Objective: To construct a Family with sequence similarity 50 member A(FAM50A) gene knockout mouse insulinoma pancreatic β-cell line Beta-TC-6 using CRISPR/Cas9 gene editing technology and to prepare polyclonal antibodies specifically recognizing FAM50A. Methods: Two guide RNAs(sgRNAs) targeting the FAM50A gene were designed,and a recombinant plasmid expressing blue fluorescent protein(BFP) was constructed for gene knockout.The successfully constructed plasmid was transfected into Beta-TC-6 cells,and BFP-positive single cells were isolated for clonal expansion.The expanded monoclonal cell lines were genotyped by Sanger sequencing,and FAM50A protein expression was assessed by Western blot.Purified human recombinant FAM50A protein was used to immunize New Zealand rabbits for the preparation of a polyclonal antibody.The specificity of the prepared antibody was then validated using the successfully established FAM50A knockout cell line. Results: A monoclonal cell line with a successful knockout of the FAM50A gene was identified.Sanger sequencing confirmed base deletions at the target site.Western blot analysis showed a complete absence of FAM50A protein expression in this cell line.The prepared polyclonal antibody successfully recognized endogenous murine FAM50A protein in wild-type Beta-TC-6 cells and in hTERT-RPE1 cells overexpressing human FAM50A-GFP fusion protein,while no signal was detected in the FAM50A knockout cells. Conclusion This study successfully established a FAM50A gene knockout Beta-TC-6 cell model and generated a FAM50A polyclonal antibody,providing powerful tools for future research.

ObjectiveTo investigate the correlation between the color， odor and volatile components of Paeoniae Radix Alba（PRA） and its processed products， and to examine the effects of different processing methods on the odor and color formation of PRA. MethodThe odor and chromaticity information of PRA， honey chaff-fried PRA and honey bran-fried PRA were identified by electronic nose and colorimeter， and the volatile components in the different processed products of PRA were identified and relatively quantified by headspace gas chromatography-mass spectrometry（HS-GC-MS）， and analyzed using principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）， then the differential key flavor components among the three were screened according to the principles of variable importance in the projection（VIP） value>1 and relative odor activity value（ROAV）≥1. Pearson correlation analysis was used to investigate the association between the differential flavor components and the colorimetric values and electronic nose sensors， respectively. ResultAfter being fried with honey chaff and honey bran， the lightness value（L^*） of PRA decreased， and red-green value（a^*） and yellow-blue value（b^*） increased significantly（P<0.05， P<0.01）. The odor differences were mainly reflected in the S1， S2， S4， S5， S6， S8 and S11 sensors， and the results of PCA of the electronic nose indicated that the odor differences among PRA， honey chaff-fried PRA and honey bran-fried PRA were obvious， among which the overall odor intensity of honey bran-fried PRA was higher than that of honey chaff-fried PRA. A total of 47 volatile components were identified from PRA and its processed products， including 21 for PRA， 36 for honey chaff-fried PRA， and 37 for honey bran-fried PRA. Odor analysis revealed that 12， 24 and 22 volatile components may be the key flavor components in PRA， honey chaff-fried PRA and honey bran-fried PRA， respectively. Correlation analysis showed that the L^* of the decoction pieces was negatively correlated with the content of the Maillard reaction products， the a^* and b^* were positively correlated with the content of the Maillard reaction products， the S1 and S8 sensors were negatively correlated with the content of the Maillard reaction products， and the S2， S4， S5， S6 and S11 sensors were positively correlated with the Maillard reaction products. ConclusionThe color of PRA is deepened after being stir-fried with honey chaff and honey bran， and 5-hydroxymethylfurfural， furfural and other components are generated at the same time， which is in line with the theory of burning aroma strengthens the spleen of stir-fried with honey chaff and honey bran. Honey bran-fried PRA has a stronger Maillard reaction than honey chaff-fried PRA， which makes honey bran-fried PRA with a burnt flavor and a dark yellow color， while honey chaff-fried PRA has a sweet flavor and a bright yellow color.

