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Proline is a well-known proteinogenic amino acid, which features a unique structure among the canonical amino acids: its side chain forms a ring with the backbone nitrogen atom. There are many other cyclic secondary amino acids of different ring size, which are non-proteinogenic. Many of their derivatives are natural products and have high potential in pharmacology, biotechnology, and synthetic biology. Mathematical and computational approaches in biochemistry complement the analysis of the physicochemical and functional properties of compounds, and provide an overview of both known and hitherto unknown, yet hypothetically possible, molecules. Here, we present a mathematical enumeration procedure for all possible cyclic secondary α-amino and imino acids, depending on the number of atoms in the ring, in terms of saturated and unsaturated congeners. A recursion formula is given, which results in a modified Lucas sequence. Moreover, explicit formulas for determining the number of such substances are derived. This enumeration is helpful for the construction of virtual compound databases and computer-assisted design of chemical syntheses. Additionally, we provide an overview of detected and characterised structures in this class, as well as some of their derivatives, along with exemplary applications. Moreover, we discuss the conformational and functional properties of compounds with different ring sizes.

Jumbo bacteriophages are bacterial viruses with double-stranded DNA genomes exceeding 200 kb. These viral giants feature exceptionally large virions, expansive genetic repertoires, and in some cases, remarkable eukaryotic-like traits. Jumbo phages challenge long-standing notions of phage simplicity, redefining the boundaries of what a phage can be. In this Review, we examine the biology of jumbo bacteriophages, highlighting their diversity, evolutionary origins, distinctive morphologies and lifecycles, complex interactions with bacterial hosts, and their potential for biotechnology and therapy, with a focus on, but not limited to, the Chimalliviridae phages.

The protein design field is rapidly advancing, with frequent emergence of new models and pipelines for designing de novo proteins with tailored properties and functions not found in nature. However, the current tool landscape is fragmented, tools are hard to install and deploy, and require significant computational expertise to integrate into end-to-end, scalable pipelines. A particular challenge is managing many sequences, structures, and metrics for downstream testing and retrospective analysis of input parameters. To address this need, we introduce Ovo, an open-source de novo protein design ecosystem that consolidates models, workflows, data management, and interactive visualization into a scalable, infrastructure-agnostic platform. Ovo features Nextflow-based workflow orchestration, a storage layer, and both command-line and graphical interfaces that democratize scaffold design, binder design and diversification, and validation workflows. Ovo's novel ProteinQC module computes comprehensive sequence and structure descriptors, contextualizing designs against reference sets. Ovo plugins let the community add new workflows and user interfaces to accelerate adoption of emerging methods and facilitate community-driven benchmarking. Ovo lowers engineering barriers and demystifies the design process, allowing experts and non-technical users to design proteins at scale. With community-driven development, Ovo can accelerate de novo protein design and advance discovery in therapeutics and biotechnology.

This article reappraises brain function by reconsidering the role of cerebral fluid dynamics in cognition. Tracing a lineage from early modern thinkers like Descartes-who invoked hydraulic metaphors and 'animal spirits'-to J.C. Bose's pioneering studies on electro-mechanical plant physiology, and culminating in contemporary findings on cerebrospinal fluid (CSF), extracellular space (ECS), and neurovascular coupling, we reveal a forgotten yet vital computational substrate. We argue that alongside the well-studied digital operations of neural circuits exists a slower, analog, evolutionarily older layer of electrofluidic computation. This system-comprised of ionic diffusion, convective CSF flow, and vascular modulation-not only supports but dynamically shapes neural activity. The brain emerges as an electromechanical ecology of electric pulses and fluid flows, where the mind is both a pattern of neural activity and a choreography of fluid flows.

We developed a scalable pipeline for extracellular miRNA (ex-miRNA) profiling that integrates automated exRNA extraction, small RNA sequencing, and bioinformatic analysis including data processing and normalization. Automated extraction protocols, including doubling input volume or lyophilization to increase RNA yield, were benchmarked against leading manual methods, with donor pregnancy status serving as the primary biological variable. Small RNA sequencing was performed across conditions, enabling systematic evaluation of data quality and comparison of normalization strategies. Across methods and biofluids, DESeq2 most effectively reduced technical variability while preserving biological signal. Comparing specimen types, plasma exhibited the highest reproducibility and retention of biological signal, followed by serum, while urine exhibited greater variability and less differentially expressed miRNAs. Pregnancy-associated ex-miRNA signatures, including C19MC miRNAs, were consistently detected in both plasma and serum. Together, this study establishes a robust framework for scalable exRNA extraction and profiling, supporting standardized assay development for biomarker discovery and clinical applications.

