Journal of Biomedical Science

IntroductionSevere dengue infection is characterized by endothelial injury and systemic inflammatory complications. To better understand the mechanisms underlying disease severity, we investigated a broad panel of circulating inflammatory and endothelial mediators in patients with clinical dengue infection. MethodsA prospective cross-sectional case-control study was carried out involving 111 dengue patients and 42 healthy controls. Among the dengue cases, 85 were identified as primary, while 26 were classified as secondary dengue infections. Serum levels of endothelial markers (Ang-2, CXCL10, MCP1, TRAIL), acute-phase and liver dysfunction and acute-phase markers (CRP, galectin 3, and serum amyloid protein), systemic inflammatory mediators (MIF, TNF-, IL-1{beta}), mast cell-derived proteases (chymase, tryptase), and tissue repair markers HGF, IL-10, IL-1Ra) were quantified using ELISA and Luminex multiplex assays. Correlations among serum analytes, severity indicators, and haematological markers were also explored ResultsSeveral biomarkers, Ang-2, CXCL10, TRAIL, CRP, MIF, IL-1Ra, TNF-, and chymase showed differential expression across severity groups, indicating coordinated endothelial and inflammatory activation. Stratification of patients with primary-secondary dengue also followed a similar pattern except IL-1{beta}, which had significant differential expression across the cohorts. Ang-2 showed strong positive correlations with markers of hepatic dysfunction, including ALT, AST, and bilirubin, suggesting a link between endothelial injury and liver involvement. ConclusionsSevere dengue is driven by the coordinated activation of endothelial dysfunction, acute-phase responses, mast cell mediators, and counter-regulatory pathways. These processes collectively contribute to vascular leakage and organ injury, reinforcing the value of biomarkers such as Ang-2, CXCL10, CRP, and chymase for severity assessment.

The Ube2J1 enzyme that mediates the ubiquitination and proteasomal degradation of misfolded proteins at the ER is phosphorylated at serine S184. Following anisomycin treatment of HEK293T cells, we observed an inverse relationship between phosphorylation and dephosphorylation at this site. This suggested a dynamic interchange between the two forms, and we show that S184 is a target for protein phosphatase 2A. The S184-phosphorylated protein is known to exhibit increased sensitivity to proteasomal degradation, and we found that mutation at K186R increased the ratio of S184-phosphorylated to S184-dephosphorylated protein. Although the K186R mutant retained some sensitivity to proteasomal inhibition, our results show that Ube2J1 steady state expression can be exercised at multiple levels, and can involve dynamic phosphorylation and dephosphorylation at S184.

Dengue virus (DENV) infection is a major global health threat, affecting more than half of the worlds population. Severe dengue is a life-threatening condition characterised by systemic bleeding, vascular leakage, and interstitial fluid accumulation that can progress to hypovolaemic shock. Circulating DENV non-structural protein 1 (NS1) has long been implicated in driving vascular hyperpermeability through its disruptive effects on endothelial cell junctions and the glycocalyx. The lymphatic system, which runs alongside the vascular network, plays a critical role in resorbing and recirculating interstitial fluid and immune cells extravasated from blood vessels. Despite its importance in maintaining tissue fluid homeostasis, the impact of dengue disease on lymphatic vessels has not previously been explored. Here, we present the first evidence that DENV-2 NS1 induces marked hyperpermeability in lymphatic endothelial cells, as measured by transendothelial electrical resistance, and impairs lymphangiogenesis in vitro. These effects were not attributable to changes in cell viability, morphology, or metabolic activity, as assessed by live/dead and metabolic assays and image analysis. Instead, we observed a defect in lymphatic endothelial cell migration, measured by scratch assay, which may underlie the reduced lymphangiogenic potential. Bulk RNA-seq, immunocytochemistry, and advanced image analysis further demonstrated pronounced reorganisation of cell-cell junctions, the cytoskeleton, and focal adhesions. Notably, junctional proteins including VE-cadherin, ZO-1, and Claudin-5 were not downregulated but instead displayed disorganised distribution along the cell junctions or aberrant cytoplasmic localisation. These structural disruptions became even more pronounced under flow conditions produced using a microfluidic system. Together, these findings demonstrate for the first time that DENV-2 NS1 directly disrupts lymphatic endothelial cell function, leading to junctional disorganisation and hyperpermeability. Such impairment of lymphatic drainage may contribute to the pathophysiology of severe dengue. Author SummaryDengue is a rapidly expanding mosquito-borne disease that now affects many tropical and subtropical regions worldwide. Severe cases can lead to extensive fluid leakage from blood vessels, which causes tissue swelling and, in the most dangerous situations, shock. Although much research has focused on how dengue damages the blood vascular system, almost nothing is known about its impact on the lymphatic system, which is responsible for removing fluid from tissues and returning it to the bloodstream. Because both systems work together to maintain fluid balance, understanding how dengue affects lymphatic vessels is important for explaining why fluid accumulation becomes so severe in critical disease. In our study, we examined whether the viral protein NS1, which circulates during infection, directly affects the cells that line lymphatic vessels. We found that NS1 increases the permeability of these cells and reduces their ability to form new vessel structures. These effects were not caused by cell death but by disruptions in how the cells organise their junctions, internal scaffolding, and interactions with neighbouring cells. By showing that NS1 can directly impair lymphatic vessel function, our work identifies a previously overlooked mechanism that may contribute to fluid build-up in severe dengue and suggests new avenues for future therapeutic research.

