Objective: : To explore the effect of PD-1 gene knockout by CRISPR/Cas9 system on the proliferation and IFN-γ secretion in human T cells. Methods: : The sequence of sgRNA targeting PD-1 was designed. The PD-1-sgRNA and Cas9 mRNA were synthesized by T7 RNApolymerase in vitro, and then the mixture of PD-1-sgRNAand Cas9 mRNAwas delivered into activated T cells by nucleofection. The efficiency of gene knockout was confirmed by sequencing. The phenotypes of T lymphocytes and the expression of PD-1 after gene knockout were analyzed by Flow cytometry. The proliferation of T lymphocytes was calculated by trypan blue counting. The level of IFN-γ secreted by T lymphocytes was detected by ELISA. Results： ：PD-1-sgRNA and Cas9 mRNA were successfully synthesized in vitro and delivered into T cells by nucleofection. Sequencing technology confirmed that the PD-1 gene sequence was edited and the editing efficiency was 58.3%. The expression of PD-1 on T lymphocyte surface was down-regulated successfully by CRISPR/Cas9 system [(9.6±1.85)% vs (16.2±2.05)%, P<0.05]. The knockout of PD-1 gene did not affect the proliferation and phenotype of T lymphocytes(P<0.05); However, compared with the control group, the level of IFN-γ secreted by T lymphocytes in the PD-1sgRNA group was significantly increased (P<0.01). Conclusion: : CRISPR/Cas9 system can successfully ablate PD-1 gene in human T lymphocytes, which could block the negative regulation of PD-1／PD-L1 and further promote the IFN-γ secretion in T cells.

Objective To explore the utilization and effectiveness of ultrasound screening for neural tube defects (NTDs),so that to provide scientific evidences for the secondary prevention of NTDs.MethodsFour hundred and fifty-nine women who delivered or gestated NTDs babies or fetuses were randomly selected from Shandong Province and Shanxi Province,and the related information was collected with structured questionnaire by trained interviewers.Results Of the 459 cases,the ultrasonography utilization rate was 98.7%,and 6 cases (1.3%) never took examinations by ultrasonography during the whole pregnancy period.The total diagnosis rate of ultrasound screening for NTDs was 85.9%,and those of anencephalus,spina bifida and encephalocele were 96.4%,79.6% and 73.8% respectively (P<0.05).The average diagnosis week of NTDs was 24.0 and those of anencephalus,spina bifida and encephalocele were 21.2,27.1 and 24.7 respectively (P<0.05).The detection rates of NTDs before 16 weeks,16-20 weeks,20-24 weeks,24-28 weeks and after 28 weeks were 14.1%,49.4%,46.3%,49.2% and 52.1% respectively (P<0.05).The detection rates of NTDs in hospitals,maternal and child care service centers and family planning centers were 46.4%,52.0% and 28.1% respectively (P<0.05).The detection rate of NTDs by two-dimensional ultrasound equipment was 41.3% and 83.3% by three-dimensional ultrasound equipment (P>0.05).Conclusions The detection rates of NTDs and the subtypes by ultrasonography are low at different pregnant periods and in different medical institutions.It is important to increase the utilization rate of ultrasound screening by pregnant women and improve the NTDs diagnostic level of primary health care institutions,so that to improve the efficacy of secondary prevention strategy for NTDs in China.

OBJECTIVETo construct γ-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells(HUVECs) in vitro.

METHODSTotal RNA was extracted from colon cancer cell line HT29 and the cDNA of γ-synuclein was amplified using RT-PCR. The digested fragment of cDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA.

RESULTSHuman γ-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-γ-synuclein protein in vitro. γ-synuclein facilitated SW1116 cell passing through matrigel and filter membrane(198.4±20.7 vs. 98.8±13.2, P<0.05) and elevated the adherence of SW1116 cells to HUVECs(3.08±0.36 vs. 1.22±0.21, P<0.05).

CONCLUSIONExpression of γ-synuclein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.

Cell Adhesion ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Genetic Vectors ; biosynthesis ; Human Umbilical Vein Endothelial Cells ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; gamma-Synuclein ; genetics

