Monocyte-derived macrophages play a significant role in the initiation and progression of atherosclerosis by transforming into lipid-laden foam cells and regulating vascular inflammation. However, the molecular mechanisms that regulate macrophage lipid accumulation, phenotype, efferocytic capacity, and atherosclerosis are incompletely understood. Our preliminary studies revealed increased expression of LGR4 (leucine-rich repeat-containing G protein-coupled receptor 4) in human atherosclerotic arteries. Additional experiments demonstrated increased LGR4 levels in atherogenic oxidized low-density lipoprotein-treated macrophages. However, the macrophage-specific role of LGR4 in atherogenesis has never been investigated. To investigate the role of myeloid cell Lgr4 in atherosclerosis development, myeloid cell-specific Lgr4 knockout (Lgr4f/f LysM Cre±, Lgr4ΔM) and littermate control Lgr4f/f (Lgr4WT) mice were injected intraperitoneally with hPCSK9-AAV8 and fed a Western diet for 16 weeks. Various in vitro cell-based assays, molecular biology techniques, and immunohistological approaches were used to evaluate the functional roles of macrophage Lgr4 and underlying signaling mechanisms. Oil red O staining of whole aortas and aortic root sections demonstrated reduced atherosclerosis in Lgr4ΔM mice compared with sex-matched Lgr4WT mice. However, no changes in circulating monocyte frequencies were detected. Histochemical staining performed on aortic root sections revealed smaller necrotic cores and higher collagen content in Lgr4ΔM mice. Additionally, Lgr4ΔM mice exhibited lower fat mass and blood glucose levels, while plasma total cholesterol was comparable to that of control mice. Further in vitro and ex vivo studies demonstrated reduced lipid accumulation, enhanced efferocytic capacity, a suppressed proinflammatory phenotype, and attenuated expression of different low-density lipoprotein uptake genes in Lgr4-deficient macrophages. Moreover, Lgr4 knockout macrophages displayed increased cholesterol efflux capacity and reduced activation of oxidized low-density lipoprotein-induced Wnt/β-catenin signaling. These findings suggest that myeloid cell Lgr4 contributes to atherosclerotic lesion formation via stimulating macrophage lipid accumulation, impairing efferocytic capacity, and promoting a proinflammatory phenotype. Collectively, these results identify macrophage LGR4 as a novel therapeutic target for atherosclerosis.
This study aimed to identify allergenic epitopes of tropomyosin by characterizing peptide interactions with major histocompatibility complex class II (pMHC-II) using semiempirical quantum computation (Geometry, Frequency, Noncovalent, extended Tight Binding, GFN2-xTB), Molecular Mechanics Poisson Boltzmann surface area (MMPBSA) and GFN-FF, and verified their correlation with experimental allergenicity. The results clarified that nine species-conserved peptides were identified, six of which were validated as antigenic epitopes. Interactive interface within pMHC-II complexes identified critical residue pairs and structural motifs for antigen presentation mechanism. By capturing electronic and dispersion effects, GFN2-xTB performed better than MMPBSA and GFN-FF in charged-ligand system, exhibiting a Pearson correlation of -0.74 between ΔGbind and IgE inhibition. Furthermore, partial least squares regression using interactive isosurface areas in GFN2-xTB revealed a correlation between MHC-II' anchors and IgE binding (R2 = 0.77). Conclusively, molecular dynamics combined with GFN2-xTB can elucidate epitope peptides' distribution, specific anchors, and correlation between ΔGbind and IgE.
To assess clinical effectiveness, safety and in vivo effects on cytokines, chemokines and low-density neutrophils (LDNs) in patients with active systemic lupus erythematosus (SLE) treated with anifrolumab over a 12-month real-world follow-up. We established a longitudinal, multicentre, observational cohort of adult patients with active SLE receiving anifrolumab 300 mg intravenously every 4 weeks. Patients with active lupus nephritis or central nervous system involvement were excluded. Clinical and laboratory measures were recorded at baseline and at months 3, 6, 9 and 12. Chemokines, cytokines, LDNs and neutrophil extracellular trap degradation products were assessed at the respective time points. Longitudinal changes were analysed using generalised additive models with patient-level random intercepts. Twenty patients were recruited. Baseline mean Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) was 10.9±5.0, with frequent musculoskeletal, immunological and cutaneous involvement. Over 12 months, mean SLEDAI-2K declined to 3.1 (95% CI 1.1 to 5.1; p<0.001). SLE Responder Index-4 response rates were 64.7% at month 12 in intention-to-treat analyses with non-responder imputation. By month 12, 86% of patients reached Lupus Low Disease Activity State and 50% achieved Definition of Remission in SLE remission. Physician Global Assessment improved from 1.6 to 0.2 (95% CI 0.0 to 0.5; p<0.001). Daily prednisolone equivalents were significantly reduced (p=0.036); a dose ≤5 mg/day was achieved in 11/13 patients receiving glucocorticoids at baseline, including three who discontinued glucocorticoids. Drug persistence at 12 months was 65%. Fifty-five adverse events were recorded, of which four were serious. Anifrolumab significantly reduced circulating LDNs (p=0.032), interferon-driven chemokines and inflammatory cytokines. This real-world cohort provides clinical and mechanistic evidence that anifrolumab improves clinical outcomes, enables glucocorticoid tapering and reduces LDNs as well as interferon-driven chemokines in patients with active SLE. Safety and drug persistence were consistent with clinical trial data. DRKS00024360.
