Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

ACE2 is a major receptor for cell entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2s binding, how to efficiently and flexibly control ACE2 levels for prevention of SARS-CoV-2 infection has not been explored. Here, we revealed Vitamin C (VitC) administration as an effective strategy to prevent SARS-CoV-2 infection. VitC reduced ACE2 protein levels in a dose-dependent manner, while partial reduction of ACE2 can greatly restrict SARS-CoV-2 infection. Further studies uncovered that USP50 is a crucial regulator of ACE2 protein levels, and VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination at Lys788 and degradation of ACE2, without disrupting ACE2 transcriptional expression. Importantly, VitC administration reduced host ACE2 and largely blocked SARS-CoV-2 infection in mice. This study identified an in vivo ACE2 balance controlled by both USP50 and an essential nutrient VitC, and revealed a critical role and application of VitC in daily protection from SARS-CoV-2 infection. HighlightsO_LIVitC reduces ACE2 protein levels in a dose-dependent manner C_LIO_LIVitC and USP50 regulate K48-linked ubiquitination at Lys788 of ACE2 C_LIO_LIVitC blocks the interaction between USP50 and ACE2 C_LIO_LIVitC administration lowers host ACE2 and prevents SARS-CoV-2 infection in vivo C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/499651v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@196682borg.highwire.dtl.DTLVardef@190f14dorg.highwire.dtl.DTLVardef@d22b59org.highwire.dtl.DTLVardef@1c0faa_HPS_FORMAT_FIGEXP M_FIG C_FIG The deubiquitinase USP50 controls ACE2 protein stability and levels, while Vitamin C blocks the USP50-ACE2 interaction and therefore results in ACE2 degradation, offering a flexible and efficient approach to protection of the host from SARS-CoV-2 infection.

The expression and identification of NS3-NS4 gene of hepatitis Cvirus / 西安交通大学学报(医学版)

Objective To construct and expr es s a recombinant plasmid of nonstructural protein NS3-NS4 of hepatitis C virus ( HCV), and to identify the antigenicity of the expressed protein. Methods A gene region encompassing the nonstructural protei n NS3-NS4 of HCV was amplified by polymerase chain reaction (PCR) from the pUC1 9/HCV template. The recombinant expression plasmid containing the pBV220/NS3-NS 4 sequence was constructed, and the nonfused NS3-NS4 recombinant protein was ex pressed in E.coli DH5? efficiently. The recombinant protein was det ected by SDS-PAGE and ELISA. Results We successfully constructed and expressed the recom binant plasmid in prokaryote. Its antigenicity was detected with 50 standard ser a. Compared with the second-generation diagnostic Kit, the total detection rate was 96%. Conclusion The whole NS3-NS4 protein, a region of dominant immunogenicity, should be the effective component of the HCV diagnostic Kit and provide the clue for developing HCV DNA vaccine.

Primary study on immunomodulatory effect of ursolic acid in mice / 西安交通大学学报(医学版)

Objective To confirm the immunomodulatory effect of ursolic acid on normal and immunosuppressive mice caused by cyclophosphamide.Methods We assayed the function of T lymphocyte transformation with MTT test,examined the function of B lymphocyte with hemolysis spectrophotography,determined the function of non-specific immunity with phagocytosis of macrophages,and measured the content changes of Th_(1) cytokine IL-2 and Th_(2) cytokine IL-6 in serum with ELISA.Results Ursolic acid had a suppressant effect on the function of humoral immunity and enhanced cell-mediated immune response at the low dosage but presented opposite effect at the high dosage in normal mice.Ursolic acid significantly improved immune function on immunosuppressive mice caused by cyclophosphamide(P

Construction of a DNA vaccine encoding herpes simplex virus type 1 glycoprotein D / 新乡医学院学报

Objective To construct a recombinant plasmid DNA containing herpes simplex virus type 1(HSV-1) glycoprotein D (gD) gene.Methods The HSV-1 gD gene was obtained by polymerase chain reaction (PCR) and inserted into TA cloning vector pGEM-T, then cloned into the eukaryotic expression vector pcDNA3.1 to generate pLy-D. The recombinant plasmid pLy-D, which was confirmed by partial sequencing and restriction endonuclease analysis, was transfected into Cos-7 cells and used to inoculate ICR mice via muscular injection. Immunohistochemistry and enzyme-linked immunoabsorbent assay (ELISA) were employed to test the gD expression in transfected cells and the specific anti-HSV-1 antibody in the serum of immunized mice, respectively.Results The gD eukaryotic expression plasmid pLy-D was constructed. Using the immunohistochemistry technique, the gD expression in pLy-D-transfected cells was detected. The ELISA demonstrated that specific anti-HSV-1 antibody could be induced in immunized mice after three times injection.Conclusions We constructed HSV-1 gD eukaryotic expression plasmid pLy-D which could express gD protein in transfected cells and could induce humoral immune response in mice. This observation will be helpful in designing HSV prophylactic vaccine.

