Objective To investigate the effect of nuclear translocation of E2p45 related factor 2 (Nrf2)on the biological activity of melanocytes.Methods Plasmid vectors containing wild-type nrf2 gene (pcDNA-nrf2) and nls-deleted nrf2 gene (pcDNA-nrf2△nls) were constructed.B10BR normal murine melanocytes were classified into three groups,i.e.,untransfected group,wild-type nrf2 group transfected with pcDNA-nrf2,and mutated nrf2 group transfected with pcDNA-nrf2△nls.Each of the above groups were further divided into three subgroups:control subgroup receiving no treatment,hydrogen peroxide (H2O2) subgroup treated with H2O2 of 200 μmol/L for 24 hours,and combined subgroup pretreated with tert-butyl hydroquinone (TBHQ) followed by treatment with H2O2 of 200 μmol/L for 24 hours.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of cells,dopa oxidation assay to determine tyrosinase activity,Transwell assay to estimate cell migration ability,Western blot to quantify the expressions of Nrf2 and his tag fusion protein.Results TBHQ significantly enhanced the nuclear expression of Nrf2 in B10BR cells transfected with pcDNA-nrf2 or pcDNA-nrf2△nls (both P ＜ 0.01).No significant difference was observed in tyrosinase activity between untreated wild-type nrf2 group,mutated nrf2 group,and untransfected group (P ＞ 0.05).There was a statistical decrease in tyrosinase activity in the two H2O2-treated transfected groups compared with the untreated transfected groups (both P ＜ 0.05),and the decrease was reversed by TBHQ pretreatment in the wildtype nrf2 group (P ＜ 0.05),but not in the mutated nrf2 group (P ＞ 0.05).Further more,the proliferative activity of B10BR cells experienced no obvious changes in the wild-type nrf2 group (P ＞ 0.05),but was significantly reduced in the untransfected group (P ＜ 0.05) and mutated nrf2 group (P ＜ 0.01) after the H2O2 treatment compared with the corresponding untreated groups.TBHQ could protect the pcDNA-nrf2-transfected B10BR cells,but not pcDNA-nrf2△nls-transfected B10BR cells,from H2O2-induced oxidative damage.Transwell assay showed no significant difference in migration ability among these nine groups (P ＞ 0.05).Conclusions Abnormal nuclear translocation of Nrf2 could affect antioxidant activity of,proliferative activity of and tyrosinase activity in melanocytes.TBHQ may enhance the tyrosinase activity in,proliferative activity and antioxidant activity of melanocytes via activating the nuclear expression of wild type Nrf2.

Objective To express and purify the epitope peptide of human melanin-concentrating hormone receptor 1, and to evaluate its performance in the detection of autoantibodies in vitiligo patients. Methods The target gene encoding the epitope peptide of human melanin-concentrating hormone receptor 1 was synthesized, cloned to prokaryotic expression vector pGEX-4T-2 which was then transferred to E. coli BL21. The protein expression was induced by isopropy-β-D-thiogalactoside (IPTG) and identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Blocking ELISA was carried out with membrane proteins extracted from melanocytes as the blocking antigen. The antigenicity of the peptide was detected in sera from 100 patients with progressive vitiligo and 30 healthy human controls. Results The recombinant expression vector was successfully constructed, and the target protein was successfully expressed in E.coli, which was evidenced by SDS-PAGE and Western blot. With the glutathione S-transferase (GST) purification kit, the purity of the recombinant protein reached 100% when the sampling weight was less than 0.625 μg.The binding of the target protein with serum IgG antibodies from vitiligo patients could be blocked by natural membrane antigen of melanocytes. Of the 100 sera from patients with progressive vitiligo, 36 were reactive with the target protein. Conclusions The epitope peptide of human melanin-concentrating hormone receptor 1 has been successfully expressed and purified. The purified protein can bind with serum IgG antibodies from vitiligo patients, and may be applied to the detection of autoantibodies against human melanin-concentrating hormone receptor 1.

