1.Cross-linking and immunoprecipitation assay and its development
Lingyue XING ; Yan ZHANG ; Jing HUANG ; Ping LI ; Yang WANG ; Minglei SHI ; Zhihu ZHAO
Military Medical Sciences 2013;(11):867-869
It′s reported that RNA-binding proteins ( RBP) play key roles in post-transcriptional regulation of eukaryotic genes.The aberrations of RBP are associated with a large number of human disorders , particularly autoimmune and neuro-logic diseases .The interaction between RNA and proteins has been widely explored since the development of the method known as RNA immunoprecipitation with differential display or microarray analysis (RIP-ChIP) around the year of 2000. Since then, diverse derivatives of the RIP-ChIP, such as ultraviolet crosslinking and immunoprecipitation ( CLIP), high-throughput sequencing of CLIP cDNA library (HITS-CLIP), photoactivatable -ribonucleoside-enhanced crosslinking and immunoprecipitation ( PAR-CLIP) , and individual nucleotide resolution CLIP ( iCLIP ) have been developed .All these methods have some advantages over the original RIP-ChIP and greatly facilitate the study of RBP-RNA interactions .Addi-tionally , aided by the next-generation sequencing , transcriptome-wide identification of RBP target sites has become possible and the RNA-binding site resolution of RBP has also improved to some degree .We introduced the basic principles and processes of the interactions between proteins and RNA , focusing on the advantages , disadvantages and prospect of the present genome-wide version of CLIP .
2.Non-fusion soluble expression of broad-spectrum antivirus protein in Escherichia coli by translational-coupling with SUMO
Lingyue XING ; Dejian XIE ; Bingyu YE ; Zhang ZHANG ; Ping LI ; Wenlong SHEN ; Minglei SHI ; Yan ZHANG ; Zhihu ZHAO
Military Medical Sciences 2015;(8):597-601
Objective To design and construct a new non-fusion soluble expression vector pTIG-mSUMO(small ubiq-uitin-related modifier) using the widely used solubility promoting protein SUMO and based on the translational coupling phenomenon in order to enable the non-fusion soluble expression of the broad-spectrum antiviral protein RA in Escherichia coli by pTIG-mSUMO.Methods The smt3 gene coding for SUMO protein was cloned from yeast genome DNA by PCR. After directed-site silent mutation to eliminate the EcoRⅠsite, the mutant mSUMO was inserted into pET-22b to obtain the translational coupling expression vector pTIG-mSUMO.The RA was subject to PCR amplification and cloned into the pTIG-mSUMO to obtain the expression plasmid pTIG-mSUMO/RA which was supposed to direct the soluble expression of RA by the translational coupling with mSUMO.Results A translational coupling expression vector pTIG-mSUMO which could di-rect/drive the SUMO and heterogonous protein non-fusion expression simultaneously was designed and constructed.The Western blotting result indicated that pTIG-mSUMO could direct the high-level expression of RA, around 40%of which was soluble.Conclusion A translational coupling expression vector pTIG-mSUMO is obtained.After coupling with SUMO, RA is highly expressed in E.coli and both the expression level and solubility are greatly improved.pTIG-mSUMO might contrib-ute to soluble expression of other proteins.
3.The Wear on a Novel Motion Mode Hinged Knee Prosthesis: A Finite Element Analysis
Lingyue KONG ; Jing ZHANG ; Dongmu TIAN ; Jingyu ZHANG ; Yongcheng HU
Journal of Medical Biomechanics 2023;38(1):E097-E103
Objective To perform finite element analysis on a novel motion mode hinged knee prosthesis, and investigate the method of wear simulation on hinged prosthesis and the influence of motion mode on wear of the prosthesis. Methods Based on the finite element model of contact stress on spherical axis prosthesis, the finite element model of wear was established according to Archard wear theory. The kinematics data during different motions were input as loading condition to simulate mechanical environment of the knee arthroplasty in physiological activities. The wear results of spherical axis prosthesis were studied. Results For tibial insert, the average and maximum contact stresses during upstairs and downstairs climbing were higher than those during walking, and the cumulative wear volume during upstairs climbing was larger than that during downstairs climbing and walking. The wear mainly occurred on lower surface of tibial insert during all 3 motions. For rotating bushing, there was only a short period of contact and wear during walking, and the cumulative wear was 0. 19 mm3. Conclusions The spherical axis motion of hinged knee prosthesis can improve the mechanical environment of knee, reduce the wear of rotating bushing, and prolong the prosthesis survival. The finite element simulation can predict the wear of hinged prosthesis effectively, and provide the theoretical basis for design and improvement of the prosthesis.
