Objective To investigate the significance of miR-103 as noninvasive biomarker for diagnosis of nonalcoholic fatty liver disease (NAFLD).Methods The serum samples were collected from healthy subjects and NALFD patients to detect biochemical parameters.NucleoZOL method was used to isolate serum miRNA.SYBR Green qPCR was used for relatively quantitative analysis of miR103a-2.The correlations between miR-103a-2 and biochemical parameters were analyzed by Spearman method.Results Compared with healthy control group,the levels of total cholesterol (TC),triacylglyceride (TG),alanine aminotransferase (ALT) and glutamyl transpeptidase (γ-GGT) were all significantly increased in NAFLD group (P ＜ 0.01).Compared with the NAFLD group accompanying ortholiposis,the levels of TC,TG and low density lipoprotein-cholesterol (LDL-C) were all significantly increased in the NAFLD group accompanying dyslipidemia (P ＜0.01).Compared with the healthy control group,the level of serum miR-103a-2 increased in both NAFLD with ortholiposis group (P ＜ 0.05) and NAFLD with dyslipidemia group (P ＜ 0.01).The level of serum miR-103a-2 was positively correlated with serum TC (r =0.495,P =0.001).Conclusion Serum miR-103a-2 may be a more sensitive parameter for noninvasive screening of NAFLD than the parameters of blood lipid.

Objective To test the mutation locus of uridine diphospho-glucuronosyltransferase gene (UGT1A1) in a Chinese patient with Crigler-Najjar syndrome type Ⅰ and her family members,analyzing the genetic characteristics of the pedigree.Methods Genomic DNA was extracted from the patient and her family members and other 50 full-term infants with normal serum bilirubin as a healthy control group.Fifty cases of full-term newborn whose serum bilirubin level were nomal were study as controls.The promoter and all exons of UGT1A1 gene were amplified by the method of polymerase chain reactions (PCR),and mutations were identified by direct sequencing.Results The propositus and her miscarriage sister were homozygous for a nonsense mutation at nucleotide number 715 (715C ＞ T) in exon 1 of gene UGT1A1,substituting of stop codon (TAG) for glutamine (CAG) at position 239 (Q239X).The other 5 members were heterozygous in the same mutation locus.A TA insertion mutation and a G71R mutation in exon 1 were observed in the family members.The patient and her sister were homozygous of A(TA)7TAA mutation while other four were heterozygous.Propositus,grandmother,mother and her younger brother were heterozygous of G71 R mutation.No mutation was found in exons 2-5.No mutation was found in other fifty healthy cases in the healthy control group.Conclusions Q239X homozygous mutations is considered to be the lethal gene in this Crigler-Najjar syndrome family.Collaborative G71 R and A(TA)7TAA mutations may further reduce the enzyme activity of UGT1A1,causing varying degrees of bilirubin disorder.

Objective To construct GST/HIF1α fusion protein expression vector and induce its expression in Escherichia coli (E.coli). Methods The coding sequence of hypoxia inducible factor-la (HIF-la) and its deletion fragments were amplified from the plasmid pcD-NA3.1-HIF-la by PCR and inserted into pGEX-4T-2 by BamHI and Not I. The positive recombinanls were identified by restriction endonu-clease digestion and DNA sequencing. Then they were transformed into E.coli BL21, induced by IPTG and identified by SDS-PAGE and Western blot. Results The prokaryotic expression plasmid pGEX-4T-2-HIF-lα and its deletion mutants were successfully constructed and confirmed by enzyme digestion and sequencing. The desired CST/HIF-1α fusion proteins were expressed and confirmed by Western blot. Conclusion The prokaryotic expression plasmid of HIF-la and its deletion mutants were successfully constructed and the expression of fu-sion proteins was confirmed. This study provides the basis for the further research on purifying HIF-la protein and the biological function of HIF-lα.

Objective To construct the expression plasmid of human PAK4 gene and identify its recombinant protein expression.Methods Total RISA was extracted from human breast cancer MCF-7 cells.The hPAKA coding sequence was amplified by polymerase chain reaction (PCR) method and subcloned into pEBG vector.After the target region was sequenced.the plasmid was transfected into HEK293 cell line.The expression of the recombinant plasmid in HEK293 cells was proved by Western blot.Results hPAKA had been constructed into the expressing vector pEBG successfully.The length of the fragment was 1 800 bp,identified by restriction enzymes digestion.The expression of pEBG-hPAK4 fusion protein was detected by Western blot,with a molecular weight 94 KDa and was pulled down by Qutathione Sepharose 4B.Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector,and the expression of pEBG-hPAK4 fusion protein was identified and pulled down by Glutathione Sepharose 4B.

