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

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

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

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

Autoimmune diseases (ADs) increase the risk of non-Hodgkin's lymphoma and contribute to poor prognosis of patients. However, the association between immunologic markers and clinical outcome has rarely been investigated. This study aims to analyze the prognostic value of pretreatment immunologic markers in newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL). We retrospectively reviewed the data on 502 patients with DLBCL treated in our institution from January 2013 to March 2018. Survival functions were estimated using Kaplan-Meier method and Cox regression model. The 3-year progression free survival (PFS) and overall survival (OS) rates were 70.2% and 80.9%, respectively, and the complete remission (CR) rate was 78.1%. Among the patients, those with multiple ( ⩾ 3) abnormal immunologic markers had significantly shorter 3-year PFS (52.7% vs. 77.3%, P < 0.001) and OS (68.5% vs. 85.8%, P = 0.001) than those without multiple abnormal immunologic markers. Multivariate analysis revealed that the presence of multiple abnormal immunologic markers and the elevated serum levels of lactate dehydrogenase were the independent adverse prognostic factors for PFS (P = 0.008, P < 0.001) and OS (P = 0.003, P < 0.001). Meanwhile, advanced Ann Arbor stage was an independent adverse prognostic factor for PFS (P = 0.001) and age > 60 years for OS (P = 0.014). In conclusion, the immunologic status was closely related to lymphoma progression, and this study provides new insights into the risk stratification of patients with DLBCL.
Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Biomarkers ; China ; Disease Progression ; Female ; Humans ; Immunotherapy ; methods ; Lymphoma, Large B-Cell, Diffuse ; mortality ; therapy ; Male ; Middle Aged ; Multivariate Analysis ; Prognosis ; Retrospective Studies ; Survival Analysis ; Survival Rate ; Young Adult

OBJECTIVEChinese allergic subjects have high levels of sensitization to house dust mite (HDM) and other indoor allergens. This study quantifies common indoor allergen levels in Chinese households.

METHODSDust samples were collected from nine cities. Major allergens Der p 1 and Der f 1 from Dermatophagoides pteronyssinus and D. farinae, and specific antigens of Blomia tropicalis, Tyrophagus putrescentiae, Acarus siro, and cockroach species Blattella germanica and Periplaneta americana were measured by ELISA.

RESULTSHDM allergens were found in dust samples from bedding in 95% of the Chinese households. The median levels varied from <0.006 to 9.2 µg/g of dust, depending on the city. The percentages of households having HDM allergen levels associated with the risk of developing allergy sensitization and asthma were 65% and 25%, respectively. Specific antigens of the storage mite and cockroach were only found in samples from the southern and tropical regions of China. Levels of mite allergens were generally higher in samples from bedding compared to samples from the living room, even for storage mites, whereas levels of cockroach antigens were higher in the living room samples.

CONCLUSIONHDM allergens are present in bedding dust samples from most Chinese households. Cities in southern and central China have relatively high levels of HDM major allergens compared to cities in northern and western China. Antigens of storage mites and cockroaches are not as common as HDM allergens.

Air Pollution, Indoor ; statistics & numerical data ; Allergens ; chemistry ; Animals ; Bedding and Linens ; China ; Cockroaches ; Dust ; analysis ; Housing ; Pyroglyphidae ; Seasons