The innate immunity, linked to macrophage polarization, plays a critical role in the progression of tuberculosis (TB) disease caused by M. tuberculosis (Mtb) infection. Within a single patient with active pulmonary TB, Mtb infection leads to the formation of extensive lung pathology with a spectrum of morphologically and physiologically distinct TB lesions at different stages of progression, where macrophages are believed to represent varying functional phenotypes. Understanding the dynamics of macrophage polarization with identification of the cytokine network, molecular pathways, and polarizing stimuli in the lung TB lesions of patients, depending on various aspects of the host-pathogen interactions, is crucial for developing better treatment strategies, based on immune-modulatory therapeutic approaches, especially in the context of the ongoing spread of drug-resistant TB. The expression of pro-inflammatory/anti-bacterial M1 cytokines (IFNγ, TNFα, IL-1β, and IL-12) and anti-inflammatory/immunosuppressive M2 cytokines (IL-4, IL-10, and FGF2) as well as activation of the AhR and NF-κB pathways were estimated in an immunofluorescence assay and analyzed in relation to Mtb infection in macrophages in the ex vivo cell cultures and/or on the histological sections obtained from various lung TB lesions of the same patients (n = 25) with clinically active pulmonary, predominantly drug-resistant TB. Macrophage polarization was determined by local tissue microenvironments with varying fibrosis severity but was independent of Mtb's genetic and phenotypic diversity in human lungs, the anti-TB treatment of patients before surgery and AhR signaling activity in smoker's macrophages. Pathogen control (without complete Mtb clearance) correlated with co-expression of cytokines of both cytokine types in the mixed M1/M2-polarized macrophages and, presumably, with NF-κB-mediated activation of smoker's alveolar macrophages in the lung TB lesions with local/minimal fibrosis and the preserved alveoli. In contrast, suppressed macrophages (lacking cytokine production and NF-κB-activated mediators and, in parallel, harboring higher Mtb loads) exhibited an M0-like polarization state in tuberculoma walls and extensively fibrotic lung tissues. The host cells' inability to eradicate Mtb reflected suboptimal development of the immune responses in the patients' lungs. Defining the complexity and functional diversity of macrophage phenotypes and signaling pathways involved in the pathogen control and tissue pathology across various lung TB lesions within the same patients with pulmonary TB is essential for designing novel host-directed therapies that modulate the patients' immune responses to optimize protection against Mtb infection and prevent the pathogen transmission.
Metabolically healthy non-obese (MHN) individuals are considered at low cardiometabolic risk, yet a subset may harbor unfavorable visceral adiposity not captured by conventional anthropometric measures, including waist circumference (WC) and BMI. We conducted a prospective cohort study of 22,040 UK Biobank participants (median follow-up 4.2 years [interquartile range 3.4-5.6]) defined as MHN (BMI < 30 kg/m2, absence of diabetes or concurrent hypertension and hyperlipidemia). Visceral (VAT) and subcutaneous adipose tissue (SAT) volumes were quantified from whole-body MRI using a validated deep learning framework. Sex-specific VAT/SAT ratio cutoffs were derived from the German National Cohort based on prevalent cardiometabolic disease and applied to the UK Biobank. The primary outcome was incident major adverse cardiovascular events (MACE); the secondary outcome was incident type 2 diabetes. Categorical net reclassification improvement (NRI), quantifying the net proportion of individuals correctly reclassified between predefined risk categories, compared VAT/SAT ratio versus WC as competing classification approaches. Cox proportional hazards models assessed associations with outcomes after stepwise adjustment for age, sex, smoking, WC, and BMI. Nested models with and without VAT/SAT ratio were compared to test for added value beyond other factors. The VAT/SAT ratio improved risk classification over WC for MACE (NRI 0.088, 95%CI 0.019-0.158, p = 0.013) and diabetes (NRI 0.102, 95% CI 0.024-0.181, p = 0.010). High VAT/SAT ratio independently predicted MACE (adjusted hazard ratio [aHR] 1.30, 95%CI 1.02-1.66, p = 0.037) and diabetes (aHR 1.77, 95% CI 1.34-2.33, p < 0.001) after full adjustment. Adding VAT/SAT to fully adjusted models improved discrimination for MACE (C-index 0.694 vs. 0.690, p = 0.036) and diabetes (C-index 0.723 vs. 0.715, p < 0.001). The VAT/SAT ratio identifies MHN individuals at elevated cardiometabolic risk beyond conventional anthropometric measures, with particularly strong associations for incident diabetes. These findings support the concept of opportunistic imaging-based risk assessment and provide the prognostic foundation for future trials investigating whether targeted intervention in VAT/SAT-reclassified individuals improves outcomes.