A new method using on-line cleanup technology combined with liquid chromatography tandem mass spectrometry ( UHPLC-MS/MS) was developed for the determination of seven kinds of β2-agonists residues, Formoterol, Salmeterol, Carbuterol, Clenisopenterol, Clenpenterol, Clencyclohexerol and Clenbuterol-hydroxymethyl in livestock manure. The sample was sufficiently extracted by acidic acetonitrile and diluted by 0. 2% formic acid. The extract was online purified on HyperSep Retain CX column where the sample matrix was washed away and the analytes were retained. The analytes were eluted into Hypersil Gold C18 column by 2% Ammonia-methanol solution. The seven β2-agonists were detected in selected reaction monitoring ( SRM) mode via positive electrospray ionization ( ESI+) by liquid chromatography-tandem mass spectrometry. The results showed good linearity correlation coefficients over 0 . 9928 for the seven kinds of β2-agonists in 0 . 5-100 μg/L concentration range. The LOD of seven kinds of β2-agonists in livestock mature is 1 μg/kg, while the LOQ is 5 μg/kg. When 5-50 μg/kg of the seven kinds ofβ2-agonists were added into the blank livestock manure, an average recovery of 67% -112% was obtained with the relative standard deviations of 2. 9%-10. 2%. The method is simple, rapid and has good reproducibility for quantitative and confirmatory analysis ofβ2-agonist residues.

To determine the residue of 14 sulfonamides in milk, a high performance liquid chromatography-tandem mass spectrometry ( HPLC-MS/MS ) method with on-line soild phase extraction ( SPE ) in cation exchange mode was established. 5 g of milk was extracted with 15 mL acetonitrile. Then the extraction was evaporated by 50 ℃ nitrogen and dissolved by 1. 00 mL 0. 2% formic acid. The dissolution was enriched and purified by MS/MS cation exchange on-line SPE column on a double ternary liquid chromatography, and eluted by the mixed solution of 2% ammonia methanol and 0. 2% formic acid (50:50, V/V). The compounds were separated by an octadecyl silica bonded column and determined by the tandem mass spectrometry. The results showed that the linearity of 14 sulfonamides was good in the range of 0 . 1–10 μg/kg ( r≥0 . 9995 ) .
The LOD of the method was 0. 05 μg/kg, while the LOQ was 0. 1 μg/kg. The recoveries of the 14 sulfonamides were in the range of 60 %-90 %, while the inter-batch and intra-batch RSDs were all lower than 10%. The method was proved to be more convenient, economical and stable than the traditional SPE column method.

Surgical shunt is still an effective method in managing portal hypertension related gastrointestinal bleeding.To minimize the invasive trauma and adverse effect on transplantation remains to be the unsolved problem.Herein we present the use of a newly designed surgical shunt to cure massive refractory gastrointestinal tract hemorrhage in a patient,who was critically ill because of the extensive thrombus in portal venous system.The procedure is named gastrojugular shunt.For the sake of its simple operation and effective outcome,the procedure was performed on four other patients.All the patients were well treated and recovered uneventfully with good follow up results.

Objective:To investigate the antiviral efficacy of direct-acting antiviral agents (DAAs) in the treatment of liver transplantation (LT) recipients with hepatitis C virus (HCV) infection.Methods:Twenty-two HCV-infected LT recipients treated with DAAs at Fifth Medical Center of Chinese PLA General Hospital from December 2014 to June 2018 were retrospectively analyzed, Twenty cases of HCV RNA gene type 1b were treated with sofosbuvir (400 mg/d) + ledipasvir (90 mg/d) or sofosbuvir (400 mg/d) + daclatasvir (60 mg/d) for 12 weeks or 24 weeks; 2 cases of gene type 2a were treated with sofosbuvir (400 mg/d) for 12 weeks. The effect of antiviral treatment, adverse reactions during treatment, and laboratory indicators such as HCVRNA quantification, blood routine, liver and kidney function during treatment and follow-up were studied.Results:The LT recipients of HCV infection included 16 males and 6 females, with a median age of 61.5 (36-71) years old, and the median time of antiviral treatment was 48 (2-117) months after transplantation. Among the 22 patients, 16 received a 12-week course of treatment. Except for 2 patients who did not get HCVRNA negative conversion at 4-week, all achieved a negative HCV RNA at 4-week and the end of the treatment. Six LT recipients received a 24-week course of treatment (gene type 1b), and HCVRNA was negative at 4-week and the end of treatment. All patients achieved end of treatment virological response and a sustained virological response (SVR) rate of 100% at 12 weeks and 24 weeks after the end of treatment. The serum levels of alanine aminotransferase (ALT) and creatinine were 71.5 (30, 110) U/L and (89.4±25.7) mmol/L before treatment, respectively. ALT decreased to 22 (17.8, 28.5) U/L after 4 weeks of treatment, and serum creatinine decreased to (77.4±11.5) mmol/L at 24 weeks after the end of treatment. The differences before and after treatment were statistically significant (all P<0.05). No serious adverse events occurred during the treatment. Conclusions:DAAs have a definite antiviral effect in the treatment of LT recipients with HCV infection, and long-term SVR can be obtained.