Objective To detect 10 kinds of human milk oligosaccharides (HMOS) and compare their amounts during lactational stage.Methods Breast milk samples in different stages of lactation as colostrum (day 0-7 postpartum),transitional milk (day 8-15 postpartum),and mature milk (day 16-180 postpartum) were collected and 10 HMOS in those samples were detected and quantified by ultra high performance liquid chromatography-fluorescence detection after fluorescence labeling by using standard curves.Correlations between HMOS and lactation day were conducted by Person correlation analysis method,while the differences among three stages were calculated by ANOVA test.Results Ten HMOS were successfully separated and quantified under chosen chromatographic parameters.2'FL,3'SL,6'SL,LNT,LNnT and LNFP-I were negatively correlated and 3'FL was positively correlated with lactation days.They were different in three lactational stages (P ＜ 0.05),while P1,LNFP-V and LNnFP-V showed no correlation and difference (P ＞ 0.05).Conclusion The amount of HMOS changed during lactational stages.Seven HMOS were correlated with lactation days and different in three lactational stages (P ＜ 0.05).

Objective To find out the distribution of TCM constitution on the patients with stroke in Beijing. Method The TCM Constitution Questionnaire was used to evaluate 489 patients with stroke in three hospitals in Beijing. Results The qi-deficiency constitution (140 patients, 28.63%), yin-deficiency constitution (95 patients, 19.43%), blood-stasis constitution(89 patients, 18.20%) and phlegm-heat constitution (73 patients, 14.93%) were the most common constitution, while qi-depression constitution (22 patients, 4.50%) and yang-deficiency constitution (20 patients, 4.09%) were the less common. The top three constitution in middle aged wereqi-deficiency (33 patients, 27.27%), phlegm-heat (23 patients, 19.01%) and blood stasis (22 patients, 18.18%). The top three constitution in the aged groupwere qi-deficiency (107 patients, 29.08%), yin-deficiency (76 patients, 20.65%) and blood-stasis (67 patients, 18.21%). Conclusions Stroke is closely related with imbalance of TCM constitution. The prevention and treatment of stroke patients with different ages should vary according to the constitutions.

Objective To undertake an updated genetic spectrum analysis in patients with hereditary spinocerebellar ataxia (SCA) in mainland China. Methods SCA 1, 2, 3, 6, 7, 8, 10, 12, 17 and dentatorubral-pallidoluysian atrophy (DRPLA) nucleotide repeat mutations were detected in 430 families with autosomal dominant SCA (ADCA) and 237 patients with sporadic ataxias by PCR and DNA sequencing. Subsequently, point and Indel (Insertion/deletion) mutation analyses of SCA5, SCA11, SCA13, SCA14, SCA15/16/29, SCA27, SCA31 and SCA35 were detected in 91 families with ADCA and 196 patients with sporadic ataxias excluded from SCA1, 2, 3, 6, 7, 8, 10, 12, 17 and DRPLA genotypes via PCR and Denaturing High Performance Liquid Chromatography (PCR-DHPLC), Multiplex ligation-dependent probe amplification and DNA direct sequencing analysis. Results Among the 430 ADCA families, there were 25 SCA1 (5.81%), 27 SCA2 (6.28%), 267 SCA3/MJD (62.09%), 8 SCA6 (1.86%), 8 SCA7 (1.86%), 1 SCA12 (0.23%), 1 SCA17 (0.23%) and 2 SCA35 (0.47%), and the remaining 91 families (21.16%) were genetically unidentified. Among the 237 sporadic SCA patients, there were 6 SCA1 (2.53%), 9 SCA2 (3.80%), 23 SCA3/MJD (9.70%) and 3 SCA6 (1.27%), and the remaining 196 (82.7%) were genetically unidentified. No pathogenic point mutation causing SCA5, SCA11, SCA13, SCA14, SCA27 or SCA31 subtypes was found. Conclusion SCA3/MJD is substantially the most common subtype in patients with ADCA and sporadic forms in mainland China, followed by SCA2, SCA1, SCA6 and SCA7. While SCA12, SCA17 and SCA35 are seldom found, SCA5, SCA8, SCA10, SCA11, SCA13, SCA27, SCA31 and DRPLA are very rare. The high proportion of genetically unidentified cases further verify that SCAs are of highly genetic heterogeneity, suggesting that other disease-causing genes might be involved in the negative ADCA pedigrees, and other etiological factors may involve in those sporadic cases other than genetics.

