Clofazimine broadly inhibits coronaviruses including SARS-CoV-2.

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.

Pseudorabies virus gM and its homologous proteins in herpesviruses induce mitochondria-related apoptosis involved in viral pathogenicity.

Apoptosis is a critical host antiviral defense mechanism. But many viruses have evolved multiple strategies to manipulate apoptosis and escape host antiviral immune responses. Herpesvirus infection regulated apoptosis; however, the underlying molecular mechanisms have not yet been fully elucidated. Hence, the present study aimed to study the relationship between herpesvirus infection and apoptosis in vitro and in vivo using the pseudorabies virus (PRV) as the model virus. We found that mitochondria-dependent apoptosis was induced by PRV gM, a late protein encoded by PRV UL10, a virulence-related gene involved in enhancing PRV pathogenicity. Mechanistically, gM competitively combines with BCL-XL to disrupt the BCL-XL-BAK complex, resulting in BCL-2-antagonistic killer (BAK) oligomerization and BCL-2-associated X (BAX) activation, which destroys the mitochondrial membrane potential and activates caspase-3/7 to trigger apoptosis. Interestingly, similar apoptotic mechanisms were observed in other herpesviruses (Herpes Simplex Virus-1 [HSV-1], human cytomegalovirus [HCMV], Equine herpesvirus-1 [EHV-1], and varicella-zoster virus [VZV]) driven by PRV gM homologs. Compared with their parental viruses, the pathogenicity of PRV-ΔUL10 or HSV-1-ΔUL10 in mice was reduced with lower apoptosis and viral replication, illustrating that UL10 is a key virulence-related gene in PRV and HSV-1. Consistently, caspase-3 deletion also diminished the replication and pathogenicity of PRV and HSV-1 in vitro and in mice, suggesting that caspase-3-mediated apoptosis is closely related to the replication and pathogenicity of PRV and HSV-1. Overall, our findings firstly reveal the mechanism by which PRV gM and its homologs in several herpesviruses regulate apoptosis to enhance the viral replication and pathogenicity, and the relationship between gM-mediated apoptosis and herpesvirus pathogenicity suggests a promising approach for developing attenuated live vaccines and therapy for herpesvirus-related diseases.

Exploiting the CD200-CD200R immune checkpoint axis in multiple myeloma to enhance CAR T-cell therapy.

ABSTRACT: Patients with multiple myeloma (MM) treated with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells usually relapse with BCMA+ disease, indicative of CAR T-cell suppression. CD200 is an immune checkpoint that is overexpressed on aberrant plasma cells (aPCs) in MM and is an independent negative prognostic factor for survival. However, CD200 is not present on MM cell lines, a potential limitation of current preclinical models. We engineered MM cell lines to express CD200 at levels equivalent to those found on aPCs in MM and show that these are sufficient to suppress clinical-stage CAR T-cells targeting BCMA or the Tn glycoform of mucin 1 (TnMUC1), costimulated by 4-1BB and CD2, respectively. To prevent CD200-mediated suppression of CAR T cells, we compared CRISPR-Cas9-mediated knockout of the CD200 receptor (CD200RKO), to coexpression of versions of the CD200 receptor that were nonsignaling, that is, dominant negative (CD200RDN), or that leveraged the CD200 signal to provide CD28 costimulation (CD200R-CD28 switch). We found that the CD200R-CD28 switch potently enhanced the polyfunctionality of CAR T cells, and improved cytotoxicity, proliferative capacity, CAR T-cell metabolism, and performance in a chronic antigen exposure assay. CD200RDN provided modest benefits, but surprisingly, the CD200RKO was detrimental to CAR T-cell activity, adversely affecting CAR T-cell metabolism. These patterns held up in murine xenograft models of plasmacytoma, and disseminated bone marrow predominant disease. Our findings underscore the importance of CD200-mediated immune suppression in CAR T-cell therapy of MM, and highlight a promising approach to enhance such therapies by leveraging CD200 expression on aPCs to provide costimulation via a CD200R-CD28 switch.

