OBJECTIVE To establish HPLC methods with different separation principles to analyze the relevant impurities in the APIs of bivalirudin from seven enterprises, to provide a basis for the comprehensive control of related substances of bivalirudin. METHODS Reversed-phase high performance liquid chromatography(RP-HPLC) was used to separate and analyze 11 kinds of impurities. Hydrophilic chromatography(HILIC)-HPLC was used to control four process impurities. Polymers were determined by size exclusion chromatography(SEC)-HPLC. RESULTS The established RP-HPLC could effectively separate the principal component and 11 impurities, the correction factors of 11 impurities were between 0.8−1.2, the detection concentration of bivalirudin was 0.1 μg·mL−1, and the detection limit was 0.004%. The established HILIC-HPLC could effectively separate the principal components and four process impurities, and the detection concentration of bivalirudin was 0.3 μg·mL−1, and the detection limit was 0.01%. Under SEC-HPLC conditions, the polymer and bivalirudin peaked sequentially, the resolution of the two was 2.9, the detection concentration of bivalirudin was 6 ng·mL−1, and the detection limit was 0.000 6%. Fifteen kinds of known impurities and polymers in 15 batches of samples from 7 enterprises were calculated by the self-control method of principal components, and the impurity contents from different enterprises had a certain correlation with their production processes. CONCLUSION The three different principles of the method have good specificity, high sensitivity, good durability, and reliable results, and can be used for quality control of substances related to bivalirudin.

Objective:To explore the curative effect of surgical treatment for Crohn′s disease (CD), to investigate the timing of surgical intervention and the choice of surgical methods.Methods:From January 1, 2016 to August 31, 2020, at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, the clinical data of 123 patients with CD and receiving surgical treatment were retrospectively analyzed, which included the type of lesion, the location of lesion, clinical manifestation, surgical method, preoperative inflammatory and nutritional indicators, postoperative recovery of digestive tract function, and the development and treatment of postoperative complications. CD was diagnosed according to Consensus opinion on diagnosis and treatment of inflammatory bowel disease ( Beijing 2018). Patient was classitied according to the Montreal Classification. Postoperative complications were graded according to the Clavien-Dindo Criteria. Mann-Whitney U test was used for statistical analysis. Results:Among 123 patients, according to the Montreal classification, two cases (1.6%) were diagnosed at ≤16 years old (type A1), 66 cases (53.7%) were diagnosed at 17 to 40 years old (type A2), and 55 cases (44.7%) were diagnosed at >40 years old (type A3). The lesions were 52 cases (42.3%) of terminal ileum (L1) type, 20 cases (16.3%) of colon (L2) type, and 51 cases (41.5%) of ileocolon (L3) type. Four cases (3.2%) were non-stenosis and non-penetrating (B1) type, 87 cases (70.7%) were stenosis (B2) type, and 32 cases (26.0%) were penetrating (B3) type. Eighteen patients (14.6%) underwent emergency surgery due to complete intestinal obstruction (10 cases), gastrointestinal perforation (five cases), gastrointestinal bleeding (two cases), and rectovesical fistula complicated with septic shock (one case). One hundred and five patients (85.4%) received selective surgery due to poor conservative treatment effects. 51 cases (41.5%) underwent traditional open surgery and 72 cases (58.5%) underwent laparoscopic surgery. Nineteen patients (15.4%) received temporary or permanent ostomy. The preoperative C reactive protein level of patients with emergency surgery was higher than that of patients undergoing selective surgery ((39.23±24.13) mg/L vs. (11.48±2.68) mg/L), while the levels of plasma albumin (ALB) and pre-ALB were lower than those of patients receiving selective surgery ((29.90±10.60) g/L vs. (38.38±8.30) g/L, (146.00±125.49) mg/L vs. (209.06±61.19) mg/L), and the differences were statistically significant ( Z=9.603, 8.754 and 7.111, all P<0.01). During the follow-up, a total of 23 cases (18.7%) developed postoperative complications, including one case of postoperative intra-abdominal hemorrhage and underwent re-operation (Clavien-Dindo grade Ⅲ complication); four cases of anastomotic leakage after operation; six cases of postoperative paralytic ileus; 11 cases of surgical site infection, all of which were Clavien-Dindo grade Ⅱ complications, and one case of deep venous thrombosis of lower extremity. No patient with severe intraoperative complication was observed, and no patients died during the operation or hospitalization. The postoperative exhaust time of patients was (3.2±1.4) d, the time of open fluid diet was (5.8±0.8) d, the length of hospital stay was (18.0±14.1) d, and the length of postoperative hospital stay was (11.2±8.8) d. Conclusions:The concept of multidisciplinary collaboration should be emphasized in the treatment of CD. Surgical treatment can effectively control the complications and improve the quality of life of patients, but the timing of operation and the choice of surgical methods should be decided prudently after perioperative treatment, multi-disciplinary participated and regulation of the internal environment. The standardized and targeted treatments for the surgical difficulties of inflammatory bowel disease should be conducted.