Objective:To investigate the protective effect of berberine hydrochloride on intestinal mucosal barrier damage in sepsis rats and its mechanism.Methods:Forty-eight male SD rats were divided into a control group (Sham group, 6 cases), a sepsis model group (LPS group, 14 cases), a berberine hydrochloride intervention group (Ber group, 14 cases), and a Notch signaling pathway inhibition group (DAPT group, 14 cases) according to random number table method. The DAPT group was intraperitoneally injected with 5 mg/kg Notch signaling pathway inhibition DAPT 2 hours before modeling. The sepsis model was established by intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS); Sham group was injected with an equal amount of saline (2 mL). The Ber group and DAPT group were treated with gavage of 50 mg/kg berberine hydrochloride 2 hours after modeling; Sham group and LPS group were treated with gavage of an equal amount of saline (2 mL). The temperature, weight, behavior and survival rate of rats were observed at 0, 6, 12 and 24 hours of modeling. After 24 hours of modeling, abdominal aortic blood was collected under anesthesia, and intestinal tissues were obtained after euthanasia. The pathological changes of ileum were observed under light microscope. The ultrastructure of ileum was observed under transmission electron microscope. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of serum diamine oxidase (DAO), intestinal fatty acid binding protein (iFABP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Real time-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expressions of tight junction proteins (Occludin and Claudin1), Notch1 and their downstream target signals in the ileum tissue.Results:After 24 hours of modeling, compared with the Sham group, the LPS group, Ber group, and DAPT group showed a decrease in weight and an increase in temperature. Among them, the LPS group showed the most significant changes, followed by the DAPT group, and the Ber group showed the least significant changes. The survival rates of the LPS group, Ber group, and DAPT group were all lower than those of the Sham group [42.9% (6/14), 57.1% (8/14), 57.1% (8/14) vs. 100% (6/6)], and six rats were taken from each group for subsequent testing. Macroscopic observation of the intestine showed that the LPS group had the most severe edema in the ileum tissue and abdominal bleeding, with significant improvement in the Ber group and followed by the DAPT group. Under the light microscope, the LPS group showed disordered arrangement of glandular tissue in the ileum mucosa, significantly reduced goblet cells, and extensive infiltration of inflammatory cells, which were significantly improved in the Ber group but less improved in the DAPT group. Under electron microscopy, the LPS group showed extensive shedding of ileal microvilli and severe damage to the tight junction complex structure of intestinal epithelial cells, which was significantly improved in the Ber group but less improved in the DAPT group. The levels of serum DAO, iFABP, TNF-α, IL-6 in the LPS group were significantly higher than those in the Sham group, while the above indicators in the Ber group were significantly lower than those in the LPS group [DAO (μg/L): 4.94±0.44 vs. 6.53±0.49, iFABP (ng/L): 709.67±176.97 vs. 1 417.71±431.44, TNF-α (ng/L): 74.70±8.15 vs. 110.36±3.51, IL-6 (ng/L): 77.34±9.80 vs. 101.65±6.92, all P < 0.01], while the above indicators in the DAPT group were significantly higher than those in the Ber group. The results of RT-PCR and Western blotting showed that the mRNA and protein expressions of Occludin, Claudin1, Notch1, and Hes1 in the ileum tissue of LPS group rats were decreased compared to the Sham group, which were significantly increased in the Ber group compared with the LPS group [mRNA expression: Occludin mRNA (2 -ΔΔCt): 1.61±0.74 vs. 0.30±0.12, Claudin1 mRNA (2 -ΔΔCt): 1.97±0.37 vs. 0.58±0.14, Notch1 mRNA (2 -ΔΔCt): 1.29±0.29 vs. 0.36±0.10, Hes1 mRNA (2 -ΔΔCt): 1.22±0.39 vs. 0.27±0.04; protein expression: Occludin/GAPDH: 1.17±0.14 vs. 0.74±0.04, Claudin1/GAPDH: 1.14±0.06 vs. 0.58±0.10, Notch1/GAPDH: 0.87±0.11 vs. 0.56±0.09, Hes1/GAPDH: 1.02±0.13 vs. 0.62±0.01; all P < 0.05], while those in the DAPT group were significantly lower than those in the Ber group. Conclusion:Early use of berberine hydrochloride can significantly improve intestinal mucosal barrier damage in sepsis rats, and its mechanism may be related to inhibiting inflammatory response and regulating the expression of intestinal mechanical barrier tight junction protein through Notch1 signal.