Dengue virus (DENV) is a major burden to global public health, affecting hundreds of millions annually. Children represent the major proportion of global dengue cases, ranging from asymptomatic or subclinical presentation to dengue fever (DF) and severe dengue hemorrhagic fever or shock syndrome (DHF/DSS). The factors that distinguish this range of disease severity are still poorly understood. To identify biomarkers of severity, we analyzed the plasma proteome of acute DENV infected children including both subclinical and hospitalized cases. Proteins associated with the acute-phase response, innate immune and lysosomal activation, and components of the coagulation cascade showed marked differences between hospitalized and subclinical cases during early infection. Longitudinal profiling demonstrated that endothelial dysfunction emerges early, with PTX3 showing the strongest and most rapid upregulation in hospitalized patients, supporting its potential role as a marker of imminent vascular involvement. When comparing severe (DHF/DSS) and classical DF hospitalized cases, CLEC11A displayed the highest fold change at hospital admittance. We used machine-learning analysis to predict disease severity at the acute phase of infection, distinguishing subclinical from hospitalized cases and patients that develop classical dengue fever or severe disease based on the identified complement regulators and inflammatory markers. The panel of identified plasma proteins shed light on the mechanisms of dengue related disease progression and may provide a handle to predict disease severity based on blood markers present during the acute phase of infection.

BackgroundPreeclampsia, a maternal hypertensive syndrome affect fetal brain development and cerebral angiogenesis, with potential acute and long-term consequences. Underlying mechanisms of these brain vascular alterations are unknown. This study investigates the role of thrombospondin-1 (TSP-1), an antiangiogenic glycoprotein, as a key mediator of communication between the fetoplacental and fetal brain endothelium in the context of preeclampsia. MethodsConditioned media (CM) of human umbilical vein endothelial cells (HUVECs) from normal pregnancies (NP-CM) and preeclamptic pregnancies (PE-CM), were used to treat human (hCMEC/D3) and murine brain microvascular endothelial cells (BMECs). A proteomic analysis was performed in plasma of the umbilical cord of normal pregnancy and preeclampsia. TSP-1 was identify using proteomic analysis and confirmed by Western blot. PE-CM depleted of TSP-1, using immunoprecipitation, was used to evaluate protein-protein interaction with vascular endothelial growth factor (VEGF). Antibody-mediated blockage of TSP-1 was used to investigate antiangiogenic effect and pro-angiogenic signaling pathways in brain endothelial cells exposed to PE-CM. ResultsPE-CM significantly reduced angiogenesis, migration, and invasion of brain endothelial cells and altered cytoskeletal organization. These effects were accompanied by reduced VEGFR2 and AKT signaling, indicating impaired angiogenic pathways. Proteomic analysis of umbilical cord plasma revealed elevated TSP-1 levels in preeclampsia, which was confirmed by Western blotting. TSP-1 was also increased in PE-CM, and immunoprecipitation assays suggested a protein-protein interaction with VEGF. Antibody-mediated blockade of TSP-1 restored angiogenesis, as reflected by increased total tube length, and rescued VEGFR2 and AKT signaling in brain endothelial cells exposed to PE-CM. ConclusionTSP-1-mediated endothelium-endothelium communication between placenta-brain axis in offspring of mothers with preeclampsia. This communication mediated by TSP-1 may contribute to acute and long-lasting cerebrovascular dysfunction observed in infants exposed to preeclampsia.

