Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide, 2',5' cGAMP, that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-ß gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-ß reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and that site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2',5' cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization.

Therapeutic Strategies Targeting Mitochondrial Dysfunction in Sepsis-induced Cardiomyopathy.

Sepsis is an increasingly worldwide problem; it is currently regarded as a complex life-threatening dysfunction of one or more organs as a result of dysregulated host immune response to infections. The heart is one of the most affected organs, as roughly 10% to 70% of sepsis cases are estimated to turn into sepsis-induced cardiomyopathy (SIC). SIC can be defined as a reversible myocardial dysfunction characterized by dilated ventricles, impaired contractility, and decreased ejection fraction. Mitochondria play a critical role in the normal functioning of cardiac tissues as the heart is highly dependent on its production of adenosine triphosphate (ATP), its damage during SIC includes morphology impairment, mitophagy, biogenesis disequilibrium, electron transport chain disturbance, molecular damage from the actions of pro-inflammatory cytokines and many other different impairments that are major contributing factors to the severity of SIC. Although mitochondria-targeted therapies usage is still inadequate in clinical settings, the preclinical study outcomes promise that the implementation of these therapies may effectively treat SIC. This review summarizes the different therapeutic strategies targeting mitochondria structure, quality, and quantity abnormalities for the treatment of SIC.

The Interaction of Bluetongue Virus VP6 and Genomic RNA Is Essential for Genome Packaging.

The genomes of the Reoviridae, including the animal pathogen bluetongue virus (BTV), are multisegmented double-stranded RNA (dsRNA). During replication, single-stranded (ss) positive-sense RNA segments are packaged into the assembling virus capsid, triggering genomic dsRNA synthesis. However, exactly how this packaging event occurs is not clear. A minor capsid protein, VP6, unique for the orbiviruses, has been proposed to be involved in the RNA-packaging process. In this study, we sought to characterize the RNA binding activity of VP6 and its functional relevance. A novel proteomic approach was utilized to map the ssRNA/dsRNA binding sites of a purified recombinant protein and the genomic dsRNA binding sites of the capsid-associated VP6. The data revealed that each VP6 protein has multiple distinct RNA-binding regions and that only one region is shared between recombinant and capsid-associated VP6. A combination of targeted mutagenesis and reverse genetics identified the RNA-binding region that is essential for virus replication. Using an in vitro RNA-binding competition assay, a unique cell-free assembly assay, and an in vivo single-cycle replication assay, it was possible to identify a motif within the shared binding region that binds BTV ssRNA preferentially in a manner consistent with specific RNA recruitment during capsid assembly. These data highlight the critical roles that this unique protein plays in orbivirus genome packaging and replication.IMPORTANCE Genome packaging is a critical stage during virus replication. For viruses with segmented genomes, the genome segments need to be correctly packaged into a newly formed capsid. However, the detailed mechanism of this packaging is unclear. Here we focus on VP6, a minor viral protein of bluetongue virus, which is critical for genome packaging. We used multiple approaches, including a robust RNA-protein fingerprinting assay, to map the ssRNA binding sites of recombinant VP6 and the genomic dsRNA binding sites of capsid-associated VP6. By these means, together with virological and biochemical methods, we identify the viral RNA-packaging motif of a segmented dsRNA virus for the first time.

Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages.

Coronaviruses are positive-sense RNA viruses that generate double-stranded RNA (dsRNA) intermediates during replication, yet evade detection by host innate immune sensors. Here we report that coronavirus nonstructural protein 15 (nsp15), an endoribonuclease, is required for evasion of dsRNA sensors. We evaluated two independent nsp15 mutant mouse coronaviruses, designated N15m1 and N15m3, and found that these viruses replicated poorly and induced rapid cell death in mouse bone marrow-derived macrophages. Infection of macrophages with N15m1, which expresses an unstable nsp15, or N15m3, which expresses a catalysis-deficient nsp15, activated MDA5, PKR, and the OAS/RNase L system, resulting in an early, robust induction of type I IFN, PKR-mediated apoptosis, and RNA degradation. Immunofluorescence imaging of nsp15 mutant virus-infected macrophages revealed significant dispersal of dsRNA early during infection, whereas in WT virus-infected cells, the majority of the dsRNA was associated with replication complexes. The loss of nsp15 activity also resulted in greatly attenuated disease in mice and stimulated a protective immune response. Taken together, our findings demonstrate that coronavirus nsp15 is critical for evasion of host dsRNA sensors in macrophages and reveal that modulating nsp15 stability and activity is a strategy for generating live-attenuated vaccines.

Structural and Functional Attributes of the Interleukin-36 Receptor.