Clinical data from 11 previously diagnosed and treated patients with hyperthyroidism(Graves′ disease) complicated by liver failure were collected. Among them, 4 cases were drug-induced liver injury leading to liver failure, 1 case had a history of schistosomal liver cirrhosis combined with hyperthyroidism, and 6 cases had hyperthyroidism-induced liver injury(HILI) leading to liver failure. During hospitalization, all patients received supportive therapy and symptomatic treatment with β-blockers. Nine patients were treated with glucocorticoids and artificial liver support therapy. Among the 11 patients, 2 died, 8 patients achieved normal thyroid and liver function within 1-12 months after treatment, and 1 patient with liver cirrhosis had stable liver function in the later stage. After improvement in liver function, 7 patients received isotope therapy, 1 patient underwent total thyroidectomy, and 1 patient received medication. These results indicate that the clinical characteristics differ for drug-induced liver injury and HILI-related liver failure. Early initiation of artificial liver support therapy, in addition to β-blockers and glucocorticoids, is important in alleviating thyroid toxicity and liver damage, thus creating an opportunity for subsequent radioactive iodine or surgical treatment.

Objective:To explore the metabolic outcomes of type 2 diabetes patients with different durations after 1 year treatment under the standardized metabolic disease management model.Methods:(1)From September 2017 to September 2018, 345 type 2 diabetes patients in the Standardized Metabolic Management Center(MMC) of Shanghai General Hospital were recruited and included in this research. They were divided into newly-diagnosed type 2 diabetes(duration≤1 year) and long-term groups(duration>1 year). The general characteristics, blood pressures, glycemic levels, lipids levels, control rates and comprehensive compliance rates(blood glucose, pressure and lipids all reached targets) were compared at baseline between 2 groups.(2)All patients underwent one year standardized management, and metabolic indicators mentioned above and control rates at the time were compared as well.Results:(1) At baseline, compared with long-term group, patients in newly-diagnosed type 2 diabetes group were younger ( P<0.01), and 2 h blood glucose level after glucose loading were higher [(15.20±5.26 vs 13.68±4.94) mmol/L, P<0.01]. (2) After one year standardized management, body weight, blood pressure, glucose and lipids metabolism in all patients were significantly improved. Compared with patients in long-term group, newly-diagnosed type 2 diabetes patients achieved better glycemic level [fasting blood glucose(6.27±1.56 vs 7.63±2.08) mmol/L, P<0.01; glycated hemoglobin(6.33±0.96 vs 7.23±1.37) %, P<0.01] , and had higher HOMA-β [(74.01±56.45 vs 40.17±37.07) %, P<0.01]. The glycemic control, blood pressure and blood lipids control rates in both groups increased significantly in one year. Comprehensive compliance rate of the whole patients increased from 5.80% to 24.06%. The metabolism indexes of the newly-diagnosed type 2 diabetes group were better than those of the long-term group[comprehensive compliance rate: (24.73% vs 17.18%, P=0.087, glycemic control rate(84.62% vs 53.37%, P<0.01)]. Conclusion:Standardized metabolic disease management promoted the overall improvement in blood glucose, blood pressure, and lipids levels in type 2 diabetes patients, especially in terms of blood glucose and those of the newly-diagnosed type 2 diabetes. In the future, we should focus on the early diagnosis and treatment of type 2 diabetes, actively promote the MMC model and stress the integrated management of blood glucose, blood pressure, and blood lipid levels. We should pay more attention to the long-term patients, to improve their awareness and treatment compliance.