To evaluate and compare the diagnostic performance of [68Ga]Ga-DOTA-FAPI-04 with [18F]F-FDG and [68Ga]Ga-DOTATATE PET/CT for detecting recurrent disease in patients with medullary thyroid carcinoma (MTC) and additionally evaluate their role for prognostication and theranostics in management of MTC. In this prospective single-centre study, 56 patients with suspected recurrent MTC underwent [68Ga]Ga-DOTA-Fibroblast activation protein inhibitor (FAPI)-04, [18F]F-FDG, and [68Ga]Ga-DOTATATE PET/CT. The primary endpoint was patient-based diagnostic accuracy for recurrent disease. Secondary endpoints included lesion-based detection rates across pre-defined anatomical regions, correlation with biochemical and clinicopathological parameters, prognostic assessment for progression-free survival, and a novel exploratory FAPI-SSTR Theranostic (FAST) score was developed to guide personalized radiopharmaceutical therapy (RPT). [68Ga]Ga-DOTA-FAPI-04 PET/CT demonstrated higher sensitivity 95.92% and accuracy 94.64% compared to [18F]F-FDG-PET/CT (sensitivity: 89.80%, accuracy: 89.29%) and [68Ga]Ga-DOTATATE (sensitivity: 77.55%, accuracy: 76.79%) on patient-based analysis for detecting recurrent disease. [68Ga]Ga-DOTA-FAPI-04 was showed high detection rates for disease recurrence in mediastinal nodal (91.9%), liver (91.7%), and skeletal (86.5%) metastases.[68Ga]Ga-DOTA-FAPI-04 uptake was associated with a modestly shorter mean progression-free survival and altered clinical management in 35.7% of total patients. Additionally, the pre-defined exploratory FAST scoring system stratified 34 patients (60.7%) for [177Lu]Lu-FAPI-RPT, 8 (14.3%) for [177Lu]Lu-DOTATATE PRRT, and 9 (16.1%) for either or combined RPT. [68Ga]Ga-DOTA-FAPI-04 PET/CT showed higher diagnostic accuracy for the identification of recurrent MTC over standard [18F]F-FDG and [68Ga]Ga-DOTATATE PET/CT, with higher regional detection rates for liver and osteoblastic skeletal metastases. Furthermore, while the exploratory FAST score provides a framework for patient stratification and personalizing targeted theranostic strategies, it requires future validation through larger multicentre trials prior to clinical adoption.
To evaluate the real-world reliability, safety, and effectiveness of a novel platform for robotic microsurgery when used to assist with microsurgical anastomoses. The physical demands associated with advanced supermicrosurgical techniques strain human limitations. The Symani Surgical System® is a robotic platform designed to support these challenging procedures with extreme motion scaling, tremor reduction, and miniaturized, wristed instruments. This was a non-randomized, multicenter, post-market clinical follow-up study (NCT04843436) evaluating use of Symani for microsurgical anastomosis in adults (age 18+) requiring free-flap (FF), nerve-repair, or lymphovenous anastomosis (LVA) procedures. Cases were enrolled both prospectively and retrospectively. The primary endpoints were robotic-procedure technical success and incidence of procedure-associated complications. Key secondary endpoints were subjective usability, intraoperative patency, and FF viability at discharge. Between May 2021 and February 2025, 412 patients at 10 sites underwent at least one microsurgical robotic anastomosis. The procedure technical success rate was 94.1% (507/539 robotic anastomoses; 95% CI: 91.7%-95.9%). Freedom from device-related events was 99.8%. Intraoperative patency at first attempt was 91.7% for FF (331/361, 95% CI: 88.3%-94.3%) and 96.2% (225/234, 95% CI: 92.8%-98.2%) for LVA. Intraoperative revision rates were 8.4% for FF (31/367, 95% CI: 5.8%-11.8%), 3.4% for LVA (8/234, 95% CI: 1.5%-6.6%), and 0% for nerve repair (95% CI: 0.0%-26.5%). FF survival at discharge was 97.8% (268/274, 95% CI: 95.3%-99.2%). The study results support the safety, reliability, and efficacy of Symani when used for robotic-assisted, microsurgical reconstructions.
