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.

cAMP-response element binding protein mediates podocyte injury in diabetic nephropathy by targeting lncRNA DLX6-AS1.

BACKGROUND: Progressive proteinuria is one of the earliest clinical features of diabetic nephropathy (DN). In our previous study, lncRNA DLX6-AS1 (DLX6-AS1, Dlx6os1 in the mouse) was found to be associated with the extent of albuminuria in DN patients. Furthermore, the lack of Dlx6os1 was pivotal in switching off the inflammatory response in db/db mouse model. However, the regulatory factors responsible for elevated DLX6-AS1 in DN remains unknown. METHODS: To identify potential regulatory factors for DLX6-AS1, JASPAR database and DNA pull down combined subsequent liquid chromatography-tandem mass spectrometry were used. Dual-luciferase reporter assay and chromatin immunoprecipitation were then performed to confirm binding sites. We also investigated the effects of the regulatory factors on DN progression in db/db mouse model and cultured human podocytes. RESULTS: Our analyses demonstrated that cAMP-response element binding protein (CREB) was highly expressed and closely associated with DLX6-AS1 in DN. In db/db mouse and in cultured podocytes, CREB silencing significantly reduced the level of DLX6-AS1 or Dlx6os1 and attenuated renal damage. Mechanistically, CREB overexpression aggravated renal inflammation and destroyed the structure of podocytes by targeting DLX6-AS1. The damaging role of CREB in podocyte injury was also inhibited by 666-15, a selective inhibitor, in a dose-dependent manner. In vivo, the inhibition of CREB by 666-15 significantly attenuated albuminuria and ameliorated inflammatory infiltration in podocytes. CONCLUSIONS: Our findings indicated that CREB is a key mediator of podocyte injury and acts by regulating DLX6-AS1. Thus, CREB may be an effective and potential therapeutic target for the treatment of DN.

GAB1 alleviates septic lung injury by inhibiting the TLR4/ NF-κB pathway.

Sepsis, with its high morbidity and mortality, is a difficult problem in critical care medicine. The purpose of this study is to investigate the involvement of GRB2-associated binding protein 1 (GAB1) in septic lung injury. Lipopolysaccharide (LPS)-induced mouse model and A549 cell model were used to simulate septic lung injury. Haematoxylin and eosin (H&E) staining was used to observe the pathological changes. The terminal-deoxynucleotidyl transferase/(TdT)-mediated dUTP-biotin nick end labelling (TUNEL) staining and flow cytometry were used to detect apoptosis. The levels of inflammatory factors in the bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA). In LPS-induced sepsis mice, GAB1 expression was markedly reduced, and GAB1 overexpression significantly attenuated cell apoptosis and decreased levels of macrophages, neutrophils, and inflammatory factors in the BALF. Our results also demonstrated that GAB1 overexpression significantly reduced LPS-induced apoptosis and inflammation of A549 cells. More importantly, GAB1 overexpression significantly inhibited the Toll-like receptor/ NFkappaB (TLR4/NF-κB) pathway, while silencing GAB1 significantly activated the TLR4/NF-κB pathway and induced apoptosis and increased expression of inflammatory factors. However, the TLR4 inhibitor TAK-242 eliminated the effect of GAB1 silencing on A549. In conclusion, GAB1 is a key regulator of sepsis by inhibiting TLR4/NF-κB mediated apoptosis and inflammation.

The PCT to Albumin Ratio Predicts Mortality in Patients With Acute Kidney Injury Caused by Abdominal Infection-Evoked Sepsis.

Purpose: Considerable evidence suggests that inflammation and malnutrition are common in patients with acute kidney injury (AKI) and correlated with mortality of various diseases. Despite this, few studies have reported the underlying predictive effects of inflammatory and nutritional markers in combination on the mortality of AKI patients. Herein, we aimed to explore the values of PCT and CRP as well as the ratios of PCT/Alb and CRP/Alb in the poor prognosis of patients with sepsis-induced AKI. Patients and Methods: A total of 171 patients with AKI, caused by abdominal infection-evoked sepsis, were retrospectively studied and divided into a survival group (107 cases) and a non-survival group (64 cases). Univariate analysis was used to compare the clinical data of the two groups. Multivariate logistic regression analysis was used to analyze the independent risk factors of poor prognosis in patients with sepsis-induced AKI. The ROC curve was used to evaluate the diagnostic value of PCT, CRP, PCT/Alb, and CRP/Alb in the poor prognosis of patients with sepsis-induced AKI. Results: Univariate analysis revealed that the values of PCT, CRP, CRP/Alb, and PCT/Alb were significantly different between AKI survival and non-survival groups, and further analysis found that PCT and PCT/Alb were independent risk factors for poor prognosis in patients with sepsis-induced AKI after adjusting with age and gender. Of note, the predictive accuracy (0.864 vs. 0.807), specificity (83.2 vs. 69.2), and sensitivity (79.7 vs. 76.6) of PCT/Alb were all higher than that of the simple PCT. Conclusions: The ratio of PCT to Alb is an independent risk factor possessing a robust and accurate risk assessment for the poor prognosis of patients with sepsis-induced AKI, and it offers the potential to improve the management of this type of disease and a lower resultant mortality.

