Objective:To construct the eukaryotic cell translation initiation factor 4A3(EIF4A3)short hairpin RNA(shRNA)lentiviral vector,and to establish the Neuro-2a-EIF4A3-shRNA stable transfection cell line.Methods:The EIF4A3 gene sequence was retrieved from the National Center for Biotechnology Information(NCBI)database;the PCR identification primers were designed and synthesized,and connected to the lentiviral GV493 vector digested with Eco R I and Age I enzymes to construct the GV493-EIF4A3-shRNA lentiviral plasmid;PCR method was used to screen the positive clones,which were sequenced for the identification;the GV493 empty plasmid and GV493-EIF4A3-shRNA recombinant plasmid were transfected into the HEK293T cells,regarded as GV493 control lentivirus and GV493-EIF4A3-shRNA lentivirus,respectively.After 48 h of transfection,the lentiviruses were collected for packaging and the viral titer was determined.The Neuro-2a cells were divided into blank group,GV493 control group,and GV493-EIF4A3 shRNA group.The Neuro-2a cells in blank group were untreated,and the Neuro-2a cells in GV493 control group and GV493-EIF4A3 shRNA group were infected with the respective lentiviruses at a multiplicity of infection(MOI)of 100.The infected Neuro-2a cells were selected by 10 mg·L-1 puromycin,and the growth status and green fluorescence expression of the Neuro-2a cells in various groups were observed under fluorescence microscope;real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of EIF4A3 mRNA and protein in the Neuro-2a cells in various groups.Results:The PCR sequencing results showed that the gene sequence of the GV493-EIF4A3-shRNA recombinant plasmid was consistent with the designed EIF4A3-shRNA sequence,indicating successful construction of the GV493-EIF4A3 lentiviral vector.The fluorescence microscope observation results showed that there was strong fluorescence expression and good growth status in the HEK293T cells,confirming successful lentiviral packaging.The viral titers for GV493 control lentivirus and GV493-EIF4A3-shRNA lentivirus both were 2×108 TU·mL-1.The growth status of the Neuro-2a cells in GV493 control group and GV493-EIF4A3 shRNA group was good,and they expressed green fluorescence,indicating successful construction of the stable transfection cell line.The RT-qPCR results showed that compared with blank group and GV493 control group,the expression level of EIF4A3 mRNA in the cells in GV493-EIF4A3 shRNA group was significantly decreased(P<0.01).The Western blotting results showed that the specific bands was at a relative molecular mass of 49 000,indicating successful EIF4A3 protein expression in the Neuro-2a cells.Compared with blank group and GV493 control group,the expression level of EIF4A3 protein in the cells in GV493-EIF4A3 shRNA group was significantly decreased(P<0.01).Conclusion:The GV493-EIF4A3-shRNA lentiviral vector is succfssfully constructed,and the Neuro-2a-EIF4A3-shRNA stable transfection cell line is established;the results provide the reference for the study of the effect of EIF4A3 on the intracranial atherosclerosis.

Objective:To construct an over-expression lentiviral vector of the dedicator of cytokinesis 4(DOCK4),and to establish DOCK4 stably over-expressing Neuro-2a cells.Methods:The DOCK4 sequence was searched in the National Center for Biotechnology Information(NCBI)and primers were designed and synthesized;polymerase chain reaction(PCR)method was used to amplify the DOCK4 gene sequences.After digestion with BamH Ⅰ and Age Ⅰ restriction endonucleases,the DOCK4 gene sequences were ligated with the digested lentiviral vector GV492 to construct the GV492-DOCK4 over-expression recombinant plasmid.The positive clones with a similar length to the target gene fragment were screened and identified by PCR method.The GV492-control plasmid and GV492-DOCK4 over-expression recombinant plasmid were transfected into the HEK293T cells,and the lentivirus was collected and titered 48 h after transfection.The Neuro-2a cells were divided into GV492-control group and GV492-DOCK4 group,and the cells were infected with GV492-control lentivirus and GV492-DOCK4 over-expression lentivirus,respectively,and the multiplicity of infection(MOI)was 100.After 72 h of infection,the successfully infected Neuro-2a cells were screened by using puromycin(10 mg·L-1).The growth status of Neuro-2a cells and the expression of green fluorescent protein in various groups were observed under fluorescence microscope.Real-time quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of DOCK4 mRNA and DOCK4 protein in the Neuro-2a cells in various groups.Results:The PCR results showed that the gene fragment length of the GV492-DOCK4 over-expression recombinant plasmid was approximately 691 bp.The sequencing results showed that the gene sequence of the GV492-DOCK4 over-expression recombinant plasmid was consistent with the designed over-expression sequence of DOCK4.The titers of the lentiviruses in GV492-control group and GV492-DOCK4 over-expression group were 2.5×108 TU·mL-1 and 2.5×108 TU·mL-1,respectively.The fluorescence microscope observation results showed that Neuro-2a cells in various groups grew well and expressed green fluorescent protein.The RT-qPCR results showed that compared with GV492-control group,the expression level of DOCK4 mRNA in the Neuro-2a cells in GV492-DOCK4 group was significantly increased(P＜0.01).The Western blotting results showed the specific bands near the relative molecular mass of 225 000 in various groups.Compared with GV492-control group,the expression level of DOCK4 protein in the Neuro-2a cells in GV492-DOCK4 group was significantly increased(P＜0.01).Conclusion:This study successfully constructs the DOCK4 over-expression lentiviral vector and establishes the Neuro-2a cells stably over-expressing DOCK4.