Objective:To explore the tumor microenvironment (TME) module associated with pancreatic ductal adenocarcinoma (PDAC) and identify prognostic biomarkers and potential immunotherapeutic targets.Methods:The genetic expression profile data were collected and selected from a dataset of 142 PDAC patients in The Cancer Genome Altas (TCGA) database and 2 microarray datasets (GSE2150, GSE62452) of 168 PDAC patients in Gene Expression Omnibus (GEO) database, and the cell type enrichment analysis of PDAC gene expression data was analyzed by xCell network tool. According to the median cell enrichment score, 142 patients from TCGA were divided into high score group and low score group, and the cell types with prognostic value were determined by univariate survival analysis and validated by GEO datasets. According to the cell type, the differential expression gene analysis and univariate survival analysis were performed to determine the prognosis related differential expression genes (DEGs), and the prognostic DEGs were analyzed by function enrichment analysis and protein-protein interaction (PPI) network analysis. At the same time, GEO dataset was used to verify the prognosis related DEGs of TCGA datasets. Finally, TISIDB database was searched for the common DEGs of TCGA and GEO database, and its correlation with immune system was analyzed.Results:Cell type enrichment analysis showed that Th1 cell and keratinocyte had the same prognostic value in both TCGA and GEO dataset; the overall survival rate of patients with high score was lower than that of those with low score, and the differences were statistically significant (all P values <0.05). 216 prognosis related DEGs were identified, and the results of functional enrichment showed that 9 of the 21 biological process items were closely related to the immune process, and 4 of the 5 KEGG (Kyoto Encyclopedia Of Genes and Genomes) pathways were closely related to the immune process. Through PPI network analysis, CCR7, CD 27, CD 5, CXCL13, ZAP70, MS4A1 and CCL19 were proved to be possibly closely associated with central genes. Through the validation of GEO datasets, there were 15 DEGs with similar prognostic value in GEO and TCGA datasets, which was searched in TISIDB dataset, and the result showed that GIMAP7 was closely related with the immune process of PDAC. Conclusions:A group of 216 TME genes and 7 central genes related to the prognosis of PDAC were identified, and 5 potential targets for immunotherapy of PDAC were provided, including CCR7, CCL19, CD 27, CXCL13 and GIMAP7.

Lymphocytic hypophysitis(LYH) is a rare autoimmune inflammatory disorder of the pituitary gland, usually affecting young women in late pregnancy or postpartum period. To enhance the knowledge of LYH, herein we reported a case of LYH in a female during postpartum who presented with pituitary crisis.

Objective: To explore the glycemic control of newly-diagnosed type 2 diabetes with different levels of baseline body mass index (BMI) after 6 months treatment under the standardized metabolic disease management model. Methods: (1) 163 patients of newly-diagnosed type 2 diabetes were divided into normal weight (BMI 18.5-23.9 kg/m²), overweight (BMI 24.0-27.9 kg/m²), and obese (BMI≥28 kg/m²) groups according to baseline BMI, the blood glucose and lipids levels were compared among 3 groups. (2) The blood glucose levels were compared among 3 groups after 6 months of standardized management. (3) The overweight and obese patients were divided into group weight loss≥5% and group weight loss<5% or weight gain in 6 months. The blood glucose levels were compared. Results: (1) At baseline, overweight and obese groups had higher homeostasis model assessment for insulin resistance and lower high density lipoprotein-cholesterol compared with normal weight group. (2) After 6 months of treatment, HbA_1C and HbA_1C reduction showed no difference among 3 groups (normal, overweight and obese) after adjusted by baseline HbA_1C. The rate of HbA_1C<7% among 3 groups were 77.78%, 83.95%, and 80.43% (P>0.05). (3) After 6 months of treatment, 32.28% overweight and obese patients lost weight by ≥ 5%, while HbA_1Cand HbA_1Creduction showed no difference between 2 groups (weight loss≥5% and weight gain or weight loss<5%) after adjusted by baseline HbA_1C. Both groups achieved good glycemic control [(6.27±1.38 vs 6.43±0.66)%], but have no significantly(P>0.05). Group weight loss≥5% had better glucose control (92.68% vs 77.91%, P<0.05). Conclusions As BMI increased, insulin resistance and lipid disorders were more serious in newly-diagnosed type 2 diabetes. After 6 months of standardized metabolic management, newly-diagnosed type 2 diabetes with different baseline BMI and weight changes both achieved good glycemic control. In addition, patients losing weight equal to or more than 5% achieved higher attainment of HbA_1C targets.

Objective: To propose a reasonable and feasible system of performance audit indicators for New Rural Cooperative Medical System(NCMS). Methods:Using the balanced score card as amended to perform the evaluation. Results:From 5 dimensions, 37 indicators were used to perform comprehensive evaluation for the NCMS. Conclusion: The indicator system is suit for the current situation, as well as audit evidence through questionnaires to obtain the required data of the system;it provides a better evaluation of the NCMS performance.

Visfatin was expressed in rat anti mouse islets,as well as in MIN6 cells. The visfatin expression was affected by various concentrations of environmental glucose (5.5 and 33.3 mmoL/L) and palmitate(0.5 mmol/L). As compared with low-level glucose medium (5.5 mmol/L, 1.0±0.11) , visfatin expression increased in media with high glucose and palmitate (1.32 ±0. 18, 1. 33±0. 15,1.72±0.27, all P<0. 05). The result suggests that visfatin seems to be involved in the regulation of insulin secretion.