To ascertain current situation of wild Marsdenia tenacissima resources in Honghe, Yunnan province, the distribution, habitat characteristic and resources reserves of M. tenacissima were surveyed based on interviews and investigation. The results showed that M. tenacissima was found in 7 counties such as Jinping, Mengzi etc, and distributed mainly on the mountainsides from 800 m to 1 200 m. And distribution was affected by many factors, such as light, heat, topography, soil, and vegetation. M. tenacissima grew well in distribution areas. M. tenacissima had averagely a weight of 2.8 kg per plant. Resources reserve of M. tenacissima in Honghe was estimated to 1 300 tons by now but it reduced rapidly in resent years, the wild resources reserve may not meet demand of market. Resources protection and wildlife tending would be conducted to deal with increasing medication requirements.
China ; Ecosystem ; Marsdenia ; classification ; growth & development ; Plants, Medicinal ; classification ; growth & development ; Soil ; chemistry

Humans ; Homicide ; Forensic Pathology

OBJECTIVETo construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.

METHODSWe firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.

RESULTSOur results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.

CONCLUSIONOur protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.

Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Histones ; genetics ; Humans ; Polymerase Chain Reaction ; methods

Classical Swine Fever Virus (CSFV) E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The plasmid pVAXE2 was transformed into Salmonella choleraesuis C500 (S. C500) attenuated vaccine strain by electroporation to generate Salmonella choleraesuis engineering strain S. C500/pVAXE2. The characterization of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated that it retained the same properties as its original strain S. C500 with exception of kanamycin resistance originated from the plasmid pVAXE2. The plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits were vaccinated three times at two weeks interval with S. C500/pVAXE2 in oral and intramuscular routes at the dosage of 1 x 10(8) CFU for mice and 2 x 10(9) CFU for rabbits each time. The specific antibody response against CSFV and Salmonella choleraesuis was detected by ELISA. Two weeks after the third boost the immunized rabbits were challenged with 20 ID50 of hog cholera lapinized virus (HCLV), followed by a virulent strain of Salmonella choleraesuis two week later than HCLV challenge. The results showed that all immunized mice and rabbits produced significant antibodies against CSFV and Salmonella choleraesuis, and the immunized rabbits demonstrated the effective protection against the challenge of HCLV and virulent Salmonella choleraesuis. These results indicated the potential of developing multiplex swine DNA vaccine by using this bacteria as the vector.
Animals ; Classical Swine Fever ; immunology ; prevention & control ; virology ; Classical swine fever virus ; genetics ; immunology ; Mice ; Rabbits ; Salmonella arizonae ; genetics ; Swine ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology ; Viral Vaccines ; immunology

This study was aimed to evaluate the effect of triptolide (TPL) on the reversal of multidrug resistance in K562/A02 cell line. The sensitivity of K562 and K562/A02 to adriamycin (ADM) and reversal of drug resistance were determined with MTT method. The concentration of intracellular ADM and P-glycoprotein expression were detected by flow cytometry. Luciferase reporter gene assay was used to detect the transcriptional activity of MDR1 promoter. The results showed that TPL significantly decreased the resistance degree of K562/A02 cells, inhibited P-glycoprotein expression (mean fluorescent intensity decreased from 123 ± 13 to 39 ± 13) and increased the intracellular concentration of ADM (mean fluorescent intensity increased from 18 ± 5 to 34 ± 6) in K562/A02 cells. Luciferase reporter gene assay demonstrated that TPL inhibited the transcriptional activity of MDR1 promoter by 75%. It is concluded that TPL may effectively reverse the multidrug resistance in K562/A02 cells via modulating P-glycoprotein expression and increasing intracellular ADM accumulation.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Diterpenes ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Epoxy Compounds ; pharmacology ; Humans ; K562 Cells ; Phenanthrenes ; pharmacology ; Promoter Regions, Genetic

