Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection.

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-κB signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection.

NLRP2 in health and disease.

NLR family pyrin domain containing 2 (NLRP2) is a novel member of the Nod-like receptor (NLR) family. However, our understanding of NLRP2 has long been ambiguous. NLRP2 may have a role in the innate immune response, but its 'specific' functions remain controversial. Although NLRP2 can initiate inflammasome and promote inflammation, it can also downregulate inflammatory signals. Additionally, NLRP2 has been reported to function in the reproductive system and shows high expression in the placenta. However, the exact role of NLRP2 in the reproductive system is unclear. Here, we highlight the most current progress on NLRP2 in inflammasome activation, effector function and regulation of nuclear factor-κB. And we discuss functions of NLRP2 in inflammatory diseases, reproductive disorders and the potential implication of NLRP2 in human diseases.

Free heme induces neuroinflammation and cognitive impairment by microglial activation via the TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND: Red blood cells (RBCs) transfusion is related to perioperative neurocognitive disorders. The toxic effect of free heme has been identified in many pathologies. However, the underlying mechanisms of RBCs transfusion or free heme in cognitive impairment have not been clearly explored. Therefore, this research was conducted to determine the mechanism of free heme-induced neuroinflammation and cognitive impairment. METHODS: Rats were received intraperitoneal injection of hemin alone or combined with intracerebroventricular injection of Hemopexin (HPX), and MWM test was conducted to measure cognitive function. The amount of heme-HPX complexes was evaluated by flow cytometry for CD91 + cells. The microglial inflammatory response in rat brain was observed by immunofluorescence staining of Iba-1, and the inflammatory factors of TNF-α, IL-1ß and IL-6 in rat brain and BV2 cells were detected by ELISA analysis. Furthermore, neuronal apoptosis in HT22 cells alone and in HT22 + BV2 coculture system was detected by flow cytometry and immunofluorescence staining. Finally, western blot was conducted to detect TLR4/MyD88/NF-κB proteins in rat brain and BV2 cells treated with hemin or combined with pathway inhibitors. Additionally, the M1 surface marker CD86 was observed in BV2 cells to further confirm neuroinflammation. RESULTS: Intraperitoneal injection of hemin induced cognitive impairment, increase of CD91 + cells, up-regulation of TNF-α and IL-1ß, down-regulation of IL-6, activation of microglia, and activation of the TLR4/MyD88/NF-κB signaling pathway in rat brain. Significantly, intracerebroventricular injection of HPX reduced the above effects. Hemin induced boost of TNF-α, IL-1ß and IL-6 in BV2 cells, as well as apoptosis in HT22 cells. Notably, when HT22 cells were cocultured with BV2 cells, apoptosis was significantly increased. Hemin also induced activation of the TLR4/MyD88/NF-κB signaling pathway and increased the M1 surface marker CD86 in BV2 cells, and inhibiting this pathway reduced the inflammatory responses. CONCLUSIONS: Free heme induces cognitive impairment, and the underlying mechanism may involve neuronal apoptosis and microglial inflammation via the TLR4/MyD88/NF-κB signaling pathway. HPX may have potential therapeutic effects. Video Abstract.

Mitofilin in cardiovascular diseases: Insights into the pathogenesis and potential pharmacological interventions.

The impact of mitochondrial dysfunction on the pathogenesis of cardiovascular disease is increasing. However, the precise underlying mechanism remains unclear. Mitochondria produce cellular energy through oxidative phosphorylation while regulating calcium homeostasis, cellular respiration, and the production of biosynthetic chemicals. Nevertheless, problems related to cardiac energy metabolism, defective mitochondrial proteins, mitophagy, and structural changes in mitochondrial membranes can cause cardiovascular diseases via mitochondrial dysfunction. Mitofilin is a critical inner mitochondrial membrane protein that maintains cristae structure and facilitates protein transport while linking the inner mitochondrial membrane, outer mitochondrial membrane, and mitochondrial DNA transcription. Researchers believe that mitofilin may be a therapeutic target for treating cardiovascular diseases, particularly cardiac mitochondrial dysfunctions. In this review, we highlight current findings regarding the role of mitofilin in the pathogenesis of cardiovascular diseases and potential therapeutic compounds targeting mitofilin.

