A full-length glycoprotein mRNA vaccine confers complete protection against severe fever with thrombocytopenia syndrome virus, with broad-spectrum protective effects against bandaviruses.

Highly pathogenic viruses from family Phenuiviridae, which are mainly transmitted by arthropods, have intermittently sparked epidemics worldwide. In particular, tick-borne bandaviruses, such as severe fever with thrombocytopenia syndrome virus (SFTSV), continue to spread in mountainous areas, resulting in an average mortality rate as high as 10.5%, highlighting the urgency and importance of vaccine development. Here, an mRNA vaccine developed based on the full-length SFTSV glycoprotein, containing both the receptor-binding domain and the fusion domain, was shown to confer complete protection against SFTSV at a very low dose by triggering a type 1 helper T cell-biased cellular immune response in rodents. Moreover, the vaccine candidate elicited long-term immunity and protection against SFTSV for at least 5 months. Notably, it provided complete cross-protection against other bandaviruses, such as the Heartland virus and Guertu virus, in lethal challenge models. Further research revealed that the conserved epitopes among bandaviruses within the full-length SFTSV glycoprotein may facilitate broad-spectrum protection mediated by the cellular immune response. Collectively, these findings demonstrate that the full-length SFTSV glycoprotein mRNA vaccine is a promising vaccine candidate for SFTSV and other bandaviruses, and provide guidance for the development of broad-spectrum vaccines from conserved antigens and epitopes. IMPORTANCE: Tick-borne bandaviruses, such as SFTSV and Heartland virus, sporadically trigger outbreaks in addition to influenza viruses and coronaviruses, yet there are no specific vaccines or therapeutics against them. mRNA vaccine technology has advantages in terms of enabling in situ expression and triggering cellular immunity, thus offering new solutions for vaccine development against intractable viruses, such as bandaviruses. In this study, we developed a novel vaccine candidate for SFTSV by employing mRNA vaccination technology and using a full-length glycoprotein as an antigen target. This candidate vaccine confers complete and durable protection against SFTSV at a notably low dose while also providing cross-protection against Heartland virus and Guertu virus. This study highlights the prospective value of full-length SFTSV-glycoprotein-based mRNA vaccines and suggests a potential strategy for broad-spectrum bandavirus vaccines.

Identification of tanshinone I as cap-dependent endonuclease inhibitor with broad-spectrum antiviral effect.

IMPORTANCE: The spread of avian-borne, tick-borne, and rodent-borne pathogens has the potential to pose a serious threat to human health, and candidate vaccines as well as therapeutics for these pathogens are urgently needed. Tanshinones, especially tanshinone I, were identified as a cap-dependent endonuclease inhibitor with broad-spectrum antiviral effects on negative-stranded, segmented RNA viruses including bandavirus, orthomyxovirus, and arenavirus from natural products, implying an important resource of candidate antivirals from the traditional Chinese medicines. This study supplies novel candidate antivirals for the negative-stranded, segmented RNA virus and highlights the endonuclease involved in the cap-snatching process as a reliable broad-spectrum antiviral target.

Protection of the receptor binding domain (RBD) dimer against SARS-CoV-2 and its variants.

IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants achieved immune escape and became less virulent and easily transmissible through rapid mutation in the spike protein, thus the efficacy of vaccines on the market or in development continues to be challenged. Updating the vaccine, exploring compromise vaccination strategies, and evaluating the efficacy of candidate vaccines for the emerging variants in a timely manner are important to combat complex and volatile SARS-CoV-2. This study reports that vaccines prepared from the dimeric receptor-binding domain (RBD) recombinant protein, which can be quickly produced using a mature and stable process platform, had both good immunogenicity and protection in vivo and could completely protect rodents from lethal challenge by SARS-CoV-2 and its variants, including the emerging Omicron XBB.1.16, highlighting the value of dimeric recombinant vaccines in the post-COVID-19 era.

Advances in mRNA vaccines for viral diseases.

