Biomarkers, Tumor ; metabolism ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; metabolism ; pathology ; Humans ; Lung Neoplasms ; drug therapy ; metabolism ; pathology ; Oncogene Proteins, Fusion ; chemistry ; metabolism ; Protein Kinase Inhibitors ; therapeutic use ; Pyrazoles ; therapeutic use ; Pyridines ; therapeutic use ; Pyrimidines ; therapeutic use ; Smoking

Genomic DNA of two fungi Thamnidium elegans and Umbelopsis isabellina were extracted with an amended Cetyltrimethyl Ammonium Bromide (CTAB) method. This modified method uses repeated freezing in liquid nitrogen and thawing with combination of shocking with glass beads to replace of the tra- ditional method. Quality and concentration of DNA extracted by the modified methodwere tested. Compared with the traditional method, higher yield and purity of genomic DNA were obtained with less amount ofmy- celium. The result indicted that this is a simple and highly efficient method, which is suitable to treat many samples at one time and for basic molecular experiments, such as restriction endonuclease reaction and PCR.

Objective To find the mutation of disease-causing genes of sudden unexplained death syn-drome (SU D S ) in the young by whole exome sequencing in one case. Methods O ne SU D S case was found no obvious fatal pathological changes after conventional autopsy and pathological examination. The whole exome sequencing was performed with the Ion Torrent PGMTM Systemwith hg19 as reference se-quence for sequencing data. The functions of mutations were analyzed by PhyloP, PolyPhen2 and SIFT. A three-step bioinformatics filtering procedure was carried out to identify possible significative single nu-cleotide variation (SN V ), which was missense mutation with allele frequency <1% of myocardial cell. Results Four rare suspicious pathogenic SN V were identified. C ombined with the analysis of convention-al autopsy and pathological examination, the mutation MYOM 2 (8_2054058_G/A ) was assessed as high-risk deleterious mutation by PolyPhen2 and SIFT, respectively. Conclusion Based on the second genera-tion sequencing technology, analysis of whole exome sequencing can be a newmethod for the death cause investigation of SU D S. The gene MYOM2 is a newcandidate SU D S pathogenic gene for mecha-nismresearch.

OBJECTIVETo explore the mechanism of Shangke Jiegu Tablet (SJT)in repairing the mandibular defect.

METHODSTotally 72 healthy male New Zealand rabbits were randomly divided into the normal control group (n = 24), the model group (n = 24), and the SJT group (n = 24). Then the mandibular defect model was established. Animals in the normal control group and the model group were fed with normal forage, while those in the SJT group were fed with SJT forage. On the day 7, 14, 28, and 56 after model establishment, 6 rabbits were killed in each group. The bone was collected from the mandibular defect. The gene expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) were detected by means of RT-PCR. The positive dyeing strength and area of the bone tissue were detected by means of immunohistochemical technique.

RESULTSCompared with the normal control group, the degree of OPGmRNA expression was remarkably up-regulated on day 7 after model establishment (P < 0.05) and the degree of OPGLmRNA expression was remarkably up-regulated on day 14 after model establishment (P < 0.05) in the model group. Compared with the model group, the degree of OPGmRNA expression was remarkably up-regulated (P < 0.05), and the positive dyeing strength and area of bone tissue were stronger and broader on day 14, 28, and 56 after model establishment in the SJT group. The degree of OPGLmRNA expression was remarkably down-regulated (P < 0.05), and the positive dyeing strength and area of bone tissue were weaker and smaller on day 14 after model establishment in the SJT group. The ratio of OPGmRNA/OPGLmRNA was remarkably up-regulated on day 14, 28, and 56 after model establishment (P < 0.05).

CONCLUSIONThe effect mechanism of promoting mandibular defect repairing by SJT may be correlated to regulating the expressions of OPGmRNA and OPGLmRNA.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression ; drug effects ; Ligands ; Male ; Mandibular Injuries ; genetics ; metabolism ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; Rabbits ; Wound Healing ; drug effects

OBJECTIVETo investigate the inhibition of COX-2 gene expression and its effects on malignant proliferation of human lung adenocarcinoma A549 cells after interfering at different target sites in vitro.

METHODSThe 3rd, 7th and 10th exon of COX-2 were selected as the targets and three COX-2 siRNA expression vectors with human U6 promoter were constructed. Three siRNA expression vectors and two vacant vectors were transfected into A549 cells expressing COX-2 with lipofectamine, respectively. The transfected cell strains were constructed and the change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A549 cells after interfering at different target sites were studied by cell growth curve and colony formation assay in vitro.

