Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

Objective To investigate the relationship between cyclin A2 (CCNA2) and the prognosis of colon cancer, and its possible mechanism from the perspective of immune infiltration. Methods We downloaded the transcriptome data of colon cancer patients from The Cancer Genome Atlas database. Clinicopathological feature analysis and survival analysis were performed based on the expression levels of CCNA2. A total of 75 specimens of colon cancer and normal tissues were collected, and the expression level of CCNA2 was analyzed using immunohistochemical methods. Multivariate analysis was conducted to explore its relationship with clinicopathological features. Gene Set Enrichment Analysis (GSEA) was used to assess the potential molecular functions of CCNA2 in colon cancer. CIBERSORT algorithm was applied to calculate the correlation between CCNA2 and immune-cell infiltration in colon cancer. Results Database and immunohistochemical analyses indicated that CCNA2 was expressed at a significantly higher level in colon cancer tissues than normal tissues (P<0.001). The overall survival, disease-specific survival, and progression-free interval were all longer in the group with high CCNA2 expression than the group with low expression (all P<0.05). In tumor tissues, the expression level of CCNA2 decreased with increased pathological and TNM stages (P<0.05). The expression level of CCNA2 in normal tissues was consistently lower than that in colon cancer tissues across all clinical stages (all P<0.001). GSEA suggested that Wnt/β-catenin, KRAS, and other signaling pathways were enriched when CCNA2 was lowly expressed. CIBERSORT analysis revealed an increase in the infiltration of immune cells such as regulatory T cells and macrophages M0 when CCNA2 expression was low. Conclusion CCNA2 is highly expressed in colon cancer and closely associated with grade of pathology and TNM stage. It may recruit regulatory T cells through the KRAS and Wnt/β-catenin pathways, thereby reducing immune-cell infiltration and promoting colon cancer progression, leading to poor prognosis.

Purpose To explore the clinical value of screening for partial agenesis of the corpus callosum(PACC)via measuring the distance from the end of the fetal corpus callosum to the occipital bone.Materials and Methods A Prospective study were performed from October 2017 to April 2023 in Ji’an Maternal and Child Health Care Hospital and Jiangxi Maternal and Child Health Hospital.A total of 33 PACC fetuses(abnormal group)and 396 normal fetuses(normal group)were selected as the research subjects.The distance(Z value)from the terminal posterior edge of the corpus callosum to the occipital bone was measured,Z value was calculated and compared between groups.The truncation value and related diagnostic efficiency indexes were calculated by receiver operator characteristic curve analysis,and the positive rate of Z value of abnormal group was compared with that of indirect signs.Results The distance from the terminal posterior edge of the corpus callosum to the occipital bone was positively correlated with the gestational age(r=0.913,P<0.001).The best regression equation was that the distance from the terminal posterior edge of the corpus callosum to the occipital bone was 3.879+1.115×gestational age,and the standard deviation was 1.670.The results of mean comparison showed that the Z value of the abnormal group was significantly higher than that of the normal group(t=11.223 9,P<0.001).When Z value 2.199 7 was used as the cut-off value for the diagnosis of PACC,the area under the curve was 0.9981,the Yoden index was 0.959 6,and the sensitivity,specificity,positive and negative predictive values were 96.97%,98.99%,88.89%and 99.75%,respectively.The positive rate of Z value in abnormal group was significantly higher than that of indirect signs(96.97%vs.63.64%,χ2=7.692 3,P<0.01).Conclusion The distance from the terminal posterior edge of corpus callosum to the occipital bone of PACC fetus is larger than that of normal fetus.The increase of Z value indicates that the end of corpus callosum moves forward,which can be used as one of the basis for screening PACC,and it has high clinical value when Z value 2.199 7 is used as the cut-off value for PACC screening.

Purpose To explore the clinical value of screening for partial agenesis of the corpus callosum(PACC)via measuring the distance from the end of the fetal corpus callosum to the occipital bone.Materials and Methods A Prospective study were performed from October 2017 to April 2023 in Ji’an Maternal and Child Health Care Hospital and Jiangxi Maternal and Child Health Hospital.A total of 33 PACC fetuses(abnormal group)and 396 normal fetuses(normal group)were selected as the research subjects.The distance(Z value)from the terminal posterior edge of the corpus callosum to the occipital bone was measured,Z value was calculated and compared between groups.The truncation value and related diagnostic efficiency indexes were calculated by receiver operator characteristic curve analysis,and the positive rate of Z value of abnormal group was compared with that of indirect signs.Results The distance from the terminal posterior edge of the corpus callosum to the occipital bone was positively correlated with the gestational age(r=0.913,P<0.001).The best regression equation was that the distance from the terminal posterior edge of the corpus callosum to the occipital bone was 3.879+1.115×gestational age,and the standard deviation was 1.670.The results of mean comparison showed that the Z value of the abnormal group was significantly higher than that of the normal group(t=11.223 9,P<0.001).When Z value 2.199 7 was used as the cut-off value for the diagnosis of PACC,the area under the curve was 0.9981,the Yoden index was 0.959 6,and the sensitivity,specificity,positive and negative predictive values were 96.97%,98.99%,88.89%and 99.75%,respectively.The positive rate of Z value in abnormal group was significantly higher than that of indirect signs(96.97%vs.63.64%,χ2=7.692 3,P<0.01).Conclusion The distance from the terminal posterior edge of corpus callosum to the occipital bone of PACC fetus is larger than that of normal fetus.The increase of Z value indicates that the end of corpus callosum moves forward,which can be used as one of the basis for screening PACC,and it has high clinical value when Z value 2.199 7 is used as the cut-off value for PACC screening.

