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Treatment strategies that target host entry factors have proven an effective means of impeding viral entry in HIV and may be more robust to viral evolution than drugs targeting viral proteins directly. High-throughput functional screens provide an unbiased means of identifying genes that influence the infection of host cells, while retrospective cohort analysis can measure the real-world, clinical potential of repurposing existing therapeutics as antiviral treatments. Here, we combine these two powerful methods to identify drugs that alter the clinical course of COVID-19 by targeting host entry factors. We demonstrate that integrative analysis of genome-wide CRISPR screening datasets enables network-based prioritization of drugs modulating viral entry, and we identify three common medications (spironolactone, quetiapine, and carvedilol) based on their network proximity to putative host factors. To understand the drugs real-world impact, we perform a propensity-score-matched, retrospective cohort study of 64,349 COVID-19 patients and show that spironolactone use is associated with improved clinical prognosis, measured by both ICU admission and mechanical ventilation rates. Finally, we show that spironolactone exerts a dose-dependent inhibitory effect on viral entry in a human lung epithelial cell line. Our results suggest that spironolactone may improve clinical outcomes in COVID-19 through tissue-dependent inhibition of viral entry. Our work further provides a potential approach to integrate functional genomics with real-world evidence for drug repurposing.
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Although vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been successful, there are no good treatments for those who are actively infected. While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from sensory organs and the brain are also susceptible to infection. While many patients suffer from diverse neurological symptoms, the viruss neuronal entry remains mysterious. To discover host factors involved in SARS-CoV-2 viral entry, we performed CRISPR activation (CRISPRa) screens targeting all 6000+ human membrane proteins in cells with and without overexpression of ACE2 using Spike-pseudotyped lentiviruses. This unbiased gain-of-function screening identified both novel and previously validated host factors. Notably, newly found host factors have high expression in neuronal and immune cells, including potassium channel KCNA6, protease LGMN, and MHC-II component HLA-DPB1. We validated these factors using replication-competent SARS-CoV-2 infection assays. Notably, the overexpression of KCNA6 led to a marked increase in infection even in cells with undetectable levels of ACE2 expression. Analysis of human olfactory epithelium scRNA-seq data revealed that OLIG2+/TUJ1+ cells--previously identified as sites of infection in COVID-19 autopsy studies-- have high KCNA6 expression and minimal levels of ACE2. The presence of KCNA6 may thus explain sensory/neuronal aspects of COVID-19 symptoms. Further, we demonstrate that FDA-approved compound dalfampridine, an inhibitor of KCNA-family potassium channels, suppresses viral entry in a dosage-dependent manner. Finally, we identified common prescription drugs likely to modulate the top identified host factors, and performed a retrospective analysis of insurance claims of ~8 million patients. This large cohort study revealed a statistically significant association between top drug classes, particularly those targeting potassium channels, and COVID-19 severity. Taken together, the potassium channel KCNA6 facilitates neuronal entry of SARS-CoV-2 and is a promising target for drug repurposing and development.
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Objective:To explore the differences of wear resistance of three kinds of glass ceramics and Wieland Zenostar zircona (Zenostar) , and to clarify their influencing factors.Methods:Zenostar were made into flat-shaped specimens (zirconia base sample group) and hemisphere-shaped specimens (zirconia pair grinding group) .There kinds of glass ceramics IPS Empress (Empress) , IPS e.max CAD (e.max) , VITA Suprinity (Suprinty) were used as base specimens.Each group was exposed to UMT-2testing machine to simulate the clinical service.The wear depthes of base specimens were detected by laser confocal scanning.Scanning electron microscope (SEM) was used to evaluate the wear surfaces.Results:In zirconia base sample group, there were no significant differences in the maximum wear depthes to Zenostar between the three kinds of glass ceramics (P＞0.05) .In zirconia pair grinding group, the maximum wear depthes ranked as follows:Zenostar group＜e.max group≈Empress group＜Suprinity group;there was no significant difference between e.max group and Empress group (P＞0.05) , but there were significant differences between other groups (P＜0.01) .The SEM results showed that the wearing surface of the Zenostar in zirconia base sample group was relatively smooth;whereas the wearing surface of Empress in zirconia pair grinding group was rougher with alarge area of clebris desquamation surface.Conclusion:The wear resistance of the three kinds of glass ceramics to Zenostar is related to the compositions and the chemical structures of materials.
