Ascorbyl palmitate ameliorates inflammatory diseases by inhibition of NLRP3 inflammasome.

The aberrant activation of NLRP3 inflammasome contributes to pathogenesis of multiple inflammation-driven human diseases. However, the medications targeting NLRP3 inflammasome are not approved for clinic use to date. Here, we show that ascorbyl palmitate (AP), a lipophilic derivative of ascorbic acid (AA) and a safe food additive, is a potent inhibitor of NLRP3 inflammasome. Compared with AA, AP inhibited the activation of NLRP3 inflammasome with increased potency and specificity. Mechanistically, AP directly scavenged mitochondrial reactive oxygen species (mitoROS) by its antioxidant activity and blocked NLRP3-NEK7 interaction and NLRP3 inflammasome assembly. Moreover, AP showed more significant preventive effects than AA in LPS-induced systemic inflammation, dextran sulfate sodium (DSS)-induced colitis and experimental autoimmune encephalomyelitis (EAE). Thus, our results suggest that AP is a potential therapeutic combating NLRP3-driven diseases.

Tegument protein UL3 of bovine herpesvirus 1 suppresses antiviral IFN-I signaling by targeting STING for autophagic degradation.

Bovine herpesvirus 1 (BoHV-1) is a highly contagious pathogen which causes infectious bovine rhinotracheitis in cattle worldwide. Although it has the ability to evade the host's antiviral innate immune response and establish persistent latent infections, the mechanisms are not fully understood, especially the function of the tegument protein to escape innate immunity and participate in viral replication. In this study, we showed that overexpression of tegument protein UL3 facilitates BoHV-1 replication and suppresses the expression of type-I interferon (IFN-I) and IFN-stimulated genes. Then, STING was identified as the target by which UL3 inhibits the IFN-I signaling pathway, and STING was degraded through the UL3-induced autophagy pathway. Furthermore, overexpression of UL3 promotes the expression of the autophagy-related protein ATG101, thereby inducing autophagy. Further study showed that UL3 enhances the interaction between ATG101 and STING, and then the degradation of STING was reversed following ATG101 silencing in UL3-overexpressing cells during BoHV-1 infection. Our research results demonstrate a novel function of UL3 in regulating host's antiviral response and provide a potential mechanism for BoHV-1 immune evasion.

The host protein CALCOCO2 interacts with bovine viral diarrhoea virus Npro, inhibiting type I interferon production and thereby promoting viral replication.

Bovine viral diarrhoea-mucosal disease caused by bovine viral diarrhoea virus (BVDV) is a major infectious disease that affects the cattle industry. The nonstructural protein Npro of BVDV antagonizes the type I interferon (IFN-I) pathway, thereby escaping the host immune response. The exact mechanism by which Npro uses host proteins to inhibit IFN-I production is unclear. The host protein CALCOCO2 was identified as a binding partner of Npro using a yeast two-hybrid screen. The interaction between Npro and CALCOCO2 was confirmed by yeast co-transformation, co-immunoprecipitation assays, and GST pull-down assays. The stable overexpression of CALCOCO2 markedly promoted BVDV propagation, while the knockdown of CALCOCO2 significantly inhibited BVDV replication in MDBK cells. Interestingly, CALCOCO2 inhibited IFN-I and IFN-stimulated gene production in BVDV-infected cells. Ectopic expression of CALCOCO2 effectively reduced IRF3 protein levels via the proteasome pathway. CALCOCO2 was found to promote Npro-mediated ubiquitination degradation of IRF3 by interacting with IRF3. Our results demonstrate the molecular mechanism by which Npro recruits the CALCOCO2 protein to regulate IRF3 degradation to inhibit IFN-I production.

CD97 negatively regulates the innate immune response against RNA viruses by promoting RNF125-mediated RIG-I degradation.

The G protein-coupled receptor ADGRE5 (CD97) binds to various metabolites that play crucial regulatory roles in metabolism. However, its function in the antiviral innate immune response remains to be determined. In this study, we report that CD97 inhibits virus-induced type-I interferon (IFN-I) release and enhances RNA virus replication in cells and mice. CD97 was identified as a new negative regulator of the innate immune receptor RIG-I, and RIG-1 degradation led to the suppression of the IFN-I signaling pathway. Furthermore, overexpression of CD97 promoted the ubiquitination of RIG-I, resulting in its degradation, but did not impact its mRNA expression. Mechanistically, CD97 upregulates RNF125 expression to induce RNF125-mediated RIG-I degradation via K48-linked ubiquitination at Lys181 after RNA virus infection. Most importantly, CD97-deficient mice are more resistant than wild-type mice to RNA virus infection. We also found that sanguinarine-mediated inhibition of CD97 effectively blocks VSV and SARS-CoV-2 replication. These findings elucidate a previously unknown mechanism through which CD97 negatively regulates RIG-I in the antiviral innate immune response and provide a molecular basis for the development of new therapeutic strategies and the design of targeted antiviral agents.