CLCF1 signaling restrains thermogenesis and disrupts metabolic homeostasis by inhibiting mitochondrial biogenesis in brown adipocytes.

Great progress has been made in identifying positive regulators that activate adipocyte thermogenesis, but negative regulatory signaling of thermogenesis remains poorly understood. Here, we found that cardiotrophin-like cytokine factor 1 (CLCF1) signaling led to loss of brown fat identity, which impaired thermogenic capacity. CLCF1 levels decreased during thermogenic stimulation but were considerably increased in obesity. Adipocyte-specific CLCF1 transgenic (CLCF1-ATG) mice showed impaired energy expenditure and severe cold intolerance. Elevated CLCF1 triggered whitening of brown adipose tissue by suppressing mitochondrial biogenesis. Mechanistically, CLCF1 bound and activated ciliary neurotrophic factor receptor (CNTFR) and augmented signal transducer and activator of transcription 3 (STAT3) signaling. STAT3 transcriptionally inhibited both peroxisome proliferator-activated receptor-γ coactivator (PGC) 1α and 1ß, which thereafter restrained mitochondrial biogenesis in adipocytes. Inhibition of CNTFR or STAT3 could diminish the inhibitory effects of CLCF1 on mitochondrial biogenesis and thermogenesis. As a result, CLCF1-TG mice were predisposed to develop metabolic dysfunction even without external metabolic stress. Our findings revealed a brake signal on nonshivering thermogenesis and suggested that targeting this pathway could be used to restore brown fat activity and systemic metabolic homeostasis in obesity.

The emerging roles of MARCH8 in viral infections: A double-edged Sword.

The host cell membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, regulates intracellular turnover of many transmembrane proteins and shows potent antiviral activities. Generally, 2 antiviral modes are performed by MARCH8. On the one hand, MARCH8 catalyzes viral envelope glycoproteins (VEGs) ubiquitination and thus leads to their intracellular degradation, which is the cytoplasmic tail (CT)-dependent (CTD) mode. On the other hand, MARCH8 traps VEGs at some intracellular compartments (such as the trans-Golgi network, TGN) but without inducing their degradation, which is the cytoplasmic tail-independent (CTI) mode, by which MARCH8 hijacks furin, a cellular proprotein convertase, to block VEGs cleavage. In addition, the MARCH8 C-terminal tyrosine-based motif (TBM) 222YxxL225 also plays a key role in its CTI antiviral effects. In contrast to its antiviral potency, MARCH8 is occasionally hijacked by some viruses and bacteria to enhance their invasion, indicating a duplex role of MARCH8 in host pathogenic infections. This review summarizes MARCH8's antiviral roles and how viruses evade its restriction, shedding light on novel antiviral therapeutic avenues.

Uncovering neural substrates across Alzheimer's disease stages using contrastive variational autoencoder.

Alzheimer's disease is the most common major neurocognitive disorder. Although currently, no cure exists, understanding the neurobiological substrate underlying Alzheimer's disease progression will facilitate early diagnosis and treatment, slow disease progression, and improve prognosis. In this study, we aimed to understand the morphological changes underlying Alzheimer's disease progression using structural magnetic resonance imaging data from cognitively normal individuals, individuals with mild cognitive impairment, and Alzheimer's disease via a contrastive variational autoencoder model. We used contrastive variational autoencoder to generate synthetic data to boost the downstream classification performance. Due to the ability to parse out the nonclinical factors such as age and gender, contrastive variational autoencoder facilitated a purer comparison between different Alzheimer's disease stages to identify the pathological changes specific to Alzheimer's disease progression. We showed that brain morphological changes across Alzheimer's disease stages were significantly associated with individuals' neurofilament light chain concentration, a potential biomarker for Alzheimer's disease, highlighting the biological plausibility of our results.

Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis.

Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography-mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.

Hypersensitivity to ferroptosis in chromophobe RCC is mediated by a glutathione metabolic dependency and cystine import via solute carrier family 7 member 11.

Chromophobe (Ch) renal cell carcinoma (RCC) arises from the intercalated cell in the distal nephron. There are no proven treatments for metastatic ChRCC. A distinguishing characteristic of ChRCC is strikingly high levels of reduced (GSH) and oxidized (GSSG) glutathione. Here, we demonstrate that ChRCC-derived cells exhibit higher sensitivity to ferroptotic inducers compared with clear-cell RCC. ChRCC-derived cells are critically dependent on cystine via the cystine/glutamate antiporter xCT to maintain high levels of glutathione, making them sensitive to inhibitors of cystine uptake and cyst(e)inase. Gamma-glutamyl transferase 1 (GGT1), a key enzyme in glutathione homeostasis, is markedly suppressed in ChRCC relative to normal kidney. Importantly, GGT1 overexpression inhibits the proliferation of ChRCC cells in vitro and in vivo, suppresses cystine uptake, and decreases levels of GSH and GSSG. Collectively, these data identify ferroptosis as a metabolic vulnerability in ChRCC, providing a potential avenue for targeted therapy for these distinctive tumors.

Genome-wide survey of KT/HAK/KUP genes in the genus Citrullus and analysis of their involvement in K+-deficiency and drought stress responses in between C. lanatus and C. amarus.

BACKGROUND: The KT/HAK/KUP is the largest K+ transporter family in plants, playing crucial roles in K+ absorption, transport, and defense against environmental stress. Sweet watermelon is an economically significant horticultural crop belonging to the genus Citrullus, with a high demand for K+ during its growth process. However, a comprehensive analysis of the KT/HAK/KUP gene family in watermelon has not been reported. RESULTS: 14 KT/HAK/KUP genes were identified in the genomes of each of seven Citrullus species. These KT/HAK/KUPs in watermelon were unevenly distributed across seven chromosomes. Segmental duplication is the primary driving force behind the expansion of the KT/HAK/KUP family, subjected to purifying selection during domestication (Ka/Ks < 1), and all KT/HAK/KUPs exhibit conserved motifs and could be phylogenetically classified into four groups. The promoters of KT/HAK/KUPs contain numerous cis-regulatory elements related to plant growth and development, phytohormone response, and stress response. Under K+ deficiency, the growth of watermelon seedlings was significantly inhibited, with cultivated watermelon experiencing greater impacts (canopy width, redox enzyme activity) compared to the wild type. All KT/HAK/KUPs in C. lanatus and C. amarus exhibit specific expression responses to K+-deficiency and drought stress by qRT-PCR. Notably, ClG42_07g0120700/CaPI482276_07g014010 were predominantly expressed in roots and were further induced by K+-deficiency and drought stress. Additionally, the K+ transport capacity of ClG42_07g0120700 under low K+ stress was confirmed by yeast functional complementation assay. CONCLUSIONS: KT/HAK/KUP genes in watermelon were systematically identified and analyzed at the pangenome level and provide a foundation for understanding the classification and functions of the KT/HAK/KUPs in watermelon plants.

Characterization of metallothionein genes from Broussonetia papyrifera: metal binding and heavy metal tolerance mechanisms.

BACKGROUND: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress. RESULTS: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues. CONCLUSION: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses.

Target-Triggered Multiple-Polarity-Switchable Multiplexed Photoelectrochemical Platform.

