OBJECTIVETo clone the glial cell line-derived neurotrophic factor (GDNF) from the mouse testis, construct the eukaryotic expression vector and transfect this vector into Sertoli cells in order to use the gdnf-transfected Sertoli cells as the feeder layer to cultivate spermatogonial stem cells (SSCs).

METHODSTotal RNA was extracted from the testes of normal mature mice and gdnf was cloned and amplified using RT-PCR, inserted into the eukaryotic expression vector and transfected into sertoli cells (TM4 cell line). Immunofluorescence with anti-GDNF antibodies was performed at 40 h following the transfection.

RESULTSgdnf cDNA was cloned successfully, and GDNF expressed after transfected into Sertoli cells.

CONCLUSIONThis study provides a basis for culturing SSCs with gdnf-transfected Sertoli cells as the feeder layer.

Animals ; Cloning, Molecular ; Gene Expression ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Male ; Mice ; Mice, Inbred Strains ; RNA ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sertoli Cells ; metabolism ; Testis ; cytology ; metabolism ; Transfection

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.
Animals ; Cryptorchidism ; metabolism ; Male ; Membrane Proteins ; analysis ; Mice ; Phosphatidylethanolamine Binding Protein ; analysis ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stathmin ; analysis ; Testis ; chemistry

OBJECTIVETo produce BMI1 polyclonal antibody, mouse Bmi1 cDNA was cloned from mouse testis and expressed in E. coli BL21.

METHODSBmi1 gene was amplified from mouse testis by RT-PCR and inserted into the prokaryotic expression vector pET-28c(+). Subsequently the recombined vector was transformed and expressed in E. coli BL21 (DE3) and the immunogenicity of recombined protein BMI1 (rBMI1) was tested by Western blot.

RESULTSMouse Bmi1 cDNA of 975 bp was successfully cloned and recombined. E. coli BL21 strains expressed rBMI1 were screened. The expression protein amounted to 12% of the total bacterial protein after induced with IPTG, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 71% of the insoluble protein. Western blot analysis showed that rBMI1 could be specially recognized by mouse monoclonal IgG1 anti-BMI1 and His-tag antibody.

CONCLUSIONThere was expression of Bmi1 gene in mouse testis. Mouse Bmi1 cDNA was successfully cloned and expressed prokaryoticly.

Animals ; Antibodies, Monoclonal ; immunology ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Gene Expression ; Male ; Mice ; Nuclear Proteins ; biosynthesis ; genetics ; immunology ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; immunology ; Testis ; metabolism

AIMTo investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice.

METHODSMice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured.

RESULTSLow doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia.

CONCLUSIONE2 has a dose-related mitogenic effect on spermatogonia.

Animals ; Cell Division ; drug effects ; Cryptorchidism ; physiopathology ; Disease Models, Animal ; Estradiol ; blood ; pharmacology ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Mice ; Spermatogonia ; cytology ; drug effects ; pathology ; Testosterone ; blood

Objective:To analyze the clinical features and imaging findings of chest computed tomography (CT) of patients with corona virus disease 2019 (COVID-19), and to provide imaging guidance for early diagnosis and clinical treatment.Methods:A total of 74 patients diagnosed with positive nucleic acid test of nasopharyngeal swab were retrospectively collected from January 23 to February 22, 2020 in the inpatient department of Rui′an People′s Hospital. Their clinical features were analyzed in combination with CT image characteristics.Results:Of the 74 patients, 62 (83.8%) had fever, 59 had abnormalities in CT examinations and 15 patients had no imaging abnormalities. The average interval from onset to CT examination was 4.6 days. Among 59 patients, 58 cases (98.3%) were mainly peripherally distributed, 53 cases (91.4%) had multiple lesions and five cases (8.6%) had single nodules. The main manifestation was ground glass opacities (45 cases, 76.3%). The lesions were mainly distributed in the middle and lower lobe of the lungs, accounting for 76.3% (45 cases). Air bronchogram was shown in 21 cases (35.6%), and reversed halo sign was shown in 10 cases (16.9%). Other rare abnormalities were small cavities in three cases (5.1%), tree-in-bud pattern in one case (1.7%). Intrathoracic lymph node enlargement was found in two cases (3.4%) by re-examination.Conclusion:The CT examination characteristics of COVID-19 patients could play an important role for early diagnosis and follow-up.

OBJECTIVETo apply and evaluate new methods regarding specific gene and antigen detection in plague surveillance program.