DLG1 promotes the antiviral innate immune response by inhibiting p62-mediated autophagic degradation of IKKε.

IMPORTANCE: The type-I interferon (IFN-I) signaling pathway is the first line of antiviral innate immunity. It must be precisely regulated against virus-induced damage. The tightly regulated mechanisms of action of host genes in the antiviral innate immune signaling pathway are still worth studying. Here, we report a novel role of DLG1 in positively regulating the IκB kinase epsilon (IKKε)-mediated IFN-I signaling response against negative-stranded RNA virus replication, whereas the RNA virus inhibits the expression of DLG1 for immune escape. Importantly, the E3 ligase March2 interacts with and promotes K27-linked polyubiquitination of IKKε, and p62 is a cargo receptor that recognizes ubiquitinated IKKε for eventual autophagic degradation. Together, the current findings elucidate the role of DLG1 in the antiviral IFN-I signaling pathway and viral infection repression.

Study on water and oxygen transfer characteristics of HT-PEM fuel cells.

In this study, a steady-state model is developed by combining mechanical, Navier-Stokes, Maxwell-Stefan, and Butler-Volmer equations. This model is then used to investigate the influences of diffusion layer thickness deformation under a specific assembly force on the porosity distribution as an indicator of fuel cell performance. The HT-PEM (high temperature proton exchange membrane) fuel cell model is built using COMSOL Multiphysics software, simulating the changes in diffusion layer porosity under different thicknesses of the diffusion layer, thus analyzing the trends in variation of water and oxygen concentration in the cathode diffusion layer. The battery has different current densities at different operating potentials. The influence of the working potential on the mass transfer concentration and the variation in the mass transfer concentration of the diffusion layer under the different areas of flow channel and flow ridge is discussed. The simulation results have a certain reference value for the optimization of mass transfer in a diffusion layer. The results reveal the combined effect of the assembly force and flow field, which makes the porosity distribution uneven and results in remarkable lateral current in the gas diffusion layer (GDL). The thicker the diffusion layer, the less oxygen consumed, and a large amount of oxygen is retained in the gaseous diffusion layer. It can be concluded that thicker diffusion layer is conducive to more uniform mass transfer and diffusion. These results can potentially be used to promote the performance and application of HT-PEMFC.

Fabrication of a magnetic metal-organic framework/covalent organic framework composite for simultaneous magnetic solid-phase extraction of seventeen trace quinolones residues in meats.

Effective capture of quinolones (QNs) in animal-derived food is a vital procedure for food safety monitoring. However, the lack of adsorption specificity and difficult to recycle in complex substrate conditions have been major problems for most of the adsorbents. In this work, a magnetic Fe3O4/MOF/COF composite (named Fe3O4@NH2-MIL-125@TpPa-SO3H) was successfully synthesized with good magnetic responsiveness and conspicuous affinity towards QNs. The Fe3O4/MOF/COF composite was used as a magnetic solid-phase extraction (MSPE) adsorbent for pretreatment and determination of QNs in meat samples. Under optimal MSPE conditions in combination with high performance liquid chromatography-quadrupole orbitrap high resolution mass spectrometer (HPLC-Q-Orbitrap HRMS), the proposed method had good linearity (R2 ≥ 0.9978) from 0.01 to 100ng g-1, low limits of detection (0.0016 to 0.0940ng g-1), good precision with relative standard deviations lower than 5.8%. This method was effectively applied to the detection of 17 QNs in the spiked pork, chicken and beef samples with satisfactory recoveries from 83.9 to 106.2%. The separation selectivity mainly due to the π-π interaction, hydrogen bonding, and electrostatic attraction between QNs and the sulfonic acid and amino functional groups of the composite. After verification, the stability and reusability of the composite meet the requirements of complex matrix sample pretreatment. The developed MSPE method based on the magnetic Fe3O4/MOF/COF composite provided an ideal sample pretreatment alternative for determining trace QNs in complex matrixes with selectivity, simplicity, rapidity, and efficiency.

IFITM3 restricts RABV infection through inhibiting viral entry and mTORC1- dependent autophagy.