Serine integrases can precisely integrate large DNA constructs into desired chromosomal sites but only if their natural target site is first installed into the recipient genome. Here, to retarget serine integrases to a desired genomic site, we develop a modular integrase (MINT) system for genome editing. Through a combination of structural modeling, single-round directed evolution and screening in human cells, we retargeted the specificity of the serine integrase Bxb1. We demonstrate the therapeutic potential of the MINT system by retargeting Bxb1 to the human AAVS1 and TRAC loci, where wild-type Bxb1 has no detectable activity. By combining MINT constructs with both known activity-increasing Bxb1 mutants and zinc-finger DNA-binding domains, we achieve efficiencies of 29% at the AAVS1 locus and 35% at the TRAC locus in K562 cells. To further demonstrate clinical potential, we achieved 29% GFP integration efficiencies at the TRAC locus in human T cells.
This work presents a low-energy, ultrafast GeTe radio frequency switch actuated by a nanosecond-pulse laser, with its key innovation being the integration of a tailored anti-reflection coating (ARC) to boost photothermal conversion efficiency. By minimizing optical reflection and confining thermal energy within the GeTe layer, the ARC addresses a critical bottleneck in laser-induced phase change RF switches - high energy consumption due to inefficient light absorption. The ARC is specifically designed to mitigate the high reflectivity of GeTe in both its crystalline and amorphous states. Coupled optical-thermal simulations reveal that a 50 nm silicon nitride ARC reduces the reflectivity of crystalline GeTe at 532 nm from ∼70% to ∼30%, thereby enhancing photothermal conversion efficiency and suppressing heat dissipation. Experimentally, the ARC-coated switch achieves a 36% reduction in switching energy density (from 132 to 84.5 mJ/cm2) during amorphization, with both phase transitions induced by a 10 ns laser pulse. Notably, the ARC integration introduces negligible degradation in RF performance, delivering an ON-state resistance of 4.5 Ω and an OFF-state capacitance of 3 fF, values comparable to those of state-of-the-art GeTe RF switches. This CMOS-compatible, compact device offers promising prospects for low-energy, high-speed, reconfigurable RF front-ends.
BackgroundCivil aviation cargo operations have expanded rapidly, but the occupational health and safety risks faced by cargo workers are still rarely examined through an integrated causal framework that captures chemical, ergonomic, psychosocial, and operational exposures together.ObjectiveThis study aims to identify, prioritize, and interpret the causal relationships among occupational health and safety risks in civil aviation cargo operations from a worker-centered perspective.MethodsThe study employs a comprehensive dataset drawn from industry professionals and applies the Fermatean Fuzzy Decision-Making Trial and Evaluation Laboratory (FF-DEMATEL) method. This approach enables the analysis of complex interrelationships among risk factors, offering a systematic framework for understanding the dynamics of aviation cargo hazards. FF-DEMATEL was applied to 16 cargo-related risk factors evaluated by three occupational safety experts. Expert weights were derived through a machine-learning-based dimensionality reduction procedure using age, occupational safety experience, and firm tenure, enabling the model to reflect both interdependence and expert heterogeneity.ResultsThe analysis reveals a network of critical risks, including improper cargo loading, closed storage conditions, hazardous substances, and unpredictable customer demands. The FF-DEMATEL method identifies both cause and effect relationships among these factors, highlighting which risks exert the greatest influence on overall safety outcomes. The model provides a clear hierarchy of risk sources that require targeted intervention. The leading weighted risks were sabotage, time pressure, incorrect loading of cargo, customer-related uncertainty, and third stakeholder effects. Prominence values showed that sabotage and time pressure were the dominant drivers of the system, while incorrect loading of cargo emerged mainly as an effect factor. A robustness check based on row sums of the normalized and total relation matrices preserved the same upper-tier risk set, supporting the consistency of the prioritization.ConclusionsThe findings indicate that security management, workload and schedule control, loading discipline, and stakeholder coordination should be prioritized together rather than addressed separately. By translating causal risk interactions into concrete priorities, the study offers practical guidance for improving worker protection and operational resilience in civil aviation cargo systems. The findings underscore the necessity of implementing proactive risk management strategies in air cargo operations. Emphasizing the role of advanced analytical methods and a strong safety culture, the study offers actionable recommendations to industry stakeholders.
