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Objective: To explore the evaluation effect of ultrasonography and Pirani score on tarsal deformity, treatment effect and pseudo-correction of congenital clubfoot in infants and young children, and the correlation between the two methods. Methods: This is a retrospective case series study. The clinical data of 26 children (40 feet) with congenital clubfoot who were evaluated by ultrasonography in the Third Affiliated Hospital of Zhengzhou University from January 2020 to January 2023 were retrospectively collected. There were 16 males and 10 females. The age at the first ultrasound examination was ( M (IQR)) 9.0 (18.0) days (range: 1 to 46 days). All patients were treated with Ponseti method by the same physician. The Pirani scores before and after treatment and at the last examination, and the talonavicular angle, calcaneocuboid angle and tibiocalcaneal angle measured by ultrasound were collected, and the treatment and follow-up were recorded. Paired sample t test, repeated measures analysis of variance or Kruskal-Wallis test were used for data comparison, and Spearman correlation analysis was used for correlation analysis. The receiver operating characteristic curve was used to calculate the efficacy of ultrasound in evaluating different Pirani scores. Results: The number of plaster fixation in 26 children was 4.0 (1.0) times (range: 2 to 8 times). The medial talonavicular angle and posterior tibiocalcaneal angle were significantly improved after treatment and at the last follow-up compared with those before treatment, and the differences were statistically significant (all P <0.01). There was no difference in lateral calcaneocuboid angle before and after treatment and at the last follow-up ( F =1.971, P >0.05). Pseudo-correction occurred in 2 cases (2 feet) during the treatment, with an incidence of 5%. Correlation analysis showed that there was a moderate positive correlation between talonavicular angle and Pirani midfoot score ( r =0.480, P <0.01). There was no correlation between calcaneocuboid angle and Pirani midfoot score ( r =0.114, P =0.105). There was a moderate negative correlation between tibial heel angle and Pirani hindfoot score ( r =-0.566, P <0.01). The cut-off point of Pirani midfoot score of 1.5 was 38.78°, the sensitivity was 0.90, the specificity was 0.56, and the area under the curve was 0.75. The cut-off value of angle was 27.51 °, the sensitivity was 0.16, the specificity was 0.92, and the area under the curve was 0.44.The cut-off points of Pirani midfoot score of 3.0 were 45.08°and 9.96°, the sensitivity was 0.94 and 0.91, the specificity was 0.37 and 0.42, and the area under the curve was 0.59 and 0.62, respectively. The cut-off values of Pirani hindfoot score of 2.0 and 3.0 were 167.46° and 160.15°, respectively. The sensitivity was 0.75 and 0.67, the specificity was 0.81 and 0.83, and the area under the curve was 0.78 and 0.71, respectively. Conclusion: Ultrasound can complement with Pirani score, visually and dynamically observe the morphology and position changes of talonavicular joint, calcaneocuboid joint and tibiotalocalcaneal joint, monitor the recovery and pseudo-correction of tarsal bones, and better evaluate the therapeutic effect.

Objective: To explore the therapeutic efficacy and factors influencing the sequential combination of nucleos(t)ide analogues (NAs) with pegylated interferon alpha (Peg-IFN-α) in the treatment of patients with chronic hepatitis B (CHB). Methods: 144 CHB cases with NAs treatment for more than 1 year, HBV DNA < 20 IU/ml, hepatitis B surface antigen (HBsAg) quantification < 3 000 IU/ml, treated with a sequential combination of Peg-IFN-α treatment for 48 to 96 weeks, and followed up were selected from the Fifth Medical Center of the PLA General Hospital between May 2018 and May 2020. Intention-to-treat analysis was used to measure the HBsAg clearance rate at 96 weeks. The Kaplan-Meier method was used to compute the cumulative HBsAg clearance rate at 96 weeks. Univariate and multivariate logistic regression were used to analyze the factors influencing HBsAg clearance at 48 weeks of sequential combination therapy. Univariate and multifactorial COX proportional hazard models were used to analyze the factors influencing HBsAg clearance following 96 weeks of prolonged PEG-IFN-α treatment. The receiver operating characteristic curve was used to assess the predictive value of factors influencing HBsAg clearance. A Mann-Whitney U test was used to compare the measurement data between groups. The count data was compared using the χ (2) test between groups. Results: 41 (28.47%) cases achieved HBsAg clearance at 48 weeks of sequential combination therapy. The HBsAg clearance rate at 96 weeks was 40.28% (58/144) by intention-to-treat analysis. The Kaplan-Meier method computed that the cumulative HBsAg clearance rate at 96 weeks was 68.90%. Multivariate logistic regression analysis showed that HBsAg quantification at baseline ( OR = 0.090, 95% CI : 0.034-0.240, P < 0.001) and a 24-week drop in HBsAg level ( OR = 7.788, 95% CI : 3.408-17.798, P < 0.001) were independent predictors of HBsAg clearance in CHB patients treated sequentially in combination with NAs and Peg-IFN-α for 48 weeks. Receiver operating characteristic curve analysis showed that the baseline HBsAg quantification [area under the receiver operating characteristic curve (AUC), 0.911, 95% CI : 0.852-0.952)] and 24-week drop in HBsAg level (AUC = 0.881, 95% CI : 0.814-0.930) had equally good predictive value for 48-week HBsAg clearance, but there was no statistically significant difference between the two ( Z = 0.638, P = 0.523). The value of the combination of baseline HBsAg quantification and 24-week drop in HBsAg level (AUC = 0.981, 95% CI : 0.941-0.997) was superior to that of single baseline HBsAg quantification ( Z = 3.017, P = 0.003) and 24-week drop in HBsAg level ( Z = 3.214, P = 0.001) in predicting HBsAg clearance rate at 48 weeks. Multivariate COX proportional hazards model analysis showed that HBsAg quantification at 48 weeks ( HR = 0.364, 95% CI : 0.176-0.752, P = 0.006) was an independent predictor of HBsAg clearance with a prolonged course to 96 weeks of Peg-IFN-α treatment. Conclusion: The HBsAg clearance rate can be accurately predicted with baseline HBsAg quantification combined with a 24-week drop in HBsAg level in patients with CHB who are treated with a sequential combination of NAs and Peg-IFN-α therapy for 48 weeks. Prolonging the course of Peg-IFN-α treatment can enhance the HBsAg clearance rate's capability. An independent predictor of HBsAg clearance is HBsAg quantification at 48 weeks of sequential combination therapy with a prolonged course of 96 weeks of Peg-IFN-α treatment.

Objective: To compare the differences in the prevalence of mild micro-hepatic encephalopathy (MHE) among patients with cirrhosis by using the psychometric hepatic encephalopathy score (PHES) and the Stroop smartphone application (Encephal App) test. Methods: This prospective, multi-center, real-world study was initiated by the National Clinical Medical Research Center for Infectious Diseases and the Portal Hypertension Alliance and registered with International ClinicalTrials.gov (NCT05140837). 354 cases of cirrhosis were enrolled in 19 hospitals across the country. PHES (including digital connection tests A and B, digital symbol tests, trajectory drawing tests, and serial management tests) and the Stroop test were conducted in all of them. PHES was differentiated using standard diagnostic criteria established by the two studies in China and South Korea. The Stroop test was evaluated based on the criteria of the research and development team. The impact of different diagnostic standards or methods on the incidence of MHE in patients with cirrhosis was analyzed. Data between groups were differentiated using the t -test, Mann-Whitney U test, and χ (2) test. A kappa test was used to compare the consistency between groups. Results: After PHES, the prevalence of MHE among 354 cases of cirrhosis was 78.53% and 15.25%, respectively, based on Chinese research standards and Korean research normal value standards. However, the prevalence of MHE was 56.78% based on the Stroop test, and the differences in pairwise comparisons among the three groups were statistically significant (kappa = -0.064, P < 0.001). Stratified analysis revealed that the MHE prevalence in three groups of patients with Child-Pugh classes A, B, and C was 74.14%, 83.33%, and 88.24%, respectively, according to the normal value standards of Chinese researchers, while the MHE prevalence rates in three groups of patients with Child-Pugh classes A, B, and C were 8.29%, 23.53%, and 38.24%, respectively, according to the normal value standards of Korean researchers. Furthermore, the prevalence rates of MHE in the three groups of patients with Child-Pugh grades A, B, and C were 52.68%, 58.82%, and 73.53%, respectively, according to the Stroop test standard. However, among the results of each diagnostic standard, the prevalence of MHE showed an increasing trend with an increasing Child-Pugh grade. Further comparison demonstrated that the scores obtained by the number connection test A and the number symbol test were consistent according to the normal value standards of the two studies in China and South Korea ( Z = -0.982, -1.702; P = 0.326, 0.089), while the other three sub-tests had significant differences ( P < 0.001). Conclusion: The prevalence rate of MHE in the cirrhotic population is high, but the prevalence of MHE obtained by using different diagnostic criteria or methods varies greatly. Therefore, in line with the current changes in demographics and disease spectrum, it is necessary to enroll a larger sample size of a healthy population as a control. Moreover, the establishment of more reliable diagnostic scoring criteria will serve as a basis for obtaining accurate MHE incidence and formulating diagnosis and treatment strategies in cirrhotic populations.

