Immune dysfunction following COVID-19, especially in severe patients.

The coronavirus disease 2019 (COVID-19) has been spreading worldwide. Severe cases quickly progressed with unfavorable outcomes. We aim to investigate the clinical features of COVID-19 and identify the risk factors associated with its progression. Data of confirmed SARS-CoV-2-infected patients and healthy participants were collected. Thirty-seven healthy people and 79 confirmed patients, which include 48 severe patients and 31 mild patients, were recruited. COVID-19 patients presented with dysregulated immune response (decreased T, B, and NK cells and increased inflammatory cytokines). Also, they were found to have increased levels of white blood cell, neutrophil count, and D-dimer in severe cases. Moreover, lymphocyte, CD4+ T cell, CD8+ T cell, NK cell, and B cell counts were lower in the severe group. Multivariate logistic regression analysis showed that CD4+ cell count, neutrophil-to-lymphocyte ratio (NLR) and D-dimer were risk factors for severe cases. Both CT score and clinical pulmonary infection score (CPIS) were associated with disease severity. The receiver operating characteristic (ROC) curve analysis has shown that all these parameters and scores had quite a high predictive value. Immune dysfunction plays critical roles in disease progression. Early and constant surveillance of complete blood cell count, T lymphocyte subsets, coagulation function, CT scan and CPIS was recommended for early screening of severe cases.

[Effects of dihydromyricetin on high fat diet induced obesity in mice and its mechanism].

OBJECTIVE: To observe the effects of dihydromyricetin (DHM) on obesity induced by high-fat diet in mice, and to explore whether its mechanism of action is related to the promotion of WAT browning. METHODS: Sixty c57bl/6j mice were randomly divided into 6 groups (n=10): ①normal control group (ND group): normal feed feeding; ②Normal control + low dose DHM group (ND+L-DHM group): normal feed feeding was treated with low dose DHM (125 mg/(kg·d)); ③Normal control + high dose DHM group (ND+H-DHM group): normal feed feeding was treated with high dose DHM (250 mg/(kg·d)); ④High-fat diet group (HFD): high-fat diet; ⑤high-fat diet + low-dose DHM group (HFD+L-DHM group): high-fat diet feeding with low-dose DHM; ⑥High-fat diet + high-dose DHM group (HFD+H-DHM group): High-fat diet was treated with high-dose DHM. After 16 weeks, the mice were fasted overnight, blood samples were collected for fasting blood glucose and blood lipids, then the animals were sacrificed, body length was measured, and Lee's index was calculated. After weighing the adipose tissue in the scapula, groin and epididymis, formaldehyde fixation and HE staining were used to observe the fat cells size, immunohistochemistry was used to detect the expression of uncoupling protein 1 (UCP1). The body weight was measured every 4 weeks during the experiment. RESULTS: Compared with the ND group, the body weight of the mice in the HFD group was increased significantly, suggesting that the obese mouse model replicated successfully. In addition, the body fat weight, fat cell diameter, Lee's index and blood glucose of the HFD group were increased significantly, and the expression of UCP1 in the adipocytes was increased. Body weight, fat cell diameter, Lee's index and blood glucose of HFD mice treated with L-DHM and H-DHM were reversed significantly, while the expression of UCP1 in adipocytes was more significantly increased; however, L-DHM and H-DHM had no significant effects on the above indicators in normal mice. CONCLUSION: Dihydromyricetin inhibited high fat diet induced mouse obesity; the mechanism might be associated with promoting WAT browning.

Cellular context- and protein level-dependent interaction of pluripotency factor OCT4A with multiple octamer motifs of the same target gene.

