80.5%), recurrent fever (63.3%), gastrointestinal symptoms (58.6%), cytopenias (56.6%), arthritis/arthralgia (46.7%), and recurrent infections (35.5%). Compared with adults and patients from the U.S. cohort, intestinal ulcers were significantly more frequent in children and patients from the Chinese cohort (P = 0.002 and P < 0.0001, respectively), whereas uveitis was less common in these groups (P = 0.001 and P < 0.0001, respectively). Hierarchical clustering of clinical disease phenotypes based on Pearson distance identified two major clusters, an autoinflammation-predominant phenotype and an autoimmune-predominant phenotype. The autoinflammation-predominant phenotype was more common in children and patients from the Chinese cohort (P = 0.033 and P = 0.003, respectively). A total of 89 pathogenic TNFAIP3 mutations were identified, including 46 novel variants. Large deletions were associated with neurologic disease and developmental delay (P = 0.0054 and P = 0.0245, respectively); however, there were no clear association between disruptions of specific functional A20 domains and onset age or phenotype. Therapeutically, TNF and IL-1 inhibitors were effective in most patients, with thalidomide and JAK inhibitors used in refractory cases; 51.5% had achieved minimal disease activity at the most recent follow-up. Conclusions: HA20 is a common dominantly inherited immune dysregulation disorder with phenotypic heterogeneity and potential age-dependent evolution. This large international cohort highlights diagnostic and therapeutic strategies to advance evaluation and management of HA20. ### Competing Interest Statement WG and GY are employees of Chigene (Beijing) Translational Medical Research Center Co. Ltd and Beijing Quanpu Medical Laboratory Co., Ltd. DMS receives consulting fees from Sobi and grant support from Sobi and Eli Lilly. The others declare no competing interest. ### Funding Statement T.H. received grant 82302056 from the National Natural Science Foundation of China. Q.Z. received grants 82225022, 32141004 and 32321002 from the National Natural Science Foundation of China and 2024YFC2511002 from the National Key Research and Development Program of China. J.Y. received grant SZSM202411012 supported by Sanming Project of Medicine in Shenzhen. D.M.S. received grants by Jeffrey Modell Foundation, Eli Lilly, Sobi, Samuel and Emma Winters Foundation. J.W received grants 82394420, 82394423, 82402121 from the National Natural Science Foundation of China and grant 2023M733104 from China Postdoctoral Science Foundation. S.W. received grant 82402118 from the National Natural Science Foundation of China. L.G. received the grant LHDMY23H100005 from Joint Funds of the Zhejiang Provincial Natural Science Foundation of China. X.Y. received the grants 82394420, 82394424 and 82471844 from the National Natural Science Foundation of China, the Hundred-Talent Program of Zhejiang University, grant 2021R01012 from Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang and Key Technology Breakthrough Program of Ningbo Sci-Tech Innovation YONGJIANG 2035 (Grant No. 2024Z221). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Shenzhen Children's Hospital and University of Pittsburgh gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study and not in the manuscript are available upon reasonable request to the authors

This study investigated dynamic changes in key volatile aroma compounds and epiphytic microbial communities during cigar tobacco fermentation. Air-cured “Dexue 1” tobacco leaves were fermented and analyzed using high-throughput sequencing and gas chromatography–mass spectrometry to track microbial community structure and volatile profiles. The contents of total sugar, nicotine, and protein decreased, while the activities of α-amylase, neutral protease, and cellulase increased. While bacterial richness and diversity increased, but fungal diversity decreased. The abundances of Sphingomonas and Massilia initially increased and then decreased, whereas Aspergillus and Alternaria progressively dominated, indicating functional microbial succession. Key metabolic pathways included sesquiterpenoid/triterpenoid biosynthesis, tryptophan metabolism, fatty acid degradation, and isoquinoline alkaloid biosynthesis. The key aroma compounds were phenethyl alcohol, β-ionone, geranyl acetone, copaene, and phenylacetaldehyde. Notably, β-ionone and geranyl acetone correlated positively with Aspergillus, while phenylacetaldehyde and phenethyl alcohol correlated positively with Pseudomonas. The fermentation of cigar tobacco leaves is characterized by the degradation of macromolecules and the accumulation of key volatile compounds, in which microorganisms play a crucial role in metabolic conversion.