Objective: To explore the value of preoperative peripheral blood inflammatory biomarkers for predicting the prognosis of intrahepatic cholangiocarcinoma (ICC) after radical resection. Methods: A total of 124 patients who underwent radical resection for ICC in the First Affiliated Hospital of Xi'an Jiaotong University from January 2010 to December 2018 were retrospectively analyzed. Receiver operating characteristic (ROC) curve was conducted to determine the best cut-off values of neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), systemic immune inflammatory index (SII), and systemic inflammatory response index (SIRI). Univariate and multivariate analyses of prognostic factors were performed using Cox proportional hazards regression model. Based on the independent prognostic factors screened by multivariate Cox regression analysis, a nomogram model of overall survival prediction for ICC patients after radical resection was established. Results: Among the 124 patients, 87 patients died and 37 patients survived during the follow-up period. The median overall survival time of the whole patients was 21 months. ROC curve analysis showed that the areas under the curve (AUC) of NLR, PLR, LMR, SII and SIRI for predicting the overall survival of ICC patients after radical resection were 57.86%, 64.21%, 60.61%, 67.57% and 66.03%, respectively. Univariate Cox regression analysis showed that the inflammatory biomarkers of NLR, PLR, SII, and SIRI were associated with overall survival of ICC after radical resection ( HR =1.787, 95% CI: 1.165-2.741; HR =1.181, 95% CI: 1.224-2.892; HR =2.412, 95% CI: 1.565-3.717; HR =1.648, 95% CI: 1.081-2.513). Multivariate Cox regression analysis showed that the inflammatory biomarker of SII was an independent prognostic factor of ICC after radical resection ( HR =1.863, 95% CI: 1.161-2.989). According to the best cut-off value of SII to predict the overall survival of ICC patients after radical resection (709.86×10(9)/L), the patients were divided into low SII group (SII≤709.86×10(9)/L) and high SII group (SII>709.86×10(9)/L). In the high SII group, the proportions of NLR>3.31, PLR>3.31, SIRI>1.30×10(9)/L, carbohydrate antigen 19-9>39.0 U/ml, Child-Pugh liver function (grade B), hemi-hepatic/extended hepatectomy, combined perineural invasion, N1 stage and TNM stage (ⅢB) were higher than those in the low SII group ( P <0.05). Based on the independent prognostic factors screened by multivariate Cox regression analysis, a nomogram model of overall survival prediction for ICC after radical resection was established, the C-index values of the training set and testing set were 0.774 and 0.737, respectively. Conclusions: Preoperative peripheral blood inflammatory marker SII is an independent risk factor for the prognosis of intrahepatic cholangiocarcinoma patients after radical resection. The nomogram model of overall survival prediction established that included SII has a good predictive ability and can be used to evaluate the prognosis of intrahepatic cholangiocarcinoma patients after radical resection.

Objective: To explore the value of preoperative neutrophil-to-lymphocyte ratio (NLR) and γ-glutamyl transpeptidase-to-platelet ratio index (GPRI) for predicting the prognosis of patients with HBV-related intrahepatic cholangiocarcinoma (ICC) after radical resection. Methods: The data of 79 patients who underwent radical resection for HBV-related ICC in the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Xi'an Jiaotong University from January 2010 to December 2018 were retrospectively analyzed. Among them, 48(60.8%) patients were male and 31 (39.2%) patients were female, (56.9±11.2) years old. X-Tile statistical software was used to determine the best cut-off values of NLR and GPRI. The χ 2 test was conducted to analyze the relationship between preoperative NLR and GPRI and the clinicopathological characteristics, and the Cox proportional hazard regression model was conducted for multivariate analysis. A nomogram prognostic prediction model was established based on independent risk factors screened by Cox regression model. Results: The best cut-off values of NLR and GPRI were 3.13 and 1.31 determined by the X-Tile software, respectively. With the best cut-off value, 79 patients were divided into NLR≤3.13 group (45 cases) and NLR>3.13 group (34 cases). GPRI≤1.31 group (54 cases) and GPRI>1.31 group (25 cases). Compared with the preoperative NLR ≤3.13 group, the proportion of patients with liver cirrhosis and atrophy, poor pathological differentiation, tumor diameter>5 cm and late TNM stage was significantly increased in the NLR>3.13 group (all P <0.05); Compared with preoperative GPRI ≤1.31 group, the proportion of patients with liver cirrhosis and atrophy was significantly increased in the GPRI>1.31 group ( P =0.025). The postoperative overall survival time of the included patients was 2 to 126 months, with the median survival time being 18 months, and the 1, 3-year overall survival rates were 63.3%, 32.8%, respectively. Multivariate analysis showed that NLR, GPRI, liver cirrhosis and atrophy, and lymphatic metastasis were independent risk factors affecting the overall survival of patients with HBV-related ICC after radical resection ( P <0.05). A nomogram prediction model was established based on independent risk factors, with the C-index of 0.750, and the prediction effect was close to the actual survival outcome of the patients. Conclusion: Preoperative peripheral blood NLR and GPRI can be used to predict the prognosis of patients with HBV-related ICC after radical resection.

Objective: To observe the dynamic changes of serum RANTES during the treatment with nucleos(t)ide analogues combined with pegylated interferon alpha (peginterferon-α), and further analyze the predictive effect of RANTES on HBsAg clearance in patients with chronic hepatitis B. Methods: 98 cases of chronic hepatitis B with quantitative HBsAg < 3 000 IU/ml and HBV DNA < 20 IU/ml after≥1 year NAs treatment were enrolled. Among them, 26 cases continued to receive NAs monotherapy, 72 cases received NAs combined with pegylated interferon alpha therapy. The changes in RANTES during treatment were observed. The receiver operating characteristic curve was used to analyze the early changes of RANTES to predict the HBsAg clearance during 48 weeks. Results: During 48 weeks, 15 cases (20.83%) had achieved HBsAg clearance in combination group, while no patient had achieved HBsAg clearance in NAs group. The overall serum RANTES level had decreased from baseline in NAs and combination group. At week 48, in the combination group, the serum RANTES level was decreased more significantly in patients with HBsAg clearance than patients without. Further analysis showed that, in combination group, HBsAg clearance rate of patients with serum RANTES decreased at week 12 and 24 was higher than patients with elevated (29.17% vs. 4.17%, P = 0.014; 28.00% vs. 4.55%, P = 0.052), and quantitative HBsAg reduction was larger significantly [(1.49 ± 1.26) log(10)IU/ml vs. (0.73 ± 0.81) log(10)IU/ml, P = 0.017; (1.54 ± 1.27) log(10)IU/ml vs. (0.57 ± 0.56) log(10)IU/ml, P = 0.004]. Receiver operating characteristic curve analysis showed that the baseline quantitative HBsAg and the reduction in quantitative HBsAg and serum RANTES during the early period were predictors of HBsAg clearance after 48-week combination therapy. Furthermore, the combination of baseline quantitative HBsAg and 12 - or 24-week reduction of serum RANTES were better predictors of HBsAg clearance than that of baseline quantitative HBsAg combined with HBsAg decrease at week 12 or 24. The area under the receiver operating characteristic curve of the former was 0.925 and 0.939, while that of the latter was 0.909 and 0.929, respectively. Conclusion: Early reduction of serum RANTES at week 12 and 24 can predict HBsAg loss in CHB patients receiving addition of peginterferon-α to ongoing NAs Therapy, so serum RANTES could be one of the key immunological markers for predicting HBsAg clearance.