Chronic diseases are often associated with increased inflammation and oxidative stress, which contribute to disease progression. Glutamine, a conditionally essential amino acid, has been studied for its potential anti-inflammatory and antioxidant properties in various chronic conditions. The present systematic review evaluates the effects of glutamine supplementation on inflammatory markers and oxidative stress indices in patients with chronic diseases. Systematic searches were performed in web databases; Web of Science, Scopus, and PubMed/Medline until May 2025, to identify related randomized controlled trials (RCTs) according to the Cochrane Library and PICOS criteria (population: individuals > 18 years, intervention: glutamine, Comparison: placebo or control, Outcomes: inflammatory and oxidative stress markers in chronic diseases). The Cochrane collaboration tool was used to assess the risk of bias in clinical trials. Six RCTs that assessed the effect of glutamine supplementation on inflammation and oxidative stress markers were included in the study. In these studies, glutamine was administered to the participants through oral or parenteral routes. In three studies improve inflammation via significant reductions in CRP were observed. However, in three studies that examined TNF-α as an inflammatory marker, only one study found its levels to be significantly reduced. Also, of the two studies that examined oxidative stress levels, only one study significantly decreased the MDA and increased SOD levels, and in the other study, glutamine supplementation had no significant effect on glutathione levels. Our findings showed that glutamine supplementation might have a positive effect on inflammation and oxidative stress indices such as TNF-α, CRP, MDA, and SOD in some chronic diseases, however, these effects have not been shown in all studies, so more carefully designed clinical trial studies with different doses of glutamine on inflammation and oxidative stress in chronic diseases are needed. PROSPERO Code: This study was registered in the PROSPERO international prospective register of systematic reviews registration number: CRD420251049112.
Native T1 mapping (T1-MAP) is a promising MRI biomarker for fibrosis but suffers critical confounding from steatosis and iron overload, leading to paradoxical negative correlations. We aimed to compare the fibrosis-specific diagnostic performance of T2-MAP and T1-MAP and evaluate their robustness against these metabolic confounders using a rigorous causal inference framework. In a dual-cohort study (exploratory n = 174; validation n = 198), we employed confounder-stratified subgroup analyses, hierarchical multivariable regression (quantifying ΔR2), causal mediation analysis, net gain metrics (ΔΔAUC, ΔIDI), and Decision Curve Analysis (DCA). (1) Paradoxical Phenomenon:With advancing fibrosis, T1-MAP paradoxically shortened while T2-MAP increased consistently.(2) Correlation Vanishes:In the non-confounded subgroup (FF ≤ 5 % & R2* ≤70 s⁻1), the apparent correlation between T1-MAP and fibrosis vanished (validation: ρ = -0.038, p = 0.681), while T2-MAP maintained a robust positive association.(3) Causal Deconstruction:Mediation analysis confirmed T1-MAP's total effect was primarily mediated by FF (∼49.9 %), with anon-significant direct effectof fibrosis.(4) Key Evidence (Variance Decomposition):Hierarchical regression revealed that T1-MAP contributedzeroindependent variance (ΔR2 = 0.000, p > 0.8) in the non-confounded subgroup, whereas T2-MAP explained highly significant variance (ΔR2 = +0.092, p < 0.001). Clinically, replacing T1-MAP with T2-MAP yielded a positive net diagnostic gain (ΔΔAUC) and net reclassification improvement (ΔIDI) in non-confounded settings. Crucially,DCA uniquely demonstrated that T2-MAP provided consistent net clinical benefit across all subgroups, while T1-MAP risked clinical harm in confounded patients. This study provides a causal deconstruction of the T1-MAP paradox. T1-MAP functions as a non-specific 'metabolic sensor' with no intrinsic signal for fibrosis. T2-MAP is a robust, fibrosis-specific biomarker that penetrates metabolic noise, offering superior net clinical benefit and safety, as proven by DCA.
