Foot-and-Mouth Disease Virus Induces Porcine Gasdermin E-Mediated Pyroptosis through the Protease Activity of 3Cpro.

Foot-and-mouth disease (FMD) is an acute, highly contagious disease of cloven-hoofed animals caused by FMD virus (FMDV). Currently, the molecular pathogenesis of FMDV infection remains poorly understood. Here, we demonstrated that FMDV infection induced gasdermin E (GSDME)-mediated pyroptosis independent of caspase-3 activity. Further studies showed that FMDV 3Cpro cleaved porcine GSDME (pGSDME) at the Q271-G272 junction adjacent to the cleavage site (D268-A269) of porcine caspase-3 (pCASP3). The inhibition of enzyme activity of 3Cpro failed to cleave pGSDME and induce pyroptosis. Furthermore, overexpression of pCASP3 or 3Cpro-mediated cleavage fragment pGSDME-NT was sufficient to induce pyroptosis. Moreover, the knockdown of GSDME attenuated the pyroptosis caused by FMDV infection. Our study reveals a novel mechanism of pyroptosis induced by FMDV infection and might provide new insights into the pathogenesis of FMDV and the design of antiviral drugs. IMPORTANCE Although FMDV is an important virulent infectious disease virus, few reports have addressed its relationship with pyroptosis or pyroptosis factors, and most studies focus on the immune escape mechanism of FMDV. GSDME (DFNA5) was initially identified as being associated with deafness disorders. Accumulating evidence indicates that GSDME is a key executioner for pyroptosis. Here, we first demonstrate that pGSDME is a novel cleavage substrate of FMDV 3Cpro and can induce pyroptosis. Thus, this study reveals a previously unrecognized novel mechanism of pyroptosis induced by FMDV infection and might provide new insights into the design of anti-FMDV therapies and the mechanisms of pyroptosis induced by other picornavirus infections.

Seneca Valley Virus Induces DHX30 Cleavage to Antagonize Its Antiviral Effects.

Seneca Valley virus (SVV) is a new pathogen associated with porcine idiopathic vesicular disease (PIVD) in recent years. However, SVV-host interaction is still unclear. In this study, through LC-MS/MS analysis and coimmunoprecipitation analysis, DHX30 was identified as a 3Cpro-interacting protein. 3Cpro mediated the cleavage of DHX30 at a specific site, which depends on its protease activity. Further study showed that DHX30 was an intrinsic antiviral factor against SVV that was dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of viral infection. RIP-seq showed comparatively higher coverage depth at SVV 5'UTR, but the distribution across SVV RNA suggested that the interaction had low specificity. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. Interestingly, DHX30 was determined to interact with 3D in an SVV RNA-dependent manner. Thus, DHX30 negatively regulated SVV propagation by blocking viral RNA synthesis, presumably by participating in the viral replication complex. IMPORTANCE DHX30, an RNA helicase, is identified as a 3Cpro-interacting protein regulating Seneca Valley virus (SVV) replication dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of virus infection. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. In addition, 3Cpro abolished DHX30 antiviral effects by inducing DHX30 cleavage. Thus, DHX30 is an intrinsic antiviral factor that inhibits SVV replication.

Porcine Sapelovirus 3Cpro Inhibits the Production of Type I Interferon.

Porcine sapelovirus (PSV) is the causative pathogen of reproductive obstacles, acute diarrhea, respiratory distress, or severe polioencephalomyelitis in swine. Nevertheless, the pathogenicity and pathogenic mechanism of PSV infection are not fully understood, which hinders disease prevention and control. In this study, we found that PSV was sensitive to type I interferon (IFN-ß). However, PSV could not activate the IFN-ß promoter and induce IFN-ß mRNA expression, indicating that PSV has evolved an effective mechanism to block IFN-ß production. Further study showed that PSV inhibited the production of IFN-ß by cleaving mitochondrial antiviral signaling (MAVS) and degrading melanoma differentiation-associated gene 5 (MDA5) and TANK-binding kinase 1 (TBK1) through viral 3Cpro. In addition, our study demonstrated that PSV 3Cpro degrades MDA5 and TBK1 through its protease activity and cleaves MAVS through the caspase pathway. Collectively, our results revealed that PSV inhibits the production of type I interferon to escape host antiviral immunity through cleaving and degrading the adaptor molecules.