Unraveling the molecular complexity: Wtap/Ythdf1 and Lcn2 in novel traumatic brain injury secondary injury mechanisms.

The primary aim of this research was to explore the functions of Wtap and Ythdf1 in regulating neuronal Lipocalin-2 (Lcn2) through m6A modification in traumatic brain injury (TBI). By employing transcriptome sequencing and enrichment analysis, we identified the Wtap/Ythdf1-mediated Lcn2 m6A modification pathway as crucial in TBI. In our in vitro experiments using primary cortical neurons, knockout of Wtap and Ythdf1 led to the inhibition of Lcn2 m6A modification, resulting in reduced neuronal death and inflammation. Furthermore, overexpression of Lcn2 in cortical neurons induced the activation of reactive astrocytes and M1-like microglial cells, causing neuronal apoptosis. In vivo experiments confirmed the activation of reactive astrocytes and microglial cells in TBI and importantly demonstrated that Wtap knockdown improved neuroinflammation and functional impairment. These findings underscore the significance of Wtap/Ythdf1-mediated Lcn2 regulation in TBI secondary injury and suggest potential therapeutic implications for combating TBI-induced neuroinflammation and neuronal damage.

Effectiveness of perioperative low-dose esketamine infusion for postoperative pain management in pediatric urological surgery: a prospective clinical trial.

BACKGROUND: Postoperative pain is common in pediatric urological surgery. The study assess the impact of perioperative intravenous infusion of low-dose esketamine on postoperative pain in pediatric urological surgery. METHODS: Pediatric patients (n = 80) undergoing urological surgery were randomized into four groups. Patients in the control group were administered an analgesic pump containing only hydromorphone at a dose of 0.1 mg/kg (Hydromorphone Group 1, H1) or 0.15 mg/kg (Hydromorphone Group 2, H2). Patients in the experimental group were injected intravenously with 0.3 mg/kg of esketamine (Esketamine group 1, ES1) or equal volume of saline (Esketamine Group 2, ES2) during anesthesia induction. Esketamine 1.0 mg/kg and hydromorphone 0.1 mg/kg were added to the analgesic pump. Face, Leg, Activity, Crying, and Comfort (FLACC) scale or the Numerical Rating Scale (NRS) and adverse effects were recorded at 2, 6, 24, and 48 h postoperatively. Additionally, total and effective PCA button presses were recorded. RESULTS: In comparison to the H1 group, the pain scores were notably reduced at all postoperative time points in both the ES1 and H2 groups. The ES2 group exhibited lower pain scores only at 24 and 48 h postoperatively. When compared to the H2 group, there were no significant differences in pain scores at various postoperative time points in the ES2 group. However, the ES1 group demonstrated significantly lower pain scores at 6, 24 and 48 h postoperatively, and these scores were also significantly lower than those observed in the ES2 group. The total and effective number of PCA button presses in the ES1, ES2 and H2 group were lower than that in the H1 group (P < 0.001). The incidence of adverse effects within 48 h after surgery was 15% in ES1, 22% in ES2, 58% in H1, and 42% in H2, respectively (P = 0.021). CONCLUSIONS: The use of low-dose esketamine infusion in analgesia pump can effectively alleviates postoperative pain in pediatric urological patients, leading to a significant reduction in the number of analgesic pump button press. The combined approach of perioperative anesthesia induction and analgesia pump administration is recommended for optimal pain management in these patients. TRIAL REGISTRATION: Chinese Clinical Trial Registry- ChiCTR2300073879 (24/07/2023).

Identification of key genes and long non­coding RNA expression profiles in osteoporosis with rheumatoid arthritis based on bioinformatics analysis.