Objective: To screen potential pan-cancer biomarkers based on The Cancer Genome Atlas (TCGA) database, and to provide help for the diagnosis and prognosis assessment of a variety of cancers., Methods: "GDC Data Transfer Tool" and "GDCRNATools" packages were used to obtain TCGA database. After data sorting, a total of 13 cancers were selected for further analysis. False disco-very rate (FDR) < 0.05 and fold change (FC) >1.5 were used as the differential expression criteria to screen genes and miRNAs that were up- or down-regulated in all the 13 cancers. In the receiver operating characteristic curve (ROC curve), the area under the curve (AUC), the best cut-off value and the corresponding sensitivity and specificity were used to reflect diagnostic significance. The Kaplan-Meier method was used to calculate the survival probability and then the log-rank test was performed. Hazard ratio ( HR ) was calculated to reflect prognostic evaluation significance. DAVID tool were used to perform GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis for differentially expressed genes. STRING and TargetScan tools were used to analyze the regulatory network of differentially expressed genes and miRNAs., Results: A total of 48 genes and 2 miRNAs were differentially expressed in all the 13 cancers. Among them, 25 genes were up-regulated, 23 genes and 2 miRNAs were down-regulated. Most differentially expressed genes and miRNAs had good ability to distinguish between the cases and controls, with AUC, sensitivity and specificity up to 0.8-0.9. Survival analysis results show that differentially expressed genes and miRNAs were significantly associated with patient survival in a variety of cancers. Most up-regulated genes were risk factors for patient survival ( HR >1), while most down-regulated genes were protective factors for patient survival (0 < HR < 1). The enrichment analysis of GO and KEGG showed that the differentially expressed genes were mostly enriched in biological events related to cell proliferation. In the regulatory network analysis, a total of 13 differentially expressed genes and 2 differentially expressed miRNAs had regulatory and interaction relationships., Conclusion: The 48 genes and 2 miRNAs that were differentially expressed in 13 cancers may serve as potential pan-cancer biomarkers, providing help for the diagnosis and prognosis evaluation of a variety of cancers, and providing clues for the development of broad-spectrum tumor therapeutic targets.

Ecological reclamation is the major method for the revegetation of acid mine wasteland worldwide. In this study the pH, fertility characteristics, and heavy metal content of soils from Laili Mountain mine wasteland were analyzed. The research also studied the morphological characteristics and the heavy metal in Juncus ochraceus Buchen as well as its resistance to acid, adaptability to soil fertility in abandoned land, and tolerance to heavy metal pollution (e.g., Zn and Cu) to determine its remediation potential as a pioneer plant for acid mine wasteland. Results showed that the pH of soils in the study area were acidic, ranging from 3.46 to 4.01.The contents of organic matter, total potassium, total phosphorus, and available phosphorus was poor, being 10.28-25.75 g·kg -1 , 8.84-9.32 g·kg -1 , 0.56-0.63 g·kg -1 , and 1.82-5.72 mg·kg -1 respectively.The contents of Zn, Cu, and Fe in the soil ranged between 54.93 and 114.49 mg·kg -1 , 92.53 and 127.59 mg·kg -1 , and 47133.60 and 112259.63 mg·kg -1 , whereby the Cu content was 1.85-2.55 times higher than the risk screening value. The height of Juncus ochraceus Buchen in the study area ranged from 43.77 cm to 55.42 cm, which was shorter than the average plant height of the control group (51.38-57.66 cm); however, this was not a significant difference, thus indicating that this plant was resistant to acidic soil and heavy metal pollution.Further analysis showed that Juncus ochraceus Buchen had accumulating capacity for both Cu and Zn, and transferring capacity for Zn as well. Thus, it had potential in heavy metal accumulation and absorption.Plant height was significantly related to the available phosphorus content in the rhizosphere soil. In the future, soil fertility could be improved by supplementing fertilizers containing available phosphorus when Juncus ochraceus Buchen is planted on the land of abandoned mines as a pioneer plant. Comprehensive analysis revealed that Juncus ochraceus Buchen had great potential as a pioneer plant to remediate acid mine wasteland.

Objective: This study aimed to compare the effects of water storage treatment and thermal cycling on the shear bond strength (SBS) of three self-adhesive dual-cure resin cements., Methods: Six cubic zirconia specimens with side length of 2 cm were obtained by cutting and sintering. Three self-adhering dual-cure resin cements (i.e., Clearfil SAC, RelyX U200, and Multilink Speed) were selected. According to their bonding modes, they were divided into three groups: direct bonding group (direct coating with resin cement), adhesive group (applying universal adhesives and then coating with resin cement), and primer group (applying Z-Prime Plus and then coating with resin cement). According to experimental conditions, each group was divided into two subgroups: subgroup a (water storage at 37 ℃ for 24 h) and subgroup b (thermalcycling for 5 000 times). SBS data were analyzed by one-way ANOVA by using SPSS 19.0 software (P<0.05). The fractured zirconia surface was observed under a stereomicroscope., Results: After water storage for 24 h, the SBS of the adhesive group and the primer group of the three resin cements was higher than that of the direct adhesive group (P<0.05), but the difference in SBS between the adhesive group and the primer group was not significant (P>0.05). After thermalcycling, the SBS of the three types of resin cements decreased (P<0.05); the SBS of the adhesive group was higher than that of the direct adhesive group and the primer group (P<0.05). Fracture mode analysis revealed that the type Ⅲ fracture mode evidently increased after the thermalcycling treatment compared with the water storage treatment., Conclusions: The universal adhesives and the primer can improve the SBS of self-adhesive dual-cure resin cement in water storage at 37 ℃ for 24 h. The universal adhesives had a better bonding durability than the zirconia primer.

Objective: To evaluate the safety and efficacy of allogeneic natural killer (NK) cells in the treatment of primary hepatocellular carcinoma (HCC), and to elucidate the mechanism of NK cells therapy., Methods: Twenty-one patients with primary HCC treated with allogeneic NK cells at the Fifth Medical Center of the PLA General Hospital were followed up for 1 year. Peripheral blood mononuclear cells (PBMCs) were isolated from patient-related donors and cultured in vitro for 15 days and infused to the patients in two consecutive days. Clinical data and laboratory data were collected and analyzed, including survival, clinical features, imaging changes, hematology, immunology, and biochemical indicators to evaluate the safety and efficacy of allogeneic NK cell therapy. The changes of peripheral blood lymphocyte subsets after treatment were also analyzed to explore the possible anti-tumor mechanisms., Results: (1) Of the 21 patients with primary HCC, 11 patients were treated once, 5 patients were treated twice, and 5 patients were treated 3 times. After allogeneic NK cells infusion, 10 patients had fever, 1 patient had slight hepatalgia and 1 patient had slight headache, no other adverse events occurred including acute and chronic graft-versus-host disease (GVHD). They resolved spontaneously within 8 hours without other treatment. (2) The total disease control rate was 76.2% during one-year follow-up. Among them, the patients with Barcelona clinic liver cancer (BCLC) stage A had a disease control rate of 100%, stable disease (SD) in 10 cases; BCLC stage B patients had a disease control rate of 60%, partial response (PR) in 1 case, and SD 2 in cases; BCLC stage C patients had a disease control rate of 50%, complete response (CR) in 1 case, and 2 cases of PR. (3) The frequencies of NK cells and CD8+ T cells in peripheral blood were significantly lower than that before at 24 hours after treatment, and the frequencies of CD4+ T cells and CD4/CD8 were significantly higher than the baseline., Conclusion: Allogeneic NK cells have good safety and efficacy in the treatment of primary HCC. The anti-tumor effect of the allogeneic NK cells may play an important role in the activation of the patient's natural immune system and delay disease progression, suggesting that allogeneic NK cells combined with sorafenib may be a very effective treatment for advanced HCC, and further large-sample multicenter randomized controlled clinical trials are needed to validate this result.

Objective: To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells. Methods: SK-N-SH cells were treated with MnCl(2) at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl(2) at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit. Results: Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl(2) treatment groups ( P <0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl(2) treatment groups were increased ( P <0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl(2) treatment groups had significant increase in the the expression of LAMP1 ( P <0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl(2), while it was decreased at the does of 1.0 mmol/L ( P <0.01) . Otherwise, it wasn't observed significant difference of the activity of CTSD between different MnCl(2) treatment groups. Conclusion: MnCl(2) could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.