The Diagnostic Value of Serum Ang, VEGF, and CRP Combined with the Chinese Medicine Antitumor Formula in the Treatment of Advanced Renal Carcinoma.

OBJECTIVE: To explore the diagnostic value of serum angiopoietin (Ang), vascular endothelial growth factor (VEGF), and C-reactive protein (CRP) combined with the Chinese medicine antitumor formula in the treatment of advanced renal carcinoma. METHODS: Retrospective analysis was performed for the data of 60 patients with advanced renal cancer admitted at Yantaishan Hospital from February 2019 to February 2020. All patients were treated with Chinese medicine antitumor formula. The serum Ang, VEGF, and CRP levels in venous blood samples were detected before and after treatment. Sensitivity, specificity, and AUC of combined serum Ang, VEGF, and CRP were analyzed utilizing the receiver operating characteristic curve (ROC) (95% CI). RESULTS: There were 52 cases of clear-cell carcinoma (86.7%), 7 cases of papillary carcinoma (11.7%), and 1 case of chromophobe renal cell carcinoma (1.7%). The average tumor diameter was (9.67 ± 0.65) cm, and the KPS score was (74.68 ± 1.52). About 75% of the patients had metastasis. After treatment, the level of serum Ang, VEGF, and CRP was immensely lower compared to that before treatment (P < 0.001). The sensitivity, specificity, and AUC (95%CI) of the combined detection of Ang, VEGF, and CRP before treatment were 86.7%, 90.0%, and 0.883 (0.817-0.950), while the sensitivity, specificity, and AUC (95%CI) of the combined detection of Ang, VEGF, and CRP were 83.3%, 86.7%, and 0.850 (0.776-0.9524), respectively. CONCLUSION: The combined detection of serum Ang, VEGF, and CRP has high diagnostic value for patients with advanced renal cancer treated with Chinese medicine antitumor formula.

The Effect of Resveratrol on Blood Glucose and Blood Lipids in Rats with Gestational Diabetes Mellitus.

BACKGROUND: Previous studies have reported that resveratrol has various biological effects such as anti-inflammatory, antioxidant, and antitumor. This study aimed to investigate the effects of resveratrol on blood glucose and blood lipids in rats with gestational diabetes mellitus (GDM). METHODS: The rat diabetes model was prepared by one-time intraperitoneal injection of streptozotocin (STZ, 35 mg/kg). Fasting blood glucose was measured by using a blood glucose meter. The ELISA method was used to detect the levels of insulin, leptin, adiponectin, resistin, TNF-α, and IL-6. The content of TC, TG, LDL-C, and HDL-C was determined by using an automatic biochemical detector. RESULTS: Compared with the GDM group, the insulin level in the resveratrol (120 and 240 mg/kg) treatment group was significantly increased. But, the blood glucose level and body weight were significantly reduced. The content of TC, TG, and LDL-C in the resveratrol (240 mg/kg) treatment group was significantly reduced, and the content of HDL-C was significantly increased. In addition, leptin, resistin, TNF-α, and IL-6 levels in the 240 mg/kg resveratrol treatment group were significantly reduced, and adiponectin was significantly increased. Also, resveratrol (240 mg/kg) was stronger than metformin hydrochloride in improving insulin secretion and regulating blood lipids and adipokine content. CONCLUSION: Resveratrol has a dose-dependent effect on GDM rats to increase insulin secretion, reduce blood glucose and body weight, and regulate blood lipids and plasma adipokines.