Objective: This study aims to analyze the relationship among the clinical features, radiologic characteristics and pathological diagnosis in patients with solitary pulmonary nodules, and establish a prediction model for the probability of malignancy. Methods: Clinical data of 372 patients with solitary pulmonary nodules who underwent surgical resection with definite postoperative pathological diagnosis were retrospectively analyzed. In these cases, we collected clinical and radiologic features including gender, age, smoking history, history of tumor, family history of cancer, the location of lesion, ground-glass opacity, maximum diameter, calcification, vessel convergence sign, vacuole sign, pleural indentation, speculation and lobulation. The cases were divided to modeling group (268 cases) and validation group (104 cases). A new prediction model was established by logistic regression analying the data from modeling group. Then the data of validation group was planned to validate the efficiency of the new model, and was compared with three classical models(Mayo model, VA model and LiYun model). With the calculated probability values for each model from validation group, SPSS 22.0 was used to draw the receiver operating characteristic curve, to assess the predictive value of this new model. Results: 112 benign SPNs and 156 malignant SPNs were included in modeling group. Multivariable logistic regression analysis showed that gender, age, history of tumor, ground -glass opacity, maximum diameter, and speculation were independent predictors of malignancy in patients with SPN(P<0.05). We calculated a prediction model for the probability of malignancy as follow: p=e^x/(1+ e^x), x=-4.8029-0.743×gender+ 0.057×age+ 1.306×history of tumor+ 1.305×ground-glass opacity+ 0.051×maximum diameter+ 1.043×speculation. When the data of validation group was added to the four-mathematical prediction model, The area under the curve of our mathematical prediction model was 0.742, which is greater than other models (Mayo 0.696, VA 0.634, LiYun 0.681), while the differences between any two of the four models were not significant (P>0.05). Conclusions Age of patient, gender, history of tumor, ground-glass opacity, maximum diameter and speculation are independent predictors of malignancy in patients with solitary pulmonary nodule. This logistic regression prediction mathematic model is not inferior to those classical models in estimating the prognosis of SPNs.

Objective To investigate the effects of anti?PD?L1 monoclonal antibody and EGFR?TKI on expression of soluble PD?L1 and function of T lymphocytes in EGFR?mutated lung cancer cells. Methods Flow cytometry was used to analyze the expression of membrane PD?LI. ELISA was performed to detect the level of sPD?L1 in the supernatant of cultured EGFR?mutated and wild type lung cancer cells before and after erlotinib treatment. After treated with anti?PD?L1 monoclonal antibody alone and in combination with erlotinib, the proliferation of T lymphocytes in co?culture system was measured using Cell Counting Kit?8 ( CCK?8) assay. The expression levels of PD?LI and IFN?γin tumor cells and T lymphocytes treated with erlotinib in co?culture system were analyzed by flow cytometry and ELISA, respectively. Results PD?L1 was highly expressed in EGFR?mutated lung cancer PC9 cells (78.7±3.1)% and HCC827 cells (82. 7±2.6)%.After treated with erlotinib, the expression rates of membrane PD?L1 in PC9 and HCC827 cells were down?regulated (64.7%±3.1% and 73.0%±2.6%, respectively),significantly lower than that in the two cell lines without erlotinib treatment ( P<0.05) , and the expression levels of sPD?L1 in the supernatant of PC9 and HCC827 cells were also down?regulated ( 0. 680 ± 0. 120) ng/ml and ( 0. 903 ± 0. 047) ng/ml, respectively, significantly lower than that in the two cell lines without erlotinib treatment ( P<0. 01 ) . However, no significant changes of membrane PD?L1 and sPD?L1 expression were found in EGFR wild type lung cancer cells ( H1299 and A549) before and after erlotinib treatment. In the co?culture system composed of T cells and EGFR?mutated lung cancer cells, treatment with erlotinib alone promoted the proliferation of T lymphocytes (P<0.05), and combined treatment of anti?PD?L1 monoclonal antibody with erlotinib had a stronger effect ( P<0.05) . In the co?culture system composed of T cells and EGFR wild type cell lines, the proliferation of T cells was not changed after using erlotinib alone or combination of erlotinib and anti?PD?L1 monoclonal antibody ( P>0.05) . Before and after treatment with erlotinib, the secretion levels of IFN?γwere (856.0± 70. 3) pg/ml and ( 1 697. 3 ± 161. 0) pg/ml, respectively, showing a significant difference ( P<0.001). The expression rates of membrane PD?L1 were (76.2±0.5)% and (50.9±0.9)%, respectively, also showing a significant difference ( P<0.001) . However, no significant changes in the expression of IFN?γ and membrane PD?L1 were found in the co?culture system composed of T cells and A549 cells. Conclusions Anti?PD?L1 monoclonal antibody combined with EGFR?TKI can effectively promote the proliferation and secretion function of T lymphocytes in the microenvironment of EGFR?mutated lung cancer cells.