Objective: To study the situation of the mutations in the A((8)) and A((9)) loci of exon 8 of retinoblastoma protein-interacting zinc finger gene (RIZ) of keloid patients. Methods: From January 2003 to December 2007, 19 outpatient and hospitalized keloid patients of our hospital were conforming to the inclusion criteria. Both 3-5 g keloid tissue and 3 mL peripheral venous blood were collected from each patient to extract their genomic DNA, and the concentration was determined. The A((8)) and A((9)) loci fragments of exon 8 of RIZ were amplified by polymerase chain reaction (PCR). The length of product was detected by agarose gel electrophoresis, and DNA sequencing was performed after column chromatography. The mutations of A((8)) and A((9)) loci fragments were searched, and the types of mutations were determined. The consistency of genetic mutations of the keloid tissue and peripheral venous blood were compared. Data were processed with McNemar test. Results: The DNA concentrations of the extracted keloid tissue and peripheral venous blood were 0.54 and 0.37 μg/μL, respectively, which were above 0.10 μg/μL. The lengths of PCR products of A((8)) locus fragment DNA of exon 8 of RIZ from keloid tissue and peripheral venous blood were 235 and 238 bp, respectively, and those of A((9)) locus were 242 and 244 bp, respectively, which were basically the same as the designed DNA fragments. PCR products purity of A((8)) locus fragment DNA of exon 8 of RIZ from keloid tissue and peripheral venous blood were 1.81 and 1.75, respectively, and those of A((9)) locus were 1.82 and 1.78, respectively, which were above 1.50. Mutations in the A((8)) locus of exon 8 of RIZ were observed in keloid tissue of 18 patients, totally 6 gene mutations, including 4 point mutations and 2 frameshift mutations. Mutations in the A((9)) locus of exon 8 of RIZ were observed in keloid tissue of 9 patients, totally 9 gene mutations, including 7 point mutations and 2 frameshift mutations. No patient had a mutation in the A((8)) or A((9)) locus of exon 8 of RIZ in peripheral venous blood. Compared with those of peripheral venous blood, the mutations in the A((8)) and A((9)) loci of exon 8 of RIZ in keloid tissue of patients were statistically significant ( χ (2)=16.06, 7.11, P <0.05). Conclusions: Point mutations and frameshift mutations occur in the A((8)) and A((9)) loci of exon 8 of RIZ in keloids of patients, which may be associated with the occurrence of keloids.

Objective: To explore the effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism., Methods: Thirty-six male ICR mice were randomly divided into three groups ( n =12):sham group, TCP wear particles (TCP) group and N-acetyl-L-cysteine (NAC) group. Aperiprosthetic osteolysis model in mouse was established by implanting 30 mg of TCP wear particles onto the surface of bilateral parietal bones following removal of the periosteum. On the 2nd day post-operation, NAC (1.0 mg/kg) was locally injected to the calvarium under the periosteum every other day for 2 weeks. Then, all the mice were sacrificed to obtain blood and the calvaria. Periprosthetic osteolysis in the mouse calvaria was observed by tartrate resistant acid phosphatase (TRAP) staining; serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6); total anti-oxidation capacity (T-AOC) and superoxide dismutase (SOD) activity were examined by ELISA and chemical colorimetry, respectively; protein levels of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α) and phospho-eIF2α (p-eIF2α) in periprosthetic bone tissue were detected by Western blot., Results: Compared with sham group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were increased obviously in TCP group ( P <0.05), and serum level of T-AOC and SOD activity were decreased significantly in TCP group ( P <0.05), GRP78 expression, the ratio of p-PERK and PERK, p-eIF2α and eIF2α in the mouse calvaria of TCP group were up-regulated markedly. Compared with TCP group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were decreased markedly in NAC group ( P <0.05), serum level of T-AOC and SOD activity were increased obviously in NAC group ( P <0.05), and GRP78 expression, the ratio of p-PERK/PERK and p-eIF2α/eIF2α were obviously down-regulated., Conclusions: Inhibition of oxidative stress can prevent periprosthetic osteolysis induced by TCP wear particles, which may be mediated by inactivation of PERK/eIF2α signaling pathway.

Objective: To investigate the effect of manganese chloride (MnCl(2)) or 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) on the neurobehavioral and histopathology in C57BL/6 mice and provide evidence for the diagnosis, treatment and prevention of manganism. Methods: Adult male C57BL/6 mice were treated with MnCl(2) and MPTP respectively by intraperitoneal injection at the doses of 5, 10, 20mg Mn/kg and 30mg MPTP/kg. Controls were injected equivalent normal saline. All animals were administrated 5 times a week for 4 consecutive weeks and sacrificed after behavior tests on the fifth week. Balance ability, anxiety and depression level and cognitive function were tested respectively by vertical pole test, open field locomotion test and Morris swim task. The neuron pathological changes of striatum and substantia nigra were examined through HE-staining pathological section by using optical microscope. Results: Compared with the control group, the high dose of MnCl(2) reduced body weight obviously ( P <0.01) . The results of vertical pole test showed that MnCl(2) and MPTP lengthened the pole-climbing time and turnaround time. Open field locomotion test showed that movement distance, stand-up time and central field time were decreased after the exposure of MnCl(2) or MPTP. In the Morris swim task, the escape latency time increased and the target quadrant activity time decreased significantly after the injection of MPTP as well as high-dose MnCl(2), comparing with controls ( P <0.05) . Moreover, the escape latency time of high dose MnCl(2) prolonged prominently comparing with MPTP grou ( P <0.05) . The results of histopathology showed that acidophilic changes elevated in MnCl(2) and MPTP group, comparing with controls. Furthermore, in striatum the oxyphil cells number increased in MnCl(2) high-dose group comparing with MPTP group ( P <0.01) . On the contrary, there were more oxyphil cells in MPTP group comparing with MnCl(2) groups in substantia nigra ( P <0.01) . Conclusion: Both manganese and MPTP can induce the impairment of dopaminergic neural system, but the symptons and injured location of manganism are inconsistent with PD models induced by MPTP.

Objective: To investigate the effect of manganese chloride (MnCl(2)) or 1-methyl-4-phenylpyridinium (MPP (+)) on oxidative stress and autophagy in human neuroblastomaSK-N-SH cells and the mechanism of the neurotoxicity of manganese. Methods: SK-N-SH cells were treated with MnCl(2) or MPP(+) at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, and 2.0 mmol/L for 24 hours, and MTT assay was used to measure cell viability. The cells weretreated with MnCl(2) or MPP(+) at doses of 0.125, 0.25, and 0.5 mmol/L for 24 hours, and flow cytometry was used to measure the content of reactive oxygen species (ROS) in cells, a laser scanning confocal microscope was used to observe autophagosome in cells, and Western blot was used to measure the expression of autophagy-related proteins P62 and LC3-II/LC3-I. Results: Compared with the control group, the 0.0625-2.0 mmol/L MnCl(2) and 0.125-2.0 mmol/L MPP (+) treatment groups had significant reductions in the viability of SK-N-SH cells, and the 0.25-2.0 mmol/L MnCl(2) treatment groups had significantly lower viability than the groups treated with the same doses of MPP(+) (all P <0.05) . Compared with the control group, the 0.125-0.25 mmol/L MnCl(2) and 0.125-0.5 mmol/L MPP(+) treatment groups had significant increases in the content of ROS, and the 0.25-0.5 mmol/L MPP(+) treatment groups had significantly higher content of ROS than the groups treated with the same doses of MnCl(2) (all P <0.05) . Compared with the control group, the 0.25-0.5 mmol/L MnCl(2) andMPP(+) treatment groups had significant increases in autophagy-related proteins LC3-II/LC3-I and significant reductions in P62 expression; the 0.125-0.5 mmol/L MPP(+) treatment groups had significantly higher LC3-II/LC3-I than the groups treated with the same doses of MnCl(2), and the 0.125 and 0.25 mmol/L MPP (+) treatment groups had significantly lower P62 expression than the groups treated with the same doses of MnCl(2) (all P <0.05) . Conclusion: Both MnCl(2) and MPP(+) can induce oxidative stress and autophagy in SK-N-SH cells, and MPP(+) has a significantly greater inductive effect on autophagy of SK-N-SH cells than MnCl(2).

In this study, we explored the effects of cadmium (Cd) on mouse sperm motility parame-ters, protein tyrosine phosphorylation and the location of tyrosine-phosphorylated targets using computer-assisted sperm analysis (CASA), western blot (WB) and immunofluorescence technique coupled to sperm in vitro culture method, respectively. The results showed sperm motility was inhibi-ted by Cd in a dose-dependent manner and when Cd increased to 1.0 μmol·L -1 , sperm motility was inhibited significantly (P<0.05). Simultaneously, protein tyrosine phosphorylation was enhanced by Cd and in particular, the tyrosine phosphorylation of ～55 kDa proteins was greatly promoted when Cd concentrations were greater or equal to 1.0 μmol·L -1 (P<0.05). Importantly, these tyrosine-phosphorylated proteins were mainly localized in the middle piece of mouse sperm. However, when sperm was incubated with 30 μmol·L -1 ethylene glycol tetraacetic acid (EGTA) and 10 μmol·L -1 Cd concurrently, both the tyrosine phosphorylation of ～55 kDa proteins and sperm motility were not changed obviously (P>0.05). These results suggested that Cd may inhibit sperm motility by inducing the tyrosine phosphorylation of ～55 kDa proteins in the middle piece and EGTA could chelate Cd ions to relieve its toxicity. This study demonstrated that Cd induced the tyrosine phosphorylation of a specific subset of proteins and thus decreased sperm motility. Interes-tingly, EGTA acted as an inhibitor to block Cd from entering the sperm, which provided a novel research method for revealing the molecular mechanisms of reproductive toxicity caused by Cd.