OBJECTIVETo evaluate the response rate and adverse reactions of xeloda, an analogue of 5-fluorouracil, in the treatment of relapsed and metastatic breast cancer.

METHODSTwenty-two breast cancer patients who had recurrent and metastatic measurable foci were treated from Dec. 1999 to Feb. 2000. Xeloda was given, as a single drug, at a dose of or 2,510 mg/m2/d, bid, for two weeks followed by one week rest as one cycle, at least for one cycle in each patient.

RESULTSAmong these 22 patients, there was no complete response. Rates of partial response 8(36.4%), stable disease 10(45.5%), progressive disease 4(18.2%), and clinical benefit response (CR + PR + SD) 18(81.8%). The response rate in patients who had failed in previous chemotherapy of taxanes and/or anthracycline was 30.0%-33.3%. The common adverse reactions were hand-foot syndrome, skin pigmentation, nausea, vomiting, anorexia and fatigue. Mild-moderate anemia and leukopenia were observed in 36.4% of patients. Stomatitis, dizziness, diarrhea and chest distress were present in some. One patient developed degree IV myelosuppression. Total bilirubin and alanine transaminase (ALAT) mild elevation occurred in a few patients.

CONCLUSIONXeloda is an effective drug in the treatment of patients with relapsed and metastatic breast cancer, especially for those who have failed in chemotherapy with taxanes and/or anthracycline. Xeloda is well tolerated but has mild adverse reactions.

Adult ; Aged ; Antimetabolites, Antineoplastic ; adverse effects ; therapeutic use ; Breast Neoplasms ; drug therapy ; pathology ; Capecitabine ; Deoxycytidine ; adverse effects ; analogs & derivatives ; therapeutic use ; Female ; Fluorouracil ; analogs & derivatives ; Humans ; Middle Aged ; Neoplasm Metastasis ; Recurrence

Objective:To investigate the efficacy and prognostic survival of pembrolizumab combined with apatinib and chemotherapy in the treatment of human epidermal growth factor receptor-2 (HER2)-negative advanced gastric cancer.Methods:Patients with HER2-negative advanced gastric cancer were selected from December 2019 to December 2022 as the study subjects. Forty-five patients who received chemotherapy therapy (fluorouracil+cisplatin) were randomly collected and included in control group, and 52 patients who were treated with pembrolizumab combined with apatinib were randomly selected and enrolled as observation group. The difference in short-term efficacy was compared. The levels of serum tumor markers and immune function (CD3 +, CD4 +, CD8 +, CD4 +/CD8 +) were recorded. The long-term efficacy and adverse reactions of patients were compared. Measurement data conforming to the normal distribution were expressed as xˉ± s, and the mean comparison between groups was performed by independent sample t test. Chi-square test was used to compare the rate or composition ratio among enumeration data. P<0.05 was considered statistically significant. Results:At 6 months after treatment, the disease control rate in observation group was significantly higher than that in control group (78.85% (41/52) vs 57.78% (26/45)) ( χ2=5.01, P=0.025), but there was no statistical significance in objective response rate between groups (36.54% (19/52) vs 24.45% (11/45)) ( χ2=1.65, P=0.199). The levels of pepsinogen I, tissue polypeptide specific antigen, carcinoembryonic antigen, carbohydrate antigen 199 and CD8 + in both groups were reduced after treatment, and the levels were lower in observation group than those in control group ( t=6.06, 6.78, 4.68, 11.21, 3.45, all P<0.001). The levels of CD3 +, CD4 + and CD4 +/CD8 + were enhanced significantly in the two groups, and the observation group had higher levels after treatment ( t values were 2.10, 3.74, and 5.19; P values were 0.028, <0.001, and <0.001). After 1 year of follow-up, the survival rate in observation group with 59.62% (31/52) was significantly higher than 37.78% (17/45) in control group ( χ2=4.60, P=0.032). The progression-free survival time ((10.22±1.62) months vs (8.13±1.57) months, t=6.43, P<0.001) and overall survival time ((11.62±1.84) months vs (9.73±1.71) months, t=5.21, P<0.001) in observation group were significantly longer compared to control group. There were no statistical differences in the incidence rates of bone marrow suppression ( χ2=1.92, P=0.165), hand-foot syndrome ( χ2=3.47, P=0.062), gastrointestinal reaction ( χ2=0.32, P=0.574), hypertension ( χ2=0.94, P=0.333) and proteinuria ( χ2=2.39, P=0.122) between the two groups. Conclusion:Compared with chemotherapy, pembrolizumab combined with apatinib shows good short-term efficacy and long-term efficacy in patients with HER2-negative advanced gastric cancer.

