Objective:To construct a fusion protein of extracellular domain peptide fragment of muscle specific kinase ( MuSK) and fluorescent protein mCherry ,and used as antigen in the detection of antibodies against MuSK ( MuSKAb ) in the sera of patients with myasthenia gravis ( MG).Methods:The mCherry gene was amplified by PCR from vector pRSET-B and cloned into pGEM-T Easy Vector,and furthermore, cloned into Eukaryotic expression vector pMT /BiP/V5-His ( MuSK), which contains MuSK extracellular domain 22-452 amino acid peptide fragment gene to construct the fluorescent fusion protein gene MuSK -mCherry.The recombinant vector was subsequently transfected into drosophila S 2 cells for expression.The expressed fusion proteins were verified in confocal mi-croscope ,and used as antigen in the detection of MuSKAb in sera of MG patents in fluorescence immunoprecipitation test .Results:The fluorescent fusion protein MuSK-mCherry was successfully constructed and expressed.The MuSKAb in sera of patents with MG could be detected in fluorescence immunoprecipitation test using the constructed MuSK-mCherry fusion protein as antigen.Conclusion: It is available to use the constructed fluorescent fusion protein MuSK-mCherry as antigen in fluorescence immunoprecipitation test for the detection of MuSKAb in sera of patents with MG.

Objective:To investigate the regulatory effects and mechanism of mannose-binding lectin(MBL) on autophagy during the differentiation of 3T3-L1 adipocytes, and provide the feasibility for targeting autophagy to prevent obesity and related pathological conditions in natural immunity.Methods:3T3-L1 preadipocytes were cultured in vitro and induced to differentiation. Cell differentiation and lipid accumulation were analyzed by oil red O staining and CCK-8 was used to detect the effect of different concentrations of MBL (0, 1, 5, 10 μg/ml) on cell proliferation ability at different differentiation stages. Western blot was used to analyze the expression of MBL(10 μg/ml) on the key autophagy factors LC3B, Beclin1 and p62 protein at different stages of differentiation, and the changes of lipid droplet accumulation under the intervention of MBL were observed by oil red O staining. The protein and mRNA expression of autophagy key factors under the intervention of different concentrations of MBL were detected by Western blot and qRT-PCR. And autophagy flow analysis based on autophagic degradation was used to further illustrate the autophagic activity. The expression and phosphorylation of adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) signaling molecules were analyzed by Western blot. Results:The results of oil red O staining showed that 3T3-L1 preadipocytes could achieve complete differentiation after 10 days of induction. CCK-8 showed that the concentration of MBL (1-10 μg/ml) in the experimental group had no effect on cell proliferation at different differentiation stages. During the differentiation of 3T3-L1 preadipocytes, Western blot and qRT-PCR showed that the expression of autophagy-related proteins and mRNA levels was enhanced in the MBL treated group, and presented a concentration-dependent relationship. Oil red O staining showed that the lipid droplets in adipocytes at different stages of differentiation are reduced to varying degrees under the intervention of MBL. Fluorescence microscopy results further confirmed that MBL enhanced the autophagy activity of adipocytes by increasing the synthesis of autophagosomes. Moreover, under the intervention of MBL, the phosphorylation level of AMPK was significantly up-regulated, while the phosphorylation level of mTOR was significantly down-regulated, also showing a concentration-dependent relationship.Conclusions:MBL accelerates the autophagy process during the differentiation of 3T3-L1 adipocytes through AMPK/mTOR signaling pathway, reduces lipid accumulation, providing a possible functional pathway for the treatment of obesity and related metabolic diseases.

OBJECTIVETo assess the association of T190C polymorphism of β3 adrenergic receptor gene (β3-AR) with chronic heart failure (CHF), and to evaluate the effect of this polymorphism on clinical response to β-AR blockade among patients with CHF.

METHODSThree hundred and thirty patients with stable CHF receiving basic therapy for heart failure were included. Before initiation and 5 months after the maximal tolerated dose of carvedilol was reached, all indices including heart rate (HR), blood pressure (BP), left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), brain natriuretic peptide (BNP) level, 6 min walk distance were measured and compared with the indices of those with a T190C genotype. Distribution of the T190C polymorphisms in the control group and CHF group was compared.

RESULTSThe frequencies of T190C genotypes of the β3-AR gene have fit with the Hardy-Weinberg equilibrium. No significant difference was found between the frequencies of T190C alleles and genotypes between the two groups (P > 0.05). Compared with CC-homozygotes, TT-homozygous patients showed substantially greater improvement in LVEF and BNP (all P < 0.01).

