To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
Base Sequence ; Cloning, Molecular ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Genetic Vectors/genetics ; Hep G2 Cells ; K562 Cells ; Molecular Sequence Data ; Receptors, Transferrin/*immunology ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Single-Chain Antibodies/*biosynthesis ; Single-Chain Antibodies/genetics

OBJECTIVETo observe the effect of Yiqi Huoxue Qingre Huashi Recipe (YHQHR, a recipe capable of supplementing qi, activating blood, clearing heat, and dissipating dampness) on ulcer healing and Helicobacter pylori (Hp) eradication rate in Hp positive peptic ulcer patients, and to explore coccoid Hp occurrence in the eradication.

METHODSTotally 80 Hp positive peptic ulcer patients were assigned to the treatment group and the control groups by random digit table, 40 in each group. All patients received standard triple therapy of Western medicine for 2 successive weeks. Those in the control group additionally took omeprazole enteric coated tablet, 20 mg each time, once per day for 4 successive weeks. Those in the treatment group additionally took YHQHR, twice per day for 6 successive weeks. The ulcer healing was observed and recorded by gastroscope after discontinued medication of 14 days. The effective rate of ulcer healing under endoscope was statistically calculated. Rapid urease test (RUT) was performed in one small piece of tissue from corpora ventriculi and sinuses ventriculi using 14C breathe test (UBT). Gastric juice was collected from the stomach. Hp urease gene amplification test (urea A-PCR) was performed in living tissue from gastric antrum. Results obtained from the above three test methods were recorded and assessed to decide the final eradiation rate. Gastric mucosa tissue was observed under electron microscope,attempting to find non-eradicated Hp, which was further observed.

RESULTSThe total curative effect under gastroscope was 97.5% (39/40 cases) in the treatment group, obviously higher than that in the control group (80.0%, 32/40 cases) (P < 0.05). The eradication rate of Hp was 75.0% (30/40 cases), obviously better than that of the control group (52.5%, 21/40 cases) (P < 0.05). The total positive Hp numbers after treatment was 14C UBT (12), RUT (8), and urea A-PCR (27), respectively. The Hp positive rate detected by 14C UBT and RUT was lower than the Hp positive rate detected by urea A-PCR (P < 0.05). Rod-like and coccoid Hp bacteria could be observed under electron microscope.

CONCLUSIONYHQHR combined standard triple therapy was more effective than standard triple therapy alone in promoting ulcer healing and elevating the eradication rate of Hp.

Breath Tests ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Gastric Mucosa ; Helicobacter Infections ; drug therapy ; Helicobacter pylori ; Humans ; Omeprazole ; Peptic Ulcer ; drug therapy ; microbiology ; Urea

Objective To explore the feasibility of prediction of subacute infarct lesion volumes by processing ADC maps based on ADC cut-off values in patients with acute stroke.Methods MRI was performed in 20 patients with clinically diagnosed acute infarct less than 6 h after stroke onset.The MRI included a DWI and conventional MRI.The follow-up MR examinations of all the patients were carried out within 4-7 days after admission.The brain tissue, which showed abnormal high intensity both in DWI at the baseline and FLAIR at the reexamination , was regarded as the area of the initial ischemia core.Graphic-penumbra was regarded as the difference between initial DWI and follow-up FLAIR.The ADC values of the ischemia core, graphic-penumbra and the contralateral normal brain tissue , the relative ADC (rADC) were measured.The rADC value of the graphic-penumbra was defined as the ADC cut-off values.GE medical system based on ADC cut-off values was also tested in these patients to obtain ADC maps.The lesion volumes, the abnormal area seen on the DWI , ADC maps and follow-up FLAIR, were also measured.rADC values in different areas were analyzed by paired Student t test.Relationship between baseline DWI , ADC map and follow-up FLAIR was analyzed using Spearman rank-order correlation test , and Kruskal-Wallis H test was used to compare the volumes among three groups.Results Absolute ADC and rADC values gradually increased from the core to the periphery of the ischemic lesion.The absolute ADC values statistically differed from those on the contralateral side for both ischemia core and graphic-penumbra.The rADC values were significantly decreased in the ischemia core ( 0.620 ±0.116 ) compared with the graphic-penumbra values (0.809 ±0.097;t =8.083,P <0.01).The lesion volume of follow-up FLAIR [16.3 (4.7, 29.0) cm3 ] correlated highly with both volume predicted with the ADC map [13.5 (4.8, 25.4) cm3] (r=0.967, P<0.01) and baseline DWI volume [11.0 (4.4,30.4) cm3] (r=0.950, P<0.01).There was no difference between the volume of DWI , ADC map and follow-up FLAIR(H=0.168, P>0.05).Conclusions Data shows the high feasibility of prediction of subacute infarct lesion volumes by processing ADC maps based on ADC cut-off values in patients with acute stroke , without intravenous contrast material, and it provides a new method for outcome prediction.