METHODS1798 samples from natural foci of plague were tested, using internal quality control multiple-polymerase chain reaction, F1 antigen marked by immuno chromatographic assay and enzyme linked immunosorbent assay. Culture of Yersinia pestis and reverse indirect hemagglutination assay were used as reference diagnostic methods.

RESULTSThe overall positive rate of culture on Yersinia pestis together with gene and antigen detection was 7.34%, showing an 16.81% increase when comparing to 6.28% using Yersinia pestis culture method alone. The rate of coincidence was 97.13%.

CONCLUSIONThe new standard being used for specific gene and antigen detection could increase the positive rate of diagnosis on plague.

Animals ; Bacterial Proteins ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Mice ; Plague ; microbiology ; Polymerase Chain Reaction ; Yersinia pestis ; genetics ; immunology ; pathogenicity

Objective To investigate the antibacterial resistance in nationwide's tietiary hospitals and understand the trend of antimicrobial resistance.Methods All the clinical isolates were collected from 18 hospitals and the minimal inhibitory concentrations (MICs) were tested using agar dilution method recommended by Clinical and Laboratory Standards Institute (CLSI) in central laboratory.The susceptibilities of isolates to antimicrobial agents were determined by using CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2017 guideline.Results A total of 4333 pathogenic isolates from 18 tertiary hospitals in 18 cities nationwide over the period from July 2015 to June 2016 were studied.Based on the MIC results,Escherichia coli and Klebsiella pneumoniae showed extended spectrum β-lactamase (ESBLs) phenotype rates of 59.4％ and 27.5％,respectively;decreased by 7 to 10 percentage points comparing the last time.Carbapenems,amikacin,moxalactam,β-lactam/β-lactamase inhibitor combinations,tigecycline,and fosfomycin displayed desirable antibacterial activity against Enterbacteriaceae,but a significant increasing of carbapenems resistance Klebsiella pneumoniae were noted.For non-fermenting Gram-negative isolates,resistance rate of Pseudomonas aeruginosa and Acinetobacter baumannnii to imipennnem were 29.5％ and 69.8％ and multidrug-resistant (MDR) detection rate were 35.6％ and 78.3％,extensively drug-resistant (XDR) were 10.2％ and 72.5％,respectively.Klebsiella pneumoniae isolated from children were more resistant to β-lactam than those from adults and the old people,so bacterial resistance in children is an important problem in China.Conclusion Though the decline of ESBLs detection rate,carbapenem non-susceptible Klebsiella pneumoniae rates continued to increase,which should be paid more attention.

Objective To investigate the gram-positive coccus resistance in nationwide's tietiary hospitals and understand the trend of antimicrobial resistance.Methods All the clinical isolates were collected from 18 hospitals and the minimal inhibitory concentrations (MICs) were tested using agar/broth dilution method recommended by Clinical and Laboratory Standards Institute (CLSI) in central laboratory.The susceptibilities of isolates to antimicrobial agents were determined by using CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2017 guideline.Results A total of 2301 Gram-positive cocci isolated from 18 hospitals in 18 cities nationwide were studied.Based on the MIC results,the prevalence of methicillin resistant Stapylococcus aureus (MRSA) and methicillin resistant Stapylococcus epidermidis (MRSE) were 39.9％ and 86.6％ respectively.No vancomycin insensitive Staphylococcus was detected.Staphylococcus aureus were 100％ susceptibile to linezolid and teicoplanin,but resistant or insensitive for drugs other than vancomycin were observed among Coagulase Negative Staphylococci (CoNS).Antibiotic resistance rate of Enterococcus faecalis and Enterococcusfaecium to ampicillin were 4.5％ and 85.1％.The detectation rate of vancomycin resistant Enterococcus(VRE) was 2.1％.Nonsusceptibility rate of Enterococcus faecalis to linezolid was 7.8％,showing slight increase than last time.The prevalence of penicillin nonsusceptible Streptococcus pneumoniae (PNSSP) was 6.6％ based on non-meningitis and parenteral administration criterion;while for cases of oral penicillin,the rate was 70.0％,was as flat as last time.There were no significant differenees of resistance rates of Stapylococcus aureus,Stapylococcus epidermidis Enterococcus faecalis and Enterococcus faecium among various groups such as different department,age,or specimen source.Conclusion Compared with past surveillance result,VRE detection ratio was steady,while MRSA detection ratio decreased.The emergence of resistance and non-susceptible strains to new antibiotics such as linezolid,tigecycline and daptomycin should be payed more attention.