Convenient and accurate quantification of disease-relevant multitargets is essential for community disease screening. However, in the field of photoelectrochemical (PEC) sensors for multisubstance detection, research on the continuous detection of multiple targets using a polarity-switching mode is scarce. In this study, a multiplexed PEC bioassay was developed based on a target-triggered "anodic-cathodic-anodic" multiple-polarity-switchable mode. Employing miRNA-21 and miRNA-141 as model analytes, the photosensitive material combinations of Cu2O/gold nanoparticles (AuNPs)/TiO2 and CdS/AuNPs/TiO2 were successively formed through the specific binding of different whisker branches of Whisker-DNA to Cu2O-H1 and the CdS-tripod DNA ring, respectively. This process reverses the photocurrent polarity from anodic to cathodic and then back to anodic upon detecting different targets, resulting in the high-sensitivity quantification of various biological targets with reduced interference. To enhance the device's utility and affordability in community disease screening, integrating a capacitor and a multimeter-smartphone connection simplifies the assembly and reduces costs. In developing the PEC sensor, the device demonstrated linear detection ranges for miRNA-21 and miRNA-141 from 0.01 fM to 10 nM. Detection limits for miRNA-21 and miRNA-141 were established at 3.2 and 4.3 aM, respectively. The innovative target-triggered multiple-polarity-switchable mode offers adaptability for other multitarget detections by simply modifying the structure of the whisker branches and the combination of photosensitive materials.

TaWRKY31, a novel WRKY transcription factor in wheat, participates in regulation of plant drought stress tolerance.

BACKGROUND: Wheat, a crucial food crop in China, is highly vulnerable to drought stress throughout its growth and development. WRKY transcription factors (TFs), being one of the largest families of TFs, play a vital role in responding to various abiotic stresses in plants. RESULTS: Here, we cloned and characterized the TF TaWRKY31 isolated from wheat. This TF, belonging to the WRKY II family, contains a WRKYGQK amino acid sequence and a C2H2-type zinc finger structure. TaWRKY31 exhibits tissue-specific expression and demonstrates responsiveness to abiotic stresses in wheat. TaWRKY31 protein is localized in the nucleus and can function as a TF with transcription activating activity at the N-terminus. Results showed that the wheat plants with silenced strains (BSMV:TaWRKY31-1as and BSMV:TaWRKY31-2as) exhibited poor growth status and low relative water content when subjected to drought treatment. Moreover, the levels of O2·-, H2O2, and malondialdehyde (MDA) in the BSMV:TaWRKY31-induced wheat plants increased, while the activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) decreased. Compared to control plants, BSMV:TaWRKY31-induced wheat plants exhibited lower expression levels of TaSOD (Fe), TaPOD, TaCAT, TaDREB1, TaP5CS, TaNCED1, TaSnRK2, TaPP2C, and TaPYL5.Under stress or drought treatment conditions, the overexpression of TaWRKY31 in Arabidopsis resulted in decreased levels of H2O2 and MDA, as well as reduced stomatal opening and water loss. Furthermore, an increase in resistance oxidase activity, germination rate, and root length in the TaWRKY31 transgenic Arabidopsis was observed. Lastly, overexpression of TaWRKY31 in Arabidopsis resulted in higher the expression levels of AtNCED3, AtABA2, AtSnRK2.2, AtABI1, AtABF3, AtP5CS1, AtSOD (Cu/Zn), AtPOD, AtCAT, AtRD29A, AtRD29B, and AtDREB2A than in control plants. CONCLUSIONS: Our findings indicate that TaWRKY31 enhances drought resistance in plants by promoting the scavenging of reactive oxygen species, reducing stomatal opening, and increasing the expression levels of stress-related genes.

Investigating the nexus of metabolic syndrome, serum uric acid, and dementia risk: a prospective cohort study.