BACKGROUND: Although rheumatoid arthritis (RA) is a chronic systemic tissue disease often accompanied by osteoporosis (OP), the molecular mechanisms underlying this association remain unclear. This study aimed to elucidate the pathogenesis of RA and OP by identifying differentially expressed mRNAs (DEmRNAs) and long non-coding RNAs (lncRNAs) using a bioinformatics approach. METHODS: Expression profiles of individuals diagnosed with OP and RA were retrieved from the Gene Expression Omnibus database. Differential expression analysis was conducted. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) pathway enrichment analyses were performed to gain insights into the functional categories and molecular/biochemical pathways associated with DEmRNAs. We identified the intersection of common DEmRNAs and lncRNAs and constructed a protein-protein interaction (PPI) network. Correlation analysis between the common DEmRNAs and lncRNAs facilitated the construction of a coding-non-coding network. Lastly, serum peripheral blood mononuclear cells (PBMCs) from patients with RA and OP, as well as healthy controls, were obtained for TRAP staining and qRT-PCR to validate the findings obtained from the online dataset assessments. RESULTS: A total of 28 DEmRNAs and 2 DElncRNAs were identified in individuals with both RA and OP. Chromosomal distribution analysis of the consensus DEmRNAs revealed that chromosome 1 had the highest number of differential expression genes. GO and KEGG analyses indicated that these DEmRNAs were primarily associated with " platelets (PLTs) degranulation", "platelet alpha granules", "platelet activation", "tight junctions" and "leukocyte transendothelial migration", with many genes functionally related to PLTs. In the PPI network, MT-ATP6 and PTGS1 emerged as potential hub genes, with MT-ATP6 originating from mitochondrial DNA. Co-expression analysis identified two key lncRNA-mRNA pairs: RP11 - 815J21.2 with MT - ATP6 and RP11 - 815J21.2 with PTGS1. Experimental validation confirmed significant differential expression of RP11-815J21.2, MT-ATP6 and PTGS1 between the healthy controls and the RA + OP groups. Notably, knockdown of RP11-815J21.2 attenuated TNF + IL-6-induced osteoclastogenesis. CONCLUSIONS: This study successfully identified shared dysregulated genes and potential therapeutic targets in individuals with RA and OP, highlighting their molecular similarities. These findings provide new insights into the pathogenesis of RA and OP and suggest potential avenues for further research and targeted therapies.

Unmanned Aerial Vehicle Cooperative Data Dissemination Based on Graph Neural Networks.

Unmanned Aerial Vehicles (UAVs) have critical applications in various real-world scenarios, including mapping unknown environments, military reconnaissance, and post-disaster search and rescue. In these scenarios where communication infrastructure is missing, UAVs will form an ad hoc network and perform tasks in a distributed manner. To efficiently carry out tasks, each UAV must acquire and share global status information and data from neighbors. Meanwhile, UAVs frequently operate in extreme conditions, including storms, lightning, and mountainous areas, which significantly degrade the quality of wireless communication. Additionally, the mobility of UAVs leads to dynamic changes in network topology. Therefore, we propose a method that utilizes graph neural networks (GNN) to learn cooperative data dissemination. This method leverages the network topology relationship and enables UAVs to learn a decision policy based on local data structure, ensuring that all UAVs can recover global information. We train the policy using reinforcement learning that enhances the effectiveness of each transmission. After repeated simulations, the results validate the effectiveness and generalization of the proposed method.

Efficient In Vitro Regeneration System and Comparative Transcriptome Analysis Offer Insight into the Early Development Characteristics of Explants from Cotyledon with Partial Petiole in Small-Fruited Pepper (Capsicum annuum).

In our research, we utilized six small-fruited pepper germplasms as materials, selected cotyledons with the petiole and hypocotyls as explants, and conducted in vitro regeneration studies. Our outcomes specify that the most suitable explant is cotyledon with the petiole, and the suitable genotype is HNUCA341. The optimal medium for inducing and elongating adventitious buds for this genotype is Murashige and Skoog medium (MS) + 9.12 µM Zeatin (ZT) + 0.57 µM 3-Indoleacetic acid (IAA), with a bud induction rate of 44.4%. The best rooting induction medium is MS + 1.14 µM IAA, with a rooting rate of 86.7%. Research on the addition of exogenous hormones has revealed that the induction speed of buds in small-fruited pepper (HNUCA341) in the combination of ZT and IAA hormones (abbreviated as ZI) is quicker, and the induction effect is better. The histological observations indicate that ZI treatment accelerates the initiation of explant division and differentiation, causing a shorter duration of vascular-bundle tissue production. The plant hormone signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including ARR9 (LOC107843874, LOC107843885), ARR4 (LOC107848380, LOC107862455), AHK4 (LOC107870540), AHP1 (LOC107839518), LAX2 (LOC107846008), SAUR36 (LOC107852624), IAA8 (LOC107841020), IAA16 (LOC107839415), PYL4 (LOC107843441), and PYL6 (LOC107871127); these significantly enriched genes may be associated with in vitro regeneration. In addition, the carbon metabolism pathway and plant mitogen-activated protein kinase (MAPK) signaling pathway are also significantly enriched in KEGG. The results of the Gene Ontology (GO) analysis revealed that differentially expressed genes related to carbon metabolism and fixation, photosynthesis and MAPK signaling pathways were upregulated under ZI treatment. It was found that they might be associated with enhanced regeneration in vitro. Furthermore, we also screened out differentially expressed transcription factors, primarily from the MYB, bHLH, AP2/ERF, and NAC families. Overall, our work accumulated important data for the in-depth analysis of the molecular mechanism of in vitro regeneration of pepper, and provides valuable germplasm for establishing an efficient stable pepper genetic-transformation system based on tissue culture.