Signal transduction by the IL-36 receptor (IL-36R) is linked to several human diseases. However, the structure and function of the IL-36R is not well understood. A molecular model of the IL-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the IL-36R. Mutational analyses and functional assays have identified residues of the receptor subunit IL-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of Il-1Rrp2 or IL-1RAcP exhibited dominant-negative effect on IL-36R signaling. The presence of IL-36 cytokine significantly increased the interaction of IL-1Rrp2 ECD with the co-receptor IL-1RAcP. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the IL-1Rrp2 that is present in ∼2% of the human population, down-regulated IL-36R signaling by a decrease of interaction with IL-1RAcP.

Hepatitis C Virus NS4B Can Suppress STING Accumulation To Evade Innate Immune Responses.

UNLABELLED: The cyclic dinucleotide 2',3'-cGAMP can bind the adaptor protein STING (stimulator of interferon [IFN] genes) to activate the production of type I IFNs and proinflammatory cytokines. We found that cGAMP added to the culture medium could suppress the replication of the hepatitis C virus (HCV) genotype 1b strain Con1 subgenomic replicon in human hepatoma cells. Knockdown of STING expression diminished the inhibitory effect on replicon replication, while overexpression of STING enhanced the inhibitory effects of cGAMP. The addition of cGAMP into 1b/Con1 replicon cells significantly increased the expression of type I IFNs and antiviral interferon-stimulated genes. Unexpectedly, replication of the genotype 2a JFH1 replicon and infectious JFH1 virus was less sensitive to the inhibitory effect of cGAMP than was that of 1b/Con1 replicon. Using chimeric replicons, 2a NS4B was identified to confer resistance to cGAMP. Transient expression of 2a NS4B resulted in a pronounced inhibitory effect on STING-mediated beta IFN (IFN-ß) reporter activation compared to that of 1b NS4B. 2a NS4B was found to suppress STING accumulation in a dose-dependent manner. The predicted transmembrane domain of 2a NS4B was required to inhibit STING accumulation. These results demonstrate a novel genotype-specific inhibition of the STING-mediated host antiviral immune response. IMPORTANCE: The cyclic dinucleotide cGAMP was found to potently inhibit the replication of HCV genotype 1b Con1 replicon but was less effective for the 2a/JFH1 replicon and infectious JFH1 virus. The predicted transmembrane domain in 2a NS4B was shown to be responsible for the decreased sensitivity to cGAMP. The N terminus of NS4B has been reported to suppress STING-mediated signaling by disrupting the interaction of STING and TBK1 and/or MAVS. We show that 2a/JFH1 NS4B has an additional mechanism to evade STING signaling through suppressing STING accumulation.

Heterogeneous Ribonucleoprotein K (hnRNP K) Binds miR-122, a Mature Liver-Specific MicroRNA Required for Hepatitis C Virus Replication.

Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5' untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing ∼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3' portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.

Oxidized low-density lipoprotein attenuated desmoglein 1 and desmocollin 2 expression via LOX-1/Ca(2+)/PKC-ß signal in human umbilical vein endothelial cells.

Numerous studies have reported the presence of oxidized LDL (ox-LDL) and expression of its lectin-like receptor, LOX-1, have been shown in atherosclerotic regions. The present study aims to investigate the effects of ox-LDL on expression of desmoglein 1 (DSG1) and desmocollin 2 (DSC2) in endothelial cells, and to explore the role of LOX-1 mediated signal in the permeability injury associated with DSG1 and DSC2 disruption induced by oxidized lipoprotein. RT-PCR and Western blotting were applied to determine the mRNA and protein expression levels of DSG1 and DSC2 in human umbilical vein endothelial cells (HUVECs) respectively. Immunoreactivities of DSG1 and DSC2 were detected by laser scanning confocal microscope (LSCM). HUVEC monolayers permeability was evaluated by FITC-labeled LDL in transwell assay system. The possible signal was assessed using in vitro blocking LOX-1 or Ca(2+) channel or PKC. The DSG1 and DSC2 expression were decreased by ox-LDL in concentration- and time-dependent manner. The effects of ox-LDL were mediated by its endothelial receptor, LOX-1. In parallel experiments, ox-LDL increased the influx of extracellular calcium, activation of protein kinase C (PKC) and permeability to LDL, which was inhibited by the LOX-1blocking antibody (10 µg/ml), Ca(2+) channel blocker (Diltiazem, 50 µmol/L) and PKC-ß inhibitor (hispidin, 4 µmol/L). These results suggested that ox-LDL-induced decrease in DSG1 and DSC2 expression and monolayer barrier injury via calcium uptake and PKC-ß activation following up-regulation of LOX-1 is one of the mechanisms of inducing greater permeability in HUVECs.

High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2.

High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL.

Mitochondria-Associated Organelle Crosstalk in Myocardial Ischemia/Reperfusion Injury.