The sensory properties of alternative proteins are key to consumer acceptance, yet the processes shaping their odor remain unclear. Solid-state fermentation (SSF), a promising method for producing alternative proteins from agro-industrial by-products such as bagasse, brans, pomaces, husks and oil cakes, is used in this study to model odor profile development of surplus bread crusts, supplemented with perennial ryegrass protein with Rhizopus oligosporus, Aspergillus oryzae, and Neurospora intermedia at 32 °C for up to 72 h. Volatile organic compounds (VOCs) were analyzed by solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS), identifying over 150 compounds. A mechanistic model based on the Weber-Fechner law predicted odor profiles from VOC concentrations, odor descriptors and thresholds, and was validated against a quantitative descriptive analysis (QDA) performed by a trained panel using multiple factor analysis (MFA). The model reflected changes in the overall odor intensity and sweet, baked, and grass-like notes, though correlations were weaker for fungal-derived descriptors (fruity, earthy, herbal). These findings elucidate how fungal SSF alters odor profiles in alternative proteins and establish a framework for mechanistic odor prediction in food systems.

Inherited genetic variation may impact patient response and risk of toxicity following immune checkpoint inhibitor (ICI) therapy. We conducted an agnostic genome-wide analysis study for inherited genetic variants that may predict thyroiditis in patients with melanoma treated with ipilimumab and investigated Polygenic Risk Scores (PRSs) previously reported to be associated with thyroid disease. Germline DNA from 744 participants in a phase 3 adjuvant trial was analyzed following genome-wide genotyping. An agnostic genome-wide cohort-level analysis identified top associated single nucleotide polymorphisms (SNPs), and a custom 10-SNP PRS was significantly associated with thyroiditis risk and severity. Among the SNPs identified, five mapped to intronic, four intergenic and one exonic regions. The latter was the lead SNP and mapped to CNOT6L, located near CXCL13. Among the other variants, most were located within regions with potential relevance to immune regulation and autoimmunity. Separately, multiple thyroid disease-related PRSs derived from Polygenic Score Catalog weights were tested, and several were significantly associated with thyroiditis, but the custom PRS had stronger discrimination for thyroiditis risk (area under the curve 0.82). These findings indicate that inherited genetic background contributes to thyroid immune toxicity risk following ICI and support PRS-based approaches for risk-adapted monitoring during immunotherapy.

Retinoic acid (RA), a bioactive metabolite of vitamin A, plays roles in early embryogenesis and hematopoietic development. However, its precise function in directing the hematopoietic lineage outcomes of human pluripotent stem cells (hPSCs) remains unclear. Here, we uncovered a distinct, stage-specific role for RA as a lineage-specifying modulator during late-stage hematopoietic differentiation, rather than as a promoter of hematopoietic progenitor generation. Using a stepwise hPSC differentiation system, we demonstrated that RA exerted minimal or inhibitory effects when applied during early mesoderm or hemogenic endothelial stages. In contrast, RA treatment during days 13-15 significantly enhanced progenitor maturation, proliferation, and functional output. Notably, RA acted cooperatively with external cytokines to modulate lineage fate. In the presence of erythropoietin (EPO), RA strongly promoted erythroid differentiation by activating EPOR signaling and upregulating erythroid transcriptional programs, including GATA1, KLF1, and globin gene expression. Conversely, under GM-CSF/M-CSF stimulation, RA biased progenitor differentiation toward macrophages, consistent with its role as an amplifier of the prevailing cytokine-directed lineage fate rather than an independent suppressor of erythropoiesis. These effects were highly dose- and context-dependent, with low-dose RA optimally enhancing lineage bias without cytotoxicity. Importantly, RA modulated the transcriptional and proliferative dynamics of committed progenitors. Taken together, our findings reveal a previously unrecognized role of RA as a versatile and tunable modulator of hematopoietic lineage fate that offers a novel strategy for in vitro blood cell engineering. This study advances approaches for lineage-specific blood production relevant to disease modeling, drug screening, and regenerative medicine.