Diet is a modifiable risk factor for cognitive decline, yet evidence regarding the association between plant-based diets and cognitive aging remains limited. We examined the association between plant-based diets and cognitive decline among older adults from China. A total of 6,721 participants (mean age: 81.41 ± 10.40 years) from the Chinese Longitudinal Healthy Longevity Survey (CLHLS) were included. Dietary intake was assessed using a simplified food frequency questionnaire to derive the plant-based diet index (PDI), healthy PDI (hPDI), and unhealthy PDI (uPDI). Global cognitive function and six specific domains were assessed using the Mini-Mental State Examination (MMSE). Z-scores were calculated using baseline mean and standard deviation. Linear mixed-effects models and Cox proportional hazards models were used to evaluate the associations between plant-based diet indices and cognitive decline and impairment. Over 6.07 years of follow-up, 1,580 participants developed cognitive impairment. Participants in the highest tertiles of PDI and hPDI exhibited 32.04

Objective This study aims to characterize pathogenic somatic mutations in patients with autoinflammatory or autoimmune diseases lacking disease-causing germline mutations, explore their contribution to disease pathogenesis and progression, and evaluate their implications for diagnosis and targeted therapy.Methods We performed a systematic analysis of somatic mutations in a selected panel of 185 immune-related genes in 2,912 patients with autoinflammatory or autoimmune diseases, recruited from 41 medical centers across China, who were previously negative for germline mutations based on whole-exome sequencing.Results We identified both previously reported and novel somatic mutations in genes such as UBA1, KRAS, and NLRP3. Pathogenic somatic mutations in TNFAIP3 were discovered first in patients with autoinflammatory diseases. The pathogenic somatic mutation detection rate was 1.35% in adults and 0.97% in children, emphasizing the importance of genetic diagnosis and novel gene discovery for somatic mutations. In addition, somatic mutations in Ras-related genes were identified in seven patients, and 39 clonal hematopoiesis-associated mutations were identified in 36 adult patients. Moreover, myeloid cells harboring somatic mutations expanded during disease flare and reduced during remission. Disregarding the dynamic elevation of the variant allele fraction during disease progression led to therapeutic failure.Conclusion This study delineated the genetic landscape of pathogenic somatic mutations underlying autoinflammatory and autoimmune diseases, offering valuable insights for genetic diagnosis and targeted therapies.

Background Hippocampal synaptic deficits constitute a core pathology of depression. Mitophagy, an essential mitochondrial quality-control process, is crucial for synaptic homeostasis. The classical antidepressant formula Kai-Xin-San (KXS) has established clinical efficacy, yet whether it rescues synaptic deficits by modulating mitophagy remains unknown. Methods We employed a chronic unpredictable mild stress (CUMS) mouse model. The effects of KXS were comprehensively evaluated through behavioral tests combined with morphological, ultrastructural, and molecular analyses of hippocampal synapses and mitochondria, complemented by mitophagy inhibition. Results KXS administration dose-dependently reversed CUMS-induced depressive-like behaviors and restored hippocampal synaptic deficits, including neuronal architecture, spine density, synapse number, and key synaptic protein levels. Mechanistically, KXS attenuated mitochondrial damage and coordinately activated mitophagy via multiple pathways, involving both the PTEN-induced putative kinase 1 (PINK1)/Parkin axis and receptor-mediated routes, thereby enhancing autophagic flux. Crucially, the mitophagy inhibitor Cyclosporin A (CsA) abolished all KXS-induced benefits on mitophagy, synaptic repair, and behavior. Conclusion This study demonstrates that KXS restores hippocampal synaptic deficits and exerts antidepressant-like effects through multi-pathway activation of mitophagy, which is essential for clearing damaged mitochondria. Our findings not only elucidate a novel mitophagy-dependent neuroprotective mechanism for KXS but also provide a mechanistic rationale for its clinical application and advance the therapeutic strategy of targeting the mitochondria-synapse axis in depression.