CONCLUSIONNo difference has been detected in the prevalence of the three genotypes between healthy and CHF subjects. The T190C variation of the β3-AR gene was not associated with increased risk for CHF. CHF patients with a T allele have greater response to carvedilol than those carrying a C allele in ethnic Han Chinese.

Adult ; Carbazoles ; therapeutic use ; Chronic Disease ; Female ; Heart Failure ; drug therapy ; genetics ; physiopathology ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Propanolamines ; therapeutic use ; Receptors, Adrenergic, beta-3 ; genetics ; Ventricular Function, Left

Free radicals are the intermediates of metabolism,widely exist in the human bodies.Under normal circumstances,the free radicals play an important role in the metabolic process on human body,cell signal pathway,gene regulation,induction of cell proliferation and apoptosis,so as to maintain the normal growth and development of human body and to inhibit the growth of bacteria,virus and cancer.However,when organic lesion occurs affected by external factors or when equilibrium of the free radicals is tipped in the human body,the free radicals will respond integratedly with lipids,protein or nucleic acid which may jeopardize the health of human bodies.This paper summarizes the research progress of the free radicals conducted in recent years,in relations to the perspective of the types,origins,test methods of the free radicals and their relationship with human's health.In addition,the possible mechanisms of environmental pollutants (such as polycyclic aromatic hydrocarbons) mediating oxidative stress and free radicals scavenging in the body were also summarized.

Objective:To investigate the regulatory effects and mechanism of mannan-binding lectin (MBL) on adipogenic differentiation of 3T3-L1 preadipocytes.Methods:3T3-L1 preadipocytes were induced to differentiate into adipocytes in vitro, and stimulated with different concentrations of MBL (0, 1, 10, 20 μg/ml). Firstly, changes in cell proliferation ability were detected by CCK-8. Then lipid accumulation was analyzed by Oil red O staining and intracellular triglyceride content determination. Further, the expression of adipogenic differentiation-related factors PPARγ and C/EBPα at protein and mRNA levels were detected by Western blot and qRT-PCR, respectively. Finally, Western blot was used to analyze the expression and phosphorylation of Akt, a signal molecule related to adipogenic differentiation. Results:MBL at the concentrations of 0, 1, 10 and 20 μg/ml had no effect on the proliferation of 3T3-L1 preadipocytes. The level of triglyceride in MBL treatment groups decreased in a dose-dependent manner on 3 d after 3T3-L1 preadipocyte differentiation. Results of Oil red O staining showed that the number of lipid droplets in MBL treatment groups reduced significantly, and the absorbance values also decreased significantly in a concentration-dependent manner. Western blot and qRT-PCR results showed that the expression of PPARγ and C/EBPα at both protein and mRNA levels in MBL treatment groups decreased significantly in a dose-dependent manner, and the phosphorylation level of Akt was significantly down-regulated as well.Conclusions:MBL regulates the adipogenic differentiation of 3T3-L1 preadipocytes via Akt signaling pathway.

Objective To investigate the regulation of Treg/Th17 axis by mannan-binding lectin (MBL) in mice with Candida albicans ( C. albicans ) infection. -ethods MBL gene-knockdown (MBL-/-) C57BL/6 mice and wild-type (WT) C57BL/6 mice were divided into four groups. C. albicans strains (2×107 CFU) were injected intraperitoneally into the mice of infection groups. Paraffin sections of mouse liver and kidney tissues were prepared on 3 d. Histopathological changes were observed with hematox-ylin and eosin ( HE) and Periodic acid-Schiff ( PAS) staining. Flow cytometry was performed to analyze Th17 and Treg cells in mice on 7 d. The levels of IL-10 and IL-17A were measured by ELISA. CD4+T cells were obtained from spleen cells by magnetic sorting. Expression of Foxp3 and RORγt at mRNA and protein levels were detected by qRT-PCR and Western blot, respectively. Results The mouse model of C. albicans infection was established successfully. After infection, the MBL-/- mice had higher percentages of Th17 cells, but lower percentages of Treg cells than the WT mice. ELISA results showed that compared with the WT mice with C. albicans infection, the MBL-/- mice had significantly increased IL-17A and decreased IL-10 after infection. Moreover, the expression of RORγt at both mRNA and protein levels was up-regula-ted, while that of Foxp3 was down-regulated in the MBL-/- mice than in the WT mice following infection. Conclusions MBL regulates the immune balance of Treg/Th17 cells in mice infected with C. albicans through promoting the differentiation of CD4+ T cells into Treg cell subsets and inhibiting the differentiation into Th17 cell subsets.

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.