Rabies, which caused by rabies virus (RABV), is a zoonotic and life-threatening disease with 100% mortality, and there is no effective treatment thus far due to the unclear pathogenesis and less of treatment targets. Interferon-induced transmembrane protein 3 (IFITM3) has recently been identified as an important anti-viral host effector induced by type I interferon. However, the role of IFITM3 in RABV infection has not been elucidated. In this study, we demonstrated that IFITM3 is a crucial restriction factor for RABV, the viral-induced IFITM3 significantly inhibited RABV replication, while knockdown of IFITM3 had the opposite effect. We then identified that IFNß induces the upregulation of IFITM3 in the absence or presence of RABV infection, meanwhile, IFITM3 positively regulates RABV-triggered production of IFNß in a feedback manner. In-depth research we found that IFITM3 not only inhibits the virus absorb and entry, but also inhibits viral replication through mTORC1-dependent autophagy. All these findings broaden our understanding of IFITM3 function and uncover a novel mechanism against RABV infection.

Apilimod activates the NLRP3 inflammasome through lysosome-mediated mitochondrial damage.

NLRP3 is an important innate immune sensor that responses to various signals and forms the inflammasome complex, leading to IL-1ß secretion and pyroptosis. Lysosomal damage has been implicated in NLRP3 inflammasome activation in response to crystals or particulates, but the mechanism remains unclear. We developed the small molecule library screening and found that apilimod, a lysosomal disruptor, is a selective and potent NLRP3 agonist. Apilimod promotes the NLRP3 inflammasome activation, IL-1ß secretion, and pyroptosis. Mechanismically, while the activation of NLRP3 by apilimod is independent of potassium efflux and directly binding, apilimod triggers mitochondrial damage and lysosomal dysfunction. Furthermore, we found that apilimod induces TRPML1-dependent calcium flux in lysosomes, leading to mitochondrial damage and the NLRP3 inflammasome activation. Thus, our results revealed the pro-inflammasome activity of apilimod and the mechanism of calcium-dependent lysosome-mediated NLRP3 inflammasome activation.

A Chinese soil conservation dataset preventing soil water erosion from 1992 to 2019.

Soil conservation service (SC) is defined as the ability of terrestrial ecosystems to control soil erosion and protect soil function. A long-term and high-resolution estimation of SC is urgent for ecological assessment and land management on a large scale. Here, a 300-m resolution Chinese soil conservation dataset (CSCD) from 1992 to 2019, for the first time, is established based on the Revised Universal Soil Loss Equation (RUSLE) model. The RUSLE modelling was conducted based on five key parameters, including the rainfall erosivity (interpolation of daily rainfall), land cover management (provincial data), conservation practices (weighted by terrain and crop types), topography (30 m), and soil properties (250 m). The dataset agrees with previous measurements in all basins (R2 > 0.5) and other regional simulations. Compared with current studies, the dataset has long-term, large-scale, and relatively high-resolution characteristics. This dataset will serve as a base to open out the mechanism of SC variations in China and could help assess the ecological effects of land management policies.

SARS-CoV-2 ORF8 Protein Induces Endoplasmic Reticulum Stress-like Responses and Facilitates Virus Replication by Triggering Calnexin: an Unbiased Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stress-like responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or human-specific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARS-CoV-2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse.

Cross-species infection potential of avian influenza H13 viruses isolated from wild aquatic birds to poultry and mammals.

Wild aquatic birds are the primary hosts of H13 avian influenza viruses (AIVs). Herein, we performed a genetic analysis of two H13 AIVs isolated from wild birds in China and evaluated their infection potential in poultry to further explore the potential for transmission from wild aquatic birds to poultry. Our results showed that the two strains belong to different groups, one strain (A/mallard/Dalian/DZ-137/2013; abbreviated as DZ137) belongs to Group I, whereas the other strain (A/Eurasian Curlew/Liaoning/ZH-385/2014; abbreviated as ZH385) belongs to Group III. In vitro experiments showed that both DZ137 and ZH385 can replicate efficiently in chicken embryo fibroblast cells. We found that these H13 AIVs can also efficiently replicate in mammalian cell lines, including human embryonic kidney cells and Madin-Darby canine kidney cells. In vivo experiments showed that DZ137 and ZH385 can infect 1-day-old specific pathogen-free (SPF) chickens, and that ZH385 has a higher replication ability in chickens than DZ137. Notably, only ZH385 can replicate efficiently in 10-day-old SPF chickens. However, neither DZ137 nor ZH385 can replicate well in turkeys and quails. Both DZ137 and ZH385 can replicate in 3-week-old mice. Serological surveillance of poultry showed a 4.6%-10.4% (15/328-34/328) antibody-positive rate against H13 AIVs in farm chickens. Our findings indicate that H13 AIVs have the replication ability in chickens and mice and may have a risk of crossing the host barrier from wild aquatic birds to poultry or mammals in the future.

