Journal of Biomedical Science

Brutons tyrosine kinase (BTK) has been reported to be important in the inflammatory response in many diseases. However, its role and explicit mechanisms in intracerebral hemorrhage (ICH) remain unclear. Here, we used a mouse ICH model and transcriptomic datasets to explore the effect of BTK on neuroinflammation after ICH. Inhibiting BTK with ibrutinib alleviated ICH-induced neurological deficits and neuroinflammation in mice. After analyzing RNA-sequencing data of ICH and control mice by weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) analysis, we found that Btk was a hub gene in the green dynamic module. Also, 12 hub genes that closely interacted with BTK were identified in the key gene module, all having a critical role in the inflammatory process. Then, single cell RNA-sequencing data analysis showed that microglia were the immune cells that expressed the most BTK in the mouse brain. After dividing microglia in ICH mice into BTK_high and BTK_low groups, GO/KEGG enrichment analyses of differentially expressed genes (DEGs) between these two microglia groups revealed that most of the top 30 enriched pathways were immune-related. Then, gene set enrichment analysis (GSEA) of the BTK_high and BTK_low microglia showed that the expression levels of four anti-inflammatory and phagocytosis-related pathways were significantly lower in the BTK_high microglia than in the BTK_low microglia. Furthermore, gene set variation analysis (GSVA) demonstrated that multiple immune pathways were expressed differentially between the two microglia groups. Also, six microglia polarization scores were calculated, and the results showed that the BTK_high microglia tend to polarize towards M1 and M2b states, while the BTK_high microglia towards M2 (M2a, M2c) states. Finally, intercellular communication analysis was conducted, and BTK was revealed to promote communication between microglia and other immune cells both at the general level and in specific inflammatory pathways. In conclusion, our study showed that BTK is critical in promoting post-ICH neuroinflammation, at least partly by interacting with Btk-related hub genes and modulating microglias immune pathways, polarization, and intercellular communication.

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.

UNC93B1 is a crucial chaperone protein for the trafficking of TLRs and regulates antigen presentation in dendritic cells (DCs), which activates downstream immune responses. Here, we identified a novel homozygous gain-of-function (GOF) UNC93B1 variant in an early-onset lupus patient. The patient presented with elevated level of inflammation and auto-antibody, and organ damage. The Unc93b1R95L/R95L transgenic mice also exhibited with autoimmune and autoinflammatory phenotypes. The transcriptional analysis revealed increased inflammation and elevated activation of DCs in the patients PBMCs and bone marrow-derived DCs (BMDCs) from Unc93b1R95L/R95L mice. In addition to the selected TLR7/8 activation in previously reported UNC93B1 GOF variants, the single-cell transcriptome and flow cytometry of splenocytes from Unc93b1R95L/R95L mice demonstrated increased phagocytosis activity and T helper cell differentiation with altered ICAM and MHC signaling in DCs and T cells, respectively. These results suggest UNC93B1 GOF variant enhances antigen presentation from DCs to T cells in the pathogenesis of immune dysregulation. Our study expands the pathogenic variants spectrum of UNC93B1 and offers insight into the underlying mechanism of antigen presentation in immune dysregulation caused by UNC93B1 beyond its trafficking function of TLRs.

BackgroundDengue has been a major health threat globally in recent years. In particular, dengue incidences continue to increase annually and the epidemic area has expanded primarily due to global warming. Therefore, effective case detection and surveillance strategies are crucial to tackle this global health challenge. In clinical practice, the rapid test kit detecting dengue non-structural protein 1 antigen and commonly referred as NS1, is widely employed for early diagnosis. However, real-world studies revealed that the sensitivity of the NS1 test kit ranged from approximately 61% to 95%. Since early diagnosis is really critical for disease surveillance in the early stage of a dengue epidemic, scientists have been working hard to develop novel diagnosis methods that can provide higher sensitivity levels. Methodology/Principal FindingsIn response to this challenge, in this study, we have developed a novel diagnosis procedure that integrates machine learning technologies with the NS1 test kit. Our experimental results revealed that we would be able to raise the sensitivity of the dengue diagnosis procedure to higher than 99% by incorporating machine learning based prediction models to screen the suspected patients with a negative NS1 result. Furthermore, the relative risks between the suspected patients who were predicted to be positive and those who were predicted to be negative exceeded 4.8. Conclusions/SignificanceThese results illustrate that the proposed approach provides an effective and efficient diagnosis procedure to address the global health challenge caused by spread of dengue. Author SummaryThis study has aimed to enhance surveillance of the dengue disease by integrating machine learning technologies with the rapid test kit commonly employed in early diagnosis. In clinical practice, the NS1 rapid test kit is widely employed for early diagnosis. However, real-world studies revealed that a certain percentage of the patients with a negative NS1 test result, ranging from 5% to 39%, were actually infected by dengue. Since early diagnosis is critical for disease control in the early stage of a dengue epidemic, scientists have been working hard to tackle this challenge. Based on this observation, this study was launched to investigate the effects of incorporating machine learning based prediction models to further screen those patients with a negative NS1 test result. The experimental results revealed that the proposed approach was able to identify over 99% of the patients who were infected by the dengue disease. Furthermore, the risk of the suspected patients who were predicted to be positive was 4.8 times higher than the risk of those who were predicted to be negative. The experimental results illustrate that the proposed approach provides an effective and efficient diagnosis procedure to enhance surveillance of the dengue disease.