Ceratopogonid midges of the genus Forcipomyia (subgenus Lasiohelea) are small hematophagous insects widely distributed across tropical regions. In Australia, developmental stages of Leishmania (Mundinia) macropodum have been observed in Forcipomyia (Lasiohelea), suggesting that biting midges may play a role in Leishmania transmission beyond traditional sand fly vectors. However, in Southeast Asia-where leishmaniasis caused by Mundinia species is an emerging autochthonous disease in humans-fundamental information on Forcipomyia (Lasiohelea) diversity and its association with Leishmania remains limited. An integrated morphological-molecular approach was employed to characterize Forcipomyia (Lasiohelea) midges collected using sweep nets from a leishmaniasis-endemic area in Nakhon Si Thammarat Province, Southern Thailand, near the residence of a patient with locally acquired cutaneous leishmaniasis, during January and June 2025. Morphological examination of mandibular dentition, sensory pits, cibarial armature, and spermathecal structure was combined with mitochondrial cox1 barcoding, Bayesian and maximum likelihood phylogenetic analyses, and species delimitation methods (ASAP and mPTP). Because females of the subgenus Lasiohelea possess a single spermatheca, only single-spermatheca females were selected for species identification. Specimens identified as Lasiohelea were subsequently screened for Leishmania using 18S rRNA-qPCR and ITS1-PCR, followed by nanopore-based ITS1 metabarcoding for species-level identification. Vertebrate blood meal sources were also characterized using vertebrate cox1 metabarcoding. From 264 collected midges, 72 female specimens with a single spermatheca were selected for analysis. Integrated morphological and molecular data identified seven Lasiohelea specimens forming four genetic clusters, comprising F. (L.) parvitas (n = 2) and three lineages closely related to F. (L.) peditata (n = 2), F. (L.) humilavolita (n = 2), and F. (L.) taiwana (n = 1). These were clearly separated from non-Lasiohelea taxa, including F. (Euprojoannisia) fuscimana and two unclassified Ceratopogonidae lineages. Phylogenetic and species delimitation analyses revealed cryptic genetic diversity despite morphological similarity. Leishmania DNA was detected in six of seven Lasiohelea specimens. Nanopore ITS1 metabarcoding identified autochthonous species (L. (Mundinia) martiniquensis and L. (M.) orientalis) and additional Leishmania species (L. (Leishmania) amazonensis and L. (L.) major), including mixed-species detections in four specimens. A single engorged specimen contained DNA from red junglefowl (Gallus gallus spadiceus). This study provides the first integrated characterization of Forcipomyia (Lasiohelea) diversity and associated Leishmania detection in Southeast Asia. The results identified several putatively distinct species within Lasiohelea and provide evidence of natural exposure of these midges to multiple Leishmania species, suggesting a complex parasite-midge association and parasite co-circulation in the local environment. Although vector competence was not assessed, these findings suggest that Forcipomyia (Lasiohelea) may be involved in the circulation of Leishmania parasites and may represent promising candidates for further investigation of vector competence. Future studies focusing on host associations, parasite development, and experimental transmission are needed to clarify their epidemiological significance.
Aquaculture sediments are increasingly recognized as important reservoirs of antibiotic resistance genes (ARGs). Although thermophilic fermentation is widely used to reduce ARGs and pathogens in manure, most biosafety assessments stop at the fertilizer product itself, leaving unresolved whether these benefits persist after application to aquaculture sediments. Here, we compared inorganic fertilizer (IF), raw manure (RM), and fermented fertilizer (FF) to test whether fermentation confers sustained biosafety benefits in aquaculture pond sediments. After a 6-month co-culture period, sediment samples were analyzed using shotgun metagenomic sequencing, ARG and mobile genetic element (MGE) profiling, antibiotic residue determination, and network analyses. Long-term fertilization significantly altered sediment physicochemical properties, microbial community composition, and resistome structure. Among the three groups, the RM exhibited the highest total ARG abundance and the greatest number of unique ARG subtypes, with significant enrichment of multidrug resistance genes as well as pathogen-, disease-, and host-associated mobile genetic elements (MGEs). In contrast, the FF group showed the lowest total ARG abundance and fewest unique ARG subtypes, along with suppression of pathogen-associated MGEs, indicating that FF can effectively reduce the risk of ARG dissemination. However, the potential impact of residual antibiotics still warrants attention. Redundancy analysis showed that TC and TN primarily explained bacteriome and resistome variation under RM, whereas pH, EC, AP, and AK were more strongly associated with FF. Co-occurrence analysis further suggested that fertilizer-driven microbial community shifts may regulate ARG persistence and potential cross-ecosystem dissemination. Overall, fermented fertilizer attenuated, but did not eliminate, manure-derived resistance risks in aquaculture sediments. These findings support fermented fertilizer as a safer management option than raw manure and highlight the need for integrated risk assessment combining ARGs, MGEs, microbial hosts, and antibiotic residues.
The DES gene encodes the IF (intermediate filament) protein desmin, which connects different multiprotein complexes, such as the cardiac desmosomes, and is highly important for the structural integrity of cardiomyocytes. Pathogenic DES mutations cause filament assembly defects leading to cardiomyopathies. However, most DES variants listed in genetic disease databases are currently classified as variants of unknown significance. Here, we characterized 21 different DES variants of unknown significance and 18 additional proline variants, localized in a highly conserved stretch at the C terminus of the desmin coil-2 subdomain. We inserted desmin variants via site-directed mutagenesis and investigated the filament assembly in transfected cell lines and cardiomyocytes derived from induced pluripotent stem cells by confocal microscopy. In addition, we purified recombinant wild-type and mutant desmin and analyzed the filament formation by atomic force microscopy. Coexpression with wild-type desmin delivered by adeno-associated virus was used to model the heterozygous status of cardiomyopathy patients. Twelve DES variants of unknown significance formed cytoplasmic aggregates, which were likewise verified by atomic force microscopy. Of note, these 12 variants disturb the filament assembly even when coexpressed with wild-type desmin. Using a proline screen, we showed that proline residues localized at nearly each of the positions in this stretch cause filament assembly defects. By modeling the tetrameric structure of desmin, we demonstrated that specific heptad positions, as well as positions of intramolecular and intermolecular ion bridge sites, are particularly susceptible to mutations that promote desmin aggregation. In summary, our study demonstrated that the highly conserved stretch at the C terminus of the coil-2 subdomain is a hotspot region, where several pathogenic DES mutations cause an aberrant desmin aggregation. Based on our molecular data, we suggest reclassifying the aggregate-forming variants as likely pathogenic mutations rather than variants of unknown significance. Our study may have relevance for the genetic counseling of cardiomyopathy patients with similar DES variants.