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Objective:To observe the development of dentinal microcracks after root canal preparation with there kinds of reciprocating nickel-titaninm instruments using an in situ cadaver model by means of micro-computed tomography (Micro CT),and to provide the reference for their clinical application.Methods:A total of 15 mandible bone specimens having at least the anterior teeth (n=60) with single root were selected and there was no dentina microcrack,then they were randomly divided into 4 groups (n=15) according to the preparation protocol:Wave One,Reciproc,One File and ProTaper Universal groups,and ProTaper Universal was used as control group.The root canals were prepared up to 25 # instruments in Wave One,Reciproc and One File groups and F2 instruments in ProTaper Universal groups.After the preparation procedures,the specimens were scanned by Micro CT again,and the number of dentinal microcracks were investigated and analyzed statistically.Results:Compared with ProTaper Universal group,the incidence rates in Wave One,Reciproc and One File groups were significantly reduced (P＜0.05).There were no statistical differences in the incidence rates of dentinal microcracks between Wave One,Reciproc and One File groups (P＞0.05).Conclusion:Root canal preparation can cause the dentinal microcracks,so it is important to choose the types of instruments.Root canals prepared with Reciproc,Wave One and One File produce less dentinal microcracks,which can decrease the risk of vertical root fracture.
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Objective To construct human canstatin gene eukaryotic expression vector and investigate the therapeutic effect of intramuscular canstatin gene delivered by electroporation on tumor growth.Methods Canstatin cDNA was amplified from total RNA extracted from fresh fetal liver by reversing transcription polymerase chain reaction (RT-PCR).The canstatin cDNA fragment was in serted into pEGFP-N1 eukaryotic expression vector.The recombination plasmid was delivered to the quadriceps of the mice with Lewis lung carcinomas by electroporation intramuscular.Fluorescence intension measured by fluorescence microscope,reverse-PCR assay,and immunohistochemistry assay were performed to detect the expression of canstatin gene in the muscle and in circulation.The tumor weight and volume were used to detect the biological effects of canstatin gene delivery.Results Recombinant eukaryotic expression vector of recombinant human canstatin was successfully constructed.The canstatin mRNA was significantly increased in the skeletal muscle and intramuscular delivery of canatatin gene by electroporation acquired the expression of enhanced green fluorescent protein (EGFP)/canstatin protein in the circulation and significantly inhibited tumor growth.The percent of the inhibition of tumor weight was 57.7 ％.Conclusions Electroporation mediated gene transfer efficiency in skeletal muscle was compared to simple plasmid injection and lasted for a long time.It was an efficient and safe,convenient and economic,gene transfer methods and might have certain clinical application value.Electroporation mediated canstatin gene transfer in skeletal muscle had obvious inhibitory effect on Lewis lung cancer in mice subcutaneous xenograft tumor growth.
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Objective To explore the role of xCT in the metastasis and drug resistance of nasopharyngeal carcinoma (NPC),to provide new evidences for the mechanism of NPC metastasis and drug resistance,and to enhance effects of chemotherapy.Methods Fluorescence quantitative polymerase chain reaction (PCR) and Western blot were used to detect the xCT expression of 5-8F and 6-10B.The xCT eukaryotic expression vector was constructed to transient transfect 6-10B cell lines.The 5-8F cell lines were treated with xCT antisense oligodeoxynucleotide (ASO).Scratching assay and transwell method were used to detect the cell metastasis and invasion abilities.The expressions of matrix metalloproteinase 1 (MMP1) of all the cell lines were surveyed by Western blot.Results The results of fluorescence quantitative PCR and Western blot showed that the expression of xCT in 5-8F was higher than 6-10B in the levels of mRNA and protein.Exogenous overexpression of xCT enhanced metastasis and invasion abilities of 6-10B cells.Silencing and inhibition of xCT could decrease metastasis and invasion abilities of 5-8F cells.The expressions of MMP1 protein in 5-8F were higher than 6-10B,and they were positively correlated with the expression of xCT.Conclusions xCT is closely related to the metastasis and invasion abilities of nasopharyngeal carcinoma.xCT could enhance the metastasis and invasion abilities of NPC cells.xCT might mediate proliferation and matastasis/invasion of NPC cells through MMP1.
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Objective To study a gastric carcinoma subcell line with higher invasive potential and its biologic characteristics screened by a transwell chamber. Methods Transwell chamber was used for the selection of tumor subline. The biological characteristics of the cell lines were studied with optics microscope, Westem blotting, Transwell, immunohistochemical staining and growth curve. Results A gastric carcinoma cell subcell line (named MKN-28S10) was established from its parent cell line MKN-28 with higher invasive potential. MKN-28S10 showed essentially the same morphous as MKN-28. The expression of E-cadherin and TIMP-1 decreased significantly in the screened subcell line(P<0.05). The expression of NM23-H1 in MKN-28S10 was significantly lower than that in MKN-28 (P<0.05). Compared with MKN-28 (61.75±2.06 per vision), the migrative ability of passing through the membrane Millipore(100.25±0.50 per vision) was obviously increased in MKN-28S10(P<0.05). Compared with MKN-28, the growth rate of MKN-28S10 was increased obviously. Conclusion MKN-28S10 cell has stronger invasive ability and more powerful proliferation than that of its mother line MKN-28.
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0.05).Conclusion MCP-1 expresses in pulp tissue in patients with pulpitis.MCP-1 could conduce the pulpitis tissue to heal in the occurrence and development of pulpitis.