The rat vestibular system plays a critical role in anti-gravity responses such as the tail-lift reflex and the air-righting reflex. In a previous study in male rats, we obtained evidence that these two reflexes depend on the function of non-identical populations of vestibular sensory hair cells (HC). Here, we caused graded lesions in the vestibular system of female rats by exposing the animals to several different doses of an ototoxic chemical, 3,3-iminodipropionitrile (IDPN). After exposure, we assessed the anti-gravity responses of the rats and then assessed the loss of type I HC (HCI) and type II HC (HCII) in the central and peripheral regions of the crista, utricle and saccule. As expected, we recorded a dose-dependent loss of vestibular function and loss of HCs. The relationship between hair cell loss and functional loss was examined using non-linear models fitted by orthogonal distance regression. The results indicated that both the tail-lift reflex and the air-righting reflexes mostly depend on HCI function. However, a different dependency was found on the epithelium triggering the reflex: while the tail-lift response is sensitive to loss of crista and/or utricle HCIs, the air-righting response rather depends on utricular and/or saccular integrity.

Vestibular schwannomas (VS) cause vestibular function loss by mechanisms still poorly understood. We evaluated the vestibulo-ocular reflex by the video-assisted Head Impulse Test (vHIT) in patients with planned tumour resection by a trans-labyrinthine approach. The vestibular sensory epithelia were collected and processed by immunofluorescent labelling for confocal microscopy analysis of sensory hair cell subtypes (type I, HCI, and type II, HCII), calyx endings of the pure-calyx afferents, and the calyceal junction normally found between HCI and the calyx (n=23). Comparing Normofunction and Hypofunction patients, we concluded that worse vestibular function associates with decreased HCI and HCII counts in the sensory epithelia and with increased proportion of damaged calyces. A decrease in the number of HCI and calyx endings of the pure-calyx afferents was recorded to associate with age increase. Partial least squares regression (PLSR) models indicated that VS and age had independent, additive effects on vestibular function. Correlation analyses indicated that lower vHIT gains associate with lower numbers of HCI and increased percentages of damaged calyces. These data support the hypothesis that the deleterious effect of VS on vestibular function is mediated, at least in part, by its damaging impact on the vestibular sensory epithelium. They also provide further evidence for the dependency of the vestibulo-ocular reflex on HCI function and for the calyceal junction pathology as a common response of the sensory epithelium to HC stress.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID are complex chronic conditions that often follow infectious triggers with overlapping clinical features but poorly defined pathophysiological relationships. This study aimed to identify disease-specific immune signatures through multiparameter immunophenotyping of monocytes, dendritic cells, and T-cell subsets. A total of 207 participants were included (ME/CFS: n = 103; long COVID: n = 63; healthy controls: n = 41). Peripheral blood mononuclear cells were analyzed using multiparameter flow cytometry. Statistical analyses included non-parametric testing, age-adjusted ANCOVA, correlation network analysis, and principal component analysis (PCA). Long COVID was characterized by increased M2-like monocyte polarization, elevated CD80 expression across monocyte subsets, expansion of dendritic cells, and reduced expression of activation markers, indicating persistent immune activation with features of immune exhaustion. In contrast, ME/CFS exhibited reduced costimulatory molecule expression, impaired CCR7-mediated immune cell trafficking, and less coordinated activation patterns, consistent with a state of immune suppression. Correlation network analysis revealed more extensive and integrated immune interactions in long COVID, while PCA identified distinct immunophenotypic components and enabled moderate discrimination between the two conditions. These findings demonstrate that ME/CFS and long COVID are characterized by distinct immune profiles, supporting the concept of divergent immunopathological mechanisms. The identified signatures may contribute to biomarker development and guide targeted therapeutic approaches.