BACKGROUND: The global dementia prevalence is surging, necessitating research into contributing factors. We aimed to investigate the association between metabolic syndrome (MetS), its components, serum uric acid (SUA) levels, and dementia risk. METHODS: Our prospective study comprised 466,788 participants without pre-existing MetS from the UK Biobank. We confirmed dementia diagnoses based on the ICD-10 criteria (F00-03). To evaluate the dementia risk concerning MetS, its components, and SUA levels, we applied Cox proportional hazards models, while adjusting for demographic factors. RESULTS: Over a median follow-up of 12.7 years, we identified 6845 dementia cases. Individuals with MetS had a 25% higher risk of all-cause dementia (hazard ratio [HR] = 1.25, 95% confidence interval [CI] = 1.19-1.31). The risk increased with the number of MetS components including central obesity, dyslipidemia for high-density lipoprotein (HDL) cholesterol, hypertension, hyperglycemia, and dyslipidemia for triglycerides. Particularly for those with all five components (HR = 1.76, 95% CI = 1.51-2.04). Dyslipidemia for HDL cholesterol, hypertension, hyperglycemia, and dyslipidemia for triglycerides were independently associated with elevated dementia risk (p < 0.01). MetS was further linked to an increased risk of all-cause dementia (11%) and vascular dementia (VD, 50%) among individuals with SUA levels exceeding 400 µmol/L (all-cause dementia: HR = 1.11, 95% CI = 1.02-1.21; VD: HR = 1.50, 95% CI = 1.28-1.77). CONCLUSIONS: Our study provides robust evidence supporting the association between MetS, its components, and dementia risk. These findings emphasize the importance of considering MetS and SUA levels in assessing dementia risk, offering valuable insights for prevention and management strategies.

Nine months of bedaquiline, linezolid, levofloxacin, clofazimine, and cycloserine chemotherapy for rifampicin/multidrug-resistant tuberculosis: a multicenter, randomized, open-label non-inferiority trial in China.

BACKGROUND: We concurrently developed a prospective study to assess clinical outcomes among patients receiving 9-month bedaquiline (BDQ)-containing regimens, aiming to provide valuable data on the use of this short-course regimen in China. METHODS: This open-label, randomized, controlled, multicenter, non-inferiority trial was conducted at sixteen hospitals, and enrolled participants aged 18 years and older with pulmonary rifampicin/multidrug tuberculosis. Participants were randomly assigned, in a 1:1 ratio. Individuals within the standard-regimen group received 6 months of BDQ, linezolid, levofloxacin, clofazimine, and cycloserine plus 12 months of levofloxacin, and any three potentially effective drugs from clofazimine, cycloserine pyrazinamide, ethambutol and protionamide, whereas individuals within shorter-regimen group received 9 months of BDQ, linezolid, levofloxacin, clofazimine and cycloserine. The primary outcome was the percentage of participants with a composite unfavorable outcome (treatment failure, death, treatment discontinuation, or loss to follow-up) by the end of the treatment course after randomization in the modified intention-to-treat population. The noninferiority margin was 10%. This trial was registered with www.chictr.org.cn , ChiCTR2000029012. RESULTS: Between Jan 1, 2020, and Dec 31, 2023, 264 were screened and randomly assigned, 132 of 264 participants were assigned to the standard-regimen group and 132 were assigned to the shorter-regimen. Thirty-three (12.55%) of 264 participants were excluded from the modified intention-to-treat analysis. As a result, 231 participants were included in the modified intention-to-treat analysis (116 in the standard-regimen group and 115 in the shorter-regimen group).In the modified intention-to-treat population, unfavorable outcomes were reported in 19 (16.5%) of 115 participants for whom the outcome was assessable in the shorter-regimen group and 26 (22.4%) of 116 participants in the standard care group (risk difference 5.9 percentage points (97.5% CI - 5.8 to 17.5)). One death was reported in the standard-regimen group. The incidence of QTcF prolongation in the shorter-regimen group (22.6%, 26/115) was similar to the standard-regimen group (24.1%, 28/116). CONCLUSIONS: The 9-month, all-oral regimen is safe and efficacious for the treatment of pulmonary rifampicin/multidrug-resistant tuberculosis. The high incidence of QTc prolongation associated with the use of BDQ highlights the urgent need of routine electrocardiogram monitoring under treatment with BDQ-containing regimens in the Chinese population.