Since the onset of the pandemic caused by severe acute respiratory syndrome coronavirus 2, messenger RNA (mRNA) vaccines have demonstrated outstanding performance. mRNA vaccines offer significant advantages over conventional vaccines in production speed and cost-effectiveness, making them an attractive option against other viral diseases. This article reviewed recent advances in viral mRNA vaccines and their delivery systems to provide references and guidance for developing mRNA vaccines for new viral diseases.

Prevalence and risk factors for depression and anxiety in adult patients with epilepsy: Caregivers' anxiety and place of residence do mater.

OBJECTIVES: This study was aimed to assess the prevalence of anxiety and depression and the possible contributions of the caregiver's anxiety and depression, disease status, and socio-demographic characteristics to psychopathological comorbidities among adult patients with epilepsy. METHODS: A total of 262 participants (131 adult patient-caregiver pairs) were enrolled in this study. The Hamilton Rating Scale for Depression (HAM-D) and the Hamilton Rating Scale for Anxiety (HAM-A) were applied to evaluate the depression and anxiety status among adult patients with epilepsy and their caregivers, respectively. We collected caregivers' anxiety and depression, patients' sociodemographic characteristic data, and disease status as independent variables using stepwise multiple linear regression analysis that were correlated to the degree of anxiety and depression among these adult patients with epilepsy. RESULTS: Among adult patients with epilepsy, 46 (35.11%) subjects showed anxiety symptoms (HAM-A scores > 6), and 48 (36.64%) had depression symptoms (HAM-D scores > 6). Caregivers' anxiety levels and place of residence were significant independent predictors of both anxiety and depression levels among adult patients with epilepsy. CONCLUSIONS: Adult patients with epilepsy are at a high risk of suffering from anxiety and depression. Caregivers' anxiety and place of residence are definite independent predictors for anxiety and depression severity among adult patients with epilepsy. Therefore, clinicians should be careful in closely monitoring the psychological status of adult patients with epilepsy and their caregivers. Furthermore, the government and medical institutions should increase educational awareness about epilepsy and its cure, especially among adult patients with epilepsy who live in rural areas and consider offering a multidisciplinary management program to improve these patients' psychological status.

Seed coat mucilages: Structural, functional/bioactive properties, and genetic information.

Seed coat mucilages are mainly polysaccharides covering the outer layer of the seeds to facilitate seed hydration and germination, thereby improving seedling emergence and reducing seedling mortality. Four types of polysaccharides are found in mucilages including xylan, pectin, glucomannan, and cellulose. Recently, mucilages from flaxseed, yellow mustard seed, chia seed, and so on, have been used extensively in the areas of food, pharmaceutical, and cosmetics contributing to stability, texture, and appearance. This review, for the first time, addresses the similarities and differences in physicochemical properties, molecular structure, and functional/bioactive properties of mucilages among different sources; highlights their structure and function relationships; and systematically summarizes the related genetic information, aiming with the intent to explore the potential functions thereby extending their future industrial applications.

Predicting seizure freedom with AED treatment in newly diagnosed patients with MRI-negative epilepsy: A large cohort and multicenter study.

OBJECTIVE: We developed and validated a prediction score for predicting the probability of 6-month and 12-month seizure freedom of antiepileptic drug (AED) treatment in newly diagnosed patients with magnetic resonance imaging (MRI)-negative epilepsy. METHODS: The development cohort included 543 consecutive patients from the Epilepsy Center of Henan Provincial People's Hospital, while the validation cohorts included 493 consecutive patients in two independent cohorts. Univariate analysis and a forward and backward elimination of multivariate Cox regression analysis were used to select predictive factors. The performance of the score was evaluated with C-index, calibration plots, and decision curve analysis. The risk stratification was also performed. RESULTS: The score included five routinely available predictors including Circadian rhythms, Electroencephalography before AED treatment, Neuropsychiatric disorders, Perinatal brain injury, and History of central nervous system infection (CENPH score). When applied to the external validation cohort, the score showed good discrimination with C-index (development group: 0.83; validation group: 0.78), and calibration plots indicated well calibration, as well as the decision curve analysis showed good predictive accuracy and clinical values in four cohorts. The points of the score were categorized to the following three probability levels for predicting seizure freedom: high probability (0-83.11 points), medium probability (83.11-122.71 points), and low probability (>122.71 points). And online calculator was established to make this score easily applicable in clinical practice. CONCLUSIONS: We established a simple, practical, and evidence-based prediction score for predicting seizure freedom with AEDs to aid in the clinical consultation and treatment decision for the newly diagnosed patients with MRI-negative epilepsy.