Continuously Flow Photothermal Catalysis Efficiently CO2 Reduction Over S-Scheme 2D/0D Bi5 O7 I-OVs/Cd0.5 Zn0.5 S Heterojunction with Strong Interfacial Electric Field.

Using CO2 , water, and sunlight to produce solar fuel is a very attractive process, which can synchronously reduce carbon and convert solar energy into hydrocarbons. However, photocatalytic CO2 reduction is often limited by the low selectivity of reduction products and poor photocatalytic activity. In this study, S-scheme Bi5 O7 I-OVs/Cd0.5 Zn0.5 S (Bi5 O7 I-OVs/CZS-0.5) heterojunction with strong interfacial electric field (IEF) is prepared by in situ growth method. The performance of reduction CO2 to CO is studied by continuous flow photothermal catalytic (PTC) CO2 reduction platform. 12.5% Bi5 O7 I-OVs/CZS-0.5 shows excellent CO yield of 58.6 µmol g-1  h-1 and selectivity of 98.4%, which are 35.1 times than that of CZS-0.5 under visible light. The charge transfer path of the S-scheme through theoretical calculation (DFT), in situ irradiation Kelvin probe force microscope (ISI-KPFM) and in situ irradiation X-ray photoelectron spectroscopy (ISI-XPS) analysis, is verified. The study can provide useful guidance and reference for improving activity by oxygen vacancy induced strong IEF and the development of a continuous flow PTC CO2 reduction system.

The ORF7a protein of SARS-CoV-2 initiates autophagy and limits autophagosome-lysosome fusion via degradation of SNAP29 to promote virus replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is closely related to various cellular aspects associated with autophagy. However, how SARS-CoV-2 mediates the subversion of the macroautophagy/autophagy pathway remains largely unclear. In this study, we demonstrate that overexpression of the SARS-CoV-2 ORF7a protein activates LC3-II and leads to the accumulation of autophagosomes in multiple cell lines, while knockdown of the viral ORF7a gene via shRNAs targeting ORF7a sgRNA during SARS-CoV-2 infection decreased autophagy levels. Mechanistically, the ORF7a protein initiates autophagy via the AKT-MTOR-ULK1-mediated pathway, but ORF7a limits the progression of autophagic flux by activating CASP3 (caspase 3) to cleave the SNAP29 protein at aspartic acid residue 30 (D30), ultimately impairing complete autophagy. Importantly, SARS-CoV-2 infection-induced accumulated autophagosomes promote progeny virus production, whereby ORF7a downregulates SNAP29, ultimately resulting in failure of autophagosome fusion with lysosomes to promote viral replication. Taken together, our study reveals a mechanism by which SARS-CoV-2 utilizes the autophagic machinery to facilitate its own propagation via ORF7a.Abbreviations: 3-MA: 3-methyladenine; ACE2: angiotensin converting enzyme 2; ACTB/ß-actin: actin beta; ATG7: autophagy related 7; Baf A1: bafilomycin A1; BECN1: beclin 1; CASP3: caspase 3; COVID-19: coronavirus disease 2019; GFP: green fluorescent protein; hpi: hour post-infection; hpt: hour post-transfection; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERS: Middle East respiratory syndrome; MTOR: mechanistic target of rapamycin kinase; ORF: open reading frame; PARP: poly(ADP-ribose) polymerase; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; shRNAs: short hairpin RNAs; siRNA: small interfering RNA; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TCID50: tissue culture infectious dose; TEM: transmission electron microscopy; TUBB, tubulin, beta; ULK1: unc-51 like autophagy activating kinase 1.

Kynurenic acid ameliorates NLRP3 inflammasome activation by blocking calcium mobilization via GPR35.