Objective To investigate the Gram-positive coccus resistance in nationwide's tertiary hospitals and understand the trend of antimicrobial resistance.Methods All the clinical isolates were collected from 19 hospitals and the minimal inhibitory concentrations(MICs)were tested using agar/broth dilution method recommended.Results A total of 1 974 pathogenic Gram-positive coccus from 19 tertiary hospitals in 19 cities nationwide over the period from July 2021 to June 2022 were studied.Based on the MIC results,the prevalence of methicillin resistant Stapylococcus aureus(MRSA)and methicillin resistant Stapylococcus epidermidis(MRSE)were 36.4％and 79.9％respectively.No vancomycin insensitivity Staphylococcus was detected.Staphylococcus aureus were 100％susceptibility to linezolid and teicoplanin.Antibiotic resistance rate of Enterococcus faecalis and Enterococcus faecium to ampicillin were 3.1％and 92.9％.The detectation rate of vancomycin resistant Enterococcus(VRE)was 1.6％.Nonsusceptibility rate of Enterococcus faecalis to linezolid was 32.2％,two consecutive monitoring rises and nonsusceptibility rate of Enterococcus faecium(12.5％)was also significantly increased.The prevalence of penicillin non-susceptible Streptococcus pneumoniae(PNSSP)was 0.8％based on non-meningitis and parenteral administration criterion,decrease of nearly 30 percentage points from the previous surveillance.While for cases of oral penicillin,the rate was 71.8％,showing similar to last time.The results indicated that the number of strains with higher MIC value of penicillin(MIC ≥4 mg·L-1)decreased significantly.There were no significant differences of resistance rates of Stapylococcus aureus,Stapylococcus epidermidis,Enterococcus faecalis,Enterococcus faecium and Streptococcus pneumoniae among various groups such as different department,age,or specimen source.Conclusion VRE detection ratio stablized at a relatively low level.The number of Streptococcus pneumoniae with higher MIC value of penicillin decreased significantly compared with the previous monitoring.The increase of linezolidin-insensitive Enterococcus was noteworthy.

Objective To investigate the Gram-negative bacteria resistance in nationwide's tertiary hospitals and understand the trend of antimicrobial resistance.Method All the clinical isolates were collected from 19 hospitals and the minimal inhibitory concentrations(MICs)were tested using agar/broth dilution method recommended.Results A total of 4 066 pathogenic isolates from 19 tertiary hospitals in 19 cities nationwide over the period from July 2021 to June 2022 were studied.Based on the MIC results,Escherichia coli and Klebsiella pneumoniae showed extended spectrum β-lactamase(ESBLs)phenotype rates of 55.0％and 21.0％,respectively,ESBLs phenotype rate of Klebsiella pneumoniae keep going down.The ratios of carbapenems resistance Klebsiella pneumoniae increased by 5 percentage points compared with the previous monitoring.Carbapenems,moxalactam,sitafloxacin,β-lactam combination agents,fosfomycin trometamol,and amikacin displayed desirable antibacterial activity against Enterbacterales,susceptibal rates were above 75％.In addition,tigacycline,omacycline,colistin and fluoxefin maintained good antibacterial activity against their respective effective bacteria/species,and the bacterial sensitivity rates by more than 80％.Resistance rates of Pseudomonas aeruginosa and Acinetobacter baumannnii to imipenem were 26.3％and 72.1％and multidrug-resistant(MDR)detection rates were 41.1％and 77.3％,extensively drug-resistant(XDR)were 12.0％and 71.8％,respectively.Comparison of drug resistance rates from different wards,ages and specimen sources indicated that the proportion of resistance in Klebsiella pneumoniae and Acinetobacter baumannii isolated from intensive care unit(ICU)were significantly higher than non-ICU.Carbapenem resistance rates of Klebsiella pneumoniae isolated from ICU were more than 35％.Resistance rates of Haemophilus influenzae isolated in children to β-lactam,macrolide,clindamycin and ESBLs detection rate in Klebsiella pneumoniae isolated from children were more than those from adults and the old people,so bacterial resistance in children is an important problem in China.Conclusion ESBLs detection rate of Escherichia coli increased slightly after years of continuous decline.The proportion of carbapenem resistant Pseudomonas aeruginosa was stable,but the resistance rate of Klebsiella pneumoniae and Acinetobacter baumannii to carbapenems was still increased,which should be paid more attention.