The Efficacy of Tripterygium Glycosides Combined with LMWH in Treatment of HSPN in Children.

OBJECTIVE: This study aimed to explore the clinical efficacy and relevant mechanism of Tripterygium glycosides combined with low molecular weight heparin calcium (LMWH) in the treatment of Henoch-Schönlein purpura nephritis (HSPN) in children. METHODS: 64 cases of children patients with HSPN treated at Qilu Hospital (Qingdao) from January 2015 to May 2020 were selected and randomly divided into the control group and the observation group and 32 cases in each group. Conventional medical treatment was applied in the two groups, besides which the control group was given LMWH while the observation group was given Tripterygium glycosides based on the control group. The clinical efficacy and the indexes of clinical symptoms of the two groups were compared. Immune globulin level, fibrinogen content (FIB), prothrombin time (PT), platelet level (PLT), and activated partial thromboplastin time (APTT) level of the two groups were compared before and after the treatment. RESULTS: The total effective rate in the observation group was significantly higher than that of the control group, and the recurrence rate in the observation group was lower than that in the control group. After treatment, urine red blood cell count and 24 h urine protein were obviously better than those of the control group. There was no statistically significant difference in PT between the two groups of children before and after treatment. The levels of PLT and FIB in the two groups of patients after treatment were significantly lower than before treatment, and the PLT levels in the observation group were lower than those in the control group. CONCLUSION: The combination of Tripterygium glycosides and LMWH had good clinical effects in the treatment of children with HSPN, and it could improve the clinical symptoms, the mechanism of which might be related to the increase of PT, a decrease of PLT, and the improvement of coagulation function.

Seneca Valley Virus 3C Protease Induces Pyroptosis by Directly Cleaving Porcine Gasdermin D.

Seneca Valley virus (SVV), a newly emerging virus belonging to the Picornaviridae family, has caused vesicular disease in the swine industry. However, the molecular mechanism of viral pathogenesis remains poorly understood. This study revealed that SVV infection could induce pyroptosis in SK6 cells in a caspase-dependent and -independent manner. SVV may inhibit caspase-1 activation at late infection because of 3Cpro cleavage of NLRP3, which counteracted pyroptosis activation. Further study showed that 3Cpro targeted porcine gasdermin D (pGSDMD) for cleavage through its protease activity. 3Cpro cleaved porcine GSDMD (pGSDMD) at two sites, glutamine 193 (Q193) and glutamine 277 (Q277), and Q277 was close to the caspase-1-induced pGSDMD cleavage site. pGSDMD1-277 triggered cell death, which was similar to N-terminal fragment produced by caspase-1 cleavage of pGSDMD, and other fragments exhibited no significant inhibitory effects on cellular activity. Ectopic expression of pGSDMD converted 3Cpro-induced apoptosis to pyroptosis in 293T cells. Interestingly, 3Cpro did not cleave mouse GSDMD or human GSDMD. And, both pGSDMD and pGSDMD1-277 exhibited bactericidal activities in vivo. Nevertheless, pGSDMD cannot kill bacteria in vitro. Taken together, our results reveal a novel pyroptosis activation manner produced by viral protease cleavage of pGSDMD, which may provide an important insight into the pathogenesis of SVV and cancer therapy.

Selective autophagy receptor SQSTM1/ p62 inhibits Seneca Valley virus replication by targeting viral VP1 and VP3.