OBJECTIVE:Knee adduction moment and knee adduction angular impulse enlargement is the main biomechanical risk factor of knee osteoarthritis.According to the survey,a change in the foot progression angle could effectively change the motion mode of patients with knee osteoarthritis.However,the impact of toe-in and toe-out on knee adduction moment and knee adduction angular impulse in young and elderly patients did not reach a consensus.Therefore,this study comprehensively discussed the effect of foot progression angle on knee adduction moment and knee adduction angular impulse in different populations through meta-analysis and provided a reference for the treatment of knee osteoarthritis. METHODS:By June 2022,searches were conducted on Web of Science,EBSCO,PubMed and CNKI databases using"foot progression angle,knee adduction moment,knee adduction angular impulse,gait"as Chinese and English search terms.Self-controlled randomized controlled studies analyzing the effects of toe-in and toe-out on knee adduction moment bimodality and knee adduction angular impulse were included.The cochrane bias risk assessment tool was utilized to make a quality evaluation of the literature.Stata 15.1 software was used for subgroup analysis to determine the effect of foot progression angle on knee adduction moment and knee adduction angular impulse.Meta-regression analysis was used to further determine characteristics of outcome indicators(knee adduction moment,knee adduction angular impulse)changing with foot progression angle. RESULTS:(1)A total of 15 self-control trials and 2 randomized controlled trials(455 subjects)were included in the meta-analysis.All of the included articles were of medium to high quality.(2)The meta-analysis results showed that the toe-in gait could reduce the first peak of knee adduction moment(SMD=-0.380,95%CI:-0.710 to-0.060,P=0.022)and knee adduction angular impulse(SMD=-1.470,95%CI:-2.160 to-0.770,P<0.001)in young patients.The toe-out gait reduced the second peak of knee adduction moment(SMD=-0.720,95%CI:-1.010 to-1.440,P<0.001)in young patients.In addition,toe-in gait could reduce the first peak of knee adduction moment in elder patients(SMD=-0.550,95%CI:-0.800 to-0.300,P<0.001),but increase the second peak knee adduction moment of elderly(SMD=0.280,95%CI:-0.010 to 0.560,P=0.047).The toe-out gait could decrease the second peak knee adduction moment in this population(SMD=-0.510,95%CI:-0.830 to-0.190,P=0.002).(3)Meta-regression showed that the greater the toe-out in elderly patients,the lower the second peak knee adduction moment. CONCLUSION:(1)Toe-in reduced the first peak knee adduction moment and knee adduction angular impulse in young knee osteoarthritis patients aged 18 to 34 years.Since knee adduction moment and knee adduction angular impulse were associated with medial knee loading and knee osteoarthritis incidence,toe-in gait intervention may be a suitable rehabilitation strategy for young patients.(2)Toe-in increased the second peak of knee adduction moment in older knee osteoarthritis patients over 60 years of age,which may exacerbate knee osteoarthritis in this population.However,the second peak of knee adduction moment during walking in this population decreases as the toe-out increases,contributing to a reduction in medial knee loading,suggesting that older patients may consider using toe-out gait during walking.

Objective To investigate the effect of trunk control on the biomechanical characteristics of lower limb movements during Asian squats(AS)and Western squats(WS)in young adults to provide empirical support for the application and promotion of deep squat training.Methods Twenty-four healthy young male collegiate students performed AS and WS with and without bar control,and their lower limb kinematic and kinetic characteristics were collected using an infrared light-point motion capture system and a three-dimensional(3D)dynamometer.The 3D angles of the lower limbs were obtained using Cortex-642.6.2 software,based on the calculation of Euler angles,and the 3D moments were obtained by applying the inverse dynamics method.The effects of trunk control and deep squatting posture on the lower limb kinematic characteristics were examined using a two-factor analysis of variance with a 2×2 repeated design.Results There was no significant interaction between trunk control and the deep squatting posture for either kinematic or kinetic parameters(P>0.05).The WS group had a large knee flexion angle,peak patellofemoral contact force,and ratio of peak hip and knee extension moments,and small ankle dorsiflexion and hip flexion angles(P<0.05).The deep squat with a bar had a large ankle dorsiflexion angle,peak patellofemoral contact force,and hip flexion angle as well as a small knee flexion angle and ratio of peak hip and knee extension moments(P<0.05).Conclusions WS is helpful for training hip extension muscle groups,whereas AS is helpful for training knee extension muscle strength.The peak patellofemoral joint contact force of the WS is significantly greater than that of the AS;therefore,it is recommended that patients with patellofemoral joint pain use the AS.A squat with a bar can compensate for the body's balance;thus,people with limited ankle dorsiflexion range of motion or anterior tibial muscle weakness may consider trunk control training,such as a deep squat with a bar.This may help improve lower limb stability during squats.