A scale for prediction of response to AEDs in patients with MRI-negative epilepsy.

OBJECTIVES: Antiepileptic drugs (AEDs) are the first choice in magnetic resonance imaging (MRI)-negative patients with epilepsy, although the responses to AEDs are diverse. Preoperative evaluation and postoperative prognosis in MRI-negative epilepsy have been reported. However, there are few tools for predicting the response to AEDs. Herein, we developed an AED response scale based on clinical factors and video-electroencephalography (VEEG) in MRI-negative patients with epilepsy. METHODS: A total of 132 consecutive patients with MRI-negative epilepsy at the Epilepsy Center of Henan Provincial People's Hospital between August 2016 and August 2018 were included. Patients were further divided into drug-responsive epilepsy ([DSE-MRI (-)]; n = 101) and drug-resistant epilepsy ([DRE-MRI (-)]; n = 31) groups. The clinical and VEEG factors were evaluated in univariate analyses and multivariate logistic regression analyses. A scale was derived and the scores categorized into 3 risk levels of DRE-MRI (-). RESULTS: A scale was established based on 4 independent risk factors for DRE-MRI (-). The scale had a sensitivity of 83.87%, specificity of 80.20%, positive likelihood ratio of 4.24, negative likelihood ratio of 0.20, and showed good discrimination with the area under the curve (AUC) of 0.886 (0.826-0.946). The categorization of the risk score based on this scale was: low risk (0-3 points), medium risk (3-5 points), and high risk (>5 points). CONCLUSION: We established a DRE-MRI (-) scale with a good sensitivity and specificity, which may be useful for clinicians when making medical decisions in patients with MRI-negative epilepsy.

High risk of anxiety and depression in caregivers of adult patients with epilepsy and its negative impact on patients' quality of life.

OBJECTIVE: The objective of this study was to assess the anxiety and depression of caregivers of adult patients with epilepsy (PWE) and evaluate its effect on patient quality of life (QOL). METHOD: One hundred sixty pairs of adult PWE and their caregivers were enrolled in our study. Quality of life in adult PWE was evaluated with the Quality of Life in Epilepsy Inventory-31 scale (QOLIE-31). Symptoms of anxiety and depression in caregivers were assessed with the Hamilton Anxiety Rating Scale (HAM-A) and the Hamilton Depression Rating Scale (HAM-D) respectively. Correlation and stepwise multiple liner regression analyses were used as statistical analysis. RESULTS: Of the caregivers, 41 (31.30%) had anxiety symptoms (HAM-A scores > 6) and 44 (33.59%) had depression symptoms (HAM-D scores > 6). Caregiver anxiety was significantly associated with poorer adult PWE QOL scores in four of the seven subscales and the QOLIE-31 total score. Caregiver depression was significantly associated with poorer adult PWE QOL in all seven subscales as well as the QOLIE-31 total score. Caregiver depression was an independent predictor of the QOLIE-31 total score and five subscales: seizure worry, emotional wellbeing, energy/fatigue, cognitive, and medication effects. CONCLUSION: Caregivers of adult PWE are at high risk of experiencing anxiety and depression. Caregiver psychological status, especially depression, was an independent predictor of poorer QOL for adult PWE.

A competitive aptamer chemiluminescence assay for ochratoxin A using a single silica photonic crystal microsphere.