The nasal epithelium is a complex tissue composed of both respiratory and olfactory tissue, and is constantly exposed to environmental insults, including toxins and pathogens. The main olfactory epithelium (MOE) serves as the critical site for olfaction, or sense of smell. Dysfunction at this critical barrier tissue can result in partial or total loss of olfactory function, resulting in significant impact to quality of life. The MOE is heterogeneous, comprised of many cell types including olfactory sensory neurons, support cells, and immune cells. It is not well understood how these diverse cell types in the MOE interact to regulate this tissue during homeostasis, and during times of injury and inflammation. We investigated how environmental olfactory exposures impact cell type specific transcriptional responses in the mouse MOE. We performed single-cell RNA sequencing (scRNA-seq) of the MOE following controlled environmental exposure to both well-known odorants and allergens. We identified major cell types and subtypes within the MOE, and identified transcriptional changes in response to the olfactory exposures. We identified transcriptional changes in OSNs, sustentacular cells, and resident immune cells to each condition. This indicated that environmental olfactory exposures drive changes to multiple cell types in the MOE. To our knowledge, this is the first study to identify effects of environmental olfactory exposures on cell-type specific transcription at homeostasis. These findings highlight the potential importance of multi-cellular interactions and communication in regulation of the olfactory epithelium.

Osteoporosis affects millions of women globally. In this study, we applied bioinformatics methods to screen for novel diagnostic biomarkers of osteoporosis in women using the GSE62402 and GSE56814 datasets. PCSK5, ZNF225, and H1FX were used to construct a diagnostic model. ROC, calibration, and decision curve analyses were performed to assess the diagnostic performance on the training (GSE56814) and external (GSE56815) datasets. The expression level of model genes was validated in GEO datasets. Furthermore, five transcription factors (ETS1, NOTCH1, MAZ, ERG, and FLI1) were identified as common upstream regulators of model genes. PCSK5, ZNF225, and H1FX serve as novel diagnostic biomarkers, providing new insights into the pathogenesis of and treatment strategies for osteoporosis in women.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

Long noncoding RNAs (LncRNAs) have emerged as a class of important regulatory ncRNAs and are known to fine-tune numerous cellular processes including proliferation, differentiation and development; however, their role in quiescence still remains largely unexplored. A miRNA host gene lncRNA, MIR503HG, has been reported to play important role in cancer development. Here, we demonstrate the role of MIR503HG lncRNA in regulating cellular quiescence. MIR503HG displays elevated levels in human diploid fibroblasts induced to undergo quiescence. Depletion of MIR503HG in HDFs affects the entry of cells into quiescence but has no effect on cell cycle progression, suggesting its role in quiescence attainment and/or maintenance. Additionally, MIR503HG depletion led to a drastic decrease in the levels of miR508 target, PTEN with a concomitant increase in pAkt levels, indicating its role in negative regulation of miR508. Further, we demonstrate that the lncRNA MIR503HG regulates PTEN levels by acting as a ceRNA for miR508 to maintain cellular quiescence. Our studies illustrate that MIR503HG can function synergistically with miR503 to maintain cells under quiescence and both the miRNA-HG and the miRNA encoded by its gene locus synergistically control the same biological process in different ways by regulating different downstream genes.