To evaluate intratumoral fat in hepatocellular carcinoma (HCC) using both qualitative and quantitative approaches based on routine chemical-shift magnetic resonance imaging (MRI), and to investigate its potential value in predicting histological grade. This retrospective study included 282 patients with pathologically confirmed HCC between January 2015 and November 2025. Tumors were classified into low-grade and high-grade groups according to the Edmondson-Steiner grade. Intratumoral fat was assessed on in-phase and opposed-phase MRI images. For qualitative assessment, intratumoral fat pattern was categorized as none, heterogeneous, or homogeneous. For quantitative assessment, regions of interest were manually delineated on three consecutive slices showing the largest tumor area, and the mean fat fraction (FF) was calculated. Logistic regression analysis was performed to identify risk factors associated with high-grade HCC. Furthermore, models incorporating clinicoradiological factors were developed for the preoperative prediction of HCC histological grade. Homogeneous intratumoral fat was more frequently observed in low-grade tumors than in high-grade tumors, and FF was significantly higher in low-grade tumors than in high-grade tumors. Both homogeneous intratumoral fat (odds ratio [OR] = 0.230 [0.097-0.514], P = 0.001) and FF (OR = 0.861 [0.811-0.907], P < 0.001) were identified as independent predictors of high-grade HCC. When combined with other clinicoradiological factors, the FF-based model showed better performance than the intratumoral fat pattern-based model (area under the receiver operating characteristic curve: 0.792 vs. 0.744, P = 0.024). Intratumoral fat assessed using chemical-shift imaging provides a simple and noninvasive imaging biomarker for predicting the histological grade of HCC. Both homogeneous intratumoral fat and higher FF were associated with a lower risk of high-grade HCC, and the model based on quantitative assessment outperformed that based on qualitative evaluation.
Sustained low-grade inflammation is increasingly recognized as a central element in how polycystic ovary syndrome (PCOS) develops. Here we set out to clarify what role follicular fluid (FF)-borne exosomal miR-424-5p plays in shaping ovarian inflammation and how this ties in with the ovulatory problems seen in PCOS. Across FF samples taken from patients, miR-424-5p levels tracked inversely with pro-inflammatory cytokine concentrations to a striking degree. When granulosa cells (GCs) were challenged with lipopolysaccharide (LPS), delivering exosomal miR-424-5p dampened cytokine release and restored the expression of ovulation-linked genes. Using a dual-luciferase reporter assay, we traced this effect to FGF2, which miR-424-5p binds directly, and we found FGF2/FGFR1 signaling to be conspicuously raised in PCOS. In dehydroepiandrosterone (DHEA)-treated rats modeling the syndrome, giving exosomal miR-424-5p eased ovarian inflammation and brought back part of the lost ovulatory capacity. Taken together, the data suggest that exosomal miR-424-5p tempers ovarian inflammation in PCOS by holding the FGF2/FGFR1 axis in check, which casts fresh light on the follicular abnormalities that mark this condition.
This study aimed to explore the chain mediating effect and cross-lagged associations between pregnancy-related anxiety and hope in the relationship between family function (FF) and blood glucose management behavioral decision-making (BDM) among patients with gestational diabetes mellitus (GDM). This study also aimed to provide insights into enhancing self-management efficacy and optimizing maternal and infant outcomes. A convenience sample of 245 GDM patients was selected. Surveys were conducted using the Pregnancy-related Anxiety Questionnaire (PAQ), the Herth Hope Index (HHI), the Family Adaptation, Partnership, Growth, Affection, and Resolve (APGAR) Index, and the Blood Glucose Management Decision-Making Behavior Questionnaire at three time points: 24-28 weeks of gestation (T1), 32 weeks of gestation (T2), and 36-42 weeks of gestation (T3). Statistical analyses included equivalence testing and cross-lagged modeling. A total of 220 valid questionnaires were collected. Equivalence testing indicated that all variables met the requirements for measurement invariance, ensuring cross-temporal comparability of the tools. Cross-lagged path analysis revealed that T1 family function significantly predicted T2 pregnancy-related anxiety (β = -0.26, p < 0.01), T2 hope (β = 0.18, p < 0.01), and T2 behavioral decision-making (β = 0.24, p < 0.001). T1 pregnancy-related anxiety significantly predicted T2 hope (β = -0.11, p < 0.01) and T2 behavioral decision-making (β = -0.28, p < 0.001). T2 family function positively predicted T3 hope (β = 0.18, p < 0.001), while T2 pregnancy-related anxiety negatively predicted T3 behavioral decision-making (β = -0.25, p < 0.001). T2 hope positively predicted T3 behavioral decision-making (β = 0.18, p < 0.001), and T2 behavioral decision-making positively predicted T3 family function (β = 0.11, p < 0.01). Bootstrap analysis demonstrated significant indirect effects of T2 pregnancy-related anxiety (β = 0.065, p < 0.01) and T2 hope (β = 0.032, p < 0.01) in the prediction of T3 behavioral decision-making by T1 family function. Clinical practice should develop family-centered intervention models, assess family function, screen for anxiety, and enhance hope through psychological support and health education to promote positive behavioral decision-making and improve blood glucose control.