4.A nomogram predictive model of ileus after radical cystectomy
Yunzhao AN ; Heng ZHANG ; Ye TIAN ; Lingyue AN ; Guangheng LUO
Journal of Modern Urology 2023;28(10):882-887
【Objective】 To analyze the risk factors of ileus after radical cystectomy, and to construct a nomogram predictive model accordingly. 【Methods】 Clinical data of patients who underwent radical cystectomy during Jan.2018 and Dec.2021 were collected. The risk factors related to postoperative ileus were assessed with Logistic univariate and multivariate regression analysis. After that, the predictive model was constructed and its specificity and accuracy were verified. 【Results】 A total of 326 patients were included, 65 of whom developed ileus. Statistical analysis showed that gender, lymph node dissection, serum creatinine and albumin were correlated with postoperative ileus. The area under the receiver operating characteristic curve of the model was 0.769 (95%CI:0.724-0.802). Bootstrap correction curve showed that the model had good prediction accuracy. 【Conclusion】 Male, lymph node dissection, elevated postoperative serum creatinine and postoperative blood albumin decrease are predictors of ileus. The nomogram predictive model based on these predictors can predict the probability of ileus after radical cystectomy.
5.Effects of Danggui Sini Granules (当归四逆颗粒) on Cardiomyocyte Apoptosis and Vasoactive Factors in Rat Models of Coronary Heart Disease with Cold Congealing and Blood Stasis Syndrome
Qianchi GUO ; Lingyue ZHANG ; Kangyu WANG ; Jiyu GONG ; Wenyi GAO
Journal of Traditional Chinese Medicine 2024;65(13):1375-1382
ObjectiveTo explore the possible mechanism of Danggui Sini Granules (当归四逆颗粒) in treatment of coronary heart disease with cold congealing and blood stasis syndrome. MethodsFifty SD rats were randomly divided into a blank group, a model group, a Danshen Pill (丹参滴丸) group, and a low- and high-dosage Danggui Sini Granules group, with 10 rats in each group. Except for the blank group, the rat model of coronary heart disease with cold congealing and blood stasis syndrome was established by repeated cold stimulation at low temperature combined with intraperitoneal injection of posterior pituitary hormone in all other groups. In the 6th week of modelling, 0.073 g·kg-1·d-1 of compound Danshen Pill was given to the Danshen Pill group, 20.2 and 40.4 g·kg-1·d-1 of Danggui Sini Granules were given to the low- and high-dose Danggui Sini Granules groups, respectively, and 0.2 ml/10 g of sterile water was given to the blank group and the model group, all for 2 consecutive weeks. The general conditions of the rats were recorded, and the body mass was compared weekly. At the end of the intervention, electrocardiogram, blood rheological indexes, including whole blood low-cut viscosity, whole blood high-cut viscosity, erythrocyte aggregation index, and cardiac index were detected to evaluate the effect of the medication, and HE staining was used to observe the myocardial histopathological changes, TUNEL staining to detect the apoptotic situation of cardiomyocytes, and ELISA to detect the serum thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), free tetraiodothyronine (FT4) levels and thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (6-Keto-PGF1α), endothelin 1 (ET-1), and nitric oxide (NO) levels. ResultsCompared with the blank group at the same time point, the body mass of rats in the remaining groups decreased at all time points (P<0.05 or P<0.01); compared with the model group at the same time point, the body mass of rats in high-dose Danggui Sini Granules group and Danshen Pill group increased at the 6th and 7th weeks (P<0.05 or P<0.01). In the blank group, the S-T segment of the electrocardiogram was close to the isoelectric line, the myocardial structure was regular, the fibres were closely arranged, and the nuclei of the cells were intact and neatly aligned; in the model group, the S-T segment of the electrocardiogram significantly elevated, and the arrangement of the myocardial fibres was obviously disordered, with myocardial cells appearing to be swollen, necrotic, and infiltrated by inflammatory cells, and the apoptosis-positive cells of the cardiac muscle cells obviously increased; in each of the medication groups, the electrocardiogram had a lowered S-T segment, and myocardial fibres were aligned, myocyte structure and morphology were improved, inflammatory cells reduced, and the number of apoptosis-positive cells significantly reduced. Compared with the blank group, the cardiac index, whole blood high cut viscosity, whole blood low cut viscosity, erythrocyte aggregation index increased in the model group, and the serum levels of TSH, FT3, FT4, NO, 6-Keto-PGF1α significantly reduced, and the levels of ET-1 and TXB2 significantly increased (P<0.05 or P<0.01). Compared with the model group, the levels of cardiac index, whole blood high cut viscosity, whole blood low cut viscosity, erythrocyte aggregation index, ET-1 and TXB2 significantly decreased and the levels of NO and 6-Keto-PGF1α significantly increased in each medication group, and the serum levels of TSH, FT3, and FT4 elevated in the high-dose Danggui Sini Granules group and Danshen Pill group (P<0.05 or P<0.01). The serum TSH level of rats in the low-dose Danggui Sini Granules group was lower than that in Danshen Pill group and the high-dose Danggui Sini Granules group (P<0.01). ConclusionDanggui Sini Granules have the effect of alleviating myocardial injury in coronary heart disease with cold congealing and blood stasis syndrome, and its mechanism may be related to reducing myocardial cell apoptosis, improving energy metabolism, and regulating the level of vasoactive factors.