Objective To discuss the sleep quality of hospitalized patients with rheumatic diseases and the factors affecting the quality of sleep. Methods With Pittsburgh sleep quality index ( PSQI) questionnaire and the self-designed questionnaire about the factors affecting sleep were used to survey 125 patients with rheumatic disease from November 2013 to October 2014 in a level three class A hospital in Beijing city. Results Rheumatoid disease inpatients PSQI scores between 7 to 12 points had 35 cases, accounted for 28. 0%, more than 12 points having 6 cases, accounted for 4. 8%, PSQI various dimensions of 93 patients with seven hours of sleep time and accounted for 74. 4%, sleep efficiency more than 85% having 115 patients and accounted for 92. 0%, but the poor quality of sleep group with 113 cases and accounted for 90. 4%, sleep time group 101 cases of patients and accounted for 80. 8%, patients with sleep disorders in 120 cases, accounted for 96%, 111 cases of patients with daytime dysfunction, accounted for 88. 8%, and used hypnotic drugs in patients with only 13 cases, accounted for 10. 4%, group of patients with rheumatic disease of the Pittsburgh sleep quality index was higher than normal group (P<0. 01); there was no significant difference in the total score of PSQI and in patients with glucocorticoid (P <0. 05). The percentage of poor sleep quality was in patients with more than sixty years old, not continue to work or study and poor sleep before hospitalization, and sleep quality of patients affected by physical discomfort (P<0. 01);the primary factors affected patients′sleep was psychological factors. Conclusions The hospitalized patients with rheumatoid disease sleep quality are poor, while psychological factors are the main factors affecting sleep and corticosteroids is not absolutely the influencing factors of sleep. The elderly, the length of sleep before good quality patients should attract the attention of nurses. Using your own sleep drugs can improve sleep quality. Evaluation and assessment of patients′sleep quality can find out potential problems.

0bjective To translate and revise the Assessment of Interprofessional Team Collaboration Scale (AITCS) into Chinese, then to evaluate the reliability and validity of the Chinese version of AITCS. Methods The Chinese version of AITCS was translated from the original one, back-translated and adjusted for cultural adaptation. The reliability and validity were tested among 288 nurses, 81 physicians, 25 respiratory therapists, 10 physical therapists and 3 nutritionists from a tertiary hospital in Hangzhou using convenience sampling. Results The average of scale-level content validity index was 0.98, unanimity of scale-level content validity index was 0.84 and item-level content validity index was 0.89-1.00. Exploratory factor analysis extracted three common factors,which explained 61.427% of the total variance,and each item had high factor loading quality (>0.4). The correlations coefficients between each dimension score and the total score ranged from 0.801 to 0.898 (P<0.05),and the correlations coefficients between each dimension score ranged from 0.607 to 0.698 (P<0.05). The Cronbach α of AITCS was 0.909, split-half reliability was 0.835 and test-retest reliability was 0.763. Conclusion The Chinese version of the AITCS has been proved to be reliable and valid. It is a valuable tool for evaluating interprofessional team collaboration among the health professional providers in mainland China.

OBJECTIVE: To determine the effect of elastic strain rate ratio method and virtual touch tissue quantification (VTQ) on the diagnosis of breast masses.
 METHODS: Sixty female patients with breast cancer, who received surgical treatment in Daqing Oilfield General Hospital, were enrolled. All patients signed the informed consent paperwork and they were treated by routine ultrasound examination, compression elastography (CE) examination, and VTQ examination in turn. Strain ratio (SR) was checked by CE and shear wave velocity (SWV) value was measured by VTQ. The diagnostic values of different methods were evaluated by receiver operating characteristic (ROC) curves in the diagnosis of benign and malignant breast tumors.
 RESULTS: The maximum diameter and SWV value of the benign tumors were lower than those of the malignant tumors, and the SR ratio of benign masses was higher than that of malignant tumors (P<0.01). The AUC, sensitivity and specificity for elastic strain rate and VTQ for single or combined use were higher than those of conventional ultrasound (0.904, 97.5%, 69.2%; 0.946, 87.5%, 87.2%; 0.976, 90%, 97.4% vs 0.783, 85%, 61.5%). The AUC and specificity of VTQ were higher than those of the elastic strain rate (0.946, 87.2% vs 0.904, 69.2%), but the sensitivity of VTQ was higher than that of the latter (87.5% vs 97.5%). The AUC and specificity for combination of both methods were higher than those of single method, but the sensitivity was lower than that of the elastic strain rate. 
 CONCLUSION Combination of elastic strain rate ratio method with VTQ possesses the best diagnostic value and the highest diagnostic accuracy in the diagnosis of breast mass than that used alone.
Breast ; Breast Neoplasms ; Elasticity Imaging Techniques ; Female ; High-Energy Shock Waves ; Humans ; Microsurgery ; ROC Curve ; Touch

OX40 is a costimulatory receptor that is expressed primarily on activated CD4⁺, CD8⁺, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.
Mice ; Animals ; Receptors, Tumor Necrosis Factor/physiology* ; Receptors, OX40 ; Membrane Glycoproteins ; Ligands ; Antibodies, Monoclonal/pharmacology* ; Antineoplastic Agents/pharmacology*