The emergence and spread of Plasmodium falciparum resistance to artemisinin and its partner drugs pose a major challenge to malaria elimination efforts. Continuous monitoring of resistance-associated molecular markers provides valuable insights into the emergence of resistant genotypes and their geographical distribution. In this study, we investigated mutations linked to antimalarial drug resistance in the pfcrt, pfmdr1, and pfmrp1 genes among 100 P. falciparum isolates collected from southeastern Iran during 2022-2023 and compared the findings with data from the same region a decade earlier. Point mutations were genotyped using PCR-RFLP. Molecular analysis demonstrated a high prevalence (95%) of the pfcrt K76T mutation. In pfmdr1, no mutations were observed at codons 1034, 1042, or 1246; however, the N86Y and Y184F mutations were detected in 3.75% and 51.25% of isolates, respectively. Comparative analysis between the previous (2007-2010) and current (2022-2023) studies revealed a significant increase in the wild-type allele for N86Y and the mutant allele for Y184F. In pfmrp1, mutations at H191Y, S437A, I876V, and F1390I were identified in 68.75%, 85%, 78.75%, and 46.25% of isolates, respectively, while no mutation was observed at K1466R. The predominant pfmdr1 haplotype shifted from wild-type (50%) in the previous study to the single-mutation haplotype N86F184S1034N1042D1246 (50%) in the current study. Combined haplotype analysis of all three genes revealed a decrease in one major haplotype T76N86Y184S1043N1042D1246Y191A437V876F1390K1466 (from 23.5 to 3.75%) and an increase in haplotype T76N86Y184S1043N1042D1246Y191A437V876I1390K1466 (from 7 to 23.75%) over the same period. The persistence of the pfcrt K76T mutant allele despite the withdrawal of Chloroquine from the treatment policy in Iran may be attributable to the co-endemicity of P. vivax in the study area. The increasing prevalence of the N86F184S1034N1042D1246 pfmdr1 haplotype is of concern, given its reported association with reduced susceptibility to lumefantrine. Although mutant pfmrp1 alleles were detected at relatively high frequencies, current evidence does not suggest an immediate threat to the efficacy of artemether-lumefantrine in the studied population.

Activation of endothelial Tie2 signaling has emerged as a potential therapeutic strategy for ameliorating vascular abnormalities and hyperpermeability in ocular diseases. Early therapeutic strategies have focused on inhibiting angiopoietin-2 (Ang2), an antagonistic ligand of Tie2, thereby indirectly promoting Tie2 activation. However, accumulating evidence indicates that indirect Tie2 activation via Ang2 blockade is insufficient for enhanced therapeutic efficacy, underscoring the need for Tie2 agonists. In addition, since it is still necessary to inhibit neovascularization induced by vascular endothelial growth factor (VEGF), appropriate therapeutic efficacy could be achieved by direct Tie2 agonism and VEGF neutralization. An affinity-matured Tie2-activating antibody, MT-101, was identified by phage display panning using a complementarity-determining region-targeted mutagenesis library. Tie2xVEGF bispecific antibodies were generated with MT-101 fused to five different anti-VEGF modules, and their functionality in Tie2 and VEGF signaling was compared using endothelial cells. The therapeutic efficacy of the bispecific antibody fusion MT-103, comprising MT-101 and VEGFR domains, was evaluated in mouse oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (LI-CNV) models compared with anti-VEGF agent Aflibercept or Ang2xVEGF bispecific antibody. MT-101 activated the Tie2 signaling pathway, including AKT-eNOS and ERK cascades, and exhibited efficacy comparable to that of Aflibercept in OIR and LI-CNV models. By directly comparing five different Tie2xVEGF bispecifics, we selected the most potent construct, MT-103, generated by fusing VEGFR1/2 domains to MT-101. MT-103 demonstrated approximately four- and five-fold greater potency than the Ang2xVEGF bispecific antibody in inhibiting VEGF signaling and reducing permeability, respectively, in retinal endothelial cells. MT-103 further demonstrated improved efficacy, reducing neovascularization by 14% compared with Aflibercept in the LI-CNV model and suppressing vascular leakage by 20% compared with Ang2xVEGF bispecific antibody in the OIR model. Moreover, MT-103 elicited robust Tie2 activation and vessel stabilization by enhancing pericyte coverage relative to Ang2xVEGF bispecific antibody. These findings demonstrate that a therapeutic strategy combining direct Tie2 activation and VEGF blockade may provide improved therapeutic potential compared to neutralizing VEGF alone or Ang2 and VEGF, representing a promising therapeutic strategy that warrants further validation for the treatment of various ocular diseases.