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.

The inositol triphosphate-associated, ER transmembrane proteins IRAG and LRMP are isoform specific regulators of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, while IRAG mimics the effect of cAMP on the channel. We previously showed that regulation by LRMP requires both the N-terminus of HCN4 and a unique orientation of the HCN4 cAMP transduction center, which is comprised of the N-terminal HCN domain, the C-linker, and the S4-S5 linker. However, it remains unknown if the homologous IRAG requires similar structural features to mimic cAMP-dependent potentiation, or if the site and mechanism of action are different between the two regulators. Using patch clamp electrophysiology, we determined that the initial 43 amino acids of IRAG are necessary and sufficient to confer regulation of HCN4. Similar to LRMP, IRAG also requires a portion of the N-terminus of HCN4 to confer its regulatory effects. Also similar to LRMP, two point mutations in the C-linker region, which are the only sequence differences in that region between HCN4 and the other HCN isoforms, were able to eliminate the effect of IRAG suggesting the unique orientation of the cAMP transduction center in HCN4 is likely important for IRAG function. Taken together, these findings suggest a model whereby IRAG and LRMP interact with the channel in similar regions, although potentially in unique ways, and act on the cAMP transduction center with LRMP inhibiting the coupling of this region to gating and IRAG strengthening it. SUMMARYThe ER transmembrane protein IRAG binds to and potentiates HCN4 channels. This study demonstrates that IRAG regulation of HCN4 requires only the first 43 amino acids of IRAG and involves contributions from the N-terminus and cAMP transduction center of HCN4.

Administration of ciliary neurotrophic factor (CNTF) reduces food intake and body weight in both humans and experimental animals, where it also ameliorates hyperglycemia, hyperinsulinemia, and dyslipidemia. To exert its anti-obesogenic and anti-diabetogenic effects, CNTF targets brain feeding centers as well as multiple peripheral organs inducing the phosphorylation of the transcription factor signal transducer and activator of transcription 3 (p-STAT3). However, data showing which peripheral cytotypes are specifically targeted by exogenous CNTF in vivo in metabolically relevant organs are currently lacking. Here, we first evaluated the gene expression levels of the subunits of the tripartite CNTF receptor (Cntfr) complex, i.e., the Cntfr, the leukemia inhibitory factor receptor {beta} (Lifr{beta}) and the glycoprotein 130 (gp130), by quantitative real-time PCR in metabolically relevant organs of adult male mice: gastrointestinal (GI) tract, pancreas, liver, visceral and subcutaneous white (WAT) and interscapular brown adipose tissue (iBAT), skeletal muscle and the sciatic nerve. We then quantified p-STAT3 by Western blotting in these organs after intraperitoneal administration of CNTF (0.3 mg/kg) or saline. Finally, we mapped CNTF-responsive cells by immunohistochemistry, followed by morphometric quantification and confocal microscopy in both CNTF- and saline-treated mice. Lifr{beta} and gp130 were ubiquitously detected across all the investigated organs; the Cntfr showed the highest expression levels in the skeletal muscle, sciatic nerve, and iBAT, whereas it was found to be expressed to a lesser extent in the other sites. Administration of CNTF led to a significant increase of p-STAT3/STAT3 protein ratio in all organs examined, except the duodenum, and induced a distinctive pattern of cell nuclear p-STAT3 immunoreactivity. Notably, along the analyzed GI tract CNTF induced nuclear STAT3 phosphorylation in neurons of the submucosal and myenteric plexuses of the enteric nervous system and in contractile cells of the muscularis externa, where the response peaked in the mesenteric gut and colon. In the pancreas, CNTF triggered a higher activation within the endocrine component compared to the exocrine parenchyma. In the liver, CNTF induced STAT3 phosphorylation not only in parenchymal cells but also in sinusoids and resident macrophages. The cytokine activated p-STAT3 in subcutaneous and visceral white adipocytes, but also in brown adipocytes, with a prominent response observed in the beige subcutaneous adipocytes; adipose resident macrophages and endothelial cells of numerous blood vessels were also CNTF-responsive. Lastly, in skeletal muscle, a major site for glucose/lipid utilization, CNTF induced widespread nuclear p-STAT3 immunoreactivity in muscle fibers and in connective and Schwann cells of the peripheral nerves, including the sciatic nerve, supplying the gastrocnemius. In conclusion, our data indicate that CNTF acts across diverse cytotypes within metabolically relevant organs and tissues, likely fostering its peripheral metabolic effects through this cellular heterogeneity.