Organelle damage is a significant contributor to myocardial ischemia/reperfusion (I/R) injury. This damage often leads to disruption of endoplasmic reticulum protein regulatory programs and dysfunction of mitochondrial energy metabolism. Mitochondria and endoplasmic reticulum are seamlessly connected through the mitochondrial-associated endoplasmic reticulum membrane (MAM), which serves as a crucial site for the exchange of organelles and metabolites. However, there is a lack of reports regarding the communication of information and metabolites between mitochondria and related organelles, which is a crucial factor in triggering myocardial I/R damage. To address this research gap, this review described the role of crosstalk between mitochondria and the correlative organelles such as endoplasmic reticulum, lysosomal and nuclei involved in reperfusion injury of the heart. In summary, this review aims to provide a comprehensive understanding of the crosstalk between organelles in myocardial I/R injury, with the ultimate goal of facilitating the development of targeted therapies based on this knowledge.

Cell death in atherosclerosis.

A complex and evolutionary process that involves the buildup of lipids in the arterial wall and the invasion of inflammatory cells results in atherosclerosis. Cell death is a fundamental biological process that is essential to the growth and dynamic equilibrium of all living things. Serious cell damage can cause a number of metabolic processes to stop, cell structure to be destroyed, or other irreversible changes that result in cell death. It is important to note that studies have shown that the two types of programmed cell death, apoptosis and autophagy, influence the onset and progression of atherosclerosis by controlling these cells. This could serve as a foundation for the creation of fresh atherosclerosis prevention and treatment strategies. Therefore, in this review, we summarized the molecular mechanisms of cell death, including apoptosis, pyroptosis, autophagy, necroptosis, ferroptosis and necrosis, and discussed their effects on endothelial cells, vascular smooth muscle cells and macrophages in the process of atherosclerosis, so as to provide reference for the next step to reveal the mechanism of atherosclerosis.

Insight into modulators of sphingosine-1-phosphate receptor and implications for cardiovascular therapeutics.

Cardiovascular disease is the leading cause of death worldwide, and it's of great importance to understand its underlying mechanisms and find new treatments. Sphingosine 1-phosphate (S1P) is an active lipid that exerts its effects through S1P receptors on the cell surface or intracellular signal, and regulates many cellular processes such as cell growth, cell proliferation, cell migration, cell survival, and so on. S1PR modulators are a class of modulators that can interact with S1PR subtypes to activate receptors or block their activity, exerting either agonist or functional antagonist effects. Many studies have shown that S1P plays a protective role in the cardiovascular system and regulates cardiac physiological functions mainly through interaction with cell surface S1P receptors (S1PRs). Therefore, S1PR modulators may play a therapeutic role in cardiovascular diseases. Here, we review five S1PRs and their functions and the progress of S1PR modulators. In addition, we focus on the effects of S1PR modulators on atherosclerosis, myocardial infarction, myocardial ischaemia/reperfusion injury, diabetic cardiovascular diseases, and myocarditis, which may provide valuable insights into potential therapeutic strategies for cardiovascular disease.

Turmeric-Derived Nanoparticles Functionalized Aerogel Regulates Multicellular Networks to Promote Diabetic Wound Healing.

Regulation of excessive inflammation and impaired cell proliferation is crucial for healing diabetic wounds. Although plant-to-mammalian regulation offers effective approaches for chronic wound management, the development of a potent plant-based therapeutic presents challenges. This study aims to validate the efficacy of turmeric-derived nanoparticles (TDNPs) loaded with natural bioactive compounds. TDNPs can alleviate oxidative stress, promote fibroblast proliferation and migration, and reprogram macrophage polarization. Restoration of the fibroblast-macrophage communication network by TDNPs stimulates cellular regeneration, in turn enhancing diabetic wound healing. To address diabetic wound management, TDNPs are loaded in an ultralight-weight, high swelling ratio, breathable aerogel (AG) constructed with cellulose nanofibers and sodium alginate backbones to obtain TDNPs@AG (TAG). TAG features wound shape-customized accessibility, water-adaptable tissue adhesiveness, and capacity for sustained release of TDNPs, exhibiting outstanding performance in facilitating in vivo diabetic wound healing. This study highlights the potential of TDNPs in regenerative medicine and their applicability as a promising solution for wound healing in clinical settings.

T7 expression plasmids for producing a recombinant human G1P[8] rotavirus comprising RIX4414 sequences of the RV1 (Rotarix , GSK) vaccine strain.

The live oral rotavirus RV1 (Rotarix) vaccine is formulated from the human G1P[8] RIX4414 virus. Based on RIX4414 sequences, T7 expression plasmids were constructed that supported recovery of recombinant RIX4414-like viruses by reverse genetics. These plasmids will advance the study of the RV1 vaccine, possibly allowing improvements to its efficacy.

[Retracted] Oxidized­low density lipoprotein accumulates cholesterol esters via the PKCα­adipophilin­ACAT1 pathway in RAW264.7 cells.

