To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Objective This study aims to explore the application value of tuberculosis T lymphocytes enzyme-linked immune SPOT test (T-SPOT.TB) on early diagnosis of tuberculosis.Methods The TB infection in 189 inpatients suspected tuberculosis in pneumology department of Shaanxi Provincial People's Hospital was detected with T-SPOT.TB,fluorescence RQPCR,tuberculosis (TB-Ab)protein chip and PPD methods.Results The sensitivity of four methods was 91.54％ (119/130),73.85％(96/130),63.08％(82/130) and 57.69％ (75/130) respectively and the specificity of those was 89.83％ (53/59),86.44％(51/59),67.79％(40/59) and 66.10％(39/59),respectively.The sensitivity of T-SPOT.TB method was statistically higher than those of other three tests,respectively (P＜0.05).The specificity of T-SPOT.TB was significantly higher than those of TB-AB and PPD (P＜0.05),but there was no statistical difference between RQ-PCR and T-SPOT.TB (P＞0.05).The positive predictive values of T-SPOT.TB,fluorescent quantitative PCR,TB-Ab and PPD assays were 95.2％ (119/1250),92.3％ (96/104),81.2％ (82/101) and 78.9％ (75/95) respectively while the negative predictive values of those were 82.8％ (53/64),60％ (51/85),45.5％ (40/88) and 41.5％ (39/94),respectively.The false-positive rates (misdiagnosis rate) of four assays were 10.2％ (6/59),13.6％ (8/59),32.2％ (19/59) and 33.9％ (20/59) respectively and the false-negative rates (rates of missed diagnosis) of those were 8.5％ (11/130),26.2％ (34/130),36.9％ (48/130)and 42.3 ％ (55/130),respectively.The negative likelihood ratios of T SPOT.TB,fluorescent quantitative PCR,TB-Ab and PPD assays were 0.11,0.16,0.48 and 0.51 respectively,meanwhile the positive likelihood ratios of T-SPOT.TB,fluorescent quantitative PCR,TB-Ab andPPD assays were 9.0,5.4,2.0 and 1.7,respectively.What' s more,the diagnostic accordance rates of the four assays were 91.0％ (172 189),77.8％ (147 189).64.6％ (122/189) and 60.3％ (114/189),respectively.Conclusion T-SPOT.TB test is a more sensitive and specific method and of great significance to the early diagnosis of TB,which has more clinical value in different stages of tuberculosis diagnosis.

Objective To identify the rate of hepatitis B virus (HBV) reactivation and potential risk factors in hepatitis B surface antigen negative/hepatitis B core antibody positive patients with lung cancer receiving adjuvant chemotherapy without concomitant antiviral prophylaxis.Methods The records of 3280 patients with lung cancer who received adjuvant chemotherapy were retrospectively reviewed from January 2003 to December 2011.Among these patients,367 hepatitis B surface antigen negative/hepatitis B core antibody positive patients were analyzed for the HBV reactivation in this study.The HBV serology marker and biochemical tests of the 367 patients were performed.The data were analyzed by chi square test.Results Among 367 hepatitis B surface antigen negative/hepatitis B core antibody positive patients with lung cancer,14 patients suffered HBV reactivation.Univariate analysis showed that age≥70 years(x2 =13.003,P=0.019),abnormal liver computed tomography findings (x2 =11.225,P =0.026) and the amount of corticost eroids≥ 150 mg(x2 =7.008,P =0.033)were associated with HBV reactivation.However,gender and adjuvant chemotherapy regimens were not related with HBV reactivation.Conclusion HBV reactivation occurs in a proportion of hepatitis B surface antigen negative/hepatitis B core antibody positive patients with lung cancer during adjuvant chemotherapy.

Objective To study the feasibility of applying electronic cleaning to intestinal contents tagging by diatrizoate meglumine for single-source dual-energy CT colonography with sequential acquisitions and volume scanning.Methods Twenty-four volunteers had fine effect of intestinal contents tagging by diatrizoate meglumine,good colorectal distension effect,fine image quality of dual-energy fusion colorectal images,and with informed consents were enrolled in this study.The single-source dual-energy CT colonography with sequential acquisitions and volume scanning was performed with an Acquilion ONE 320 row CT scanner,tube voltage 135 kVp/80 kVp.The intestinal contents conducted the dual-energy electronic cleaned based on decomposition of intestinal contents tagging by diatrizoate meglumine,soft tissue and air.The intestinal contents in one segment of intestinal lumen being 100％ electronically cleaned served as the basic standard,the electronic cleaning effects were divided into the 5 grades:excellent,good,moderate,fair and poor;and grade 1-3 were effective fecal electronic cleaning.Results The grade 1,2,3,4,5 of electronic cleaning effect for solid as the main intestinal contents were 22.2％,53.3％,17.8％,6.7％ and 0％ respectively;and which of electronic cleaning effect for liquid as the main intestinal contents were 47.5％,47.5％,5.0％,0％ and 0％ respectively.The together total effective electronic cleaning of intestinal contents was 97.9％ and the electronic cleaning effect was good.Conclusion Electronic cleaning could be used in the intestinal contents tagging by diatrizoate meglumine for single-source dual-energy CT colonography with sequential acquisitions and volume scanning.

Objective:To analyze the clinical characteristics, therapeutic options and risk factors of mortality in patients with carbapenem-resistant Acinetobacter baumannii (CRAB) bloodstream infection, and to provide evidence for clinical treatment option and prognosis evaluation of CRAB bloodstream infections. Methods:A retrospective study was carried out in 224 patients with confirmed diagnosis of CRAB bloodstream infection during the period from January 2012 to December 2017 in West China Hospital, Sichuan University. The patients were divided into the death group and the survival group according to the survival status 28 days after collecting blood samples. The clinical features and therapeutic options of antibacterial drugs were reviewed. Student′s t test was used for analyzing normally distributed data and Mann-Whitney U test for non-normal data.Chi-square test was used for categorical variables. Univariate and multivariate logistic analysis were used to analyze the risk factors of mortality associated with CRAB bloodstream infection. Results:Among 224 cases of CRAB bloodstream infection, 121 cases died (54.02%). These patients were mainly in intensive care unit (ICU) and hematology department. The common underlying diseases were severe acute pancreatitis and severe cardiovascular events. The interleukin (IL)-6 level (median (interquartile range)) in the death group (480.40 ng/L (1 432.95 ng/L)) was higher than that of the survival group (107.05 ng/L (263.08 ng/L)), the difference was statistically significant ( Z=4.526, P<0.01). The procalcitionin (PCT) levels in the death group and the survival group were 3.81 μg/L (17.26 μg/L) and 2.12 μg/L (12.74 μg/L), respectively, with no difference between the two groups ( P>0.05). The death rate of empirical treatment with a single or more non-active antimicrobial agents was 57.14% (64/112), that of monotherapy with active agent was 45.68% (37/81), and that of combination therapy with at least one active drug was 64.52% (20/31). The differences had no statistical significance ( P=0.130). The logistic regression analysis showed that the risk factors of mortality associated with CRAB bloodstream infection were renal dysfunction (odds ratio ( OR)=2.181, P=0.024) and multiple organ dysfunction syndrome (MODS; OR=20.376, P<0.01). Conclusions:The fatality rate of patients with CRAB bloodstream infection is high. These patients with renal dysfunction or MODS have poor prognosis. In addition to early effective antibacterial therapy, individual comprehensive treatment should be implemented in order to improve the curative effect.