RESULTSThe three siRNAs and U6 promoter were validated by PCR, restriction endonuclease digestion, DNA sequencing and BLAST alignment, and cloned into the pEGFP vector. The cell strains transfected were named as A549-3, A549-7, A549-10, A549-p and A549-pU6, respectively. A549-p cells showed expression of GFP and A549-3, A549-7, A549-10, A549-p and A549-pU6 cells did not show at 24, 48 and 72 hours after transfection. The results of RT-PCR and Western blot showed an inhibition of COX-2 expression after interfering at three target sites (3rd, 7th and 10th exons). In contrast to A549 cells, the levels of COX-2 mRNA of A549-3, A549-7 and A549-10 cells were reduced by 10.6%, 33.4% and 61.2%, respectively. The levels of COX-2 protein of A549-3, A549-7 and A549-10 cells were reduced by 26.7%, 44.7% and 56.2%, respectively. The results of cell growth curve and colony formation assay showed a slowing down of the growth of A549-10 cells and reduction of their colony formation rate. The other two targets had no apparent effect on the growth of A549 cells.

CONCLUSIONThere is a significant inhibiting effect of RNA interference on the malignant proliferation of A549 cells in vitro, and the most striking effect can be seen when the 10th exon of COX-2 is taken as the interference target.

Adenocarcinoma ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cyclooxygenase 2 ; genetics ; metabolism ; physiology ; Exons ; Genetic Vectors ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Promoter Regions, Genetic ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

In this study, to investigate the effect on expression of IL-2 in lymphocytes from bone marrow and peripheral blood of normal donors after they were mobilized by G-CSF in allo-BMT, 7 normal donors bone marrow and peripheral blood were harvested before and after G-CSF administration. The separated lymphocytes were measured by FCM after they were stained intracellularly by anti-IL-2, and their expressions of IL-2 were compared. The degree of aGVHD in patients after bone marrow transplantation was evaluated clinically, and it was compared with the status of aGVHD of 15 patients whose donors didn't receive G-CSF administration in our department, and 2 groups of patients are comparable in age, types of diseases and status of donors. The results showed that the expression of IL-2 in lymphocytes in 7 G-CSF mobilized donors decreased significantly after G-CSF administration and more severe aGVHD than grade II didn't develop in these recipient patients, and comparing with 15 patients received the bone marrow from donors who didn't receive G-CSF, the incidence of aGVHD decreased. It is suggested that the expression of IL-2 in lymphocytes was influenced by donors' G-CSF administration, and it is likely that thereby reduces the incidence of aGVHD in patients after BMT.
Adult ; Blood Donors ; Bone Marrow Cells ; drug effects ; metabolism ; Bone Marrow Transplantation ; adverse effects ; Female ; Flow Cytometry ; Graft vs Host Disease ; etiology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Humans ; Interleukin-2 ; biosynthesis ; Lymphocytes ; drug effects ; metabolism ; Male

OBJECTIVETo study the clinical characteristics of male urethral duplication infection and offer some guidelines for the diagnosis and treatment of the disease.

METHODSWe analyzed the pathological types, clinical characteristics, therapeutic processes and follow-up results of 9 cases of male urethral duplication.

RESULTSAmong the 9 cases of urethral duplication, 7 turned out to be of Type I, 1 Type II A2 and 1 Type II B. The disease courses varied from 2 to 420 days, with an average of 77.2 +/- 141.5 days. Four cases with longer disease duration were identified with a history of repeated use of various antibiotics for treatment. Their clinical manifestations varied, with the outflow of excretions or pus from the duplicate or normal urethra as the cardinal symptoms. The pathogens detected from the secretions were mainly Neisseria gonorrhoeae, Ureaplasma urealyticum, and Chlamydia trachomatis. The consistency rate of the same pathogens detected in the vaginal or cervical secretions from the sex partners of the patients was 87.5%. All the symptoms disappeared after a sufficient-course treatment with sensitive antibiotics, and the patients' sex partners received the same medication simultaneously. No recurrence was found during a 3-month follow-up.

CONCLUSIONUrethral duplication infection has various clinical manifestations, and thus is easily missed in diagnosis. Sufficient-course treatment with sensitive antibiotics is recommended for those that prefer conservative therapy, and their sex partners should be treated simultaneously.

Adult ; Humans ; Male ; Middle Aged ; Urethra ; microbiology ; Urethral Diseases ; diagnosis ; drug therapy ; microbiology ; Young Adult

OBJECTIVETo investigate the possibility of differentiation of human mesenchymal stem cells (hMSC) into epidemic cells in vitro.

METHODShMSCs were segregated from normal adult human bone marrow by Percoll solution (1.073 g/ml) , and were cultured, purified, and amplified to 3th passage in vitro. Then the hMSCs were randomly divided into control group ( with treatment of normal L-DMEM medium) and experimental group (with treatment of L-DMEM medium containing epidermal growth factor,insulin,tretinoin, calcium chloride). After 7 days of culture, the morphologic changes of hMSCs in the 2 groups were observed with inverted phase contrast microscope. The expressions of P63 and PCK of hMSCs were assessed with immunohistochemical methods.