Following the publication of the above paper, a concerned reader drew to the Editor's attention that the "con" and "ox­LDL" panels in Fig. 1E on p. 3602, and various data panels included in Figs. 3 and 5 on p. 3604, contained apparent anomalies, including what appeared to be matching patternings of cellular data either within the same figure panels or comparing among the data panels. After having conducted an independent investigation in the Editorial Office, the Editor of Molecular Medicine Reports has determined that the above paper should be retracted from the Journal on account of a lack of confidence in the overall authenticity of the data. After having consulted the authors in this regard, they agreed with the decision to retract this paper. The Editor deeply regrets any inconvenience that has been caused to the readership of the Journal. [Molecular Medicine Reports 12: 3599­3606, 2015; DOI: 10.3892/mmr.2015.3864.

NIR-II Responsive Nanohybrids Incorporating Thermosensitive Hydrogel as Sprayable Dressing for Multidrug-Resistant-Bacteria Infected Wound Management.

Developing an effective dressing against bacterial infection and synchronously addressing wound complications, such as bleeding, long-term inflammation, and reinfection, are highly desirable in clinical practice. In this work, a second near-infrared (NIR-II) responsive nanohybrid consisting of imipenem encapsulated liposome with gold-shell and lipopolysaccharide (LPS)-targeting aptamer, namely ILGA, is constructed for bacteria elimination. Benefiting from the delicate structure, ILGA exhibits strong affinity and a reliable photothermal/antibiotic therapeutic effect toward multidrug-resistant Pseudomonas aeruginosa (MDR-PA). Furthermore, by incorporating ILGA with a thermosensitive hydrogel poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA), a sprayable dressing ILGA@Gel was prepared, which enables a quick on-demand gelation (10 s) for wound hemostasis and offers excellent photothermal/antibiotic efficacy to sterilize the infected wound. Additionally, ILGA@Gel provides satisfactory wound-healing environments by reeducating wound-associated macrophages for inflammation alleviation and forming a gel layer to block exogenous bacterial reinfection. This biomimetic hydrogel reveals excellent bacteria eradication and wound recovery effectiveness, demonstrating its promising potential for managing complicated infected wounds.

Biochemical study of the comparative inhibition of hepatitis C virus RNA polymerase by VX-222 and filibuvir.

Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC(50)s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (K(d)) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.

An involvement of SR-B1 mediated PI3K-Akt-eNOS signaling in HDL-induced cyclooxygenase 2 expression and prostacyclin production in endothelial cells.

It is well-known that sphingosine-1-phosphate (S1P), the phospholipid content of HDL, binding to S1P receptors can raise COX-2 expression and PGI(2) release through p38MAPK/CREB pathway. In the present study we assess the action of SR-B1 initiated PI3K-Akt-eNOS signaling in the regulation of COX-2 expression and PGI(2) production in response to HDL. We found that apoA1 could increase PGI(2) release and COX-2 expression in ECV 304 endothelial cells. Furthermore, SR-B1 was found to be involved in HDL induced up-regulation of COX-2 and PGI(2). Over-expressed SR-B1 did not significantly increase the expression of COX-2 and the PGI(2) levels, but knock-down of SR-B1 by siRNA could significantly attenuate COX-2 expression and PGI(2) release together with p38MAPK and CREB phosphorylation. Consistently, the declines of p-p38MAPK, p-CREB, COX-2 and PGI(2) were also observed after incubation with LY294002 (25µmol/L; PI3K special inhibitor) or L-NAME (50µmol/L; eNOS special inhibitor). In addition, we demonstrated the increases of PGI(2) release, COX-2 expression and p38MAPK phosphorylation, when nitric oxide level was raised through the incubation of L-arginine (10 or 20nmol/L) in endothelial cells. Taking together, our data support that SR-B1 mediated PI3K-Akt-eNOS signaling was involved in HDL-induced COX-2 expression and PGI(2) release in endothelial cells.

Regulation of metabolism and transport of sphingosine-1-phosphate in mammalian cells.

Sphingosine-1-phosphate (S1P), which is generated from the sphingosine kinase-catalyzed phosphorylation of sphingosine, is now recognized as a critical regulator of many kinds of physiological and pathological processes, including cancer, cardiovascular function, and diabetes. It can also trigger a wide variety of biological effect, such as cell movement, differentiation, survival, inflammation, immunity, calcium homeostasis, and angiogenesis. As we know, a number of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface or intracellular targets. However, the synthesis and the secretion of S1P are regulated by various endogenetic or ectogenous stimuli and involve many kinds of enzymes and transporters. In this review, we discuss the regulation of S1P synthesis by many kinds of enzymes and mainly introduce the process of ceramide to S1P. Moreover, S1P deterioration is important balance in physiologic adjustment. We also describe the role of verified or potential transporters in S1P release in detail.