Macroautophagy/autophagy plays a critical role in antiviral immunity through targeting viruses and initiating host immune responses. The receptor protein, SQSTM1/p62 (sequestosome 1), plays a vital role in selective autophagy. It serves as a receptor targeting ubiquitinated proteins or pathogens to phagophores for degradation. In this study, we explored the reciprocal regulation between selective autophagy receptor SQSTM1 and Seneca Valley virus (SVV). SVV infection induced autophagy. Autophagy promoted SVV infection in pig cells but played opposite functions in human cells. Overexpression of SQSTM1 decreased viral protein production and reduced viral titers. Further study showed that SQSTM1 interacted with SVV VP1 and VP3 independent of its UBA domain. SQSTM1 targeted SVV VP1 and VP3 to phagophores for degradation to inhibit viral replication. To counteract this, SVV evolved strategies to circumvent the host autophagic machinery to promote viral replication. SVV 3Cpro targeted the receptor SQSTM1 for cleavage at glutamic acid 355, glutamine 392, and glutamine 395 and abolished its capacity to mediate selective autophagy. At the same time, the 3Cpro-mediated SQSTM1 cleavage products lost the ability to inhibit viral propagation. Collectively, our results provide evidence for selective autophagy in host against viruses and reveal potential viral strategies to evade autophagic machinery for successful pathogenesis.Abbreviations: Baf.A1: bafilomycin A1; Co-IP: co-immunoprecipitation; hpi: h post-infection; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MOI: multiplicity of infection; PB1: N-terminal Phox/Bem1p; Rap.: rapamycin; Seneca Valley virus: SVV; SQSTM1/p62: sequestosome 1; SQSTM1-N355: residues 1 to 355 of SQSTM1; SQSTM1-C355: residues 355 to 478 of SQSTM1; SQSTM1-N392: residues 1 to 392 of SQSTM1; SQSTM1-C392: residues 392 to 478 of SQSTM1; SQSTM1-N388: residues 1 to 388 of SQSTM1; SQSTM1-N397: residues 1 to 397 of SQSTM1; UBA: ubiquitin association; Ubi: ubiquitin.

Seneca Valley Virus 3C Protease Inhibits Stress Granule Formation by Disrupting eIF4GI-G3BP1 Interaction.

Stress granules (SGs) are the sites of mRNA storage and related to the regulation of mRNA translation, which are dynamic structures in response to various environmental stresses and viral infections. Seneca Valley virus (SVV), an oncolytic RNA virus belonging to Picornaviridae family, can cause vesicular disease (VD) indistinguished from foot-and-mouth disease (FMD) and other pig VDs. In this study, we found that SVV induced SG formation in the early stage of infection in a PKR-eIF2α dependent manner, as demonstrated by the recruitment of marker proteins of G3BP1 and eIF4GI. Surprisingly, we found that downregulating SG marker proteins TIA1 or G3BP1, or expressing an eIF2α non-phosphorylatable mutant inhibited SG formation, but this inhibition of transient SG formation had no significant effect on SVV propagation. Depletion of G3BP1 significantly attenuated the activation of NF-κB signaling pathway. In addition, we found that SVV inhibited SG formation at the late stage of infection and 3C protease was essential for the inhibition depending on its enzyme activity. Furthermore, we also found that 3C protease blocked the SG formation by disrupting eIF4GI-G3BP1 interaction. Overall, our results demonstrate that SVV induces transient SG formation in an eIF2α phosphorylation and PKR-dependent manner, and that 3C protease inhibits SG formation by interfering eIF4GI-G3BP1 interaction.

Optical resolution and optimization of (R,S)-propranolol using dehydroabietic acid via diastereomeric crystallization.

The optical resolution of (R,S)-propranolol by the diastereomeric crystallization method was successfully performed using dehydroabietic acid (DHAA) as the resolving agent in methanol. The three important parameters: DHAA amount, solvent (methanol) amount, and crystallization temperature of diastereomeric salts were optimized employing the response surface methodology (RSM). When maintaining a lower limit of 95% for the purity of (S)-propranolol, the optimal resolution conditions were a DHAA/(R,S)-propranolol molar ratio of 1.1, solvent/(R,S)-propranolol ratio of 16.2 mL.g(-1) , and crystallization temperature of -5 °C. The desired (S)-propranolol was prepared with 94.8% optical purity and 72.2% yield under the optimal conditions.