To understand the family sex education for young children in rural areas of Sichuan province and analyze the influencing factors. A multi-stage random sampling method was used to select 2246 parents of kindergarten children from rural areas in Sichuan province for a questionnaire-based survey.The Chi-square test and Logistic regression model were used for data analysis. It was found 1132(52.33%)parents had implemented family sex education for young children and 1031(47.67%)had not.Young children having asked sex-related questions(=1.536,95%=1.257-1.878),parents thinking that early childhood sex education is necessary(=3.691,95%=2.029-6.717),and parents having the intention to know early childhood sex education(=1.700,95%=1.274-2.269),and kindergarten having implemented early childhood sex education(=3.316,95%=2.515-4.372)were promoting factors for parents to conduct early childhood sex education,whereas a total annual household income at the middle level(=0.664,95%=0.456-0.968)was a hindering factor for parents to conduct early childhood sex education. Parents of young children in rural areas of Sichuan province have poor awareness of sex education,and the proportion of parents who have never conducted sex education for children is high.The total annual income of the family,whether the children have asked about sex-related questions,parents' attitude towards early childhood sex education,and whether the kindergarten has conducted the early childhood sex education are important factors that influence the level of children's family sex education.
Child ; China ; Humans ; Logistic Models ; Parents ; Sex Education ; Surveys and Questionnaires

Objective: To establish a prediction model for high-risk cardiovascular disease (CVD) among residents aged 35 to 75 years, so as to provide the basis for improving CVD prevention and control measures. Methods: Permanent residents aged 35 to 75 years were selected from Dongcheng District, Beijing Municipality using the stratified random sampling method from 2018 to 2023. Demographic information, lifestyle, waist circumference and blood biochemical indicators were collected through questionnaire surveys, physical examinations and laboratory tests. Influencing factors for high-risk CVD among residents aged 35 to 75 years were identified using a multivariable logistic regression model, and a prediction model for high-risk CVD was established. The predictive effect was evaluated using the receiver operating characteristic (ROC) curve. Results: A total of 6 968 individuals were surveyed, including 2 821 males (40.49%) and 4 147 females (59.51%), and had a mean age of (59.92±9.33) years. There were 1 155 high-risk CVD population, with a detection rate of 16.58%. Multivariable logistic regression analysis showed that gender, age, smoking, central obesity, systolic blood pressure, fasting blood glucose, triglyceride and low-density lipoprotein cholesterol were influencing factors for high-risk CVD among residents aged 35 to 75 years (all P<0.05). The area under the ROC curve of the established prediction model was 0.849 (95%CI: 0.834-0.863), with a sensitivity of 0.693 and a specificity of 0.863, indicating good discrimination. Conclusion The model constructed by eight factors including demographic characteristics, lifestyle and blood biochemical indicators has good predictive value for high-risk CVD among residents aged 35 to 75 years.

Objective To evaluate the influence and significance of preoperative design of surgical approach on stem cell transplantation via stereotactic surgery. Methods Six patients with stroke in the basal ganglia region were selected. The transplantation target and transcranial approach point were designed by magnetic resonance examination before stem cell transplantation via stereotacfic surgery to guarantee that the line connecting the transplantation target and transcranial approach point could avoid the important functional areas, the ventricular system and the softening focus. Postoperative magnetic resonance examination was performed to observe whether the practical target and surgical approach coincided with the preoperative design or not. Results The practical transplantation target was coincided with the designed transplantation target, distributed around the softening focus without implanted cells in the softening focus. Surgical approach was coincided with the preoperative design and it successfully avoided the important brain functional area, ventricular system and softening focus.Conelnsion The preoperative design of surgical approach can not only ensure the cells being exactly transplanted into the reservation target and guarantee the curative effect, but also promise the surgical approach successfully avoiding the important brain functional area, ventricular system and softening focus and reduce the operative injury.