Objective: To investigate the autoantibody profile and its clinical implication in the patients with primary biliary cirrhosis., Methods: During the period of 2008 to 2010,123 patients with primary biliary cirrhosis (PBC) in our hospital were enrolled in this study, of whom, 70 patients were with cirrhosis and 53 without cirrhosis, The autoantibody profile was tested for each patient by using immunoblotting and indirect immunofluorescence., Results: Of the 123 PBC patients with liver cirrhosis, 49% were positive with serum ANA positive; 47%, 51%, 54%, 31% and 49% were positive with serum anti-nuclear antibodies (ANA), anti-mitochondrial antibodies-M2 (AMA-M2), anti-promyelocytic leukemia (anti-PML), anti-sp100 antibodies (anti-sp100), anti-Ro-52 antibody (anti-52KD), respectively. By contrast, of the PBC patients without liver cirrhosis, only 38%, 37%, 51%, 60%, 30% and 51% were positive with serum ANA, AMA, AMA-M2, anti-PML, anti-sp100 and anti-52KD, respectively.There was the statistical difference between the two groups. In addition, it was also found that the anti-gp210 antibody positive group had a higher Mayo risk score,lower serum albumin and severe cholestasis and impaired liver function when compared with anti-gp210 antibody negative patients., Conclusion: Our data indicate that serum AMA is helpful for early diagnosis of PBC, and in particular, serum ANA positivity can help make a diagnosis for the AMA-negative patients. These indicate that anti-gp210 antibodies appear in the late course of PBC.Anti-gp210 positive PBC patients have more severe cholestasis and liver dysfunction.

Objective: To characterize the clinical, laboratory, imaging and pathological features of primary sclerosing cholangitis (PSC) and investigate the impact of ursodeoxycholic acid (UDCA) therapy on patient prognosis., Methods: The medical records of 22 patients diagnosed with PSC between 2002 and 2011 were retrospectively reviewed. The PSC diagnosis had been made in patients with suspect biochemical abnormalities following evaluation by magnetic resonance cholangiopancreatography (MRCP) and/or endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous transhepatic cholangiography (PTC). Fibrosis and inflammation were assessed by immunohistochemical analyses of tissue biopsies. Outcome of patients treated with UDCA (13-15 mg/kg/day, oral) were compared to that of patients without UDCA treatment by the X2 or corrected X2 tests., Results: Among the 22 PSC patients, the majority was male (n=15) and presented with fatigue, dark urine, and body weight loss (n=15). Four cases had ulcerative colitis. At admission, all 22 cases showed elevated levels of alkaline phosphatase[ALP: (348+/-184) U/L], 19 cases showed elevated alanine aminotransferase [ALT: (94.0+/-67.0) U/L] and aspartate aminotransferase [AST: (98.0+/-67.0) U/L], and 15 cases showed elevated levels of total bilirubin (99.0+/-115.0) mumol/L and direct bilirubin (74.4+/-92.4 mumol/L. ERCP examination showed segmental intrahepatic bile duct stenosis with expansion, and stiff and enlarged gallbladder bile ducts, but unclear findings for the common bile ducts and pancreatic ducts. MRCP showed beading of the intrahepatic bile duct, stiffness of the bile duct wall, and dilation of the common bile duct. Fibrosis and inflammation were observed in the bile ducts, along with hyperplasia and the typical features of "onion skin" fibrosis and fibrous obliterative cholangitis. Five of the 10 patients treated with UDCA improved, and seven of the 12 patients in the non-UDCA treatment group improved. There was no statistically significant difference in outcome between the groups (paired X2=0.333, corrected X2=0.083, P more than 0.05)., Conclusion: PSC patients were predominantly male and the common clinical manifestations were fatigue, dark urine, and body weight loss. At admission, serum biochemical indicators of cholangitis were increased significantly and subsequent imaging studies confirmed the suspected diagnosis by showing obvious characteristic changes. UDCA treatment did not significantly improve patient prognosis.

Objective: To evaluate the safety of human umbilical cord derived-mesenchymal stem cell (UC-MSC) transplantation therapy in patients with decompensated liver cirrhosis., Methods: UC-MSCs were transplanted intravenously into patients with decompensated liver cirrhosis. Serum levels of glucose (GLU), total cholesterol (TC), blood urea nitrogen (BUN), alpha fetoprotein (AFP), white blood cells (WBC), and prothrombin activity (PA) were detected at different time points after UC-MSCs transplantation., Results: Most UC-MSC transplanted patients experienced an improvement in quality of life, to varying degrees. With the exception of low-grade fever in a few patients, side effects and oncogenic events were rare (treatment group: 1/38 vs. control group: 1/16; P more than 0.05). The UC-MSCs transplantation showed no effect on GLU, TC, BUN, AFP, WBC, or PA., Conclusion: UC-MSCs transplantation in patients with decompensated liver cirrhosis is safe and may improve the patient's quality of life.

Single nucleotide polymorphisms of exon 5 T32207C and exon 7 A35230G of integrin β1 gene were detected in Landrace, Large White and Duroc by CRS-RFLP. Association between the polymorphism and litter size was analyzed by the method of least square means. At 32207 polymorphic loci, there was no significant difference on TNB and NBA between the genotypes in Landrace, Large White, and Duroc. At 35230 polymorphic loci, there was significant difference (P<0.05) or greatly significant difference (P<0.01) on TNB and NBA between genotypes in the first, second, and all parities in Large White and Landrace. The effects of GG and AG genotypes were different from that of AA genotypes with the order of GG, AG>AA. These results suggested that the effect of G allele of integrin β1 gene on litter size is significant in Large White and Landrace.

Filtration and cross-flow ultrafiltration (CFUF) was applied for the effective separation of NPs in the Yangtze Estuary. The physiochemical properties of NP were characterized, and their relationships with environmental factors were further studied in the present study. The results show that NP size in Yangtze Estuary ranged from 69.5 to 263.5 nm with the average value of 157.3 nm and Zeta-potential values ranged from -40.1 mV to 196.0 mV. NOC concentrations ranged from 0.3 mg/L to 1.5 mg/L and the average value was 0.7 mg/L. NOC account for 5.1% to 30.5% of DOC, with an average of 16.7%. The binding capacity of metals with NP in the Yangtze River Estuary was in the order of Zn > Cu > total Cr > Co > Ni > Mn > Fe > Li > Al > B > K > Ba > Sr > Mg > Ca > Na, which reveals that terrigenous input may be the main source of NOC in the Yangtze River. The binding capacity of NP with trace metal is generally higher than the conventional metallic element. There was no significant correlation between NP size and salinity, DOC, NOC, SPM and Zeta-potential, respectively. Compared to NOC and UOC, better correlation was investigated between DOC, salinity and NP bound trace metals.

Aim: This study attempted to investigate the features of Treg cells in peripheral blood and liver of patients with primary hepatocellular carcinoma (HCC)., Methods: Peripheral blood samples were obtained from 30 HCC patients and 30 healthy control subjects, then were quantitatively analyzed for Treg cells by using flow cytometry. Liver infiltrating lymphocytes (LIL) isolated from resected tumor samples of 7 HCC patients were simultaneously analyzed., Results: There was a significant increase in the frequency of peripheral Treg cells in HCC patients compared with healthy controls (P < 0.01). Furthermore, we also found that there was a higher frequency of infiltrated Treg within tumor samples than the counterpart in non-tumor region (P < 0.05). In addition, CD4(+) CD25(low) and CD4(+) CD25(-) T cells isolated from resected tumor samples were found to express higher level of FOXP3 molecules., Conclusion: Our findings showed that a dramatic increasing increase of Treg in peripheral blood and liver tissue of HCC patients may be associated with the significant increased development of Treg, which favors the disease progression through the suppressive effect of Treg on host immune response in these patients.

Aim: To investigate phenotypical and functional characteristics of the CD39(+);FoxP3(+);regulatory T (Treg) cells in humans., Methods: We collected peripheral blood from 10 healthy volunteers and separated PBMC by using density gradient centrifugation, and then detected the expression of CTLA-4, HLA-DR, CD45RO and Ki-67 on CD39(+);FoxP3(+); and CD39(-);FoxP3(+);Treg cells by using flow cytometric analysis. [(3);H]-thymidine incorporation assay and ELISA were used to monitor the suppressive capacity of CD39(+); FoxP3(+); Treg cells on proliferation and IFN-gamma secretion of Treg-depleted PBMC., Results: The expression of CTLA-4, HLA-DR, CD45RO and Ki-67 was significantly higher in CD39(+);FoxP3(+); Treg cells than those in CD39(-);FoxP3(+); Treg cells. The suppressive capacity of CD39(+);FoxP3(+); Treg cells on proliferation and IFN-gamma secretion of Treg-depleted PBMC was significantly higher than those of FoxP3(+);CD39(-);Treg cells., Conclusion: CD39(+);Fxop3(+);Treg subset may play an essential role in immune regulation of Treg, and CD39 can be used as a surface marker to identify the functional Treg cells.