Objective To investigate the effects of anti?PD?L1 monoclonal antibody and EGFR?TKI on expression of soluble PD?L1 and function of T lymphocytes in EGFR?mutated lung cancer cells. Methods Flow cytometry was used to analyze the expression of membrane PD?LI. ELISA was performed to detect the level of sPD?L1 in the supernatant of cultured EGFR?mutated and wild type lung cancer cells before and after erlotinib treatment. After treated with anti?PD?L1 monoclonal antibody alone and in combination with erlotinib, the proliferation of T lymphocytes in co?culture system was measured using Cell Counting Kit?8 ( CCK?8) assay. The expression levels of PD?LI and IFN?γin tumor cells and T lymphocytes treated with erlotinib in co?culture system were analyzed by flow cytometry and ELISA, respectively. Results PD?L1 was highly expressed in EGFR?mutated lung cancer PC9 cells (78.7±3.1)% and HCC827 cells (82. 7±2.6)%.After treated with erlotinib, the expression rates of membrane PD?L1 in PC9 and HCC827 cells were down?regulated (64.7%±3.1% and 73.0%±2.6%, respectively),significantly lower than that in the two cell lines without erlotinib treatment ( P<0.05) , and the expression levels of sPD?L1 in the supernatant of PC9 and HCC827 cells were also down?regulated ( 0. 680 ± 0. 120) ng/ml and ( 0. 903 ± 0. 047) ng/ml, respectively, significantly lower than that in the two cell lines without erlotinib treatment ( P<0. 01 ) . However, no significant changes of membrane PD?L1 and sPD?L1 expression were found in EGFR wild type lung cancer cells ( H1299 and A549) before and after erlotinib treatment. In the co?culture system composed of T cells and EGFR?mutated lung cancer cells, treatment with erlotinib alone promoted the proliferation of T lymphocytes (P<0.05), and combined treatment of anti?PD?L1 monoclonal antibody with erlotinib had a stronger effect ( P<0.05) . In the co?culture system composed of T cells and EGFR wild type cell lines, the proliferation of T cells was not changed after using erlotinib alone or combination of erlotinib and anti?PD?L1 monoclonal antibody ( P>0.05) . Before and after treatment with erlotinib, the secretion levels of IFN?γwere (856.0± 70. 3) pg/ml and ( 1 697. 3 ± 161. 0) pg/ml, respectively, showing a significant difference ( P<0.001). The expression rates of membrane PD?L1 were (76.2±0.5)% and (50.9±0.9)%, respectively, also showing a significant difference ( P<0.001) . However, no significant changes in the expression of IFN?γ and membrane PD?L1 were found in the co?culture system composed of T cells and A549 cells. Conclusions Anti?PD?L1 monoclonal antibody combined with EGFR?TKI can effectively promote the proliferation and secretion function of T lymphocytes in the microenvironment of EGFR?mutated lung cancer cells.