This article takes the author's hospital as an example to explore the effective methods of discipline inspection and supervision in public hospitals.By introducing the matrix management model into discipline inspection and supervision work,integrating superior resources to form a matrix supervision system,giving full play to the professional cross-over and functional com-plementary role of all participating departments,the four-step linkage of"supervision-feedback-rectification-looking back"and the expansion and implementation of the"N+"strategy,following up the supervision throughout the process,and promoting discipline inspection and supervision work from tangible coverage to effective coverage,We effectively improve the efficiency of management and supervision,and help the hospital develop in high quality.

Gastric cancer （GC） is a digestive tract tumor that occurs in the epithelial tissues of the gastric mucosa， seriously affecting the life and health of patients， and its mortality rate ranks the third among malignancies. Although medical technology has made great progress in recent years， the progression of GC still cannot be effectively controlled by surgery， chemotherapy， and targeted therapy. The pathogenesis of GC is extremely complex and is closely related to the tumor microenvironment， chronic inflammation， and immune escape， among which the reduction of tumor cell apoptosis is one of the important mechanisms for the occurrence and development of GC. Apoptosis refers to the process of spontaneous termination of cell life caused by genes under specific physiological or pathological conditions， which is of great significance for maintaining the stability of the internal environment. Researchers have found that in the GC state， mitochondrial endogenous apoptosis， endoplasmic reticulum stress， external death receptors， and other apoptosis pathways are regulated by multiple signaling pathways and genes， which together lead to the decline of GC cell apoptosis rate and thus promote the progression of GC. Chinese medicine is advantageous and characterized by multiple components， multiple targets， synergistic effect， and few adverse reactions. A large number of studies have shown that polysaccharide components， as effective components of Chinese medicine， have biological activities such as cancer inhibition， blood sugar control， anti-inflammation， antioxidant damage， and anti-virus， and can effectively inhibit the deterioration of GC by inducing cell apoptosis， gradually becoming a hot spot in GC drug research and development. However， systematic reviews on the apoptosis of GC induced by Chinese medicine polysaccharides are rarely reported. Therefore， this paper analyzed and summarized the studies of Chinese medicine polysaccharides in promoting apoptosis and interfering with GC， in order to provide a theoretical basis for the basic research， new drug development， and clinical application of Chinese medicine polysaccharides in the intervention of GC.

Objective:Searching, generalising and summarising evidence on non-pharmacological interventions for abnormal pronunciation in thyroidectomised patients.Methods:The literature on abnormal pronunciation in patients undergoing thyroidectomy published in the past 5 years was systematically searched in British Medical Journal (BMJ) Best Practice, UpToDate, Agency for Healthcare Research and Quality, Guidelines International Network, National Institute for Health and Clinical Excellence, Medlive, Cochrane Library, PubMed, CINAHL, Web of Science, China Biomedical Literature Database, China National Knowledge Infrastructure, and Wanfang Database. The search period was from the establishment of the database to December 11, 2022. After literature screening and quality evaluation, the evidence was summarized.Results:A total of eight articles were included, including six guidelines, one expert consensus, and one systematic review. A total of 18 best pieces of evidence were extracted and categorized into four categories, including speech function assessment, neck exercise, speech training, health education and psychological support.Conclusions:This study can provide evidence-based basis for non-pharmaceutical intervention management of abnormal pronunciation in patients undergoing thyroidectomy. When applying evidence, medical and nursing staff should combine it with clinical practice and conduct targeted transformation and application.