The inflammasome has been linked to diverse inflammatory and metabolic diseases, and tight control of inflammasome activation is necessary to avoid excessive inflammation. Kynurenic acid (KA) is a tryptophan metabolite in the kynurenine pathway. However, the roles and mechanisms of the regulation of inflammasome activation by KA have not yet been fully elucidated. Here, we found that KA suppressed caspase-1 activation and IL-1ß production in macrophages by specifically inhibiting canonical and noncanonical activation of the NLRP3 inflammasome. Mechanistically, KA reduced calcium mobilization through G-protein receptor 35 (GPR35), resulting in reduced mitochondrial damage and decreased mtROS production, thus blocking NLRP3 inflammasome assembly and activation. Importantly, KA prevented lipopolysaccharide-induced systemic inflammation, monosodium urate-induced peritoneal inflammation, and high-fat diet-induced metabolic disorder. Thus, KA ameliorated inflammation and metabolic disorders by blocking calcium mobilization-mediated NLRP3 inflammasome activation via GPR35. Our data reveal a novel mechanism for KA in the modulation of inflammasome activation and suggest that GPR35 might be a promising target for improving NLRP3 inflammasome-associated diseases by regulating calcium mobilization.

Epidermal growth factor receptor (EGFR) promotes uptake of bovine parainfluenza virus type 3 into MDBK cells.

Bovine parainfluenza virus 3 (BPIV3) is an important respiratory pathogen of both young and adult cattle. The pathways of cell entry are highly related to viral transmission and pathogenicity. In previous studies, we demonstrated that macropinocytosis and clathrin-dependent endocytosis play critical roles in the entry of BPIV3 into MDBK cells. Macropinocytosis is special endocytic process which need to activate signaling pathways that remodle the actin cytoskeleton. Parainfluenza viruses (PIVs) initiate infection by binding to sialic acid receptors on cell surfaces. Nevertheless, sialic acids are not able to transmit signals across the plasma membrane, indicating the necessity for additional signaling receptors. Here, we have demonstrated that specific inhibitors and siRNAs targeting EGFR inhibit the entry of BPIV3 into MDBK cells. BPIV3 productive infection in MDBK cells led to activation of EGFR. Inactivation of EGFR suppressed BPIV3-induced rearrangement of the F-actin cytoskeleton. In addition, PI3K-Akt and ERK1/2 were activated in an EGFR-dependent manner during BPIV3 infection. Specific inhibitors targeting these canonical downstream effectors of EGFR could significantly reduce viral entry efficacy. Moreover, we also demonstrated that the important regulators of macropinocytosis, Rac1 and Pak1, are downstream mediators of EGFR during BPIV3 internalization. These results indicated that EGFR is a host-entry cofactor used by BPIV3 to enter MDBK cells.

An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups.

NLRP3 inflammasome is involved in the pathology of multiple human inflammatory diseases but there are still no clinically available medications targeting the NLRP3 inflammasome. We have previously identified RRx-001 as a highly selective and potent NLRP3 inhibitor, however, it contains high-energy nitro functional groups and may cause potential processing problems and generates highly toxic oxidants. Here, we show that compound 149-01, an RRx-001 analogue without high-energy nitro functional groups, is a potent, specific and covalent NLRP3 inhibitor. Mechanistically, 149-01 binds directly to cysteine 409 of NLRP3 to block the NEK7-NLRP3 interaction, thereby preventing NLRP3 inflammasome complex assembly and activation. Furthermore, treatment with 149-01 effectively alleviate the severity of several inflammatory diseases in mice, including lipopolysaccharide (LPS)-induced systemic inflammation, monosodium urate crystals (MSU)-induced peritonitis and experimental autoimmune encephalomyelitis (EAE). Thus, our results indicate that 149-01 is a potential lead for developing therapeutic agent for NLRP3-related inflammatory diseases.

Inhibition of the NLRP3 Inflammasome Activation by Manoalide Ameliorates Experimental Autoimmune Encephalomyelitis Pathogenesis.

The activation of NLRP3 inflammasome leads to cell pyroptosis and inflammatory cytokines secretion and gets involved in the development of many diseases, such as neuroinflammation and metabolic syndrome, but the drugs targeting NLRP3 are not clinically available for now. Through screening the small molecule library, we found that manoalide is a highly selective small molecule inhibitor of NLRP3. Mechanismly, manoalide inhibited the NLRP3 inflammasome activation by acting downstream of potassium efflux, chloride efflux and mitochondrial dysfunction. Moreover, manoalide blocked the interaction between NEK7 and NLRP3 by covalently binding to Lys 377 of the NLRP3 protein. Treatment of manoalide relieved the pathogenesis of experimental autoimmune encephalomyelitis (EAE) in mice. Thus, our results identify manoalide as a selective and covalent NLRP3 inhibitor and suggest it has the potential for the treatment of NLRP3-associated diseases.

Erratum: MiR-216a-5p inhibits tumorigenesis in Pancreatic Cancer by targeting TPT1/mTORC1 and is mediated by LINC01133: Erratum.

[This corrects the article DOI: 10.7150/ijbs.46822.].