G-quadruplexes (G4s) are critical nucleic acid secondary structures that play pivotal roles in regulating gene expression. In this study, we conducted a proteome-wide in silico analysis across multiple viruses causing hemorrhagic fevers to identify candidate proteins containing a conserved G4-binding motif. Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. We selected the NS3 protease domain of Yellow Fever virus for further validation. Biochemical assays demonstrated that the NS3 protease domain binds G4 structures with high specificity and affinity, particularly favoring the parallel conformation. Molecular docking and simulations further revealed that the NS3 protease domain interacts with the terminal G-tetrads and loop regions of G4 via key residues, including PHE40, adopting an insertion and stacking composite binding mode. These findings expand our understanding of virus - G4 interactions and offer novel potential targets for G4-based antiviral strategies. Bullet points- We screened viruses causing hemorrhagic fevers for potential G4-binding peptides. - Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. - Biochemical assays demonstrated that the NS3 protease domain of YFV binds G4 structures with high specificity and affinity.

Dengue virus (DENV) represents a growing global health challenge with billions of people at risk. Severe Dengue (SD), a complication of DENV infection that involves generalized hemorrhage, is driven, at least in part, by endothelial dysfunction. Endothelial dysfunction refers to increased permeability due to inflammation, mechanical injury and/or modification of the genetic program of endothelial cells. Previous work showed that exposure of endothelial cells to conditioned media from DENV-infected cells (CMDV) increased permeability and cellular stiffness, repressed endothelial markers and induced mesenchymal genes. However, the generality, extent, mechanism and ultimate impact of these events in the onset of SD remain elusive. Here, we integrate analysis from in vitro infection of endothelial cells with computational modeling to investigate the key features of CMDV-induced endothelial alterations and their potential impact on endothelial dysfunction. We found that CMDV increased SNA1 and CDH2 expression, while suppressing endothelial genes OCLN and CDH5. Global transcriptomics analysis revealed that CMDV triggered a transient pro-inflammatory response, followed by induction of selected tissue repair genes and matrix remodeling. A non-directed asynchronous network model (NDAM-CMDV) identified IL6 and FN1 as central nodes of DENV-induced endothelial trans-differentiation, providing new molecular insights that predict the evolution of the disease and identify potential therapeutic targets.

1Long QT syndrome Type 2 (LQT2) is a genetic disorder caused by missense mutations in the KCNH2 gene that encodes the potassium channel KV11.1. Previous studies have shown that most KV11.1 missense mutations with loss-of-function phenotypes result from impaired trafficking from the endoplasmic reticulum to the plasma membrane. To investigate the molecular basis of these defects, we used molecular dynamics simulations to analyze two sets of disease-associated missense mutations: those that suppress and those that maintain normal channel trafficking. We focused initially on the conformational and dynamics differences between wild-type and several mutants of KV11.1 via molecular dynamics simulations when two K+ were placed in the selectivity filter (SF). Our study reveals that missense mutations in the S4 helix allosterically disrupt the selectivity filter, a critical determinant for proper channel trafficking. Trafficking-competent variants largely retained a wild-type selectivity filter structure, whereas trafficking-deficient mutants exhibited pronounced structural perturbations in this region. These findings suggest that certain LQT2-associated missense mutations in KCNH2 impair channel trafficking by compromising the structural integrity of the selectivity filter. We additionally found that second-site variants Y652C in the drug binding vestibule can correct structural defects associated with some mistrafficking variants.

Maternal pancreatic {beta}-cells undergo functional and structural changes to adapt to increased metabolic demands during pregnancy. Lactogen signaling via the prolactin receptor (PRLR) contributes to these adaptations by increasing {beta}-cell mass, insulin transcription and glucose-stimulated insulin secretion[1-4]. In other lactogen-responsive tissues such as the mammary glands and specific hypothalamic nuclei, gestation induces epigenetic changes, some of which persist long after birth[5, 6]. We have previously found that prolactin treatment in islets regulates the expression of epigenetic modifiers[7, 8]. However, whether lactogen signaling in {beta}-cells mediates epigenetic changes to regulate chromatin accessibility has not been examined. Therefore, our objective was to determine whether PRLR signaling alters chromatin accessibility of {beta}-cells to facilitate transcriptional regulation. Using single-cell ATAC-sequencing, we identified differentially accessible regions (DARs) in {beta}-cells which had 718 overrepresented motifs following prolactin treatment of murine islets. Validating this approach, these included motifs bound by established PRLR signaling effectors such as the STAT family of transcription factors (TFs). Using RNA-sequencing we identified transcriptional changes in 41 TFs whose motifs were overrepresented in DARs, including several previously linked to PRLR signaling within {beta}-cells, including Myc, Mafb and Esr1. Importantly, we also identified TFs not previously associated with PRLR signaling, including OVOL2 an established regulator of epigenetic landscape within cells. OVOL2 is a transcription factor involved in EMT inhibition and energy homeostasis with unknown roles in pancreatic {beta}-cells. Here, we establish that OVOL2 acts as a negative regulator of lactogen-dependent effects on {beta}-cell proliferation, establishing a novel regulator of PRLR signaling.