Engineering and Characterization of Avian Coronavirus Mutants Expressing Fluorescent Reporter Proteins from the Replicase Gene.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes infectious bronchitis, an acute and highly contagious respiratory disease of chickens. IBV evolution under the pressure of comprehensive and widespread vaccination requires surveillance for vaccine resistance, as well as periodic vaccine updates. Reverse genetics systems are very valuable tools in virology, as they facilitate rapid genetic manipulation of viral genomes, thereby advancing basic and applied research. We report here the construction of an infectious clone of IBV strain Beaudette as a bacterial artificial chromosome (BAC). The engineered full-length IBV clone allowed the rescue of an infectious virus that was phenotypically indistinguishable from the parental virus. We used the infectious IBV clone and examined whether an enhanced green fluorescent protein (EGFP) can be produced by the replicase gene ORF1 and autocatalytically released from the replicase polyprotein through cleavage by the main coronavirus protease. We show that IBV tolerates insertion of the EGFP ORF at the 3' end of the replicase gene, between the sequences encoding nsp13 and nsp16 (helicase, RNA exonuclease, RNA endonuclease, and RNA methyltransferase). We further show that EGFP is efficiently cleaved from the replicase polyprotein and can be localized in double-membrane vesicles along with viral RNA polymerase and double-stranded RNA, an intermediate of IBV genome replication. One of the engineered reporter EGFP viruses were genetically stable during passage in cultured cells. We demonstrate that the reporter EGFP viruses can be used to study virus replication in host cells and for antiviral drug discovery and development of diagnostic assays. IMPORTANCE Reverse genetics systems based on bacterial artificial chromosomes (BACs) are the most valuable systems in coronavirus research. Here, we describe the establishment of a reverse genetics system for the avian coronavirus strain Beaudette, the most intensively studied strain. We cloned a copy of the avian coronavirus genome into a BAC vector and recovered infectious virus in permissive cells. We used the new system to construct reporter viruses that produce enhanced green fluorescent protein (EGFP). The EGFP coding sequence was inserted into 11 known cleavage sites of the major coronavirus protease in the replicase gene ORF1. Avian coronavirus tolerated the insertion of the EGFP coding sequence at three sites. The engineered reporter viruses replicated with parental efficiency in cultured cells and were sufficiently genetically stable. The new system facilitates functional genomics of the avian coronavirus genome but can also be used for the development of novel vaccines and anticoronaviral drugs.

The neuroprotective mechanism of sevoflurane in rats with traumatic brain injury via FGF2.