OBJECTIVETo detect FLG gene mutations in 10 families affected with ichthyosis vulgaris and to explore mutational hot spot of the FLG gene in Chinese Han population.

METHODSPCR and direct sequencing were carried out to identify potential mutations of the FLG gene in above families. One hundred healthy individuals were analyzed as normal controls.

RESULTSThree mutations (3321delA, 5757delCCAG and S2706X) were identified in 7 families. A homozygous mutation 3321delA was also detected in two unrelated patients. No mutations were found in the remaining three families. Neither of the null mutations (5757delCCAG and S2706X) was found in the 100 controls. However, for 3321delA, a heterozygous mutation was also found in two of the controls.

CONCLUSIONThree FLG mutations have been identified in the selected families with ichthyosis vulgaris, and the 3321delA mutation was most prevalent (46.9%). Mutations 5757delCCAG and S2706X were first found in patients with ichthyosis vulgaris. Other candidate genes may underlie the disease in those without a FLG mutation.

Asian Continental Ancestry Group ; Base Sequence ; China ; Female ; Genotype ; Humans ; Ichthyosis Vulgaris ; genetics ; Intermediate Filament Proteins ; genetics ; Male ; Mutation ; Pedigree ; Phenotype

Objective To explore the significance and the change of miRNAs expression profile in different period of HBV infection.Methods Establish the detection method of microRNA(miRNA) by RNA DNA probe liquid chip (flexible multi-analyte profiling,xMap) and estimate the specificity,repeatability and accuracy of this method.From October 2010 to October 2011,collect HBV infected patients' periphcral blood of the First affiliated hospital of SooChow University,including acute hepatitis B,chronic hepatitis B,hepatits B liver cirrhosis,liver cancer patients and health control,each group contains 40 cases.The levels of miR-191,-223,-222,-145,-21,-31,-126,-20a,-372 of peripheral blood mononuclear cell were detected by xMap liquid chiptechnology.The level of miR-103 was taken as reference.The ratio of (the mean fluorescence intensity of target miRNAs-corresponding backgroud mean fluorescence intensity)/( the mean fluorescence intensity of miR-103-corresponding backgroud mean fluorescence intensity) as a valid data and analysis the characteristics of miRNAs expression.The SPSS 17.0 was used as statistical software.The single factor analysis of variance was used as the method to analysis group comparison,and make multiple comparison by the LSD-t method if the result with a significant difference.Results The specificity of Xmap liquid chip method to detect miRNAs was 100％ ;The repeated experiment proved that the CV value was less than 5％ in the high value reference miRNA test,less than 10％ in the low value reference miRNAs test;the accruracy experiment proved that the recovery rate was ( 100 ± 5)％ in the nine miRNAs.There were no statistically differences with miR-222 ( F =1.32,P ＞ 0.05),-191 ( F =1.98,P ＞ 0.05),-145 ( F =0.78,P＞0.05),-21(F=0.64,P ＞0.05),-31 (F =0.83,P ＞0.05),-372(F =1.75,P ＞0.05)in different groups; There was statistically significant differences in miR-223 ( F =14.56,P ＜ 0.05) among different groups,with the highest expression level in actue hepatitis B group (15.37 ± 4.01),and the lowest expression level in liver cancer group (6.91 ±3.18) ; There was statistically significant differences in miR126 (F =17.43,P ＜ 0.05)among different groups,with the highest expression level in health control group (6.33 ±2.75) and the lowest expression level in liver cancer group (2.38 ± 1.07).There were statistically significant differences in miR-20a ( F =19.484,P ＜ 0.05) among different groups,with the highest expression level in health control group (0.33 ±0.18) and the lowest expression level in liver cancer group (0.81 ±0.24).Conclusion The detection method of miRNA by Xmap liquid chip has strong specificity,high accuracy and good repeatability,suitable for large throughput clinical testing.The study for miRNA in HBV infected diseases provides a new clue to the research of chronic progress mechanism.