AIMS: To compare how OCT4A proteins interact with and regulate multiple OCT4A-octamer motifs (OMs) in different regions of the FOS gene expressed in somatic cancer cells versus pluripotent stem cells. MATERIALS AND METHODS: Two FOS reporter gene systems harboring predicted OMs or their mutational counterparts were introduced into HeLa and NCCIT cells with varying OCT4A protein levels. The transcription of dsGFP reflecting FOS expression was quantitated by RT-qPCR, the OCT4A-OMs binding and the correlation between OCT4A and FOS transcription was determined by ChIP-PCR and RNA-Seq, respectively. KEY FINDINGS: In NCCIT cells, abundant OCT4A proteins bound to and inhibited OM1 and OM2 at the promoter of the FOS gene. RA-induced OCT4A down-regulation transiently increased FOS transcription. In contrast, in HeLa cells that contain much lower levels of endogenous OCT4A proteins, OCT4A primarily bound to and activate OM1 thereby promoting FOS transcription. OCT4A KO significantly reduced FOS expression. Ectopically introduced OCT4A, at its leaked or induced expression level, promoted FOS transcription by binding to OM2/OM3 or OM1/OM3, respectively. Thus, the interaction of OCT4A proteins with different OMs is cellular context- and protein level-dependent, and such complicated OCT4A binding mode can only be reflected by a dsGFP-based reporter harboring the full-length FOS gene but not by that merely having the FOS promoter. SIGNIFICANCE: Our findings unravel an additional layer of regulatory mechanisms that account for the cellular context- and dose-related versatile functions of OCT4A protein, and further underscore the importance of precise modulation of OCT4A in the regenerative medicine and anticancer therapies.

Dynamic changes of key metabolites during liver fibrosis in rats.

BACKGROUND: Fibrosis is the single most important predictor of significant morbidity and mortality in patients with chronic liver disease. Established non-invasive tests for monitoring fibrosis are lacking, and new biomarkers of liver fibrosis and function are needed. AIM: To depict the process of liver fibrosis and look for novel biomarkers for diagnosis and monitoring fibrosis progression. METHODS: CCl4 was used to establish the rat liver fibrosis model. Liver fibrosis process was measured by liver chemical tests, liver histopathology, and Masson's trichrome staining. The expression levels of two fibrotic markers including α-smooth muscle actin and transforming growth factor ß1 were assessed using immunohistochemistry and real-time polymerase chain reaction. Dynamic changes in metabolic profiles and biomarker concentrations in rat serum during liver fibrosis progression were investigated using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The discriminatory capability of potential biomarkers was evaluated by receiver operating characteristic (ROC) curve analysis. RESULTS: To investigate the dynamic changes of metabolites during the process of liver fibrosis, sera from control and fibrosis model rats based on pathological results were analyzed at five different time points. We investigated the association of liver fibrosis with 21 metabolites including hydroxyethyl glycine, L-threonine, indoleacrylic acid, ß-muricholic acid (ß-MCA), cervonoyl ethanolamide (CEA), phosphatidylcholines, and lysophosphatidylcholines. Two metabolites, CEA and ß-MCA, differed significantly in the fibrosis model rats compared to controls (P < 0.05) and showed prognostic value for fibrosis. ROC curve analyses performed to calculate the area under the curve (AUC) revealed that CEA and ß-MCA differed significantly in the fibrosis group compared to controls with AUC values exceeding 0.8, and can clearly differentiate early stage from late stage fibrosis or cirrhosis. CONCLUSION: This study identified two novel biomarkers of fibrosis, CEA and ß-MCA, which were effective for diagnosing fibrosis in an animal model.

[The effects of arecoline on the lipid metabolism of 3T3-L1 adipocytes].

OBJECTIVE: To observe the effects of arecoline on lipid metabolism in 3T3-L1 adipocytes and explore its possible mechanisms. METHODS: 3T3-L1 pre-adipocytes were induced into adipocytes with the classic "cocktail" method, subsequently, adipocytes were treated with arecoline at the concentrations of 0, 25, 50 and 100 µmol/L for 72 hours. After 72 hours, cell vability was measured with MTT method, lipid droplet accumulation in the cytoplasm was observed with oil red O staining, the protein expression of fatty acid synthase (FAS), adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were detected by Western blot assay. RESULTS: There were a large number of lipid droplets in the cytoplasm in the differentiated 3T3-L1 adipocytes. MTT results showed that 0~100 µmol/L arecoline had no significant effect on cell vability; oil red O staining found arecoline reduced lipid amount in 3T3-L1 adipocytes; Western blot results showed that compared with 0 µmol/L arecoline group (the control group), arecoline significantly reduced the protein level of FAS and increased the protein levels of ATGL and HSL, and 50 µmol/L arecoline group was the most significant. CONCLUSIONS: Arecoline significantly increased lipolysis of 3T3-L1 adipocyte, which might be associated with decreased the FAS expression of key enzyme of lipid synthesis and increased the ATGL and HSL expression of key enzyme of adipolysis.