We designed a competitive aptamer chemiluminescence assay for ochratoxin A (OTA) on the surface of a single silica photonic crystal microsphere (SPCM) in cereal samples. The structural color of SPCMs is used to recognize and trace the microspheres during process of detection. Anti-aptamer was immobilized on the surface of SPCM. OTA and anti-aptamer competed to bind to aptamer when OTA and its aptamer (labeled by biotin at 5'end) were added in the system. The chemiluminescence signal was developed by the horseradish peroxidase (HRP), luminol and H2O2. The molecules on the single SPCM can produce enough chemiluminescence signal intensity for quantitative detection for OTA. The linear detection range for OTA was from 1 pg/mL to 1 ng/mL and recovery rates were 89%-95%, 81%-92% and 94%-105% in rice, wheat and corn, respectively. The results showed that the developed method for OTA using a single SPCM has a great application potential in cereal samples.

Personalized prediction model for seizure-free epilepsy with levetiracetam therapy: a retrospective data analysis using support vector machine.

AIMS: To predict the probability of a seizure-free (SF) state in patients with epilepsy (PWEs) after treatment with levetiracetam and to identify the clinical and electroencephalographic (EEG) factors that affect outcomes. METHODS: Retrospective analysis of PWEs treated with levetiracetam for 3 years identified 22 patients who were SF and 24 who were not. Before starting levetiracetam, 11 clinical factors and four EEG features (sample entropy of α, ß, θ, δ) were identified. Overall, 80% of each the two groups were chosen to establish a support vector machine (SVM) model with 5-fold cross-validation, hold-out validation and jack-knife validation. The other 20% were used to predict the efficacy of levetiracetam. The mean impact value (MIV) algorithm was used to rank the relativity between factors and outcomes. RESULTS: Compared with SF patients, not SF patients displayed a specific decrease in EEG sample entropy in α band from the F4 channel, ß band from Fp2 and F8 channels, θ band from C3 channel (P < 0.05). The SVM model based on the clinical and EEG features yielded 72.2% accuracy of 5-fold cross-validation, 75.0% accuracy of jack-knife validation, 67.7% accuracy of hold-out validation in the training set and had a high prediction accuracy of 90% in test set (sensitivity was 100%, area under the receiver operating characteristic curve was 0.96). The feature of ß band from Fp2 weighs heavily in the prediction model according to the mean impact value algorithm. CONCLUSIONS: The efficacy of levetiracetam on newly diagnosed PWEs could be predicted using an SVM model, which could guide antiepileptic drug selection.

High-Throughput Low-Background G-Quadruplex Aptamer Chemiluminescence Assay for Ochratoxin A Using a Single Photonic Crystal Microsphere.

We reported a novel hemin-G-quadruplex aptamer chemiluminescence assay platform for ochratoxin A (OTA) using the single silica photonic crystal microsphere (SPCM). The oligonucleotide A sequence containing aptamer sequences of hemin and OTA is immobilized on the surface of SPCM. The other oligonucleotide B sequence containing a partially complementary sequence with one part OTA aptamer and one part hemin aptamer is used as a blocking chain. The hybridization between chain A and chain B will be influenced by the presence or absence of OTA in the system, which will affect the bioactivity of DNAzyme. Thus, the chemiluminescence signal depends on the concentration of OTA in the samples. In the single particle assay platform, the signal/noise is remarkably enhanced, and the background signal can be ignored by separating hemin from the surface of SPCM. The limit of detection of the new method reaches to the pg/mL scale, and the linear detection range is 4 orders of magnitude for OTA. The new assay platform can provide a sensitive, cost-efficient, simple, and high-throughput screening for OTA.

Awareness, attitudes toward epilepsy, and first aid knowledge of seizures of hospital staff in Henan, China.