Nanoemulgels are hydrocolloid-based structured systems that integrate nanoemulsions into polymeric gel networks, thereby enhancing dispersion stability, interfacial integrity, and functional performance. Hemp seed oil (HSO), rich in polyunsaturated fatty acids and γ-linolenic acid, represents a valuable lipid phase for the development of functional oil-based delivery systems. In this study, an HSO nanoemulsion (HSO-NE) and a hydrocolloid-based nanoemulgel (HSO-NE gel) were developed using a polymer matrix, and their physicochemical and structural properties were systematically investigated. Incorporation of nanoemulsion into the hydrocolloid gel network resulted in a modest increase in droplet size and polydispersity index, while inducing a pronounced increase in negative zeta potential (from -39.60 to -67.37 mV), indicating enhanced electrostatic stabilization and resistance to droplet aggregation. FTIR and TEM analyses confirmed that the hydrocolloid network physically entrapped the nanoemulsion droplets without disrupting interfacial integrity or lipid structure. As a functional validation model, the hydrocolloid-based HSO-NE gel was evaluated in an imiquimod-induced psoriasis-like mouse model. Topical application of the nanoemulgel significantly attenuated inflammatory responses, accompanied by downregulation of cytokine-cytokine receptor interaction and NF-κB signaling pathways. Collectively, this study demonstrates that hydrocolloid-based nanoemulgels represent an effective strategy for stabilizing oil-in-water nanoemulsions while preserving the functional properties of hemp seed oil, highlighting their potential as structured delivery systems for bioactive lipids.

Reaggregated early gastrula cells ("gastruloids") of the sea anemone Nematostella vectensis are able to regenerate into whole polyps within a few days. However, the cellular and molecular mechanisms underlying restoration of body axis and germ layers remains largely unknown. Here, we show that mesodermal cells sort to the periphery of the gastruloid, where they form cell clusters with hitchhiking endodermal cells. One of these clusters immigrates at one pole, forming the future pharynx and inner layer. This morphogenetic behavior that enables the symmetry break of the organizer tissue, requires a Wnt-Notch signaling feedback loop. This highlights a hitherto unknown role of Notch signaling in self-organizing gastruloids. Conservation of Notch-mediated boundary formation between germ layers mirrors similar mechanisms in bilaterians. This demonstrates how adoption of ancestral regulatory networks enables a morphospace converging to similar body plans, thus contributing to evolutionary robustness.

Obstructive sleep apnea (OSA) is common yet frequently underdiagnosed, partly because overnight polysomnography (PSG) is logistically burdensome and access to specialized testing is limited. We aimed to develop machine-learning models for OSA risk screening using multimodal digital phenotyping from consumer-grade wearable devices, smartphone-based assessments, and clinical scales. We enrolled 338 participants and collected data over four weeks. After preprocessing, 107 features were derived from wearable-derived physiological and activity measures, smartphone-based records, and questionnaire-based clinical risk profiles, and used to classify high- versus low-risk OSA groups defined by the Berlin Questionnaire. Across multiple model configurations, predictive performance was high, with the best-performing model achieving an AUC of up to 0.94 and an F1 score of 0.80 in the internal validation set. Consistently influential predictors included body mass index, Insomnia Severity Index score, Smartphone Overuse Screening Questionnaire score, resting heart rate, and heart rate recovery. These findings suggest that multimodal digital phenotyping from accessible consumer technologies may support scalable pre-screening for OSA risk in real-world settings. Further validation against PSG-confirmed OSA outcomes is needed.Trial Registration: Clinical Research Information Service (CRIS) KCT0009175 (Registration data: Feb-15-2024) (https://cris.nih.go.kr/cris/search/detailSearch.do?search_lang=E&focus=reset_12&search_page=M&pageSize=10&page=undefined&seq=26133&status=5&seq_group=26133).

