Objective To investigate the binding ability of the terminase large subunit of Pseudomonas aeruginosa bacteriophage PaP3 to the cos site. Methods The gene tls was amplified from the genome of bacteriophage PaP3 by PCR and subcloned into pMD18-T vector. Then the gene tls cut down from the vector was inserted into the plasmid pQE31 which could give a 6-His tag at the N-terminal of the expressed protein. The recombinant vector pQE-tls was transformed to E.coli. JM109, after induction with IPTG, the expressed bacteria were resuspended and sonicated, then after centrifugation, the inclusion body was obtained. The inclusion body was dissolved with lysis buffer, then the tagged protein was purified by Ni-NTA affinity chromatography and renatured by dialysis. Finally the DNA-binding ability of the fusion protein rTLS was determined by EMSA. Results The expression plasmid pQE31-tls was successfully constructed, and the target protein yield was up to 30% of the total bacterial proteins. After purification and renaturation, the fusion protein rTLS can partially bind the cos fragment. Conclusion The fusion protein rTLS was successfully expressed, purified and renatured. The rTLS has the specific DNA-binding activity. The present work lays the foundation for the further research of the gene tls.

OBJECTIVE: To determine the influence of different registration scopes and methods on kilo-voltage cone beam CT (kVCBCT) image and CT image and to estimate the appropriate clinical target volume (CTV)-to-planned target volume (PTV) margin for central-type non-small cell lung cancer in image-guided radiation therapy (IGRT). METHODS: Twenty-six patients with central-type non-small cell lung cancer (NSCLC) who received IGRT were enrolled in this study and totally 104 flames of kVCBCT scanning acquired before radiotherapy were analyzed. First, registrations were performed by gray plus manual match and were compared among different registration scopes of tumor and tumor plus vertebra. Then, the results of registration as well as time cost using tumor plus vertebra as registration scope were compared among different registration methods of gray plus manual match and bone plus manual match. At last, 200 registrations using tumor plus vertebra as the registration scope performed by bone plus manual match were recorded and the CTV-to-PTV margin was calculated. RESULTS: There was no significant difference in X, Y and Z translational and rotational movements between the registration scopes of tumor and tumor plus vertebra when gray plus manual match was used as the registration method (P>0.05). The registration results of gray plus manual match and bone plus manual match were equal when tumor plus vertebra was used as the registration scope (P>0.05), but the time cost of bone plus manual match [(1.9±0.3) min] was shorter than that of gray plus manual match [(3.1±0.2) min]. The CTV-to-PTV margins were 5.3, 4.9 and 5.7 mm in X, Y, and Z directions respectively. CONCLUSION For central-type NSCLC, tumor and vertebra can be used as registration scope and the bone plus manual match is suggested in IGRT. To avoid errors, we suggest a CTV-to-PTV margin of 6 mm.
Aged ; Carcinoma, Non-Small-Cell Lung ; radiotherapy ; Cone-Beam Computed Tomography ; Female ; Humans ; Lung Neoplasms ; radiotherapy ; Male ; Middle Aged ; Patient Positioning ; Radiotherapy Planning, Computer-Assisted ; methods ; Radiotherapy, Image-Guided ; methods

Cases of 2019-nCoV nucleic acid and antibody (IgM and IgG total antibody) after discharge from a hospital in Chongqing were continuously monitored. It was found that 5 cases of "re-positive" phenomenon, 5 cases of antibody were positive, and there was a trend of increasing with time. "Re-Positive" may be related to the following three factors. Children with asymptomatic infection had a long time of fecal detoxification.There were two consecutive nucleic acid tests "false negative" caused by various reasons.The virus clearance in patients was not complete, and the discharge standard was not conservative enough. The analysis of the causes of "Re-Positive" patients and the discussion of its infection will help us reveal more characteristics of this virus, and to provide a new basis for the discharge standard in the constantly updated diagnosis and treatment programme.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is widely used in the field of livestock breeding. However, its low efficiency, untargeted cutting and low safety have greatly hampered its use for introducing single base mutations in livestock breeding. Single base editing, as a new gene editing tool, can directly replace bases without introducing double strand breaks. Single base editing shows high efficiency and strong specificity, and provides a simpler and more effective method for precise gene modification in livestock breeding. This paper introduces the principle and development of single base editing technology and its application in livestock breeding.
Animals ; Gene Editing ; CRISPR-Cas Systems/genetics* ; Livestock/genetics* ; Mutation ; Technology

【Objective】 To evaluate the safety and efficacy of long-term indwelling of Allium ureteral stent in the treatment of ureteral stricture. 【Methods】 The clinical data of patients who underwent endoscopic Allium ureteral stent implantation for ureteral stricture in our hospital during Aug.2020 and Dec.2022 were retrospectively analyzed, and the surgical conditions and adverse events were recorded. The data of serum creatinine, blood urea nitrogen, glomerular filtration rate (GFR) and renal pelvis width under ultrasound were compared before surgery and 1, 3, 6 and 12 months after surgery. 【Results】 A total of 52 patients with ureteral stricture of 1.1 (0.7, 2.0)cm were included. All operations were successful. The operation time was 82.5 (70, 114)min, intraoperative blood loss 20 (10, 20)mL, and postoperative hospitalization stay 1 (1, 2) day. During the follow-up of (13.2±7.8) months, 14 patients had stent displacement, 5 had stone obstruction of stent tubes, 7 had occasional hematuria after movement, 9 had intermittent lumbar and abdominal pain, and 1 had recurrent urinary tract infection. The serum creatinine, blood urea nitrogen and renal pelvis width of 1 month, 3, 6 and 12 months after surgery were significantly decreased, while GFR was significantly increased. 【Conclusion】 Long-term indwelling of Allium ureteral stent is effective in the treatment of ureteral stricture, but the high incidence of stent displacement should arouse attention.

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.
Animals ; Sheep/genetics* ; Gene Editing ; Tibet ; Mutation ; Phenotype ; Mutagenesis, Site-Directed