AIM: The aim of this study was to evaluate awareness of, attitudes toward, and first aid knowledge of seizures of hospital staff in Henan, China. METHOD: Two hundred nineteen hospital staff, including doctors, nurses, medical technicians, logisticians, and executives working at tertiary hospitals in Henan, China, completed the survey from March to September in 2016. The data comprised the demographic data section, awareness of epilepsy section, attitude toward epilepsy section, and first aid knowledge of seizure attack section. RESULTS: The participants obtained a mean score of 7.48±1.705 on the awareness of epilepsy section, and a mean score of 5.32±1.165 on the first aid knowledge of seizure attacks section. There were significant correlations between educational level (r=0.187, P=0.006), occupation (r=-0.244, P=0.000), and attitudes toward patients with epilepsy (r=0.351, P=0.000) with the awareness of epilepsy. There were significant correlations between age (r=0.170, P=0.014), educational status (r=0.139, P=0.040), and professional titles (r=0.197, P=0.004) with the first aid knowledge of seizures. CONCLUSION: The study showed that hospital staff had a moderate level of knowledge regarding epilepsy, and they generally displayed a positive attitude. It was also determined that as the awareness of epilepsy increased, they displayed more positive attitudes toward patients with epilepsy. The study also suggests that specialists working on epilepsy should provide more lectures and educational sessions to improve the knowledge of and attitude toward epilepsy and first aid knowledge of seizures among hospital staff.

GR1-like gene expression in Lycium chinense was regulated by cadmium-induced endogenous jasmonic acids accumulation.

KEY MESSAGE: The G1-like gene from the Lycium chinense was cloned and transferred into N. tabacum. Evidence showed that endogenous JA accumulation was crucial to LcGR gene expression in cadmium-stressed L. chinense. Glutathione reductase (GR) plays a vital role in glutathione-ascorbate metabolism and is a key enzyme in maintaining the redox state in plants. Jasmonic acids (JA) are important hormones regulating protective responses against bacteria and mechanic damage in plants. At present, the relationship between the endogenous JA accumulation, the glutathione (GSH) content and GR gene expression in plants under cadmium (Cd) stress has not been elucidated. This study primarily aims to explore their interconnected relations. First, we isolated the GR1-like gene from Lycium chinense (LcGR). Real-time PCR showed that gene LcGR and allene oxide cyclase (LcAOC) (a JA synthesis gene) expression in L. chinense plants was significantly enhanced by CdCl2 and reduced by CdCl2 cotreatment with 12,13-epoxy-octadecenoic acid (EOA), a JA synthesis inhibitor. Meanwhile, the JA content in plants strongly increased under Cd stress and decreased under Cd + EOA treatment, which was in accordance with expression pattern of LcAOC. The function of gene LcGR was confirmed in vitro with E. coli expression system. The subcellular localization in chloroplasts of LcGR gene was proved in Nicotiana tabacum leaves with transient transfection system of Agrobacterium tumefaciens. Furthermore, the overexpression of gene LcGR in the transgenic tabacum led to great Cd-tolerance and higher GSH accumulation. Overall, the results showed that the endogenous JA accumulation in Cd-stressed plants affects the GR expression which is crucial to the GSH accumulation and GSH-dependent tolerance to cadmium in LcGR transformants.

Exosomes mediate the antibody-resistant intercellular transmission of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes severe enteritis and lethal watery diarrhea in suckling piglets, leading to tremendous economic losses. Exosomes have been reported to participate in intercellular communication by the transportation of a variety of biological materials, including RNAs, lipids, and proteins. However, PEDV transmission routes have not yet been fully elucidated, and whether exosomes function in PEDV transmission remains unclear. In this study, we extracted and purified exosomes from PEDV-infected Vero cells using a stringent isolation method with a combination of chemical precipitation, ultracentrifugation, and incubation with CD63-labeled magnetic beads. We found that exosomes from PEDV-infected Vero cells contain viral genomic RNA and viral nucleocapsid protein. Furthermore, we demonstrated that the purified exosomes from PEDV-infected cells are capable of transmitting the virus to both PEDV-susceptible and non-susceptible cells. Importantly, exosome-mediated PEDV infection was resistant to neutralization by PEDV-specific neutralizing antibodies that potently neutralized free PEDV. Our study reveals a potential immune evasion mechanism utilized by PEDV and provides new insight into the transmission and infection of this important pathogen.

Porcine Epidemic Diarrhea Virus nsp7 Inhibits MDA5 Dephosphorylation to Antagonize Type I Interferon Production.