Establishment of a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase gene / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene.</p><p><b>METHODS</b>The full length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template, and inserted into the prokaryotic expression plasmid Pgex-2T. The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits. The antibody was labeled with biotin, and a sandwich ELISA technique with biotin streptavidin amplify system was established. Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro. The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay.</p><p><b>RESULTS</b>SDS-PAGE displayed that the molecular weight of the expressed fusion protein was about 54,000. The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells. Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method.</p><p><b>CONCLUSIONS</b>The established method could sensitively reflect the activities of the upstream promoters, as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities. Therefore, it proposes a convenient assay for the studies using CAT as the reporter gene.</p>

Genetic polymorphism of D16S539, D7S820 and D13S317 loci in the Kazak ethnic population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the genetic polymorphism of D16S539, D7S820 and D13S317 in Chinese Kazak ethnic population from Xinjiang.</p><p><b>METHODS</b>One hundred and two unrelated individuals and a sample of families (n=42) were investigated by multiplex amplification, 6% denaturing PAGE and silver staining. And, the obtained allele frequencies were compared with those of other populations.</p><p><b>RESULTS</b>Eight, seven, eight alleles were observed at the 3 STR loci respectively and the genotypes distributions were in accordance with Hardy-Weinberg equilibrium. The expected heterozygosities for these loci were 0.9439, 0.9356 and 0.9304; the calculated polymorphism formation content (PIC) was 0.9905; the discrimination power (DP), 0.9998; the paternity exclusion (PE), 0.9572. In addition, significant difference was found in comparison with other populations, and in the sample of families (n=42) no new mutations could be found.</p><p><b>CONCLUSION</b>The multiplex examination of 3 STR loci can be used in forensic identification and population genetics research.</p>

THE ADAPTABLE CHANGE OF THE FUNCTION OF T LYMPHOCYTES FOR PHYSICAL EXERCISE WITH OXYGEN / 药物分析学报

Objective To find out the possible regularity and mechanism of the adaptable change of human being T lymphocytes for physical exercise with oxygen and bring the original data for the Movement of All People Improving their Health. Methods We selected 16 untrained female students in university and let them had the same amount of exercise for 8 weeks. After that, we collected the cycle blood at the time point of before exercise, the end of exercise and 1 hour after exercise at the end of the 0,first,2 nd,4 nd,6 nd and 8 nd week respectively, so as to determine its stimuli index (SI) by MTT method. Results In the different time sect, such as the early stage of exercise, quiet condition,as soon as the end of exercise and 1 hour after exercise, we found that the SI were obviously Iower than that of normal (P＜0. 05) ,especially in the time sect of the end of exercise. Continuing to 4 weeks,the function of T lymphocytes restored gradualy and it lasted to the 8 th week, the SI in quiet condition and 1 hour after exercise had restored to normal(P＞0.05),but in the end of exercise, it still was Iow,however, the extent of the cases selected was in a condition of acute excitability. Conclusion As the bodies adapting to the exercise, the function of T lymphocytes restored slowly and the rate increased faster and faster.

Preparation and molecular weight estimation of ouabain pol yclonal antibody F(ab)2 fragment / 中国生化药物杂志

Purpose　The aim is to prepare ouabain polyclonal antibody F(ab)2 fragment and to estimate its molecular weight.Methods[ KG*2 [ WTBZ]Ouabain polyclonal antibody was obtained from immunized rabbits.The antibod y was digested with pepsin.The resulting products were analyzed and the molecular weig ht of F(ab)2 fragment was estimated by HPSEC.The immune activity was detec ted by ELISA.Results　100 mg of ouabain polyclonal antibody wa s dige sted by 2 mg of pepsin for 18 hours at pH 3.0 and active ouabain polyclonal anti body F(ab)2 framgment was obtained.Its molecular weight was 107 kD.Concl usion　The active ouabain polyclonal antibody F(ab)2 fragment coul d be prepared by digesting its antibody with pepsin.

Relationships between ouabain of plasma and tissues and blood pressure in lklc hypertensive rats / 中国病理生理杂志

AIM: To explore the role of endogenous ouabain (EO) in the development of hypertension and its secretion character in 1k1c hypertensive rats(HR). METHODS: EO contents of plasma and tissues in 1k1c HR were detected by ELISA. The relationships between plasma and tissues ouabain and blood pressure were analyzed. RESULTS: EO contents of plasma, heart, kidney, adrenal gland, pituitary and hypothalamus in lklc HR were significantly higher than those of normal rats, especially in the adrenal gland and hypothalamus. EO contents of serum, kidney and hypothalamus were correlated with blood pressure. CONCLUSIONS: EO might play an important role in the development of hypertension in 1k1c hypertensive rats. Adrenal gland might be the major source of EO.