Chicken or Porcine Aminopeptidase N Mediates Cellular Entry of Pseudoviruses Carrying Spike Glycoprotein from the Avian Deltacoronaviruses HKU11, HKU13, and HKU17.

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Microglia synchronizes with the circadian rhythm of the glymphatic system and modulates glymphatic system function.

Microglia, as immune cells in the central nervous system, possess the ability to adapt morphologically and functionally to their environment. Glymphatic system, the principal waste clearance system in the brain, exhibits circadian rhythms. However, the impact of microglia on the glymphatic system function remains unknown. In this study, we explored the intricate relationship between microglia and the glymphatic system. Examining diurnal patterns, we identified synchronized behaviors in glymphatic activity and microglial morphology, peaking during sleep and exhibiting distinct changes in branching complexity. Depleting microglia using PLX5622 or in P2Y12 knockout mice enhanced glymphatic function. Chemogenetic manipulation of microglia demonstrated that activating HM3D improved glymphatic function, while inhibiting HM4D unexpectedly increased microglial complexity. These findings highlight the dynamic influence of microglia on the glymphatic system.

Vesicle-associated membrane protein 8 knockdown exerts anti-proliferative, pro-apoptotic, anti-autophagic, and pro-ferroptotic effects on colorectal cancer cells by inhibition of the JAK/STAT3 pathway.

Vesicle-associated membrane protein 8 (VAMP8), a soluble n-ethylmaleimide-sensitive factor receptor protein, acts as an oncogenic gene in the progression of several malignancies. Nevertheless, the roles and mechanisms of VAMP8 in colorectal cancer (CRC) progression remain unknown. The expression and prognostic significance of VAMP8 in CRC samples were analyzed through bioinformatics analyses. Cell proliferation was detected using CCK-8 and EdU incorporation assays and apoptosis was evaluated via flow cytometry. Western blot analysis was conducted to examine the protein expression. Ferroptosis was evaluated by measurement of iron metabolism, lipid peroxidation, and glutathione (GSH) content. VAMP8 was increased in CRC samples relative to normal samples on the basis of GEPIA and HPA databases. CRC patients with high level of VAMP8 had a worse overall survival. VAMP8 depletion led to a suppression of proliferation and promotion of apoptosis in CRC cells. Additionally, VAMP8 knockdown suppressed beclin1 expression and LC3-II/LC3-I ratio, elevated p62 expression, increased Fe2+, labile iron pool, lipid reactive oxygen species, and malondialdehyde levels, and repressed GSH content and glutathione peroxidase activity. Moreover, VAMP8 knockdown inhibited the activation of janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in CRC cells. Mechanistically, activation of the JAK/STAT3 pathway by JAK1 or JAK2 overexpression attenuated VAMP8 silencing-mediated anti-proliferative, pro-apoptotic, anti-autophagic, and pro-ferroptotic effects on CRC cells. In conclusion, VAMP8 knockdown affects the proliferation, apoptosis, autophagy, and ferroptosis by the JAK/STAT3 pathway in CRC cells.

Human MARCH1, 2, and 8 block Ebola virus envelope glycoprotein cleavage via targeting furin P domain.

Membrane-associated RING-CH (MARCH) family proteins were recently reported to inhibit viral replication through multiple modes. Previous work showed that human MARCH8 blocked Ebola virus (EBOV) glycoprotein (GP) maturation. Our study here demonstrates that human MARCH1 and MARCH2 share a similar pattern to MARCH8 in restricting EBOV GP-pseudotyped viral infection. Human MARCH1 and MARCH2 retain EBOV GP at the trans-Golgi network, reduce its cell surface display, and impair EBOV GP-pseudotyped virions infectivity. Furthermore, we uncover that the host proprotein convertase furin could interact with human MARCH1/2 and EBOV GP intracellularly. Importantly, the furin P domain is verified to be recognized by MARCH1/2/8, which is critical for their blocking activities. Besides, bovine MARCH2 and murine MARCH1 also impair EBOV GP proteolytic processing. Altogether, our findings confirm that MARCH1/2 proteins of different mammalian origins showed a relatively conserved feature in blocking EBOV GP cleavage, which could provide clues for subsequent MARCHs antiviral studies and may facilitate the development of novel strategies to antagonize enveloped virus infection.