Abstract Rapid and accurate pathogen identification is crucial for the clinical management of infectious diseases, particularly sepsis and severe respiratory infections, yet standard clinical workflows remain slow and resource-intensive. Here, we developed an automated, high-throughput imaging platform built on standard, clinically accessible bright-field microscopy, and generated a large dataset comprising 24.9 million label-free bacterial cells across six focal pathogens. Leveraging this resource, we trained a neural network (ESKAPe-ResNet) to identify ESKAPe species at the single-bacterium level. The model achieved >92% accuracy in species-level classification and >82% accuracy in quantifying ESKAPe abundance in mock mixtures, with high specificity against non-ESKAPe bacteria. In clinical validation using sputum, bronchoalveolar lavage fluid and blood samples from patients with respiratory infections and sepsis, the approach correctly identified the dominant ESKAPe pathogen in >78% of samples after minimum broth culture enrichment. The imaging-to-identification pipeline was completed in under 10 minutes, and coupled with brief cultivation, the median time to accurate identification was reduced to 5–6 hours, compared with days for conventional blood culture-based workflows. This work establishes the proof-of-principle for label-free, hardware-minimal rapid pathogen identification, providing a clinically deployable workflow to expedite diagnosis and reduce mortality in severe bacterial infections.

Interleukin-1 receptor-associated kinase 2 (IRAK2) is essential for the Myddosome complex formation downstream of Toll-like receptors. We identify twelve patients with a homozygous loss-of-function copy number variant in IRAK2, designated IRAK2-∆ex2. Most patients present with recurrent infections, autoantibody production, and gastrointestinal ulceration. Two patients were clinically diagnosed with primary immunodeficiency, while the majority fulfill diagnostic criteria for autoimmune or autoinflammatory diseases. The IRAK2-∆ex2 protein fails to interact with IRAK4, leading to impaired activation of nuclear factor kappa B signaling via the Myddosome complex. An elevated type I interferon signature is observed in the patients, which is confirmed in bone marrow-derived macrophages from knock-in mice and knockout cell lines. Mechanistically, our data are consistent with engagement of a TRIF-dependent interferon pathway. Baricitinib attenuates the elevated interferon signature in patient-derived cells ex vivo and cell lines. Here, we show IRAK2 deficiency as a monogenic immune dysregulation disorder.

IntroductionGlobally, respiratory infections remain a leading cause of mortality, with treatment efficacy increasingly challenged by antimicrobial resistance. This study aimed to investigate the role of serum metabolites in the prognosis of severe human pneumonia.MethodsUntargeted and targeted serum metabolomics were performed on intensive care unit (ICU) patients. Experimental validation was conducted in a murine bacterial infection model and cellular models. RNA sequencing was used for mechanistic exploration to identify the signaling pathways regulated by the key metabolite.ResultsValeric acid, a short-chain fatty acid, was significantly elevated in survivors compared with non-survivors of severe pneumonia. In the murine Klebsiella pneumoniae model, valeric acid treatment alleviated infection severity, reduced body weight loss, lung inflammation, and bacterial load. Mechanistically, RNA sequencing revealed that valeric acid suppresses IL-17-associated inflammation and upregulates pathways related to mucociliary clearance. We further delineated the underlying mechanism, finding that valeric acid acts as a histone deacetylase (HDAC) inhibitor, specifically targeting HDAC3. This inhibition activates the canonical Wnt/β-catenin signaling pathway, leading to the upregulation of the master transcriptional regulator Foxj1 and subsequent promotion of cilia assembly and function in airway epithelia.DiscussionThe findings establish a protective role for the gut microbiome-derived valeric acid in respiratory infections via the novel HDAC-Wnt-FOXJ1 axis, revealing its potential as a therapeutic agent to improve clinical outcomes.