Research on dendritic cells to soak gallbladder carcinoma tissue / 中国免疫学杂志

Objective:Explore the dendritic cells to soak gallbladder carcinoma tissue,to explain the relation dendritic cells and tumor immunity.Methods:Adopt immune histochmistry S-P method.Results:The extent of dendritic cells to soak gallbladder carcinoma tissue is not relate to age,gender and types of pathhistology but negative contact with degree of pathological differentiation (P

Construction and identification of adenovirus-recombined vector of CTLA4Ig-IRES2-I?B? / 第三军医大学学报

Objective To construct adenovirus vector harboring CTLA4Ig-IRES2-I?B? gene for the study of enhancement blockade of T cell costimulatory pathway. Methods The IRES2 fragment was cloned in to the shuttle plasmid pAdtrack-CMV-CTLA4Ig, and then the human I?B? fragment was constructed with the link of the IRES2 to the pAdtrack-CMV-CTLA4Ig. Subsequently, adenovirus vector harboring CTLA4Ig-IRES2-I?B? was constructed by homologous recombination in E. coli BJ5183, and recombinant vector was packaged and propagated in 293 cells. Results The recombinant CTLA4Ig-IRES2-I?B? adenovirus was constructed by homologous recombination and identified by PCR and restrictive enzyme digestion methods. Conclusion The recombinant CTLA4Ig-IRES2-I?B? adenovirus may be used as a novel immunosuppressive agent for gene therapy in organ transplantation.

The changes of resistin, blood fat and hepatic function in patients with diabetes mellitusⅡ / 西安交通大学学报(医学版)

Objective To study the changes of resistin, blood fat and hepatic function in patients with type 2 diabetes mellitus. Methods Enzyme linked immunosorbent assay (ELISA) was used to determine the content of resistin in peripheral blood. Radio immunoassay was used to determine the content of insulin . Oxydase or chemical method was used to determine blood sugar,and automatic analytical instrument was used to determine blood sugar, blood fat and hepatic function. Results Blood-fasting sugars in patients with type 2 diabetes mellitus (8.85?3.21 mmol/L) increased significantly (P0.05). Conclusion Disorders of internal environment exist in patients with type 2 diabetes mellitus. Contents of resistin, ALB, and blood fat are higher in the patients than those in the controls.

SELECTION AND SUBCLONE OF THE TRANSFORMING GENES OF HUMAN PAPILLOMAVIRUS TYPE 16 / 西安交通大学学报(医学版)

The transformation of human papillomavirus type 16 (HPV16) may play somc important roles in the oncogenesis of cervical carcinoma Studies showed that the transforming gence mainly located in the E6. E7 open reading frames (ORFs). We digested HPV16 DNA with PstI+EcoRI. Using pUC-19 as the vector, E. coli JM103 strain as the host cell, after two times of directive subelone, plasmid pEP-8 was construeted, confirmed by RE analysis and Southern blot hybridization, the inserted band of pEP-8 was 1.43Kb long contained the intact E6 E7 ORFs, and also a cat-box region, two TATA-boxics and a cell-type specific cnhancer in the upstream swquence. The pEP-8 could be used as the specific probe of HPV-16 E region for identifing virol mRNAs in the HPV infected cells. Meanwhile it was a basework for studying the transformation and transforming proteins of HPV-16.

DETECTION AND ANALYSIS OF HUMAN PAPILLOMAVIRUS DNA IN CONDYLOMA ACUMINATUM / 西安交通大学学报(医学版)

Condyloma acuminatum is closely associated with human papillomavirus (HPV). In this study the method of slotting hybridization was used to detect the Specimens of 28 cases of condyloma acuminatum. Fourteen out of the 28 samples were positive for HPV 11 probe, the positive rate was 50% (14/28). Nine out of the 14 positive samples were digested with Bamll Ⅰ and Pst Ⅰ respectively to study the present state of DNA after using Southern blotting hybridization. It was shown that HPV 11 DNA were mainly present in a simple monometric episomal state in the condyloma acuminatum cells.

The influence of chronic neuropathic pain on cellular immune function of mice / 西安交通大学学报(医学版)

Objective To explore the influence of chronic neuropathic pain on cellular immune function of mice models.Methods The mice models were established by ligating tibeal nerve and common peroneal nerve on one side,and the influence of chronic neuropathic pain on cellular immune function and tumor necrosis factor-?(TNF-?) and interleukin-6(IL-6) was observed with lymphocyte cell increase experiment(MTT method) and enzyme linked immunosorbent assay(ELISA).Results The increased reactivity of T lymphocyte of the model group was significantly higher than that of the sham group and the control group.At the same time,content of TNF-? and IL-6 in blood of the model group also increased.Conclusion Cellular immune function of mice is increased in the state of chronic neuropathic pain.