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.

BackgroundThe Hump-nosed pit viper is a recognized but neglected medically significant species causing morbidity and mortality, with non-availability of a specific antivenom. There are many gaps in our understanding of its envenomation, including burden, clinical syndrome, complications and management. MethodologyThe study is a retrospective sub analysis of the Prospective VENOMS registry and hospital records of Hump Nosed Pit Viper envenomation from a single tertiary care center in coastal Karnataka from May 2018 to March 2024. Epidemiology, syndrome, complications and treatment strategies have been described. A linear mixed model analysis was conducted to study the effect of different therapeutic interventions in combating venom induced consumptive coagulopathy (VICC) Principal FindingsOf 46 cases, 24 patients had VICC. The most common complications were AKI (21.7%), TMA (10.9%) and stroke (4.4%). Anaphylaxis to ASV (23.9%) was the most common therapeutic complication. Therapeutic interventions included ASV, administration of blood products and therapeutic plasma exchange along with supportive care. The linear mixed model revealed that administration of blood products (p=<0.001) had the strongest influence on the INR value, however, often resulting in a transient decline in INR value. ASV (p=0.052) caused only marginally significant change in INR. The role of TPE could not be statistically inferred, however, individual cases with severe VICC improved without complications, therefore it required further study but can be considered in critical cases. Conclusions/SignificanceThis study describes the syndrome of hump-nosed pit viper envenomation, while highlighting the urgent need for a species-specific antivenom, recommends treatment strategies that can be used in the interim. Additionally, geo-spatial mapping draws attention to hotspots and the hypothesis that HNPV in coastal Karnataka have regionally distinct toxicity trends. Author SummaryIndia is often known as the snakebite capital of the world, with recent literature suggesting that not all death and disability is attributable to the "Big four" and highlighting regionally significant species of snake. In the Western Ghat region of India, envenomation by the Hump-nosed pit viper is increasingly being reported. In coastal Karnataka, it has been reported as the second most common cause of envenomation following the Russells Viper, causing systemic envenomation and death. However, little is known about why envenomation is common in this region, is it increasing, how to diagnose envenomation, its clinical syndrome, the anticipated complications, and most importantly, an effective treatment strategy. This study reports envenomation in 46 patients, resulting in 3 deaths, and 24 patients developing derangement in coagulation parameters.

The voltage-gated potassium channel hERG (Kv11.1) plays a central role in cardiac repolarisation by mediating the rapid delayed rectifier K+ current (IKr). Blockage of hERG by small molecules can lead to delayed repolarisation, QT interval prolongation, and potentially fatal arrhythmias, making the channel a critical focus in drug safety screening. Despite extensive pharmacological and electrophysiological characterisation, a complete structural understanding of hERG gating remains limited by the absence of an experimentally determined closed-state structure. Here, we use AI-based structural modelling to predict and compare candidate closed conformations of hERG. Building on recent work in which AlphaFold2 (AF2) predictions guided by engineered structural templates captured closed and inactivated states, we applied the emerging protein structure predictor, Chai-1, which employs a single-sequence, language model-based approach independent of multiple-sequence alignments. The resulting Chai-1 hERG model was compared with the AF2-derived closed structure, a homology model based on the Rattus norvegicus EAG channel, and an experimentally resolved open-state cryo-EM structure. We assessed these models using a combination of all-atom and coarse-grained molecular dynamics simulations, analysing protein dynamics, pore geometry, gating residue orientation, hydration, and lipid interactions. The Chai-1 and AF2 models displayed strong structural and dynamic agreement, both adopting compact, non-conductive conformations consistent with a physiologically closed state. Our data reveal insights into VSD dynamics, as well as suggesting a state dependence for ceramide binding at the previously identified M651 residue. Our findings support the validity of AI-derived closed-state hERG models and underscore the growing potential of deep learning-based protein structure prediction to identify previously uncharacterised, pharmacologically relevant conformations of membrane proteins. Further, our Chai-1 derived closed state model expands our structural insights into hERG gating and may have utility for investigation of drug-hERG interactions.