ObjectiveTo investigate the clinical promoting effect of crossing nape electroacupuncture on the recovery of swallowing function and recovery from pulmonary infection in post-cerebral infarction patients with tracheotomy.MethodSixty post-cerebral infarction patients with cough reflex disorder and swallowing dysfunction associated with pulmonary infection receiving tracheotomy and tracheal intubation were subjects. They were allocated, using a random number table, to three groups, 20 cases each. In each group, the patients were enrolled in order of visits. The three groups were given the same basic treatment for fighting inflammation, resolving phlegm and improving blood supply. The crossing nape electroacupuncture group received bilateral points Fengchi (GB20), Yifeng (TE17), Dicang (ST4)-to-Jiache (ST6) and Lianquan (CV23) acupuncture with electrodes connected by left-right crossing. The acupuncture group received bilateral points Fengchi, Yifeng, Dicang-to-Jiache and Lianquan acupuncture without electrodes connected. The control group received basic treatment with Western drugs without acupuncture therapy. Observations were carried out using the Kubota’s water drinking test, the Toshima Ichiro Swallowing Assessment and the Clinical Pulmonary Infection Score. The clinical therapeutic effects were evaluated in the three groups.ResultThe therapeutic effects evaluated using the Kubota’s water drinking test and the Toshima Ichiro Swallowing Assessment were better in the crossing nape electroacupuncture group than in the acupuncture group and better in the acupuncture group than in the control group (P<0.05). The score of the Clinical Pulmonary Infection Score decreased in all the three groups. The promoting effect on recovery from pulmonary infection was marked in the crossing nape electroacupuncture group (P<0.01).ConclusionCrossing nape electroacupuncture has a marked improving effect on dysphagia in post-cerebral infarction patients with tracheotomy and tracheal intubation. It can promote recovery from pulmonary infection in post-cerebral infarction patients with cough reflex disorder receiving tracheotomy and tracheal intubation.

Objective:To investigate the relationship between adiponectin (ADIPOQ) gene polymorphism and postpartum type 2 diabetes mellitus (T2DM) in pregnant women with gestational diabetes mellitus (GDM).Methods:A retrospective study was conducted on 236 GDM postpartum women admitted to the Affiliated Hospital of Jining Medical College from June 2020 to June 2021 as observation subjects. They were divided into a T2DM group and a non T2DM group based on the occurrence of T2DM after delivery. The clinical data of the two groups were compared. The double deoxygenation end termination method was used to detect the single nucleotide polymorphism (SNP) of the ADIPOQ gene, and the four loci rs17366568, rs822395, rs1501299, and rs2241766 were classified. The relationship between ADIPOQ genotype polymorphism and postpartum T2DM was analyzed using a logistic regression model.Results:The G allele carrying the rs2241766 locus in ADIPOQ gene was negatively correlated with the occurrence of T2DM ( OR=0.71, 0.68, P<0.05). Compared with T2DM patients with TT genotype, the GT+ GG genotype at the rs2241766 locus had a lower risk of occurrence for gestational age ≥2 and HbA 1c>85%. Similarly, T2DM patients with pre pregnancy body mass index (BMI)>25 kg/m 2 were more likely to be carriers of the rs2241766 TT genotype ( P=0.026). The (GT+ TT) genotype carrying the T allele at the rs1501299 locus was a protective factor for gestational age and HbA 1c in T2DM patients. Conclusions:The rs2241766 and rs1501299 polymorphisms of the ADIPOQ gene are associated with susceptibility to postpartum T2DM in GDM women. Individuals with rs2241766 and rs1501299 mutant genotypes belong to the high-risk population for T2DM.