Objectives: To investigate the relationship between the birth weight of fetus and the P(70)S6K from ribosome in cells of placental tissues and investigate whether insulin can affect the P(70)S6K by the PI3K/AKT/mTOR (P(70)S6K) signaling pathway in the cells of the placental tissues and more regulate the birth weight of fetus., Methods: (1) 60 cases of mature placental tissues after uterine-incision delivery without complications in the gestational period were selected as the investigated subjects, they were divided into three groups according to their birth weights. group 1. the weigh of the fetus > or = 4000 g, group 2. 3000 g < or = the weight of the fetus < 4000 g, group 3. the weight of the fetus < 3000 g, (2) 10 cases of mature placental tissues and 20 cases of villus in the early pregnant the fetus that were divided into insulin group, rapamycin group and control group according to stimulating factors. investigations on the insulin affection activities of P(70)S6K were carried out by immunoprecipitation, incorporation of [r-(32)P]ATP, tissue cell culture, Western blotting., Results: (1) P(70)S6K activities in mature placental tissues (2546 +/- 357), (2333 +/- 318), (2111 +/- 315) cpm/mg, there were significant statistical differences in the comparison every two groups. (2) The P(70)S6K activities in the placenta from the insulin stimulation group was significantly elevated, the P(70)S6K activities in the placenta from the rapamycin stimulation group was significantly declined, there were statistical significance when each was compared with control group (P < 0.01). (3) P(70)S6K activities in the nutrient cells of earlier pregnant period and protein expression will be elevated under insulin stimulating. However, that would be declined under rapamycin stimulating, it was statistical significant when each group compared with the control group (P < 0.01)., Conclusion: The activities of P(70)S6K increased with the increase in the birth weight of the fetus, there was certain relationship between the activities of P(70)S6K in the placental tissues and the birth weight of the fetus and Insulin can regulate the activities of P(70)S6K in the placenta and earlier nutrient cells.

Objective: To evaluate the safety and efficacy of Wuling Capsule combined with Deanxit in treating post-stroke depression (PSD)., Methods: One hundred and fourteen patients with PSD were randomly divided into Wuling Capsule-treated group (n=39), Deanxit-treated group (n=37) and Wuling Capsule plus Deanxit-treated group (n=38). Patients in Wuling Capsule-treated group were administered with three Wuling Capsules three times a day, and patients in Deanxit-treated group were administered with Deanxit 10.5 mg twice daily, while patients in the Wuling Capsule plus Deanxit-treated group were administered with both Wuling Capsule and Deanxit. Patients in the three groups were all treated for six weeks. Treatment efficacy was evaluated with Hamilton Depression Scale (HAMD) and the side effects were evaluated with Treatment Emergent Symptom Scale (TESS) before treatment and after 2-, 4-, and 6-week treatment. The blood and urine routine examinations were performed, and the hepatorenal functions and electrocardiogram were examined as well., Results: There was no statistical difference in the total efficacy rate between Wuling Capsule-treated group and Deanxit-treated group (64.1% vs 64.9%, P>0.05), but the total efficacy rate of Wuling Capsule plus Deanxit-treated group was higher than that of the monotherapy (89.5% vs 64.1%, 89.5% vs 64.9%, P<0.05). There were no significant side effects in Wuling Capsule-treated group, while the incidence of side effects was 9% in both groups administered with Deanxit., Conclusion: The efficacy of Wuling Capsule plus Deanxit is better than that of the monotherapy in treating PSD.

Objective: To investigate the frequencies of immunologically competent cells (ICCs) in the liver-infiltrating lymphocytes (LILs) and peripheral blood and their possible role in pathogenesis in patients with chronic severe hepatitis B (CSHB)., Methods: LILs were isolated from the liver tissue samples from 11 CSHB patients and 5 normal controls (NCs) by the method of combined grinding with semi-frosted microscopic slides and sedimentation of hepatic cells. The frequency of isolated ICCs, including CD3(+), CD4(+), and CD8(+) T-cells, NK cells, NKT cells, and B cells was examined and compared with that of the circulating ICCs in the CSHB patients. Comparison was conducted between the CSHB patients and the controls., Results: (1) In the CSHB patients, the frequencies of CD4(+) T cells and B cells in LILs were 17% +/- 6% and 3.0% +/- 1.0% respectively, both significantly lower than those in the circulating blood (32% +/- 8% and 21.4% +/- 12.2% respectively, both P < 0.01); however, the frequencies of CD8(+) T cells, NK cells, and NKT cells in LILs were 38% +/- 13%, 34% +/- 18%, and 10% +/- 4% respectively, all significantly lower than those in the circulating blood (26% +/- 6%, 15% +/- 9%, and 6% +/- 4%, all P < 0.05). (2) The frequencies of infiltrating CD3(+) T cells and CD4(+) T cells of the CSHB patients were both significantly higher than those of the NCs (P = 0.042 and P = 0.001); and the frequency of infiltrating CD8(+) T cells of the CSHB patients was higher than that of the NCs, and the and the frequencies of infiltrating NK cells and NKT cells in LILs were lower than those of the NCs, however, not significantly. (3) Compared with the liver tissues from the NCs, the liver tissues from the CSHB patients exhibited a significantly higher ratio of liver-infiltrating CD4(+) T cells to peripheral blood CD4(+) T-cell subsets (P = 0.001), and significantly lower ratios of liver-infiltrating NKT cells and B cells to the peripheral blood NKT-cells and B cells (P = 0.029 and P = 0.001 respectively)., Conclusion: The abundant infiltrating immune active cells, especially the CD4(+) T cells, CD8(+) T cells, and NK cells, may be the causal factors that drive the progressive development of CSHB.

Objective: To investigate the difference in the CD4+T lymphocytes activation between long term non progressors (LTNP) and typical progressors (TP) of HIV-1 infected patients., Methods: Twenty-four HIV-1 infected patients and 15 heathy control adults were tested and flow cytometry was used to detect the activation marker CD38 and CD4 count in blood samples taken from the patients and control. bDNA method was used to test the virus load in the plasma of patients., Results: The activation of CD4+T cells was positively correlated with virus load and negatively correlated with CD4 counts. Compared with normal controls, the activation of CD4+T cells was obviously increased in TP patients but not obviously changed in LTNP patients., Conclusion: Compared with healthy controls, the activation of CD4+T cells in LTNP did not obviously increase. This maybe partially accounts for LTNP patients keeping a good state for a long time.

Objective: Our aim was to investigate the frequency, phenotype and function of CD4+ CD25 high regulatory T cells (Treg) in patients with acute and chronic hepatitis B (AHB, CHB)., Methods: Peripheral blood mononuclear cells (PBMCs) from 16 AHB patients at acute phase (week 1 of illness), 72 CHB patients, and 32 health subjects were analyzed for Treg frequency and CD45RO, CD45RA, CD95 and HLA-DR phenotype by flow cytometry. Intracellular expression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) was examined by intracellular cytokines staining. Forkhead/winged helix transcription factor (FoxP3) mRNA was detected by a real-time RT-PCR assay. The effects of MACS magnetic beads-purified Treg cells on the proliferation of PBMCs were examined by a [3H]-thymidine incorporation assay. The effect of Treg cells on IFN gamma secretion of autologous PBMCs was examined by ELISA., Results: CHB patients presented a higher fraction of circulating CD4+ CD25 high Treg frequency than AHB patients (P < 0.05), but had no significant difference compared with healthy controls. CD4+ CD25 high Treg expressed high levels of CD45RO, HLA-DR and CTLA-4, low level of CD45RA, and expressed FoxP3 mRNA specifically. We also observed that Treg cells could suppress the expansion and IFN gamma secretion of autologous PBMCs when stimulated with HBV antigen or anti-CD3 antibody, and the suppressive effect was stronger when HBV antigen was used as stimulator., Conclusions: CHB patients presented a higher fraction of circulating Treg frequency than AHB patients at acute phase, but had no significant difference compared with healthy controls. FoxP3 mRNA was specifically expressed in CD4+ CD25+ cell population. Treg could suppress HBV antigen-specific T cell response in vitro. The study furthers our understanding of Treg's role in immunopathogenesis of hepatitis B.