BACKGROUND: Traumatic brain injury (TBI) is a kind of acquired brain injury, which is caused by external mechanical forces. Moreover, the neuroprotective role of sevoflurane (Sevo) has been identified in TBI. Therefore, this research was conducted to figure out the mechanism of Sevo in TBI via FGF2. METHODS: The key factors of neuroprotective effects of Sevo in TBI rats were predicted by bioinformatics analysis. A TBI model was induced on rats that then inhaled Sevo for 1 h and grouped via lentivirus injection. Modified Neurological Severity Score was adopted to evaluate neuronal damage in rats, followed by motor function and brain water content measurement. FGF2, EZH2, and HES1 expression in brain tissues was evaluated by immunofluorescence staining, and expression of related genes and autophagy factors by RT-qPCR and Western blot analysis. Methylation-specific PCR was performed to assess HES1 promoter methylation level, and ChIP assay to detect the enrichment of EZH2 in the HES1 promoter. Neuronal damage was assessed by cell immunofluorescence staining, and neuronal apoptosis by Nissl staining, TUNEL staining, and flow cytometry. RESULTS: Sevo diminished brain edema, improved neurological scores, and decreased neuronal apoptosis and autophagy in TBI rats. Sevo preconditioning could upregulate FGF2 that elevated EZH2 expression, and EZH2 bound to the HES1 promoter to downregulate HES1 in TBI rats. Also, FGF2 or EZH2 overexpression or HES silencing decreased brain edema, neurological deficits, and neuronal autophagy and apoptosis in Sevo-treated TBI rats. CONCLUSIONS: Our results provided a novel insight to the neuroprotective mechanism of Sevo in TBI rats by downregulating HES1 via FGF2/EZH2 axis activation.

The reduction reaction of carbon dioxide on a precise number of Fe atoms anchored on two-dimensional biphenylene.

In recent accomplishments, a new two-dimensional allotrope of carbon-biphenylene (BP) was experimentally synthesized [Fan et al., Science, 372, 852-856 (2021)]. The BP sheet is composed of four-, six-, and eight-membered carbon rings constructed using periodically arranged sp2-hybridized carbon atoms. Unlike semi-metallic graphene, BP is metallic with quite active atoms and chemical bonds, and the binding strength with reaction intermediates will be enhanced, which means that it may exhibit good catalytic activity in some electrochemical catalytic reactions. Using spin-polarized density functional theory based on first-principles simulations and ab initio molecular dynamic calculations, we systematically investigated the structure, thermodynamic stability, CO2 reduction reaction (CO2RR) activity and product selectivity of a precise number of Fen (n = 1-3) atoms embedded on a BP monolayer. The calculated results indicate that our designed Fe1@BP, Fe2@BP and Fe3@BP complexes possess good thermodynamic and electrochemical stabilities and strong absorption for CO2, which promotes the activation of CO2. Furthermore, the Fe2@BP catalyst possesses good catalytic ability for the CO2RR to CH3OH due to a small rate determining potential of -0.48 V. In addition, Fe2- and Fe3@BP catalysts demonstrate superior catalytic performance for the CO2RR to CH4 with low rate-limiting steps. More importantly, both the Fe2 and Fe3@BP catalysts can effectively suppress the hydrogen evolution reaction (HER) during the entire CO2RR process. The electronic structure analysis shows that the enhanced ability of Fe1-3@BP catalysts for effective CO2 reduction can be attributed to the establishment of strong hybridization between Fe-3d and O-2p or C-2p states, which is conducive to the transfer of strong electrons to the anti-bond orbital of CO2. This work provides an in-depth insight into the intrinsic catalytic mechanisms of the CO2RR on Fe1-3@BP catalysts, and highlights the excellent performance of the BP sheet as a substrate material for the polyatomic catalyst.

Mesenchymal stem cells for the treatment of cognitive impairment caused by neurological diseases.

Patients with neurological diseases often have cognitive impairment, which creates a substantial emotional and economic burden for patients and their families. This issue urgently needs to be addressed. The pathological mechanism of this cognitive impairment is a complicated process that involves a variety of cells and molecules, central nervous system inflammatory reactions, oxidative stress, free radical damage and nerve protection factor-related metabolic disorders. Traditional treatments include neuroprotective agents and analgesic therapy. However, analgesic therapy cannot improve cognitive function, and the blood-brain barrier (BBB) largely blocks neuroprotective agents from entering the central nervous system; therefore, it is very important to find a more effective treatment. Mesenchymal stem cells (MSCs) have anti-inflammatory, anti-apoptotic and immunomodulatory properties and have been proven to play an important role in the treatment of many neurodegenerative diseases. Most importantly, MSCs are likely to cross the BBB. Therefore, MSC therapy is regarded as an important means of ameliorating neurological impairment. The purpose of this review is to summarize recent researches on the treatment of cognitive dysfunction caused by neurological diseases with MSCs.