Background: The global burden of dengue infection has rising, yet limited data exists on its impact in the Caribbean. We describe the incidence and associates of acute kidney injury in adults and children with dengue at a teaching hospital in Jamaica. Methods: A single-centre retrospective cohort study of admissions with laboratory confirmed dengue infection at University Hospital of the West Indies, Mona Jamaica between January 2023 to November 2024. AKI was defined using Kidney Disease Improving Global Outcomes definitions. Patients were included if aged >1year and had at least 2 creatinine values. Clinical, demographic and laboratory data were abstracted by chart review. Summary statistics were used to describe continuous and categorical data, and logistic regression to determine AKI associations. Stratified analysis was performed by age-group (adults-aged [&ge;] 16, and paediatric-aged <16 years). Results: Analyses included 167 persons, 62% (103) were male, mean age was 26.1{+/-}19.5 years. AKI occurred in 25.8%, 65.1% were KDIGO stage 1. AKI incidence was 30.2% and 18.0% among adults and children respectively. There were 3 in-hospital deaths. People with AKI were older 32{+/-}21.4 vs 24 {+/-}18.4 (p=0.021), and had longer duration of stay [6 vs 4 days (p <0.001)]. Male sex [OR 2.09 (95% CI:0.96-4.59), p=0.064], age per year [OR 1.02 (95% CI:1.01-1.04), p=0.015] symptom duration [OR1.11 (CI 0.99-1.24), p = 0.058], admission bilirubin [OR 1.02 (CI: 1.00-1.04), p = 0.022], NLR [OR 1.09 (CI 1.00-1.18), p = 0.037] were associated with AKI. In adults admission potassium was inversely associated with AKI [OR 0.46 (95% CI 0.21-1.01), p 0.056], while in children admission potassium [OR 3.00 (95% CI 0.88-10.6), p 0.088] was associated with AKI. Conclusion: AKI in dengue hospitalizations is higher than most reports at 25.8%. Targeted public health policy on vector control and early symptom recognition may be needed to improve outcomes.

BackgroundInterferon-gamma (IFN-{gamma}) is the primary effector cytokine of adaptive anti-tumor immunity, yet it paradoxically induces a potent immunosuppressive tumor microenvironment (TME). The full mechanistic scope of this paradox in head and neck squamous cell carcinoma (HNSC) has not been characterized at the transcriptomic scale. MethodsUsing TCGA HNSC RNA-seq data (n = 522), we applied an integrated computational pipeline: Spearman correlation analysis, principal component analysis (PCA), UMAP, K-means clustering (k = 4), Random Forest regression, deep neural networks, permutation importance, JAK-STAT cascade mapping, and DNN-based transcriptome-wide mediation analysis across 57 IFN-{gamma} pathway and 78 immunosuppressive genes. ResultsIFN-{gamma} pathway activity was universally and positively correlated with six immunosuppressive axes, including checkpoints (CD274; LAG3; IDO1), Tregs, myeloid suppression, and tryptophan catabolism. K-means clustering identified four immunologically distinct tumor subgroups. DNN models predicted suppressive TME. Permutation importance identified IRF8 as the dominant mediator linking IFN-{gamma} signaling to immunosuppression. DNN mediation analysis identified PDCD1LG2 (PD-L2) as the strongest intermediary between IFNG and PD-L1 regulation, followed by JAK2 and GBP5. ConclusionsIFN-{gamma} orchestrates coordinated immunosuppression in HNSC through JAK-STAT-IRF8 signaling. PDCD1LG2 and JAK2 are actionable mediators of this paradox, supporting combination strategies co-targeting IFN-{gamma}-induced checkpoint induction and direct checkpoint blockade in HNSC immunotherapy. GRAPHICAL ABSTRACT