Objective:To analyze the false positive rate and false negative rate of layered mammography in diagnosis of mammary gland diseases.Methods:120 patients with malignant breast diseases and 120 patients with benign breast diseases treated in Huadong Hospital Affiliated to Fudan University from February 2015 to October 2016 were selected.All patients were examined by both traditional mammography and layered mammography.Taking surgical pathology as the gold standard,the diagnostic value of the two methods on patients with different ages,different lesion sizes,and different gland texture were compared.Results:The false positive rate and false negative rate of patients with mammary gland diseases in layered mammography group aged less than 45 years,45-60 years,and more than 60 years were lower than those of the conventional group,and the differences were statistically significant (P＜0.05).The false positive rate and false negative rate of patients with mammary gland diseases in layered mammography group with lesion sizes less than 1 cm,1 2 cm,and more than 2 cm were significantly lower than those of the conventional group,and the differences were statistically significant (P＜0.05).As for the patients with different gland texture,the false positive rate and false negative rate of the two groups were not statistically different.Conclusions:The false positive rate and false negative rate of layered mammography in diagnosis of breast diseases are lower than the conventional mammography,so it has higher clinical diagnostic value.

OBJECTIVETo construct a cell model of 4.1R gene knockout in murine macrophage cell line RAW264.7 using CRISPR/Cas9 technique.

METHODSThree high?grade small?guide RNAs (sgRNAs) that could specifically identify 4.1R gene were synthesized and inserted into lentiCRISPRv2 plasmid. RAW264.7 cells were infected with sgRNA?Cas9 lentivirus from 293T cells transfected with the recombinant sgRNA?lentiCRISPRv2 plasmid, and the positive cells were screened using puromycin and the monoclonal cells were obtained. The expression of 4.1R protein in the monoclonal cells was measured by Western blotting, and the mutation site was confirmed by sequence analysis. Result A 4.1R gene knockout RAW264.7 cell line was obtained, which showed a 19?bp deletion mutation in the 4.1R gene sequence and obviously enhanced proliferation.

CONCLUSIONWe successfully constructed a 4.1R gene knockout macrophage cell line using CRISPR/Cas9 technique, which may facilitate further investigation of the function of 4.1R in macrophages.

OBJECTIVEThis study was to clarify E-cadherin expressions in non-small cell lung cancer (NSCLC) and its correlation with patients' prognosis.

METHODSTissue microarrays (TMAs) containing specimens from 365 different NSCLC were constructed, covering all stages and almost all histological types of this disease. Slides were immunohistochemically stained with antibodies against E-cadherin. Expression pattern of the protein was analyzed with relation to the clinicopathological. Correlations of the results with patients' overall survival were also examined.

RESULTSImmunohistochemical staining revealed that E-cadherin protein was localized mainly on membranes and the cytoplasm of NSCLC tumors cells. Reduced E-cadherin expression was evident in 32.1%. Reduced E-cadherin expression significantly correlated with lymph nodes metastasis (chi(2) = 16.430, P = 0.001), histological dedifferentiation (chi(2) = 9.243, P = 0.010) and advanced clinical stage (chi(2) = 9.421, P = 0.024). There was no significant difference in E-cadherin expression between squamous cell carcinoma and adenocarcinoma. E-cadherin reduced expression correlated with a poor prognosis (P < 0.0001) in univariate analysis. Multivariate analysis showed a significantly lower survival probability for patients with reduced E-cadherin (P < 0.001), and E-cadherin was an independent prognostic factor for survival of NSCLC patients.

CONCLUSIONSIt suggests that dysfunction of E-cadherin has an important impact in the progression of lung cancer. As an independent prognostic factor, expression of E-cadherin can predict outcome of different group, together with conventional prognostic factors, and subsequently make appropriate management.

Adult ; Aged ; Aged, 80 and over ; Cadherins ; biosynthesis ; Carcinoma, Non-Small-Cell Lung ; metabolism ; mortality ; secondary ; Female ; Follow-Up Studies ; Humans ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; mortality ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; Survival Rate

OBJECTIVEAIM To establish a drug screening model based on transcriptional regulation of estrogen responsive element (ERE) and use it to screen compounds for discovering new ligands of estrogen receptor (ER) subtypes.

METHODA recombinant reporter vector pERE-TAL-SEAP was constructed by inserting a synthetic sequence composed of five tandem copies of EREs upstream of promoter of the reporter vector pTAL-SEAP. The pERE-TAL-SEAP and the internal control plasmid pCMV were transiently co-transfected into Hela cells expressing ER subtype or ER subtype, and the effects of pure ER agonists 17estradiol, phytoestrogen genistein and pure ER antagonist ICI182, 780 on reporter gene SEAP expression were observed.