Objective To determine whether the extent of airflow obstruction is associated with left ventricular function in mild-to-moderate chronic obstructive pulmonary disease (COPD) patients.Methods Left ventricle end diastolic volume ( LVEDV ),left ventricle end systolic volume ( LVESV ),left ventricle stroke volume( LVSV),left ventricle ejection fraction( LVEF),heart rate ( HR),cardiac output ( CO) and cardiac index (CI) were measured by ultrasonic cardiogram.Thirty-one patients with chronic bronchitis,42 mild-to-moderate COPD patients and 16 controls with normal lung function were recruited.The relations between the extent of airflow obstruction and the impairment of left ventricular function were analyzed.Results There were no significant differences of LVEDV,LVESV,LVSV,HR,CO or CI between the control and chronic bronchitis groups.LVEDV,LVESV,LVSV,CO and CI of chronic bronchitis group were significantly higher than those of mild-to-moderate COPD group while HR was lower.LVEDV,LVESV,LVSV,CO and CI had a positive correlation with forced expiratory volume in 1 second ( FEV1 ),forced vital capacity (FVC) and FEV1/FVC ratio.And HR had a negative correlation with FEV1 and FEV1/FVC.LVEF was positively correlated with FVC,but not with FEV1 and FEV1/FVC.LVEDV,LVESV,LVSV,HR,CO and CI were linearly related with FEV1.Conclusions Left ventricular function is maintained in chronic bronchitis patients. Left ventricular function,especially left ventricular end diastolic filling,deteriorates among the mild-to-moderate COPD patients.The extent of airflow obstruction may reflect the impairment of left ventricular function in COPD patients.

Objective Clinical strains of Shigella dysenteriae isolated from eastern parts of India in 1988, 1995 and 2002 were examined for the presence of class 1, 2 and 3 integrons which closely related with drug resistance and the distribution of resistance gene cassette in order to clarify the influence of integron system on drug resistance of Shigella dysenteriae. Methods Susceptibility to antimicrobial agents was tested by the disk agar diffusion method. Class 1, 2 and 3 integron genes (intI) were screened by polymerase chain reaction (PCR) in 16 clinical strains with drug resistance.The variable regions of gene cassette of positive strains were sequenced. Results All 16 isolates were resistant to at least 4 agents including β-lactams, aminoglycosides, tetracyclines, sulfonamides,chloramphenicols and quinolones. Class 1 integron gene was detected in 13 strains and all isolates carried class 2 integron which indicated that strains with two integron structures were detected simultaneously and class 3 integron was not detected. Class 1 integron inserted gene cassette was mainly blaara30 -aadA 1 family, conferring resistance to β-lactamase, spectinomycin and streptomycins isolates carried class 2 integron were mainly dfrAl-satl genes cassettes conferring resistance to methoxybenzyl aminopyrimidine and streptothricin, while dfrA\-sat\-aadA\ genes were present only in 4 isolates. Conclusions These data indicate that class 2 integrons are widespread in Shigella dysenteriae strains, and closely associated with multidrug resistance of Shigella.

Objective To investigate the role of the pulmonary function, arterial blood gas analysis, the St George respiratory questionnaire (SGRQ) score and nutritional status in evaluating the development of chronic obstructive pulmonary disease (COPD). Method Changes of the above-mentioned parameters were analyzed in hospitalized patients suffered from moderate to severe COPD and compared with parameters of those who had acute exacerbations when admitted to hospital. Results The forced expiratory volume in one second (FEV1)% pred and oxygenation index of the patients declined significantly with the development of the disease after one, two, three, four times in hospital [(68.43 ± 3.09)%, (61.27 ± 3.38 )%, (42.05 ± 4.16)%, (33.64 ± 3.34)% and 435.55 ± 10.23, 404.35 ± 11.56, 358.38 ± 13.21, 321.29 ± 11.78] (P < 0.05), while the level of PaCO2 increased significantly in moderate COPD [(36.23 ± 3.62), (45.44 ± 4.67), (57.82 ± 4.12), (78.28 ± 5.21 )mm Hg (1 mm Hg = 0.133 kPa) ] (P<0.05). The oxygenation index was declined significantly in severe COPD. The scores of SGRQ score declined significantly in all patients while the level of hemoglobin, albumin and body mass index did not change significantly. Conclusions Acute ex-acerbation attributes to the deterioration of pulmonary function and life quality of COPD patients. Monitoring the changes of pulmonary function, arterial blood gas analysis and SGRQ score may be useful in the evaluation of the development of COPD.