Objective: To investigate the protective effect of cordycepin( Cor) on cardiac function in aged rats and its mechanism． Methods : The aged rats were divided into Model group，Cor administration group ( Cor 5，10，20g / kg) ，and young rats were used as Control group ( Control) ．The levels of heart rate (HR) ，left ventricular e- jection fraction (LVEF) and fractional shortening (FS) were detected by echocardiography．The levels of creatine kinase MB ( CK-MB) ，myohemoglobin (Mb) ，cardiac troponin Ⅰ (cTnI) ，superoxide dismutase (SOD) ，malon- dialdehyde (MDA) and lactate dehydrogenase ( LDH) were detected by ELISA．The pathological morphology of heart tissue was observed by HE staining． The apoptosis of myocardial cells was observed by TUNEL staining. Western blot detected the protein expression of cysteine aspartic protease 9 ( Caspase 9) ，cleaved cysteine aspartic protease 9 (cleaved Caspase 9) ，cysteine aspartic protease 3 ( Caspase 3 ) ，cleaved cysteine aspartic protease 3 ( cleaved Caspase 3) ，nuclear factor E2 related factor 2 (Nrf2) ，phosphorylation nuclear factor E2 related factor 2 (p-Nrf2) ，quinone oxidoreductase 1 (NQO1) ，heme oxygenase 1 ( HO-1) . Results : Compared with the elderly model group，HR , LVEF ，FS increased ，CK-MB ，Mb ，cTnI levels decreased ，MDA ，LDH content decreased， SOD activity increased，cleaved Caspase 9 / Caspase 9，cleaved Caspase 3 / Caspase 3 expression decreased，The ex- pressions of Nrf2，NQO1 and HO-1 were increased，and the pathological morphology of cardiac tissue and myocar- dial apoptosis were improved in Cor groups． Conclusion Cor can improve cardiac function of elderly rats，relieve oxidative stress response，inhibit myocardial cell apoptosis and have a certain protective effect on cardiac tissue in- jury in rats，and the mechanism may be related to the promotion of Nrf2 / HO-1 pathway related protein expression.

Objective:To investigate the clinical significance of the transforming growth factor-β receptor I (TGF-βRⅠ) expression in na?ve CD4 + T cells from patients with systemic lupus erythematosus (SLE). Methods:Na?ve CD4 + T cells were purified using magnetic microbeads from peripheral blood mononuclear cells of SLE patients and healthy controls. Real-time quantitative PCR was used to detect TGF-βRⅠ mRNA level, and flow cytometry was used to detect the percentage of CD69 +CD4 + T cells. Data were analyzed by t test and Pearson correlation analysis. Results:The level of TGF-βR Ⅰ mRNA in na?ve CD4 + T cells from SLE patients was significantly lower than that in healthy controls [(0.674±0.873) vs (1.445±1.112), t=2.301, P<0.05]. The TGF-βR Ⅰ mRNA level was negatively correlated with systemic lupus erythematosus disease activity index (SLEDAI) ( r=-0.376, P<0.05), erythrocyte sedimentation rate (ESR) ( r=-0.376, P<0.05), serum creatinine ( r=-0.323, P<0.05) and 24 h urine protein ( r=-0.331, P<0.05), and positively correlated with serum com-plement C3 ( r=0.528, P<0.01). The level of TGF-βRⅠ mRNA level in na?ve CD4 + T cells in SLE patients with renal involvement was lower than that in SLE patients without renal involvement [(0.525±0.536) vs (1.071±1.007), t=2.198, P<0.05]. The TGF-βR Ⅰ mRNA level in the na?ve CD4 + T cells in anti-dsDNA antibody positive group was lower than that in the anti-dsDNA antibody negative group [(0.344±0.315) vs (0.958±1.076), t=2.277, P<0.05]. The expression of TGF-βRⅠ mRNA in na?ve CD4 + T cells from SLE patients was reduced after 24 h stimulation with anti-CD3/CD28 beads [(0.047±0.013) vs (1.008±0.129), t=14.38, P<0.01], which was partially reversed by dexamethasone treatment [(0.240±0.042) vs (0.047±0.013), t=7.845, P<0.01]. Meanwhile, dexamethasone significantly decreased the expression of CD69 in CD4 + T cells [(15.0±2.1)% vs(34.9±2.0)%, t=32.57, P<0.01]. Conclusion:The abnormally low expression of TGF-βRⅠ in na?ve CD4 + T cells may be involved in the pathogenesis of SLE. Glucocorticoid treatment can upregulate the expression of TGF-βRI and inhibit the activation of T cells, This suggests suggesting that TGF-βRⅠ may be a potential target for SLE treatment.