Liraglutide Attenuates Restenosis After Vascular Injury in Rabbits With Diabetes Via the TGF-ß/Smad3 Signaling Pathway.

Background: Lower limb ischemia due to arterial stenosis is a major complication in patients with diabetes mellitus (DM). Liraglutide is a long-acting analogue of a glucagon-like peptide 1 (GLP-1) receptor agonist used for lowering blood glucose in patients with DM, and is believed to possess cardiovascular protective effects. The aim of this study was to investigate whether liraglutide has a protective effect on blood vessels and alleviates vascular intimal hyperplasia in streptozotocin (STZ)-induced rabbits with DM and its molecular mechanism. Methods: Rabbits with DM were induced by STZ, and a lower limb ischemia model was established. The animals were divided into a control group, DM-injury group and liraglutide treatment group. Pathological staining was used to observe the intimal growth, analyze the oxidation levels of malondialdehyde (MDA), superoxide dismutase (SOD) and plasma glutathione peroxidase (GSH-Px), and analyze the changes in expression of marker proteins and signaling pathway proteins by Western blotting. A hyperglycemia (HG)-injured vascular smooth muscle cells (VSMCs) model was established to analyze reactive oxygen species (ROS) levels, Cell-Counting Kit-8 (CCK-8) was used to analyze cell proliferation, scratch assay and Transwell Migration Assay to analyze cell migration, flow cytometry to analyze apoptosis and Western blotting was used to analyze changes in the expression of marker and signaling pathway proteins. Results: The results of pathological staining showed that intimal hyperplasia was severe after diabetes-induced lower limb ischemia in rabbits at 4 weeks, and liraglutide treatment reduced symptoms. Liraglutide treatment significantly decreased MDA content, increased SOD, GSH-Px content, and augmented total antioxidant capacity levels in tissues. The results of Western blotting analysis showed that E-cadherin, mitochondrial membrane potential 9 (MMP-9), proliferating cell nuclear antigen (PCNA), and type I collagen protein expression levels were significantly decreased after liraglutide treatment compared with the DM injury group. The results indicated that liraglutide inhibited epithelial-mesenchymal transition (EMT) progression, vascular cell proliferation and migration and collagen production. Liraglutide inhibits transforming growth factor beta 1 (TGF-ß1)/Smad3 signaling pathway protein expression. In vitro assays have shown that liraglutide reduces cellular ROS levels, inhibits cell proliferation and migration and promotes apoptosis. Liraglutide down-regulated the expression of E-cadherin, MMP-9, PCNA, type I collagen protein as well as the TGF-ß1/Smad3 signaling pathway, but this effect could be reversed by tumor necrosis factor alpha (TNF-α). Conclusion: Liraglutide can significantly improve tissue antioxidant capacity, reduce vascular cell proliferation and migration via the TGF-ß1/Smad3 signaling pathway, inhibit the EMT and collagen production processes, and alleviate hyperglycemia(HG)-induced lower limb ischemia and intimal hyperplasia.

Structure-Based Discovery of Novel Nonpeptide Inhibitors Targeting SARS-CoV-2 Mpro.

The continual spread of novel coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), posing a severe threat to the health worldwide. The main protease (Mpro, alias 3CLpro) of SARS-CoV-2 is a crucial enzyme for the maturation of viral particles and is a very attractive target for designing drugs to treat COVID-19. Here, we propose a multiple conformation-based virtual screening strategy to discover inhibitors that can target SARS-CoV-2 Mpro. Based on this strategy, nine Mpro structures and a protein mimetics library with 8960 commercially available compounds were prepared to carry out ensemble docking for the first time. Five of the nine structures are apo forms presented in different conformations, whereas the other four structures are holo forms complexed with different ligands. The surface plasmon resonance assay revealed that 6 out of 49 compounds had the ability to bind to SARS-CoV-2 Mpro. The fluorescence resonance energy transfer experiment showed that the biochemical half-maximal inhibitory concentration (IC50) values of the six compounds could hamper Mpro activities ranged from 0.69 ± 0.05 to 2.05 ± 0.92 µM. Evaluation of antiviral activity using the cell-based assay indicated that two compounds (Z1244904919 and Z1759961356) could strongly inhibit the cytopathic effect and reduce replication of the living virus in Vero E6 cells with the half-maximal effective concentrations (EC50) of 4.98 ± 1.83 and 8.52 ± 0.92 µM, respectively. The mechanism of the action for the two inhibitors were further elucidated at the molecular level by molecular dynamics simulation and subsequent binding free energy analysis. As a result, the discovered noncovalent reversible inhibitors with novel scaffolds are promising antiviral drug candidates, which may be used to develop the treatment of COVID-19.