Infiltration of conventional immune cells has been ascribed as the fundamental drivers of innate immune signaling in the damaged myocardium. However, the emerging intrinsic immunoregulatory potential of cardiomyocytes still remains poorly understood. Interferon gamma (IFN{gamma}) is a pleiotropic cytokine with context-dependent detrimental as well protective role in regulating cardiac inflammatory circuits. The prevailing view of IFN{gamma} as a prime pro-inflammatory cytokine has been challenged due to its paradoxical actions both as an inducer as well as negative regulator of inflammation, but the players involved in these converse processes remains enigmatic. Here we show that cardiomyocytes exhibit a cell-autonomous immunocompetent response upregulating innate inflammatory signaling upon type I and type II IFN stimulus. Notably, hiPSC-derived cardiomyocytes display a robust increase in guanylate binding protein 5 (GBP5), one of the major IFN{gamma}-induced GTPase involved in inflammasome signaling, followed by upregulation of AIM2/CASP1 pathway whereas NLRP3 levels remain unaltered by IFN{gamma} stimulation. GBP5 knockdown and overexpression studies in hiPSC-derived cardiomyocytes identify GBP5/TGF{beta} axis as a non-canonical anti-inflammatory feedback regulation on the IFN{gamma}-induced inflammatory cascade.

The KCNE (KCNE1-6) proteins are single-pass transmembrane auxiliary subunits of the voltage-gated K+ channel KCNQ1. KCNQ1-KCNE complexes have been well studied in jawed vertebrates ranging from zebrafish to humans, but KCNE subunits from earlier-diverging vertebrates remain poorly characterized. Here, we functionally characterize a single KCNE-like gene in lamprey, a jawless vertebrate, and designate it kcne0 as an early-diverging member of the KCNE family. KCNE0 shows moderate amino acid sequence similarity to KCNE1-6 but is not particularly similar to any single isoform. Both kcnq1 and kcne0 transcripts were detected in multiple lamprey organs. When co-expressed with lamprey KCNQ1, KCNE0 produced a constitutively active current, similar to KCNE3. By contrast, KCNE0 modulated KCNQ1 from other species less effectively, suggesting species-specific tuning of KCNQ1-KCNE compatibility. Introducing into KCNE0 an intracellular tetra-leucine motif analogous to that in KCNE4 markedly reduced KCNQ1 current amplitude, conferring a KCNE4-like inhibitory effect. Overall, this work provides a functional reference for comparing KCNE-dependent modulation of KCNQ1 across vertebrates and suggests an underlying compatibility mechanism.

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.

BackgroundDengue virus infection remains a significant public health concern in India, with changing serotype dynamics influencing disease epidemiology. Understanding local serotype distribution and clinical characteristics is crucial for effective disease management and surveillance. ObjectivesTo determine the prevalence of dengue virus serotypes and analyze their clinical characteristics among NS1-positive patients at a tertiary-care hospital in Gujarat, India. MethodsA cross-sectional study was conducted on NS1-positive dengue patients admitted to AIIMS Rajkot from September 2023 to November 2024. Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed for serotype identification. Clinical and demographic data were collected and analyzed. ResultsNS1-positive patients (70) were confirmed by RT-PCR. DENV-2 was the predominant serotype (53 cases, 75.7%), followed by DENV-1 and DENV-3 (7 cases each, 10.0%), and DENV-4 (2 cases, 2.9%). One co-infection case (DENV-2 + DENV-3) (1.4%) was identified. The mean age was 27.7 {+/-} 14.4 years, with male predominance (58.6%). Young adults (19-35 years) were most affected (45.7%), followed by pediatric patients [&le;]18 years (32.9%). Severe dengue occurred in only one case (1.4%), while hospitalization was required in 25 cases (35.7%). All patients presented with fever, chills, headache (50%), rashes (56%), and malaise (56%), being the most common associated symptoms. ConclusionsDENV-2 showed clear predominance in the Rajkot region during the study period, with low rates of severe disease. The significant pediatric and young adult involvement highlights the need for targeted prevention strategies. These findings contribute to the understanding of regional dengue epidemiology and support evidence-based surveillance and control measures.

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.