RESULTIn the Hela cells expressing ER alpha or ER beta subtype, the expression of SEAP gene were induced in a dose dependent manner by 17-estrodiol with a maximal effect at approximately 10 nmol.L-1 and with EC50 of (80.58 +/- 8.51) pmol.L-1 and (103.90 +/- 5.29) pmol.L-1, respectively, so done by phytoestrogen genistein with a maximal effect at 1 mumol.L-1 and with EC50 of (10.86 +/- 0.75) nmol.L-1 and (39.38 +/- 2.26) nmol.L-1, respectively. The maximal level induced by estrodiol and genistein were about 7-14 fold higher than that of vehicle. The pure antiestrogen ICI182, 780 at concentration of 1 mumol.L-1 completely blocked the inductions of 17-estrodiol and genistein.

CONCLUSIONThe cellular drug screening model can be established by transfecting reporter vector pERE-TAL-SEAP in Hela cell lines expressing ER alpha or ER beta. The cell lines can be used to screen compounds with estrogenicity by testing SEAP activity in the culture media of cells growing in microtitier wells. The system should provide an efficient model for screening and analyzing the activity of large numbers of ligands of ER.

Drug Evaluation, Preclinical ; methods ; Estradiol ; pharmacology ; Estrogen Receptor alpha ; Estrogen Receptor beta ; Gene Expression Regulation ; drug effects ; Genes, Reporter ; Genistein ; pharmacology ; HeLa Cells ; Humans ; Ligands ; Promoter Regions, Genetic ; Receptors, Estrogen ; genetics ; Transfection

OBJECTIVETo evaluate the clinical effects of Chinese medicine (CM) on acute myocardial infarction (AMI) with a prospective cohort study.

METHODSA total of 334 AMI patients from January 2007 to March 2009 were consecutively enrolled, and were assigned to a treatment group (169 cases) treated with combined therapy (CM for at least one month and Western medicine) and a control group (165 cases) with Western medicine alone. Clinical data including age, gender, smoking, medical history, infarction area, heart functional classification, CM syndrome scores, blood-stasis syndrome score, primary end-point (death, nonfatal myocardial infarction, and revascularization) and secondary end-point (ischemic stroke, rehospitalization due to angina, heart failure and shock), were collected. CM syndrome scores, blood-stasis syndrome score, primary end-point and secondary end-point were collected during the 6-month follow-up. Kaplan-Meier method was used for the survival analysis. The multifactor analysis was analyzed by Cox proportional hazards regression.

RESULTSAt the end of 6-month the CM syndrome score and bloodstasis syndrome score in the treatment group were lower than those in the control group (P<0.01), especially the symptoms of chest pain, spontaneous perspiration and insomnia. Rehospitalization rate due to angina during the 6-month follow-up in the treatment group (2.96%) was lower than that in the control group (7.88%, P<0.05). Kaplan- Meier survival curve showed that event-free cumulated survival of rehospitalization due to angina during the 6-month follow-up in the treatment group was higher than that in the control group (Log rank 4.700, P=0.03). Cox regression analysis showed that heart dysfunction [hazard ratio (HR)=1.601, 95% CI=1.084-2.364, P=0.018] and diabetes mellitus (HR=1.755, 95% CI=1.031-2.989, P=0.038) were hazard factors to end-point, whereas CM (HR 0.405, 95% CI=0.231-0.712, P=0.002), percutaneous coronary intervention (PCI, HR=0.352, 95% CI=0.204-0.607, P<0.001) and angiotensin converting enzyme (ACE) inhibitors (HR=0.541, 95% CI=0.313-0.936, P=0.028) were protective factors.

CONCLUSIONSCM therapy could decrease CM syndrome scores and blood-stasis syndrome score, reduce the rehospitalization rate during 6-month follow-up due to angina. Heart dysfunction and diabetes mellitus were hazard factors to end-point, whereas CM, PCI and ACE inhibitors were protective factors.

Adult ; Aged ; Case-Control Studies ; Cohort Studies ; Female ; Hematologic Diseases ; complications ; epidemiology ; Hospitalization ; statistics & numerical data ; Humans ; Male ; Medicine, Chinese Traditional ; adverse effects ; methods ; Middle Aged ; Myocardial Infarction ; complications ; epidemiology ; therapy ; Prospective Studies ; Research Design ; Syndrome ; Treatment Outcome