Ureaplasma parvum meningitis following atypical choroid plexus papilloma resection in an adult patient: a case report and literature review.

BACKGROUND: While Ureaplasma parvum has previously been linked to the incidence of chorioamnionitis, abortion, premature birth, and perinatal complications, there have only been rare reports of invasive infections of the central nervous system (CNS) in adults. Owing to its atypical presentation and the fact that it will yield sterile cultures using conventional techniques, diagnosing U. parvum meningitis can be challenging. CASE PRESENTATION: We describe a case of U. parvum meningitis detected in an adult patient following surgical brain tumor ablation. After operation, the patient experienced epilepsy, meningeal irritation, and fever with unconsciousness. Cerebrospinal fluid (CSF) analysis showed leukocytosis (484 * 106 /L), elevated protein levels (1.92 g/L), and decreased glucose concentrations (0.02 mmol/L). Evidence suggested that the patient was suffering from bacterial meningitis. However, no bacterial pathogens in either CSF or blood were detected by routine culture or serology. The symptoms did not improve with empirical antibiotics. Therefore, we performed metagenomic next-generation sequencing (mNGS) to identify the etiology of the meningitis. Ureaplasma parvum was detected by mNGS in CSF samples. To the best of our knowledge, this case is the first reported instance of U. parvum meningitis in an adult patient in Asian. After diagnosis, the patient underwent successful moxifloxacin treatment and recovered without complications. CONCLUSIONS: As mNGS strategies can enable the simultaneous detection of a diverse array of microbes in a single analysis, they may represent a valuable means of diagnosing the pathogens responsible for CNS infections and other clinical conditions with atypical presentations.

Systems pharmacology reveals the mechanism of activity of Ge-Gen-Qin-Lian decoction against LPS-induced acute lung injury: A novel strategy for exploring active components and effective mechanism of TCM formulae.

Acute lung injury (ALI), a severe and life-threatening inflammation of the lung, with high morbidity and mortality, underscoring the urgent need for novel treatments. Ge-Gen-Qin-Lian decoction (GQD), a classic Chinese herbal formula, has been widely used to treat intestine-related diseases in the clinic for centuries. In recent years, a growing number of studies have found that GQD has a favorable anti-inflammatory effect. With the further study on the viscera microbiota, the link between the lungs and the gut-the gut-lung axis has been established. Based on the theory of the gut-lung axis, we used systems pharmacology to explore the effects and mechanisms of GQD treatment in ALI. Hypothesizing that GQD inhibits ALI progression, we used the experimental model of lipopolysaccharide (LPS)-induced ALI in Balb/c mice to evaluate the therapeutic potential of GQD. Our results showed that GQD exerted protective effects against LPS-induced ALI by reducing pulmonary edema and microvascular permeability. Meanwhile, GQD can downregulate the expression of LPS-induced TNF-α, IL-1ß, and IL-6 in lung tissue, bronchoalveolar lavage fluid (BLAF), and serum. To further understand the molecular mechanism of GQD in the treatment of ALI, we used the network pharmacology to predict the disease targets of the active components of GQD. Lung tissue and serum samples of the mice were separately analyzed by transcriptomics and metabolomics. KEGG pathway analysis of network pharmacology and transcriptomics indicated that PI3K/Akt signaling pathway was significantly enriched, suggesting that it may be the main regulatory pathway for GQD treatment of ALI. By immunohistochemical analysis and apoptosis detection, it was verified that GQD can inhibit ALI apoptosis through PI3K/Akt signaling pathway. Then, we used the PI3K inhibitor LY294002 to block the PI3K/Akt signaling pathway, and reversely verified that the PI3K/Akt signaling pathway is the main pathway of GQD anti-ALI. In addition, differential metabolites in mice serum samples indicate that GQD can inhibit the inflammatory process of ALI by reversing the imbalance of energy metabolism. Our study showed that, GQD did have a better therapeutic effect on ALI, and initially elucidated its molecular mechanism. Thus, GQD could be exploited to develop novel therapeutics for ALI. Moreover, our study also provides a novel strategy to explore active components and effective mechanism of TCM formula combined with TCM theory to treat ALI.