Pseudomonas aeruginosa is a bacterium with high antimicrobial resistance to several drugs including carbapenems. Recently, resistance to cefiderocol (FDC), a new siderophore cephalosporin, has been reported despite its rare use in clinical practice. Therefore, this study aimed to investigate the dynamics of different resistance mechanisms in P. aeruginosa that evolve under FDC pressure, as well as the interactions between these mechanisms in evolutionary trajectories to guide clinical medication. P. aeruginosa ATCC 27853 was continuously induced with subinhibitory concentrations of cefiderocol in vitro to generate strains with different resistance levels. Phenotypic adaptability and molecular regulatory networks during the evolution of resistance were systematically elucidated using growth curves, pairwise competition assays, transcriptomics, RT-qPCR and efflux inhibition assays. The minimum inhibitory concentration (MIC) of the induced FDC-resistant strain increased from 0.125 to 64 μg/mL. Simultaneously, the growth rate and peak declined below those of the parental strain. The pairwise competition assay showed that the relative fitness of resistant strains versus parental strains was < 1 in LB and ID-CAMHB broths. Transcriptome analysis revealed that the MIC of the induced FDC-resistant strain was related to the dose of efflux pumps, together with a layer-by-layer regulation of gene expression, to adapt to environmental stress. In the initial resistant strain, efflux pumps were slightly upregulated, and energy metabolism was downregulated. Conversely, in the strain with a MIC of 64 μg/mL, efflux pumps were significantly up-regulated and drove bacterial reprogramming to induce resistance, particularly the RND efflux pump component oprM, along with multiple ABC transporters. RT-qPCR validated the significant upregulation of oprM, opuC, and opuBD (p < 0.05), confirming their central role. Furthermore, in the presence of the efflux pump inhibitor phenyl-arginine β-naphthylamide (PAβN), the resistant strains exhibited significantly reduced MICs for FDC. In conclusion, these data indicate that multiple mechanisms of action are involved in the antibacterial activity of P. aeruginosa against FDC. Notably, the evolution of resistance was associated with the dose-dependent upregulation of core efflux pumps, complemented by stage-specific global physiological remodeling; however, it also came at a fitness cost.

Cyclic nucleotide-gated channel (CNGC) genes play key regulatory roles in plant immunity and abiotic stress responses. In this study, we conducted a genome-wide identification and analysis of the CNGC gene family in Suaeda glauca. A total of 44 SgCNGC genes were identified. Through phylogenetic analysis, gene structure analysis, chromosome distribution, conserved motif analysis, collinearity analysis, cis-acting element analysis, subcellular localization, and gene overexpression analysis, we systematically characterized the evolutionary relationships, structural features, and potential functions of this gene family. The results indicate that the SgCNGC gene family is evolutionarily highly conserved but exhibits functional divergence in structure and expression. Furthermore, functional assays revealed that overexpression of SgCNGC13 in Arabidopsis thaliana led to increased salt sensitivity, indicating a negative regulatory role for this gene under salt stress. These findings provide a foundation for understanding the role of the CNGC gene family in the growth, development, and stress response of S. glauca and contribute to the remediation of saline-alkali land.

Circulating brain-derived extracellular vesicles (BDEVs) have emerged as promising biomarkers for neurodegenerative diseases, including Alzheimer’s disease (AD). However, it remains unclear to what extent extracellular vesicles (EVs) proteomes reflect the molecular states and disease-associated alterations of their parent brain cell types. Here, using a multi-line human induced pluripotent stem cell (hiPSC) platform derived from three AD and three cognitively normal (CN) donors, we generated neurons, astrocytes, microglia, and oligodendrocytes, and performed paired proteomic profiling of each cell type and its secreted EVs. We systematically compared protein profiles to evaluate cell-EV similarity, disease-associated features, and concordance with proteomic datasets from human AD brain tissue. Across all four lineages, EV proteomes showed extensive overlap with parent cells (>97