RESULTSThe shape of hMSCs in experimental group became irregular or oblong in shape, while that in control group were still in spindle shape. Immunohistochemical results showed that hMSCs were P63 and PCK positive in the experimental group, while those in control group were negative.

CONCLUSIONHuman mesenchymal stem cells can differentiate into epidemic cell in vitro.

Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; cytology ; Humans ; Keratins ; metabolism ; Membrane Proteins ; metabolism ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo study the responses of peginterferon-alpha 2a antiviral therapy in chronic hepatitis B (CHB) patients.

METHODSOne hundred two CHB patients with their serum ALT values higher than 2x the upper limit of the normal (ULN) were divided into a HBeAg-positive and a HBeAg-negative group. All patients were treated with peginterferon-alpha 2a by subcutaneous injection (180 microgram once weekly). After treatment for 6 months, patients without a defined therapeutic response were dropped from the treatment group; the others completed a 12 month therapy. The sustained response and the antiviral effect of the treatment were assessed at the end of the therapy. To investigate the possible impact factors of the response to peginterferon-alpha 2a, we studied the therapeutic response of patients with different serum ALT levels, inflammation grades of liver histology, stages of fibrosis, and HBV viral load levels.

RESULTS(1) There was no statistical difference of the rates of response at the end of treatment and 6, 12, 18, 24 and 30 months after the cessation of therapy between the HBeAg-positive and the HBeAg-negative groups. (2) In the HBeAg-positive group, the rates of response of patients with serum ALT values>3*ULN were significantly higher than those with serum ALT values less than 3*ULN (x2=4.40). However, no statistical difference of serum ALT levels was found in the HBeAg-negative group. (3) In both HBeAg-positive and HBeAg-negative groups, no difference was revealed in the rates of response among patients with different levels of HBV viral loads. (4) In the HBeAg-positive group, patients with more severe liver inflammation histologically (G3 and G4) had significantly higher response rates than those with milder inflammation (G1 and G2) (x2=4.19), but no similar statistical differences were found in the HBeAg-negative group. Moreover, there was no difference in the rates of response among patients in different stages of liver fibrosis in both HBeAg-positive and HBeAg-negative groups.

CONCLUSIONSSimilar rates of response and sustained virological response to the peginterferon-alpha 2a treatment can be achieved in both HBeAg-positive and HBeAg-negative patients. Hepatic fibrosis is not a predictor of poor therapeutic response. For HBeAg-positive patients, more severe liver inflammation identified with liver biopsies (G3 or G4) and high serum ALT values (more than 3*ULN) can be considered as predictors of a good therapeutic response.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; Female ; Hepatitis B, Chronic ; drug therapy ; pathology ; Humans ; Interferon-alpha ; therapeutic use ; Liver ; pathology ; Male ; Middle Aged ; Polyethylene Glycols ; therapeutic use ; Recombinant Proteins ; Young Adult

OBJECTIVETo investigate the biological and clinical features of Chinese rhesus monkeys after intravenous (IV) and intrarectal (IR) challenge with SIVmac239 in rhesus monkeys of Chinese origin, and compare the differences between the routes of infection.

METHODSRhesus monkeys of Chinese origin were inoculated with SIVmac239 either by IV (n = 19) or IR (n = 6) routes. Simian immunodeficiency virus (SIV)-specific antibody titer, CD4 + T cell counting, plasma SIV load, lymph node pathology, and clinical manifestations were compared between these two groups 232 or 168 days after challenging.

RESULTSAll SIVmac239-inoculated animals became seropositive for SIV-specific antibodies. SIV-specific IgM was detected in IV groups as from day 10 but was not detected in IR for all the time points. Although SIV-specific IgG was detected as from day 30 in both groups, the IgG titers were ten-fold higher in IV group than in IR group after day 168. CD4 + T-cell counting decreased progressively in IV group but remained stable in IR group over time. Plasma SIV RNA loads peaked in all animals between day 10 and day 14 (10(7) copies/ml), then declined to "setpoint" (10(3) - 10(6) copies/ml) about 2 months later. Most inoculated animals manifested lymphadenopathy. Two animals in IV group and one in IR group died of simian AIDS between day 150 and day 210, as evidenced by the autopsies showing the depletion of lymph tissues, Pneumocystis carinii pneumonia and other opportunity infections. Conclusion IV or IR inoculation of SIVmac239 in Chinese rhesus monkeys will result in chronic SIV infection with a similar clinical feature of natural HIV infection, which provides an excellent experimental animal model for AIDS.

Animals ; CD4-Positive T-Lymphocytes ; metabolism ; China ; Disease Models, Animal ; Female ; Macaca mulatta ; virology ; Male ; Rectum ; virology ; Simian Acquired Immunodeficiency Syndrome ; immunology ; virology ; Simian Immunodeficiency Virus ; immunology ; pathogenicity ; Veins ; virology