Objective To obtain a thyroid cancer cell line integrated with luciferase and fluorescence,and to establish a metastatic nude mouse model of papillary thyroid cancer monitored with in vivo imaging system. Methods Lentivirus carrying recombinant plasmid containing luciferase gene and mCherry gene infected high malignant papillary thyroid cancer cell line (BHP10-3SCmice). The stable thyroid cancer cell line (BHP10-3mluc) labeled with luciferase and mCherry selected by hygromycin, analyzed for fluorescence with fluorescence microscopy and bioluminescence with in vitro analysis and in vivo imaging. 0.25 ml(5×106/ml)BHP10-3mluc cell suspension were injected into the left thigh intramuscular tissue of 5-week-old nude mice to establish BALB/C nude mice metastatic tumor model. The growth and metastasis of the tumors were monitored weekly with in vivo imaging system. The nude mice were sacrificed on the 42th day and the tissues with metastatic tumors were analyzed by fluorescent imaging ex vivo. The ectopic implanted tumors and metastatic lesions were verified by tissue sections with Hematoxylin and Eosin staining. Results BHP10-3mluc cells showed similar growth characteristics to the original BHP10-3SCmice cells and stably expressed luciferase and mCherry. At the end of the first week after ectopic implantation,the xenograft tumors were found and in the 6th week,the tumors were found to metastasized to adjacent lymph nodes and lungs,which was consistent with the results of pathology.The growth and metastasis of tumors can be accurately monitored with in vivo imaging system. Conclusions A PTC cell line stably expressing bioluciferase and fluorescence was successfully established.The nude mouse model of PTC metastatic tumor generated by intramuscular inoculation of those cells can be well monitored with in vivo imaging system, which provides ideal models for researches on tumor growth, metastasis,and drug treatment.

Objective:To investigate the clinical features and prognosis of pediatric autoimmune encephalitis associated with anti-glutamic acid decarboxylase 65 (GAD65) antibody.Methods:Clinical data of 2 patients diagnosed as autoimmune encephalitis associated with anti-GAD65 antibody at Department of Neurology, Beijing Children′s Hospital in 2019 were analyzed retrospectively. A literature search with “anti-GAD65 antibody”“encephalitis”“epilepsy” or “cerebellar ataxia” as key words was conducted at China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform and PubMed (up to January 2020). The clinical features and prognosis of pediatric cases with complete clinical data were retrieved and summarized.Results:Two patients with positive anti-GAD65 antibody of serum and cerebrospinal fluid were both females. The onset age of case 1 was 57 months and her main clinical manifestations were fever and unconsciousness. The cranial magnetic resonance imaging (MRI) showed diffuse T2 weighted imaging (T2WI) abnormal signals, and the electroencephalogram (EEG) showed slow waves. The onset age of case 2 was 80 months and her main clinical manifestations of were recurrent focal seizures, memory loss, and headache. The MRI showed high T2WI signal in bilateral hippocampus, and the EEG showed abnormal discharge involving the temporal area. Both cases were treated with methylprednisolone and intravenous immunoglobulin, the short-term symptoms of them were both improved. They were followed up for 6 months and 1 year respectively, the case 1 recovered completely, and the case 2 still had focal seizures. Six English reports which included 6 cases were retrieved. Together with these 2 cases, a total of 8 cases were analyzed. The clinical symptoms included seizures (6 cases), memory loss (4 cases), loss of consciousness (3 cases), behavioral abnormalities (3 cases), cognitive impairment (2 cases), headache (2 cases), autonomic symptoms (1 case), ataxia (1 case), dysphagia (1 case), and aphasia (1 case). There were 5 cases with cranial MRI abnormalities in the acute phase or sub-acute phase, of whom 3 cases had the limbic system involvement, and 2 cases were mainly had extra limbic area involvement. Three cases had hippocampal atrophy or sclerosis during follow-up. All 8 patients were treated with immunotherapy. After immunotherapy, all patients had short-term improvement. Follow-up for 6 months to 6 years showed that 3 cases with extra limbic encephalitis improved to baseline levels, and 5 limbic encephalitis cases had poor outcomes, including 1 death and 4 cases still had focal epilepsy.Conclusions:Pediatric anti-GAD65 antibody associated autoimmune encephalitis is a rare but treatable disease, including limbic encephalitis and extra limbic encephalitis. The most common clinical manifestations are seizures and memory impairment. Early diagnosis and immunotherapy can improve the symptoms in a short time. But patients with limbic encephalitis often had refractory epilepsy in the chronic phase, and have a poor long-term outcome.