To develop a dual-contrast deep-learning model for automated muscle segmentation on spin-echo and Dixon magnetic resonance imaging (MRI) for assessment of sarcopenia and intramuscular fat fraction (FF). One-hundred-seventy-eight MRI and liver frailty measurements were consecutively collected from patients requiring liver transplantation. The retrospective study cohort included 148 spin-echo and 90 water images with 73 FF maps. MRI images were acquired using spin-echo and 2-point Dixon sequences at 1.5T and 3.0T. Two single-contrast (spin-echo, water) and one dual-contrast U-Net models were developed for automated segmentation of paraspinal muscles and estimation of skeletal muscle index (SMI) and FF. Segmentation accuracy was evaluated using Dice similarity coefficient, sensitivity and false positive rate. Segmentation metrics for single-contrast and dual-contrast models were compared using Wilcoxon Signed-Rank test. Linear regression and Bland-Altman analyses assessed agreement between manual and model-estimated SMI and FF. One-way ANOVA and Friedman tests compared SMI and FF estimated using manual, single-contrast, and dual-contrast models. The Independent-Sample Mann-Whitney U test was used to compare differences in SMI and FF between frail and non-frail groups. All statistical tests assumed a significance level of 0.05. The dual-contrast model demonstrated improved segmentation performance for spin-echo images (single-contrast mean Dice: 0.85 ± 0.08; dual-contrast mean Dice: 0.88 ± 0.06) and comparable performance for water images (single-contrast mean Dice: 0.90 ± 0.06; dual-contrast mean Dice: 0.89 ± 0.06), along with good agreement for SMI estimation (slopes: 0.75-0.82, R2 = 0.60-0.89) and strong agreement for FF estimation (slope = 0.97 for both models, R2 = 0.97 for both models) and low mean bias (- 2.37% to 5.65% for SMI and < 1% for FF) relative to ground-truth measurements. SMI estimates obtained using both manual and model-based segmentation were lower in frail group compared to non-frail cohort (p ≤ 0.004); however, FF estimates showed no significant differences between frail and non-frail groups (p ≥ 0.995). This study demonstrates the feasibility of using a single generalized dual-contrast model for automated assessment of sarcopenia and intramuscular fatty infiltration, hence facilitating clinical adoption.
This study investigates the impact of assisted reproductive technology (ART) protocols, specifically modified natural cycle (MNC) and controlled ovarian stimulation (COS), on Kynurenine metabolism and bioactive lipid mediators. Follicular fluid (FF) samples from 28 participants (MNC: 14, COS: 14) were analyzed using targeted and untargeted liquid chromatography-tandem mass spectrometry, alongside evaluation of baseline reproductive hormones. A significant reduction in Kynurenic acid (KA) levels (p = 0.0088) was observed in the COS group, while other Kynurenine pathway metabolites such as L-Kynurenine (Kyn), 3-Hydroxykynurenine, and Tryptophan (Trp) remained unchanged. KA/Kyn and KA/Trp ratios were also unchanged, suggesting no major alteration in overall Kynurenine aminotransferase (KAT)-associated pathway activity between the groups. A targeted analysis of oxylipins revealed a significant decrease of 9,10-dihydroxy-12-octadecenoic acid (9,10-DiHOME) in COS samples (p < 0.0001), whereas levels of 12,13-DiHOME, 13-HODE, DiHETEs, and 8-Iso-prostaglandin E₂ showed no significant differences. The parallel reduction of KA and 9,10-DiHOME in COS cycles indicates that differences in the intensity of ovarian stimulation are associated with alterations in the follicular immunometabolic environment. Further research is needed to evaluate the functional relevance of these changes for oocyte quality and reproductive outcomes.