BackgroundElevated postprandial hypertriglyceridemia (PP-HTG) is a significant risk factor for development of cardiovascular diseases, however, the mechanisms underlying its exaggerated rise remains poorly understood. MicroRNAs (miRs) are known to be implicated in the regulation of lipid metabolism, thus identifying them as potential key players. We presently investigated whether miRs may control postprandial triglyceride (PP-TG) response. MethodsPostprandial changes in circulating miR expression as a function of the degree of postprandial TG response were evaluated in non-dyslipidemic healthy subjects (n=32). The impact of miR-100-5p on hepatic gene expression was evaluated in differentiated Caco2 and HepG2 cells by analysis of hepatic transcriptome (RNAseq), western blot and ELISA. In vivo studies were conducted in C57BL/6J mice overexpressing mimic miR-100-5p. ResultsPostprandial variation in circ-miR-100-5p levels inversely correlate with PP-TG response. Cir-miR-100-5p was preferentially associated with TGRL particles of intestinal origin in subjects exhibited a low PP TG response. Differential analysis of transcriptome from HepG2 cells transfected by either mimic miR-100-5p or scrambled mimic miR as control allowed us to identify PCSK9 as a down-regulated gene. Overexpression of miR-100-5p in HepG2 cells significantly decreased PCSK9 mRNA levels by 52% (p<0.0001), cellular protein content by 28 % (p<0.0001) as well as PCSK9 secretion by 39% (p<0.0001). In vivo systemic delivery of mimic miR-100-5p induced a two-fold reduction (p<0.0001) on PP-TG in mice, such effect being abolished by blocking the circulating form of PCSK9 with alirocumab. Finally, we revealed a significant inverse relationship between circulating miR-100-5p expression levels and both PCSK9 levels and the magnitude of postprandial hypertriglyceridemia. ConclusionTaken together, our observations reveal that miR-100-5p regulates postprandial hypertriglyceridemia by targeting PCSK9, thus enhancing hepatic triglyceride-rich lipoproteins (TGRL) uptake. Our findings allow us to propose circ-miR-100-5p as a potential biomarker for early identification of subjects at high cardiovascular risk, prior to appearance of classical clinical features of metabolic disorders. Postprandial clinical study, HDL-PP (NCT03109067) Lay summaryThis study examined whether miRs may control postprandial triglyceride response Key findingsOur data reveal that miR-100-5p regulates postprandial hypertriglyceridemia by targeting PCSK9 Our observations allow us to propose miR-100-5p as a potential biomarker for early identification of subjects at high cardiovascular risk

African swine fever (ASF) is a highly pathogenic disease caused by the African swine fever virus (ASFV) infection, which can affect pigs of all ages and breeds, posing significant threat to the global pig farming industry. The ASFV p30 protein is an early-expressed viral structural protein; however, its function is not fully understood. In this study, the interaction of viral p30 with host TRIM21 was identified. The ectopic TRIM21 inhibited ASFV replication, while knockdown or knockout of TRIM21 promoted ASFV replication. Further, p30 was found to interact with RIG-I-like receptor (RLR) signaling adaptor MAVS, and during ASFV infection, p30-TRIM21-MAVS interacted with each other. Mechanistically, TRIM21 activated the K27 polyubiquitination of MAVS to induce IRF3 mediated type I interferon (IFN) production, whereas p30 counteracted TRIM21 activated MAVS K27 polyubiquitination to evade RLR signaling mediated antiviral IFN induction. In summary, our study revealed a novel function of ASFV p30, and provided new insights into the immune evasion of ASFV.