LncRNA MALAT1 induces the dysfunction of ß cells via reducing the histone acetylation of the PDX-1 promoter in type 1 diabetes.

BACKGROUND: Type 1 diabetes (T1DM) severely threatens human health, and the dysfunction of insulin-secreting ß cells in islets is related to the reduced PDX-1 expression. It has been reported that long non-coding RNA MALAT1 regulates ß cell function, while the potential mechanism is unclear. METHODS: Islets were isolated from non-obese diabetic (NOD) mice and wild type (WT) mice. Mouse islets and ß cell line (Min6) were stimulated by IL-1ß. The expression of MALAT1 was determined using real-time PCR, while the PDX-1 protein expression was determined using western blotting. ChIP-qPCR was carried out to determine the histone acetylation of the PDX-1 promoter. RESULTS: In NOD islets and IL-1ß-stimulated Min6 cells, the expression of MALAT1 was increased, while the mRNA and protein levels of PDX-1 were decreased at an age/time-dependent manner. Overexpressing MALAT1 suppressed the H3 histone acetylation of the PDX-1 promoter, inhibiting both mRNA and protein expressions of PDX-1. Knocking down MALAT1 restored the decrease of the histone acetylation of the PDX-1 promoter, as well as the PDX-1 expression, which was reduced by IL-1ß stimulation. Under high glucose stimulation, the overexpression of PDX-1 alone restored the insulin secretion which was inhibited by the simultaneous overexpression of MALAT1 and PDX-1. Under high glucose and IL-1ß stimulation, the simultaneous knockdown of MALAT1 and PDX-1 reduced the enhancement of the insulin secretion which was raised by knocking down MALAT1 alone. CONCLUSION: MALAT1 induces the dysfunction of ß cells via reducing the H3 histone acetylation of the PDX-1 promoter and subsequently inhibiting the expression of PDX-1, thus suppressing the insulin secretion.

Tracheal rupture related to endotracheal intubation after thyroid surgery: a case report and systematic review.

Tracheobronchial rupture is an uncommon but potentially serious complication of endotracheal intubation. The most likely cause of tracheal injury is massive overinflation of the endotracheal tube cuff and pre-existing tracheal wall weakness. We review the relevant literature and predisposing factors contributing to this complication. Only articles that reported at least the demographic data (age and sex), the treatment performed and the outcome were included. Papers that did not detail these variables were excluded. We also focus on a case of tracheal laceration after tracheal intubation in a patient with severe thyroid carcinoma. This patient received surgical repair and recovered uneventfully. Two hundred and eight studies that reported cases or case series were selected for analysis. Most of the reported cases (57·2%) showed an uneventful recovery after surgical therapy. The overall mortality was 19·2% (40 patients). Our patient too recovered without any serious complication. Careful prevention, early detection and proper treatment of the problem are necessary when tracheal rupture occurs. The morbidity and mortality associated with tracheal injury mandate a high level of suspicion and expedient management.

Nemo-like kinase (NLK) negatively regulates NF-kappa B activity through disrupting the interaction of TAK1 with IKKß.

Stringent negative regulation of the transcription factor NF-κB is essential for maintaining cellular stress responses and homeostasis. However, the tight regulation mechanisms of IKKß are still not clear. Here, we reported that nemo-like kinase (NLK) is a suppressor of tumor necrosis factor (TNFα)-induced NF-κB signaling by inhibiting the phosphorylation of IKKß. Overexpression of NLK largely blocked TNFα-induced NF-κB activation, p65 nuclear localization and IκBα degradation; whereas genetic inactivation of NLK showed opposing results. Mechanistically, we identified that NLK interacted with IκB kinase (IKK)-associated complex, which in turn inhibited the assembly of the TAK1/IKKß and thereby, diminished the IκB kinase phosphorylation. Our results indicate that NLK functions as a pivotal negative regulator in TNFα-induced activation of NF-κB via disrupting the interaction of TAK1 with IKKß.