Human infections with Lophomonas blattarum are rare. However, the majority of the infections occurred in China, 94.4% (136 cases) of all cases in the world. This infection is difficult to differentiate from other pulmonary infections with similar symptoms. Here we reported a case of L. blattarum infection confirmed by bronchoalveolar lavage fluid smear on the microscopic observations. The patient was a 21-year-old female college student. The previous case which occurred in Chongqing was 20 years ago. We briefly reviewed on this infection reported in the world during the recent 20 years. The epidemiological characteristics, possible diagnostic basis, and treatment of this disease is discussed in order to provide a better understanding of recognition, diagnosis, and treatment of L. blattarum infection.
Female ; Humans ; Lung Diseases, Parasitic/diagnosis/*parasitology ; Parabasalidea/*isolation & purification ; Protozoan Infections/*parasitology ; Young Adult

Adolescent ; Adult ; Aged ; Blood Coagulation ; Factor XII ; genetics ; Factor XII Deficiency ; blood ; genetics ; Female ; Humans ; Male ; Point Mutation

OBJECTIVETo develop a LC-MS method for the determination of senkyunolide I (SI) in rat plasma, in order to observe whether there is significant change in the pharmacokinetics parameters of complex prescriptions of Huoluoxiaolingdan (HLXL) and single herbal extracts from Ligusticum chuanxiong Hort. in rats, and assess the effect of other components in HLXL on the pharmacokinetics of SI.

METHODTwelve male Sprague-Dawley (SD) rats were randomly divided into two groups, and orally administered with extract from HLXL and L. chuanxiong (both equal to SI 4.53 mg x kg(-1)). Their blood was collected at different time points for LC-MS, in order to detect the plasma concentration of SI. The pharmacokinetic parameters of SI were calculated by DAS 2.0 software. SPSS 16.0 software was used for independent-sample T-test and Nonparametric T-test.

RESULTA linear relationship of SI ranged from 6.750 to 675.0 microg x L(-1), and with the lowest limit of detection being 6.750 microg L(-1). Both of the plasma concentration-time curves of SI were fitted with the two-compartment model for extract of HLXL and L. chuanxiong. The detected AUC and Cmax of SI showed significant difference, with no significant difference in other parameters.

CONCLUSIONThe LC-MS determination method established in this experiment was so exclusive, accurate and sensitive that it is suitable for pharmacokinetic studies on extracts of HLXL and SI from L. chuanxion. The experiment results show that other ingredients of HLXL have noticeable effect on the absorption of SI in rat plasma.

Administration, Oral ; Animals ; Area Under Curve ; Benzofurans ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Male ; Metabolic Clearance Rate ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the significance of Rac subfamily members in the gastrointestinal carcinogenesis and progression.

METHODSThe mRNA expression of Rac1, Rac2 and Rac3 in 12 kinds of gastrointestinal cancer cell lines was examined by semi-quantitative RT-PCR. The activities of Rac1 protein in 5 kinds of gastric cancer cell lines were tested by pull-down assay.

RESULTSCompared with the normal gastric mucosa and intestinal epithelial cell line, the mRNA expression of Rac1 and Rac3 was up-regulated in most of gastrointestinal cancer cell lines. The activities of Rac1 protein increased markedly in gastric cancer cell lines.

CONCLUSIONThe increased mRNA expression of Rac1 and Rac3 in gastrointestinal cancer cell lines and the abnormal activation of Rac1 protein in gastric cancer cell lines might be correlated with the carcinogenesis of gastrointestinal cancer.

Cell Line, Tumor ; Gastrointestinal Neoplasms ; metabolism ; Humans ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; rac GTP-Binding Proteins ; genetics ; rac1 GTP-Binding Protein ; analysis ; genetics