Plasmodium falciparum malaria remains a major cause of morbidity and mortality in endemic regions, with outcomes influenced by host genetic factors. The ABO blood group system has been linked to clinical severity, though findings differ regarding blood groups and severe malaria susceptibility. This analysis aims to synthesise evidence on ABO blood group's role in severe P. falciparum malaria (SM) through meta-analysis and trial sequential analysis. A search was conducted across PubMed, Scopus, Embase, Web of Science and Science Direct databases on August 31, 2025, to identify case-control studies reporting ABO blood groups in SM and uncomplicated malaria (UM). All Statistical analysis was performed with CMAv4. Twenty-three studies comprising 3553 SM and 6112 UM cases were analysed. Results showed higher SM risk in blood groups A (OR:1.98; p = 0.00), B (OR:1.87; p = 0.00), AB (OR:1.91; p = 0.00) and non-O (OR:1.88; p = 0.00) versus group O. These associations were confirmed in African and South Asian populations, while Melanesians showed different patterns. Non-O groups showed increased risk in adults and children. Group A was linked to severe malarial anaemia (OR:1.75, p = 0.004), while group B showed protective effects (OR:0.75, p = 0.00). SM patients had lower haemoglobin (MD = -2.23; p = 0.00), with parasite density showing no correlation with severity. TSA confirmed evidence linking ABO groups with SM and haemoglobin levels. This meta-analysis and TSA confirm non-O blood groups as a susceptible factor for SM across ethnicities and ages. While ABO blood group is an inexpensive and widely available genetic marker, its clinical utility should be viewed as complementary rather than deterministic. However, at present, evidence does not support population-wide screening solely for malaria risk prediction. These findings improve understanding of host genetic susceptibility and may inform future research into targeted risk stratification.

Ovarian cancer (OC) remains one of the most lethal gynecological malignancies despite advances in conventional therapeutic approaches. The dismal approximately 30% five-year survival rate for advanced disease highlight the urgent need for innovative treatment strategies. Chimeric antigen receptor T (CAR-T) cell therapy, having revolutionized treatment paradigms in hematological malignancies, faces significant challenges when applied to the complex immunosuppressive tumor microenvironment (TME) of ovarian cancer. This comprehensive review investigates critical research questions regarding the extent to which cellular and molecular components of the ovarian cancer TME inhibit CAR-T cell cytotoxic function, and the signaling patterns associated with reduced CAR-T cell infiltration or persistence in tumor masses. We systematically examine the multifaceted immunosuppressive mechanisms within the ovarian cancer TME and evaluate breakthrough strategies designed to overcome these barriers, including next-generation CAR engineering, combinatorial therapeutic approaches, and innovative TME-modulating technologies. The insights presented here provide a crucial roadmap for translating CAR-T therapy from promising concept to clinical reality in ovarian cancer treatment, potentially transforming outcomes for patients with this recalcitrant malignancy where innovative therapeutic options are urgently needed.

Natural killer (NK) cells play a crucial role in combating gastric cancer (GC). However, they undergo ferroptosis in the tumor microenvironment of GC, which weakens their antitumor effects. Exosomal circRNA influences GC progression. Previous studies have reported the cancer-promoting role of circPDSS1 in GC, but whether it is involved in NK cell ferroptosis remains unclear. This study aimed to investigate the role of GC cell-derived exosomal circPDSS1 in NK cell ferroptosis. A noncontact coculture system of GC cells (HGC-27, AGS, and MKN-45 cells) and human NK-92 cells was established, and humanized mouse and cell-derived xenograft (CDX) tumor models were constructed for joint investigation. Cell death and viability were assessed via flow cytometry, LDH release assays and CCK-8 assays; circPDSS1 stability was determined by agarose gel electrophoresis and actinomycin D treatment; ferroptosis was determined by measuring lipid ROS, MDA, and Fe2+ levels; and key molecular interactions were determined by performing dual-luciferase reporter assays and RIP and RNA pull-down experiments. CD56 and CD16 expression was reduced in clinical tumor tissues. In vitro experiments revealed that the proportion of 7-AAD+ NK-92 cells cocultured with GC cells was significantly increased, accompanied by increased cell mortality and decreased IFN-γ and TNF-α secretion. Further studies revealed that circPDSS1 expression was abnormally elevated in GC tissues and cells and was particularly enriched in MKN-45 cell-derived exosomes (Exos). Knockdown of circPDSS1 alleviated MKN-45-Exo-induced cell damage; reduced lipid ROS, MDA, and Fe2+ levels in NK-92 cells; and delayed tumor progression. Furthermore, a ferroptosis inhibitor suppressed circPDSS1 overexpression-induced cell death. Mechanistically, circPDSS1 sponged miR-142-3p to upregulate ACSL4 expression, promoting NK cell ferroptosis. Exosomal circPDSS1 derived from GC cells contributes to NK cell ferroptosis by sponging miR-142-3p to increase ACSL4 expression and exacerbate GC.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with a broad spectrum of malignancies and immune-mediated disorders, and growing evidence highlights the importance of host glycan-lectin interactions in shaping viral persistence and immune escape. Among these, galectins have emerged as key regulators of the EBV life cycle, influencing viral attachment, latency maintenance, lytic reactivation, and the remodeling of the tumor microenvironment. Galectin-1, -3, and -9 exhibit context-dependent functions that collectively modulate oncogenic signaling pathways, T‑cell exhaustion, regulatory T‑cell expansion, and innate immune sensing. Recent clinical studies further suggest that circulating galectins and galectin-enriched exosomes may serve as non-invasive biomarkers for disease progression and prognosis in EBV-associated malignancies. Despite these advances, major knowledge gaps remain regarding member-specific functions, compensatory galectin networks, and the spatiotemporal dynamics of galectin regulation during infection. Targeting the galectin-glycan axis therefore represents a promising frontier for host-directed antiviral and anticancer therapies, with the potential to disrupt viral latency, restore antiviral immunity, and improve clinical outcomes in EBV-driven diseases.