Vigabatrin-associated brain abnormalities on magnetic resonance imaging (VABAM) is a relatively rare side effect of vigabatrin, most of which are asymptomatic. However, there will be extremely rare cases with hyperkinetic disorders, myoclonus, tremor, and acute encephalopathy under certain circumstances. VABAM is often underappreciated by physicians and its accurate incidence remains unclear. A female infant who was diagnosed with infantile spasms and required adrenocorticotropic hormone therapy accompanied by various antiseizure medicines was reported. Unfortunately, she became lethargic and her spasm deteriorated gradually after vigabatrin exposure. Her brain magnetic resonance imaging revealed abnormal signals bilaterally in the dorsal midbrain, thalamus, and rostral part of the pallidum. She had a seizure amelioration and became lively as a result of vigabatrin withdrawal. In the meanwhile, magnetic resonance imaging returned to normal. Attempts were made to discover the risk factors of VABAM and potential pathogenesis. Further understanding of the disease should contribute to decreasing misdiagnosis and making precise decisions.

Objective:To analyze the timeliness and influencing factors of antiretroviral treatment (ART) among HIV/AIDS patients from 2017 to 2019 in Shandong province.Methods:Data were collected from the Chinese HIV/AIDS Comprehensive Response Information Management System, HIV/AIDS cases enrolled in antiretroviral therapy, from January 1, 2017 to December 31, 2019, ≥15 years of age and from the mainland of China were the subjects of the study. The general demographic characteristics (gender, age, ethnicity, education level, marital status, etc), infection routes, sample sources, date of diagnosis, baseline CD4+ T cell level, and clinical stage and other information were collected from the database. Univariate analysis was conducted for the timeliness of ART of the subjects by Chi square test, and the influencing factors associated with timeliness of ART were analyzed by binary non-conditional Logistic regression.Results:Among the 7 425 cases of HIV/AIDS, 6 813(91.8%) were male, 5 236(70.5%) had homosexual transmission, the age at the ART initiation was(36.05±12.51)years. The proportion of timeliness of ART within 30 d increased from 55.9% (1145/2050) in 2017 to 65.3% (1821/2789) in 2019. The possibility of timeliness of ART among HIV/AIDS who were married or cohabiting, education of high school and above were higher, with OR values at 1.136, 1.299. The possibility of timeliness of ART among HIV/AIDS cases whose domicile place is outside Shandong province were lower, with OR values at 0.664. The possibility of timeliness of ART in group of HIV/AIDS aging≥55, 45~54 and 35~44 years old were higher than that in 15~24 years old, with OR values at 1.530, 1.505 and 1.394. The possibility of timeliness of ART among the heterosexuals were lower than that of homosexuals, with OR values at 0.803. The possibility of timeliness of ART is lower for cases in places of detention centers than cases from volunteer testing and counseling, with OR values at 0.204. The possibility of timeliness of ART among HIV/AIDS cases whose CD4+ T lymphocyte at >500 cells/μL were lower than that of the cases whose CD4+ T lymphocyte at≤500 cells/μl, with OR values at 0.545. Patients without symptoms at baseline were less likely to receive prompt treatment than those with clinical symptoms, with OR values at 0.727. Patients with baseline clinical staging of stage Ⅳ, period of HIV/AIDS treatment with timeliness was longer than the cases with baseline clinical stages ofⅠ, Ⅱ, Ⅲ, with OR values at 1.307. Conclusions:The proportion of timeliness of ART increased from 2017 to 2019. Marital status, educational level, age, sample source, infection routes, baseline clinical stage, and CD4+ T lymphocyte levels were associated with the timeliness of ART.

Objective:To understand the use of drug and its related factors among men who have sex with men, and to provide reference for the development of reasonable intervention measures.Methods:MSM was recruited from Jinan and Qingdao by means of on-site and internet recruiting from March to June in 2016. Anonymous questionnaires were conducted and HIV and syphilis serological tests were performed. The questionnaire included the general situation, sexual behavior, HIV related services and so on. Multi-factor unconditioned logistic regression model was used to explore related factors about rush poppers use.Results:The rush poppers use rate of 901 MSM was 30.1%（271/901）, the age was (29.3±8.1) years, the HIV infection rate was 4.6% (41/901) and the syphilis infection rate was 8.7% (78/901). Multivariate analysis showed that compared with those who were>25 years old, the OR (95% CI) of those who were ≤ 25 years old was 1.571 (1.110-2.224); compared with the number of anal sexual behavior was<2 times in the last week, the OR (95% CI) of those whose number of anal sexual behavior was ≥2 times was 2.991 (1.100-8.132); compared with those who had not received peer education services in the last year, the OR (95% CI) of those who received peer education services was 13.651 (7.239-25.742). Conclusion:Rush poppers are very popular in the MSM crowd, and those who aged less than 25 years old, who had anal sex more than twice in the past week, and who had received peer education services were more likely to use rush poppers. We should carry out targeted interventions according to the characteristics.