SPAG5 promotes hepatocellular carcinoma progression by downregulating SCARA5 through modifying ß-catenin degradation.

BACKGROUND: The sperm-associated antigen 5 (SPAG5) plays a key role in controlling various cellular phenomena, including cell cycle progression and proliferation. However, the role of SPAG5 in hepatocellular carcinoma (HCC) remains unknown. METHODS: This study investigated the function and clinical significance of SPAG5 protein expression in hepatocellular carcinoma. We analyzed SPAG5 expression in surgical specimens from 136 HCC patients. The correlation between the clinical characteristics and prognosis was also determined. Furthermore, the SPAG5 was overexpressed in HCC cell and silenced with shRNA in HCC cells. Moreover, cell proliferation and apoptosis were measured using Edu assay and flow cytometry and a molecular mechanism of SPAG5 promotes HCC progression was explored. RESULTS: Herein, our study showed that upregulation of SPAG5 was detected frequently in primary HCC tissues, and was associated with significantly worse survival among the HCC patients. Multivariate analyses revealed that high SPAG5 expression was an independent predictive marker for the poor prognosis of HCC. SPAG5 silence effectively abolished the proliferation abilities of SPAG5 in vivo and in vitro, while induced apoptosis in HCC cells. Furthermore, our results indicate that SPAG5 promoted cell progression by decreasing SCARA5 expression, which has been reported to control the progression of HCC, and our data demonstrated that SCARA5 is crucial for SPAG5-mediated HCC cell progression in vitro and in vivo. Moreover, we found that the expression of SPAG5 and SCARA5 are inversely correlated in HCC tissues. In addition, we demonstrated that SPAG5 promoted progression in HCC via downregulating SCARA5 depended on the ß-catenin/TCF4 signaling pathway. Interestingly, the underlying mechanism is which SPAG5 regulates SCARA5 expression by modulating ß-catenin degradation. CONCLUSIONS: Taken together, our data provide a novel evidence for the biological and clinical significance of SPAG5 as a potential biomarker, and we demonstrate that SPAG5-ß-catenin-SCARA5 might be a novel pathway involved in HCC progression.

Retrograde puncture assisted hepatic vein recanalization in treating Budd-Chiari syndrome with segmental obstruction of hepatic vein.

PURPOSE: This study aimed at investigating the feasibility and effectiveness of retrograde puncture assisted hepatic vein (HV) recanalization in management of Budd-Chiari syndrome (BCS) patients with segmental obstruction of HV. MATERIALS AND METHODS: From May 2011 to August 2014, 76 BCS patients with obstruction of HV were treated by routine transjugular HV recanalization in our center. Among them, 17 patients with segmental obstruction (obstruction length >1 cm) of HV experienced failure of the routine transjugular HV recanalization and underwent retrograde puncture assisted HV recanalization. Data on technical success, clinical success and follow-up were collected and analyzed retrospectively. RESULTS: Retrograde puncture assisted HV recanalization was technically successful in 14 of 17 (82 %) patients. Of these 14 patients, 12 patients underwent HV balloon dilation, and 2 patients underwent HV stent insertion. No procedure-related complications occurred in any of our patient. Clinical success was achieved in all of the 14 patients who experienced technical success. The mean HV pressure decreased from 43.6 ± 6.7 cmH2O before treatment to 18.4 ± 4.8 cmH2O after treatment (P < 0.001). BCS-related symptoms began to improve on the next day following the treatment. During 4-43 months (mean 17.4 ± 10.8 months) of follow-up, three patients experienced re-obstruction of HV. CONCLUSIONS: Retrograde puncture assisted HV recanalization is a simple, safe, and effective treatment for BCS patients with segmental obstruction of HV. It can serve as an additional treatment option for patients who experience the technical failure of routine transjugular HV recanalization.