Objective:To explore the application value of flexible endoscopy and rigid endoscopy in the clinical examination of chronic sinus tract wounds with different shapes.Methods:A retrospective observational study was conducted. From January 1 to December 23, 2019, a total of 46 patients with chronic sinus tract wounds, who met the inclusion criteria were admitted to the Wound Healing Center of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, including 23 males and 23 females, aged 18-81 (48±21) years. On admission, computer tomography (CT) imaging and three-dimensional reconstruction were performed to examine the shapes of wound sinus tract and classify the wounds, with the lengths of wound sinus tract by CT imaging examination (hereinafter referred to as reference lengths) recorded. The lengths of wound sinus tract were examined and measured by rigid endoscopy and flexible endoscopy. The wounds with and without obviously curved sinus tract were classified into curve group and linear group respectively, and the deviation rates between the lengths of wound sinus tract measured by flexible endoscopy or rigid endoscopy and the reference lengths (hereinafter referred to as deviation rates of lengths) in each group were calculated. The difference between the deviation rates of lengths examined by flexible endoscopy and rigid endoscopy and the differences between the above two and the deviation rate of reference lengths (0) in each group were compared. Data were statistically analyzed with paired sample t test and Wilcoxon signed rank sum test. Results:CT imaging and three-dimensional reconstruction showed that there were 4 types of wound sinus tract, including tubular (36/46), lamellar (4/46), club-mallet (4/46), and irregular (2/46) shape. Tubular wounds were further divided into type I (23/36), type L (4/36), and type Y (9/36). Wounds with type I tubular, lamellar, and club-mallet sinus tract were classified into linear group (31/46), while those with type Y tubular, type L tubular, and irregular sinus tract were classified into curve group (15/46). In linear group, the deviation rates of lengths examined and measured by rigid endoscopy and flexible endoscopy were 0. In curve group, the deviation rate of lengths examined and measured by flexible endoscopy was 0 (0, 0.58%), which was significantly lower than 41.18% (31.68%, 48.41%) examined and measured by rigid endoscopy, Z=-3.408, P<0.01; the deviation rate of lengths examined and measured by rigid endoscopy (40±19)% was significantly higher than the deviation rate of reference lengths ( t=8.343, P<0.01), while the deviation rate of the lengths examined and measured by flexible endoscopy was similar to the deviation rate of reference lengths ( Z=-1.342, P>0.05). Conclusions:Compared with rigid endoscopy, flexible endoscopy can observe the internal characteristics of chronic sinus tract wounds in a wider range in the clinical examination of this kind of wound, especially for the exploration of curved chronic sinus tract wounds. The promotion of this method will be conducive to the diagnosis and treatment of chronic sinus tract wounds.