Incretin mimetic drugs have transformed the treatment of obesity and type 2 diabetes; however, production and patient compliance challenges remain. Plasmid DNA has recently demonstrated the ability to deliver functional monoclonal antibodies for over a year in human patients. We generated plasmid-encoded long-acting incretins (pLincretins) by fusing cleavage-resistant glucagon-like peptide-1 and gastric inhibitory polypeptide analogues with IgG heavy chain (Fc) components. Plasmids were delivered to diet-induced obese mice using a clinically validated electroporation device. A single administration drove sustained expression, supporting durable reductions in body weight, food intake, and blood glucose. Building on this framework, we used AI-guided protein modeling and a synthetic consensus approach to design a dual glucagon-like peptide-1 receptor (GLP-1R)/glucose-dependent insulinotropic polypeptide receptor agonist [plasmid-encoded synthetic consensus incretin (pSynCretin)]. pSynCretin demonstrated enhanced GLP-1R avidity and induced potent weight loss in vivo. These findings establish DNA-launched incretin mimetics as a potential therapeutic tool that combines the potency of next-generation metabolic hormones with the durability, safety, and translational feasibility of plasmid delivery.

Deep vein thrombosis (DVT) remains difficult to distinguish because of its often silent presentation and the limited specificity of current diagnostic tools. We aimed to evaluate whether integrating gut microbiome profiles with routine clinical data could enhance the classification performance for identifying DVT in a case-control cohort. Stool samples were collected from individuals with DVT (n = 58), coronary artery disease (CAD, n = 56), and healthy controls (HC, n = 500). Full-length 16S rRNA gene sequencing was used to characterize the gut microbiota at species-level resolution. A random forest classifier was trained using a nested cross-validation framework, with permutation importance and SHAP (Shapley additive explanations) analyses applied to assess model interpretability. Decision curve analysis (DCA) was employed to evaluate the discriminative value of the models in an independent test set. Following linear discriminant analysis (LDA) effect size (LEfSe) screening, 95 candidate microbial features were entered into a random forest framework. Features were reduced using mutual information filtering and embedded selection to retain the final 10 for DVT vs. non-DVT classification. The integrated microbial-clinical model demonstrated substantially improved discrimination compared with the clinical-only model, achieving higher ROC-AUC [0.947 (95% CI 0.870-0.991) vs. 0.874 (95% CI 0.794-0.941)] and PR-AUC [0.793 (95% CI 0.602-0.931) vs. 0.497 (95% CI 0.274-0.724)]. Importantly, the microbiome-derived signals were robustly associated with DVT risk after adjustment for clinical covariates. Functional prediction analysis indicated enrichment of vitamin K2 and lipopolysaccharide (LPS) biosynthesis pathways in DVT, suggesting potential microbial links to coagulation and inflammation, whereas healthy controls were predominantly enriched in NAD and tetrahydrofolate (THF) biosynthesis pathways. Together, these results demonstrate that microbiome-based classification provides complementary biological insights that distinguish DVT cases from controls.