Porcine epidemic diarrhea virus (PEDV) is a reemerging enteropathogenic coronavirus that causes high mortality in piglets and has catastrophic effects on the global pig industry. PEDV-encoded nonstructural protein 7 (nsp7) is an important component of the viral replication and transcription complex, and a previous study reported that it inhibits poly(I:C)-induced type I interferon (IFN) production, but the mechanism by which this occurs remains unclear. Here, we demonstrated that ectopic expression of PEDV nsp7 antagonized Sendai virus (SeV)-induced interferon beta (IFN-ß) production, as well as the activation of transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB) in both HEK-293T and LLC-PK1 cells. Mechanistically, PEDV nsp7 targets melanoma differentiation-associated gene 5 (MDA5) and interacts with its caspase activation and recruitment domains (CARDs), which sequester the interactions between MDA5 and the protein phosphatase 1 (PP1) catalytic subunits (PP1α and PP1γ), thereby suppressing MDA5 S828 dephosphorylation and keeping MDA5 inactive. Furthermore, PEDV infection attenuated MDA5 multimerization and MDA5-PP1α/-γ interactions. We also tested the nsp7 orthologs of five other mammalian coronaviruses and found that all of them except severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp7 inhibited MDA5 multimerization and SeV- or MDA5-induced IFN-ß production. Collectively, these results suggest that the inhibition of MDA5 dephosphorylation and multimerization may be a common strategy employed by PEDV and some other coronaviruses to antagonize MDA5-mediated IFN production. IMPORTANCE Since late 2010, a reemerging porcine epidemic diarrhea virus variant with high pathogenesis has swept through most pig farms in many countries, resulting in significant economic losses. Coronavirus nonstructural protein 7 (nsp7), conserved within the family Coronaviridae, combines with nsp8 and nsp12 to form the viral replication and transcription complex that is indispensable for viral replication. However, the function of nsp7 in the infection and pathogenesis of coronaviruses remains largely unknown. Our present study demonstrates that PEDV nsp7 specifically competes with PP1 for binding MDA5 and impedes the PP1-mediated dephosphorylation of MDA5 at S828, thereby blocking MDA5-mediated IFN production, revealing the complex mechanism utilized by PEDV nsp7 to efficiently escape host innate immunity.

Porcine deltacoronavirus resists antibody neutralization through cell-to-cell transmission.

ABSTRACTPorcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that has been reported to infect a variety of animals and even humans. Cell-cell fusion has been identified as an alternative pathway for the cell-to-cell transmission of certain viruses, but the ability of PDCoV to exploit this transmission model, and the relevant mechanisms, have not been fully elucidated. Herein, we provide evidence that cell-to-cell transmission is the main mechanism supporting PDCoV spread in cell culture and that this efficient spread model is mediated by spike glycoprotein-driven cell-cell fusion. We found that PDCoV efficiently spread to non-susceptible cells via cell-to-cell transmission, and demonstrated that functional receptor porcine aminopeptidase N and cathepsins in endosomes are involved in the cell-to-cell transmission of PDCoV. Most importantly, compared with non-cell-to-cell infection, the cell-to-cell transmission of PDCoV was resistant to neutralizing antibodies and immune sera that potently neutralized free viruses. Taken together, our study revealed key characteristics of the cell-to-cell transmission of PDCoV and provided new insights into the mechanism of PDCoV infection.

Preparation and characterization of vaccarin, hypaphorine and chitosan nanoparticles and their promoting effects on chronic wounds healing.

Chronic wounds have become an important factor hindering human health, affecting tens of millions of people worldwide, especially diabetic wounds. Based on the antibacterial properties of chitosan, the angiogenesis promoting effect of vaccarin (VAC) and the anti-inflammatory effect of hypaphorine (HYP), nanoparticles with high bioavailability were prepared. VAC, HYP and chitosan nanoparticles (VAC + HYP-NPS) were used to the treatment of chronic wounds. Transmission electron microscopy (TEM) images showed the nanoparticles were spherical. ZetaPALS showed the potential of nanoparticles were -12.8 ± 5.53 mV and the size were 166.8 ± 29.95 nm. Methyl thiazolyl tetrazolium (MTT) assay showed that VAC + HYP-NPS had no toxicity and the biocompatibility was satisfactory. In the treatment of chronic wounds in diabetic rats, VAC + HYP-NPS significantly promoted the re-epithelialization of chronic wounds and accelerated the healing of chronic wounds. In the process of chronic wounds healing, VAC + HYP-NPS played the antibacterial effect of chitosan, the angiogenic effect of VAC and the anti-inflammatory effect of HYP, and finally promoted the chronic wounds healing. Overall, the developed VAC + HYP-NPS have potential application in chronic wounds healing. In view of the complexity of the causes of chronic wounds, multi-target drug administration may be an effective way to treat chronic wounds.