Two pairs of primers were designed based on the known sequence in GenBank for amplification of MTNR1A gene in a Large White and a Landrace herd. Using PCR-SSCP (single strand conformation polymorphism), we found a single nucleotide polymorphism(SNP) within the product amplified from the first pair of primers. PCR products from randomly selected different genotypes were sequenced after were recovered and purified. Results revealed a synonymous single base mutation(G-->A) at +159bp(sequence numbering based on Genbank accession number U73326) for the BB genotype. When analyzed for association with litter size traits, this MTNR1A SNP was found to have no significant effect on litter size traits.

Objective: CD4(+)CD25(high) regulatory T cells (Treg) have been shown to play an important role in maintaining peripheral tolerance against self and foreign antigens, and in suppressing T cell immune response. Our aim was to characterize circulating Treg in HBV-infected patients., Methods: Treg in peripheral blood from 72 chronic hepatitis B (CHB) patients, 16 acute hepatitis B (AHB) patients and 32 healthy subjects were quantitatively analyzed using flow cytometry. Serum HBV markers were evaluated for each subject. HBV-DNA levels were measured using real-time RT-PCR., Results: CHB patients presented a higher fraction of circulating Treg (3.9% +/- 1.4%) than those in AHB patients (3.1% +/- 0.9%) (P < 0.05), but were similar to healthy controls (3.5% +/- 0.7%). CHB patients with greater than 10(7) copies/ml of serum HBV DNA loads had a higher frequency (4.5% +/- 1.9%) of circulating Treg than health controls (P < 0.01) and the patients with less than 10(7) copies/ml of serum viral loads (3.4% +/- 0.7%). A correlation was found between circulating Treg and HBV DNA level (r = 0.32, P < 0.01). Furthermore, Treg was more frequent in convalescent phase (6.0% +/- 1.7%) than in early acute phase (3.0% +/- 0.6%)., Conclusion: Increased peripheral Treg is found to be associated with HBV replication in chronic hepatitis B. In acute HBV infection, Treg is less frequent in early phase. The related mechanisms is under further investigation.

The genetic polymorphisms of four microsatellite loci BM143, OarHH35, OarAE101, and BMS2508 were analyzed in 286 sheep of seven sheep populations (Small Tail Han sheep, Hu sheep, Ujumqin sheep, Suffolk sheep, Dorset sheep, Charolais sheep, F1 of Dorset male x Small Tail Han female sheep). The numbers of alleles for BM143, OarHH35, OarAE101, and BMS2508 are 9, 11, 14 and 9 in seven sheep populations, respectively. The polymorphism information content/number of effective alleles/ heterozygosity of BM143, OarHH35, OarAE101 and BMS2508 were 0.7073/3.7231/0.7314, 0.8267/6.4399/0.8447, 0.5743/2.5178/0.6028, 0.6172/3.0712/0.6744 in 286 sheep, respectively. The results revealed the greatest genetic variation at OarHH35 locus and the lowest at OarAE101, the greatest genetic variation in Small Tail Han sheep and the lowest in Hu sheep among seven sheep populations. In the unweighted pair group method with arithmetic mean (UPGMA) dendrograms based on Nei's D(A) distance and Nei's D(S) standard genetic distance, the Chinese native breeds (Small Tail Han sheep, Ujumqin sheep, Hu sheep) were grouped together, then with Charolais sheep. The F1 crossbred sheep, and the two British native sheep (Suffolk sheep, Dorset sheep) also clustered together. Microsatellite genotyping in sheep provided a useful tool for examining the genetic relationships among breeds (populations).

Nuclear receptors belong to a superfamily of ligand-activated transcription factors, which are involved in regulating gene expression in development, cell differentiation, physiological and metabolic processes. In this review we summarize the studies of nuclear receptor and present the progresses in the researches on nuclear receptor and lipid physiology, nuclear receptor and tumor, and nuclear receptor and co-regulators.

Objective: To observe the therapeutic efficacy of IFN or oxymatrine in combination with lamivudine in patients with lamivudine-resistant chronic hepatitis B., Methods: Forty patients ongoing treatment with lamivudine were randomized to three groups: group A, 14 patients with addition of IFN alpha-2b 3MU to ongoing lamivudine, daily, one month, followed by the same dose given every other day, five months; group B, 15 patients with addition of injectable oxymatrine 60 mg daily, three months, followed by oral oxymatrine every day, three months, and group C, 11 patients ongoing treatment with lamivudine alone. The HBV DNA level in serum, HBeAg seroconversion, and ALT level were detected at the end of the treatment., Results: After 6 months of treatment, HBV DNA became negative in 35.73% patients treated with combination with IFN, and in 13.3% patients treated with combination with oxymatrine. ALT level was normal in 85.71% or 86.66% of patients, respectively. In none of the patients under ongoing treatment with lamivudine alone HBV DNA or HBeAg became negative, and ALT level was normal in 36.36% of patients., Conclusion: These data indicated that IFN or oxymatrine in combination with ongoing lamivudine therapy provided effective antiviral therapy in patients with lamivudine-resistant HBV. The addition of IFN or oxymatrine to ongoing lamivudine therapy in lamivudine-resistant patients led to significant inhibition of viral replication and improvement in liver function after 6 months of therapy.

On the base of element and metablism balancing, the mathematical model of the cultivation process with Streptomyces aureofaciens was developed, and the unknown parameters in the model were estimated with the method of nonlinear optimization. Firstly the energetic coefficient of CTC biosynthesis was gained, which was 1.8 - 2.8 mol-ATP x C-mol(-1). The macroscopic reaction rates were predicted in the process and compared with the experimental values. The results show that the model can preferably describe the relationships between several macroscopic reaction rates in the process and can supervise the optimization of CTC fermentation process theoretically.

The human zinc finger protein ZNF191 is a krüppel-like transcription factor, which may be relevant to many diseases such as neuropsychiatric, cardiovascular and liver caner diseases. To elucidate the function of ZNF191 by gene targeting, it is necessary to clone and characterize of the homologous gene in model organisms (mice). The mouse homologous gene (ZF-12) was cloned and sequenced for the first time, the GenBank accession number is AY052495. It contains four exons and three introns; all intronic splice sites exhibited consensus GT/AG sequences. The single nucleotide polymorphisms (SNPs) in exon 2 and the alternative length of 3'-untranslated region (3'-UTR) have been found. The linkage of the ZF-12 gene and the zinc finger protein gene Zfp-35 has been found, so the ZF-12 gene can be localized to B3 to C or beside of chromosome 18. We assessed approximately 1.2 kb of 5'-flanking region of the ZF-12 gene for basal promoter activity. A series of deletion mutants of 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in COS-7 cells, NIH3T3 cells and HeLa cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found that regulatory elements sufficient for basal expression lie between -762 and +70 bp relative to the transcription start site and that a negative regulatory region lie between -824 and -762 bp. This research provides a basis for further study on ZF-12 by gene targeting.

By using the mouse zinc finger protein gene ZF-12 genomic DNA fragment, pSSC-TV-10.5 was designed and constructed as a replacement vector. Structure of pSSC-TV-10.5 was identified by restrictive digestion analysis and partly sequencing. Then linearized vector was electroporated into ES cells, and transfected cells were screened by G418 and GANC selection. Among 508 G418r/GANCr colonies, 4 were proved to have taken place the homologous recombination of ZF-12 by PCR and southern blotting analysis. This study lays the foundations of preparing mouse models of ZF-12+/- or ZF-12-/-.

This study aimed to introduce a site specific point mutation into the human coagulation factor IX gene expressing vectors (pMe4bAIXml plasmid) for microinjection and to obtain transgenic mouse containing copies of a stably integrated pMe4bAIXml plasmid on the basis of coagulation factor IX gene knock-out mouse model as an more efficient animal model of hemophilia b. The site specific point mutation was introduced into pMe4bAIXml plasmid which consists of human coagulation factor IX gene including the entire coding sequence and a shortened first intron, four copies of the mouse MCK enhancer, chicken beta-actin promotor and poly A signal sequence. The vector was linearized and injected into 817 fertilized eggs of mice in which coagulation factor IX gene has been knocked out. The manipulated embryos were transferred into the oviducts of 45 pseudopregnant females, from which 63 offsprings were obtained. The genomic DNAs of these offsprings were analyzed with PCR and genomic Southern blotting. Six mice were found to carry copies of the intact pMe4bAIXml plasmid containing a point mutation and used as founders to establish transgenic mouse lineages.

We studied a novel mutation detection method of the two Lac I target genes in pMCLac I/neo transgenic mice. The transgenic mice that contain two types of Lac I genes in pMCLac I/neo vector are different from the transgenic mice carrying only one target gene. Therefore a novel method to detect mutation quickly and efficiently has become a new project after the establishment of pMCLac I/neo transgenic mice. In this paper, a positive selection system--M9/L is used. The result showed that M9/L positive selection system was able to detect the two Lac I target genes, suggesting that it is a rapid and effective method to detect the target mutations in the pMCLac I/neo transgenic mouse.