Nephroblastoma, also known as Wilms’ tumor (WT), is the most common malignant renal tumor in children under 5 years of age. Despite a generally favorable prognosis, approximately 15% of cases experience recurrence. Currently, treatment options for refractory and recurrent WT remain limited, and targeted therapeutic strategies are still under investigation. NCBP1 is a core component of the cap-binding complex and plays a role in mRNA processing, transport, and translational regulation; however, its involvement in the initiation and progression of WT has not yet been elucidated. This study utilized bioinformatics analysis to identify that NCBP1 is highly expressed in WT tissues and is associated with a poor prognosis. Molecular experiments further confirmed that NCBP1 is significantly overexpressed in the WT cell lines 17.94 and HFWT. Functional assays demonstrated that silencing NCBP1 suppresses the proliferation, migration, invasion, and tumorigenic potential of both 17.94 and HFWT cells. Through screening for NCBP1-interacting proteins, KPNA2 was identified, and a positive correlation was observed between the expression levels of KPNA2 and NCBP1 in WT tissues. Through RNA immunoprecipitation experiments, we further validated the interaction between NCBP1 and KPNA2 in WT cells. Silencing of NCBP1 resulted in reduced stability and expression levels of KPNA2 mRNA in 17.94 and HFWT cells. Furthermore, KPNA2 overexpression not only enhanced the proliferation, migration, and invasion capabilities of 17.94 and HFWT cells, but also partially counteracted the inhibitory effects of NCBP1 silencing on these malignant cellular behaviors. In conclusion, the findings of this study elucidate the involvement of NCBP1 in the malignant progression of WT. NCBP1 enhances the stability of KPNA2 mRNA, thereby upregulating KPNA2 expression and subsequently promoting the malignant progression of WT cells.

We investigate the lineshape of the e^+e^-→ J/ψ π^+π^- cross section in the vicinity of the D^*D̅+c.c. threshold, where the “so-called" G(3900) is observed in the e^+e^-→ DD̅ channel. To take into account the possible D^*D̅+c.c. open channel effects or possible contributions from G(3900), we include the intermediate meson loop transitions in e^+e^-→ J/ψ π^+π^-. As a consequence, a triangle singularity (TS) is fulfilled which can produce nontrivial structures in the j/ψπ invariant mass spectrum. Moreover, the TS transition also allows access to exotic quantum number of (I,J^P(C))=(1,1^-(-)) in the J/ψπ invariant spectrum. We present predictions for the J/ψ π invariant-mass spectrum and our results clarify the different manifestations of the kinematic effects and genuine resonances. In particular, we show that resonance structures arising from the P-wave DD̅ scatterings or hidden charm tetraquark state with (I,J^P(C))=(1,1^-(-)) can be identified by the J/ψπ invariant mass spectrum. It can provide a theoretical guidance for future experimental search for these exotic candidates.

In this study, we investigate the isospin-violating decays ofB(c)(1P)(+)-> B-c(*)+pi(0) , which may provide additional information for the determination of the properties of B-c(1P)(+) , the first orbital excitation states of theB(c) meson. By assuming a dual relation between the U(1) anomaly soft-gluon coupling for B-c(1P)(+) -> B-c(*)+pi(0)and the intermediate meson loop transitions, we can quantify the isospin-violating decay effects for these four P-wave states. We observe that the partial decay width B*(C0 )-> B*(C0)+of is approximately three orders of magnitude larger than that for B*(C2) (+)-> B-c(+)pi(0). This indicates thatB*(C0) can be established in theB(c)(+)pi(0) decay channel as a single state. Meanwhile, the two axial-vector states B-C1(+)/B'(C1)-> B-c(+)pi(0)can be possibly identified in with comparable strengths. Although these isospin-violating decays are observed to be small, the theoretical predictions should be useful for guiding future experimental efforts.