Porcine deltacoronavirus accessory protein NS7a possesses the functional characteristics of a viroporin.

Viroporins are virus-encoded proteins that mediate ion channel (IC) activity, playing critical roles in virus entry, replication, pathogenesis, and immune evasion. Previous studies have shown that some coronavirus accessory proteins have viroporin-like activity. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that encodes three accessory proteins, NS6, NS7, and NS7a. However, whether any of the PDCoV accessory proteins possess viroporin-like activity, and if so which, remains unknown. In this study, we analyzed the biochemical properties of the three PDCoV-encoded accessory proteins and found that NS7a could enhance the membrane permeability of both mammalian cells and Escherichia coli cells. Indirect immunofluorescence assay and co-immunoprecipitation assay results further indicated that NS7a is an integral membrane protein and can form homo-oligomers. A bioinformation analysis revealed that a putative viroporin domain (VPD) is located within amino acids 69-88 (aa69-88) of NS7a. Experiments with truncated mutants and alanine scanning mutagenesis additionally demonstrated that the amino acid residues 69FLR71 of NS7a are essential for its viroporin-like activity. Together, our findings are the first to demonstrate that PDCoV NS7a possesses viroporin-like activity and identify its key amino acid residues associated with viroporin-like activity.

Vaccarin alleviates endothelial inflammatory injury in diabetes by mediating miR-570-3p/HDAC1 pathway.

Vaccarin is a flavonoid glycoside, which has a variety of pharmacological properties and plays a protective role in diabetes and its complications, but its mechanism is unclear. In this study, we aim to investigate whether histone deacetylase 1(HDAC1), a gene that plays a pivotal role in regulating eukaryotic gene expression, is the target of miR-570-3p in diabetic vascular endothelium, and the potential molecular mechanism of vaccarin regulating endothelial inflammatory injury through miR-570-3p/HDAC1 pathway. The HFD and streptozotocin (STZ) induced diabetes mice model, a classical type 2 diabetic model, was established. The aorta of diabetic mice displayed a decrease of miR-570-3p, the elevation of HDAC1, and inflammatory injury, which were alleviated by vaccarin. Next, we employed the role of vaccarin in regulating endothelial cells miR-570-3p and HDAC1 under hyperglycemia conditions in vitro. We discovered that overexpression of HDAC1 counteracted the inhibitory effect of vaccarin on inflammatory injury in human umbilical vein endothelial cells (HUVECs). Manipulation of miRNA levels in HUVECs was achieved by transfecting cells with miR-570-3p mimic and inhibitor. Overexpression of miR-570-3p could decrease the expression of downstream components of HDAC1 including TNF-α, IL-1ß, and malondialdehyde, while increasing GSH-Px activity in HUVECs under hyperglycemic conditions. Nevertheless, such phenomenon was completely reversed by miR-570-3p inhibitor, and administration of miR-570-3p inhibitor could block the inhibition of vaccarin on HDAC1 and inflammatory injury. Luciferase reporter assay confirmed the 3'- UTR of the HDAC1 gene was a direct target of miR-570-3p. In summary, our findings suggest that vaccarin alleviates endothelial inflammatory injury in diabetes by mediating miR-570-3p/HDAC1 pathway. Our study provides a new pathogenic link between deregulation of miRNA expression in the vascular endothelium of diabetes and inflammatory injury and provides new ideas, insights, and choices for the scope of application and medicinal value of vaccarin and some potential biomarkers or targets in diabetic endothelial dysfunction and vascular complications.