The effect of non-insulin-based insulin resistance indices on the prediction of recurrence in patients with atrial fibrillation undergoing radiofrequency catheter ablation.

BACKGROUND: Atrial fibrillation (AF) is acknowledged as a disease continuum. Despite catheter ablation being recommended as a primary therapy for AF, the high recurrence rates have tempered the initial enthusiasm. Insulin resistance (IR) has been established as an independent predictor for the onset of AF. However, the correlation between non-insulin-based IR indices and late AF recurrence in patients undergoing radiofrequency catheter ablation remains unknown. METHODS: A retrospective cohort of 910 AF patients who underwent radiofrequency catheter ablation was included in the analysis. The primary endpoint was late AF recurrence during the follow-up period after a defined blank period. The relationship between non-insulin-based IR indices and the primary endpoint was assessed using multivariate Cox hazards regression models and restricted cubic splines (RCS). Additionally, the net reclassification improvement and integrated discrimination improvement index were calculated to further evaluate the additional predictive value of the four IR indices beyond established risk factors for the primary outcome. RESULTS: During a median follow-up period of 12.00 months, 189 patients (20.77%) experienced late AF recurrence, which was more prevalent among patients with higher levels of IR. The multivariate Cox hazards regression analysis revealed a significant association between these IR indices and late AF recurrence. Among the four indices, METS-IR provided the most significant incremental effect on the basic model for predicting late AF recurrence. Multivariable-adjusted RCS curves illustrated a nonlinear correlation between METS-IR and late AF recurrence. In subgroup analysis, METS-IR exhibited a significant correlation with late AF recurrence in patients with diabetes mellitus (HR: 1.697, 95% CI 1.397 - 2.063, P < 0.001). CONCLUSION: All the four non-insulin-based IR indices were significantly associated with late AF recurrence in patients undergoing radiofrequency catheter ablation. Addressing IR could potentially serve as a viable strategy for reducing the late AF recurrence rate.

Highly Stable Beneficiary Attribution in Medicare's Comprehensive Primary Care Plus Model.

BACKGROUND: Advanced primary care models are key in moving primary care practices toward greater accountability for the quality and cost of a beneficiary's care. One critical but often overlooked detail in model design is the beneficiary attribution methodology. Attribution results are key inputs in calculating practice payments. Stable attribution yields predictable practice payments, fostering longer-term investments in advanced primary care. OBJECTIVE: We examine attribution stability for Medicare fee-for-service beneficiaries in Medicare's Comprehensive Primary Care Plus (CPC+) Model. DESIGN: To measure attribution stability, we calculate churn rates, which we define as the percentage of beneficiaries eligible for CPC+ who were not attributed to the same practice in a later period. Using 2017-2021 CPC+ program data and Medicare administrative data, we calculate churn rates for CPC+ overall and for beneficiary subgroups. To assess whether CPC+ attribution was responsive enough to changes in a beneficiary's practice, we calculate how long before attribution changes following a beneficiary's long-distance move. RESULTS: We find that for every 100 beneficiaries attributed to a CPC+ practice, 88 were still attributed to the same practice a year later (ie, churn rate of 12%), 79 were attributed 2 years later, 74 three years later, and 70 four years later. However, some vulnerable subgroups, such as disabled beneficiaries, had higher churn rates. Our analysis of long-distance movers reveals that only after 5 quarters did attribution change for more than half of these movers. CONCLUSIONS: Overall, high attribution stability may have encouraged CPC+ practices to make longer-term investments in advanced primary care.