The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.
Bacterial Proteins ; Genetic Engineering ; Insecticides ; Macrolides ; Saccharopolyspora

Objective To investigate the diagnostic value of superb microvascular imaging ( SM I ) , advanced dynamic flow ( ADF ) and color Doppler flow imaging ( CDFI) in breast microcarcinoma ,and to compare the correlation coefficients between these three indicators and postoperative pathological tumor microvascular density ( M VD ) . Methods A total of 85 patients ( 87 lesions ) with BI‐RADS 4 and the maximum diameter≤1 cm were selected ,of which ,46 lesions were benign ( benign group) and 41 lesions were malignant( malignant group) . All the patients and the corresponding lesions were examined by SM I , ADF and CDFI . Blood flow grading was performed on the images using Adler grade of blood flow ,and the difference of blood flow among the 3 methods was compared . T he expression level of M VD in pathological tumor tissues was detected and analyzed for its correlation with Adler classification by three detection techniques . Results T he areas under the ROC curve( AUR) of CDFI ,ADF and SM I were 0 .694 ,0 .705 and 0 .776 respectively based on the gold standard with pathological diagnosis . T he sensitivity ,specificity , positive predictive value ,negative predictive value ,and accuracy of CDFI were 78 .0% ,54 .3% ,60 .4% , 73 .5% ,and 65 .5% , respectively ; those of ADF were 75 .6% , 60 .9% , 63 .3% , 73 .7% and 67 .8% , respectively ; and those of SM I were 78 .0% ,69 .6% ,69 .6% ,78 .0% and 73 .6% ,respectively . T he Adler grades of CDFI , ADF and SM I were positively correlated with M VD ( P ＜ 0 .05 ) , w hich the highest correlation coefficient between SM I and M VD ( r ＝0 .430 , P ＜0 .001 ) . Conclusions SM I is superior to ADF and CDFI in detecting the abundance of breast microcarcinoma , and has the highest correlation coefficient among those 3 detection techniques with tumor pathological M VD ,which indicates that SM I may be used for differential diagnosis of breast microcarcinoma and indirectly evaluate the prognosis of patients .

Objective: To investigate the diagnostic value of superb microvascular imaging(SMI), advanced dynamic flow(ADF) and color Doppler flow imaging(CDFI) in breast microcarcinoma, and to compare the correlation coefficients between these three indicators and postoperative pathological tumor microvascular density(MVD). Methods: A total of 85 patients(87 lesions) with BI-RADS 4 and the maximum diameter≤1 cm were selected, of which, 46 lesions were benign(benign group) and 41 lesions were malignant(malignant group). All the patients and the corresponding lesions were examined by SMI, ADF and CDFI. Blood flow grading was performed on the images using Adler grade of blood flow, and the difference of blood flow among the 3 methods was compared. The expression level of MVD in pathological tumor tissues was detected and analyzed for its correlation with Adler classification by three detection techniques. Results: The areas under the ROC curve(AUR) of CDFI, ADF and SMI were 0.694, 0.705 and 0.776 respectively based on the gold standard with pathological diagnosis. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of CDFI were 78.0%, 54.3%, 60.4%, 73.5%, and 65.5%, respectively; those of ADF were 75.6%, 60.9%, 63.3%, 73.7% and 67.8%, respectively; and those of SMI were 78.0%, 69.6%, 69.6%, 78.0% and 73.6%, respectively. The Adler grades of CDFI, ADF and SMI were positively correlated with MVD(P<0.05), which the highest correlation coefficient between SMI and MVD (r=0.430, P<0.001). Conclusions SMI is superior to ADF and CDFI in detecting the abundance of breast microcarcinoma, and has the highest correlation coefficient among those 3 detection techniques with tumor pathological MVD, which indicates that SMI may be used for differential diagnosis of breast microcarcinoma and indirectly evaluate the prognosis of patients.