Small Tail Han sheep is an excellent local sheep breed in China. Small Tail Han sheep had significant characteristics of high prolificacy. The lambing percentage averaged 260 percent in Small Tail Han sheep. The polymorphisms of 5 microsatellite loci OarAE101, BM1329, BMS2508, TGLA54 and TGLA68 which were closely linked to the fecundity genes FecB and FecXI in sheep were detected in 244 ewes of Small Tail Han sheep. The PCR amplified products of microsatellites were detected by non-denatured (natural) polyacry lamide gel electrophoresis. Allele frequency, polymorphism information content, gene homogeneity and heterozygosity for 5 microsatellite loci were calculated. The number of alleles for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 6, 9, 5, 2 and 6 in Small Tail Han sheep, respectively. The range of allele sizes for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 160 bp to 180 bp, 97 bp to 135 bp, 116 bp to 136 bp, 98 bp to 100 bp, and 93 bp to 115 bp, respectively. The alleles of the greatest frequency for BM1329, OarAE101, TGLA54 and BMS2508 were 164 bp, 97 bp, 134 bp and 99 bp, respectively. Polymorphism information content/gene homogeneity/heterozygosity for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 0.4481/0.4840/0.5160, 0.3516/0.6375/0.3625, 0.2528/0.7326/0.2674, 0.3733/0.5034/0.4966 and 0.5809/0.3581/0.6419 in Small Tail Han sheep, respectively. The results revealed the greatest genetic variation in BMS2508 and the lowest in TGLA54. These results could provide, basic molecular data for the research on the germplasm characteristics of Small Tail Han sheep.

The mouse zinc finger protein ZF-12 gene is homologous to human gene and encodes a protein of 368 amino acids, which contains four tandem C2H2-type zinc finger motifs in the N-terminal and one SCAN domain in the C-terminal. Some recent studies suggest that ZNF191 might be a hepatocarcinogenesis-associated gene. We screened a mouse lambda genomic library with a human ZNF191 cDNA probe and isolated a ZF-12-like gene, named ZF12p (GenBank AY040222). This intronless gene closely resembles ZF-12 but displays several mutations, suggesting that ZF12p represents a ZF-12-related pseudogene. RT-PCR analysis on total RNA from mouse tissue and bioinformatis analysis on promoter region of ZF12p gene, suggest the transcripts of ZF12p may be not synthesized. BLAST on the data of the human genome in the GenBank with ZNF191 cDNA and Southern blotting show there is no any psedogene related to ZNF191 gene in the human genome. The high similarity of ZF12p to ZF-12 might be of considerable importance for mutation and evolution analysis of ZF-12.

Small Tail Han sheep is an excellent local sheep breed in China. Small Tail Han sheep has significant characteristics of high prolificacy. The lambing percentage averaged 260 percent in Small Tail Han sheep. The genetic detection of 4 microsatellite loci OarAE101, OarHH35, BM143 and BMS2508 which were closely linked to the fecundity gene FecB in Booroola sheep was conducted in 159 sheep in Small Tail Han sheep, Dorset sheep, F1 hybzids(Dorset x Small Tail Han sheep). The results confirmed the genetic characteristics of codominance of microsatellite DNA. The PCR amplified products of microsatellites were detected by non-denatured (natural) polyacrylamide gel electrophoresis. The sequences of PCR amplification fragment of 6 clones of 4 microsatellite loci in Small Tail Han sheep were accepted by GenBank of National Center for Biotechnology Information, National Institutes of Health in USA, the GenBank accession numbers were AF394445, AF394446, AF394447, AF394448, AF394449, AF394450, respectively. The sequence homogeneity between OarAE101 in Small Tail Han sheep in this study and the ovine OarAE101 in GenBank was 98 percent. The sequence homogeneity between OarHH35 in Small Tail Han sheep in this study and the ovine OarHH35 in GenBank was 99 percent. The sequence homogeneity between BM143 in Small Tail Han sheep in this study and the bovine BM143 in GenBank was 95 percent. The sequence homogeneity between BMS2508 in Small Tail Han sheep in this study and the bovine BMS2508 in GenBank was 95 percent. OarAE101, OarHH35, BM143 and BMS2508 in Small Tail Han sheep were all perfect microsatellites. These results could provide molecular basic data for the research on the germplasm characteristics of Small Tail Han sheep.

A shuttle plasmid pMCLacI/neo with two copies of LacI was integrated into mouse genome and a novel system which could detect in vivo mutation of both expression and silence genes was constructed, enabling the comparative analysis of their mutation spectra and mutant frequencies. 486 fertilized eggs from C57BL/6 mice with microinjected pMCLacI/neo plasmid were transferred into oviducts of 18 pseudo-pregnant mice, and 32 alive offsprings were screened and identified by using PCR and Southern blotting. Genomes of 5 mice had pMCLacI/neo plasmid integrated, as verified by Southern blot after the PCR screening. Only one of the two LacI in pMCLacI/neo was in expression state; and this established a model, that the status in vivo of both gene expression and silencing could be simulated. This kind of mice might be used as a novel bifunctional mutation detection system in vivo.

INK4a/ARF locus distinguishes itself by its unusual structure and function. It contains 2 overlapping genes with exons 1 alpha, 2 and 3 encoding p16INK4a and exons 1 beta, 2 and 3 encoding p19ARF. Mice with their exons 2 and 3 of the INK4a/ARF knocked out are viable and fertile but develop spontaneous tumors at an early age and highly sensitive to carcinogenic treatment. However, mice with their exon 1 beta knocked out, without interference the expression of p16INK4a, show almost the same phenotype as those with their exons 2 and 3 knocked out. This raises a question of whether the mouse p16INK4a plays a role in tumor suppression. To investigate this problem, a targeting vector pointing to p16INK4a exon 1 alpha with 1.5 kb Eco81 I/Acc II fragment as short arm and 5.9 kb Xba I/Xho I fragment as long arm was built. After linearlization and purification, the targeting vector was introduced into ES cells through electroporation. Thirty-seven G418- and gancyclovir-resistant colonies were picked out and one of them was confirmed as positive by Southern hybridization.

Exchange plasmid pF-HPRT-F3 and Flp expression plasmid pCMV-Flp were constructed and then introduced using electroporation system into F18 ES cell line, which have an exchange cassette F-Neo-F3 at HPRT locus. After HAT selection, HAT resistant clones were obtained. Then G418 sensitivity test and Southern blotting were carried out to screen RMCE recombinants. The results indicated that RMCE had taken place in three of 12 HAT resistant clones. The frequency is 25%. The result demonstrates that it is realizable to introduce transgene to HPRT locus by using Flp recombinase mediated cassette exchange reaction.

Using the HPRT genomic DNA fragment and synthesized oligonucliotides, pSP-HPRT-F-Neo-F3 was designed and constructed as a replacement gene targeting vector by usual molecular cloning techniques. Structure of pSP-HPRT-F-Neo-F3 was identified by restrictive digestion analysis and partly sequencing. Then linearized pSP-HPRT-F-Neo-F3 DNA was electroporated into ES cells, and transfected cells were screened by being cultured in medium containing 200 micrograms/mL G418 and 2 micrograms/mL 6-GT. Twenty-four double drug resistant clones were picked up and analyzed, among them, two clones were proved to have taken place the required recombination by PCR and southern blotting analysis.

Inactivation of p16INK4a is highly related with tumors. We obtained a 14.5 kb mouse genomic DNA fragment through screening the 129/SvJ mouse genomic library. Bioinformatics study suggests that the fragment contain three exons, which encode a 17,941 dolton polypeptide consisted of 168aa. The polypeptide has 7 potential phosphorylation sites, which indicates that the function of p16INK4a itself may be under the control of phosphorylation. Many interspersed elements and simple repeats were found in the noncoding region. These elements and repeats may cause retroposition and recombination and thus be responsible for some of the p16INK4a locus deletion. We also compared our sequence of exon 1 alpha with those published elsewhere and found that polymorphism may exist.

To duplicating the regulatory subunit p35Nck5a gene of mouse neuronal cdc2-like kinase in embryonic stem (ES) cells, about 12.2 kb of pGDTV vector for p35Nck5a gene duplication was constructed. The linearized pGDTV vectors were transfected into ES cells by electroporation. 245 drug-resistant cell clones were obtained in both G418 and GANC medium and the frequency of surviving cells was 6.22 x 10(-5). The ES cell clones were identified to have duplicated the p35Nck5a gene by use of both PCR and genomic Southern blotting, and the frequency of homologous recombination is 5.08 x 10(-7). The use of negative selection gene (HSV-tk) results in 7-fold increase at selection efficiency. This study lays the foundations of preparing mouse models of Alzheimer's disease.