[The effects of dihydromyricetin on cognitive dysfunction in type 2 diabetes mice].

OBJECTIVE: To observe the effects of dihydromyricetin(DHM) on cognitive dysfunction and expression of brain derived neurotrophic factor(BDNF) protein in hippocampus of type 2 diabetic mice(T2DM). METHODS: Forty C57BL/6J mice were randomly divided into two groups, normal control group (n=8):normal diet feeding; T2DM model group (n=32):high-glucose and high-fat combined with 100 mg/kg streptozocin(STZ) treatment (five mice died during modeling and three failed). Twenty-four diabetic mice were modeled successfully and divided into three groups (T2DM group, T2DM+L-DHM group and T2DM+H-DHM group). Three groups mice were fed with high-glucose and high-fat diet, and treated with equal volume of normal saline, 125 mg/(kg·d) DHM or 250 mg/(kg·d) DHM for 16 weeks respectively. The control mice were fed with normal diet and treated with equal volume of saline (once a day, gavage) for 16 weeks. After 16 weeks, the body weight and fasting blood glucose were measured, intraperitoneal glucose tolerance test and related behavioral experiment were performed. Finally, the expression of BDNF protein in hippocampus of mice was detected by Western blot. RESULTS: The model of type 2 diabetes mellitus was established successfully with high-glucose and high-fat combined with 100 mg/kg STZ. After 16 weeks, the body weight of T2DM group was significantly decreased, the fasting blood glucose was significantly increased and the glucose tolerance was significantly abnormal compared with the normal control group. Compared with T2DM group, the body weight of T2DM+DHM groups mice was increased, while the levels of fasting blood glucose were decreased. And H-DHM could significantly improve the abnormal glucose tolerance of T2DM mice. Behavior test results showed that the ability of learning and memory of T2DM mice was significant decreased compared with control group, but these phenomena were improved in T2DM+DHM groups mice, and T2DM+H-DHM group was more obvious. Western blot analysis showed that the expression of BDNF protein in hippocampus of T2DM group was significantly lower than that of control group, while T2DM+DHM group was significant increased compared with T2DM mice. CONCLUSIONS: Dihydromyricetin can improve the cognitive dysfunction in type 2 diabetic mice. The mechanism may be through hypoglycemic effect and activation of BDNF protein expression in hippocampus.

[Ameliorative effects and the mechanism of lyceum barbarum polysaccharide on insulin resistance of HepG2 cell].

OBJECTIVE: To observe the effects of lyceum barbarum polysaccharide (LBP) on insulin resistance of HepG2 cells and investigate its possible mechanism. METHODS: IR-HepG2 cell model was induced with high glucose and high insulin in combination for 24 hours,with 104/vaccination in the 96-well plates, hole density after adherent cells (30 µg/ml、100 µg/ml、300 µg/ml) LBP cultivate 48 h, 200 µl/hole, each all had four holes. The effects of LBP at different concentrations on HepG2 cell activity and insulin resistance were tested. Intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected. The expressions of related proteins in insulin signal transduction pathways such as insulin receptor substrate-2(IRS-2), phosphatidylinositol-3-kinase(PI3-K), protein kinase B(Akt) and glucose transport-2(GLUT2) were determined. RESULTS: Compared with normal control group, the content of MDA was increased significantly and the activity of SOD and the expression levels of IRS-2,PI-3K,Akt and GLUT2 were decreased significantly in the IR model group. Compared with IR model group, medium and high concentrations of LBP decreased the content of MDA and increased the activity of SOD and the expression levels of IRS-2, PI-3K, Akt and GLUT2 in insulin-resistant HepG2 cells. MTT showed that at the same time, the OD value gradually decreased with the increase of LBP's concentration; under the same concentration of LBP, the OD value also gradually decreased with the extension of time, which indicated that LBP inhibited the proliferation of HepG2 cells with time and concentration-dependent manner. Glucose consumption experiment indicated that medium and high concentration of LBP could increase the glucose consumption of insulin-resistant HepG2 cells significantly, but low concentration of LBP had no significant impacted on glucose consumption of insulin-resistant HepG2 cells. CONCLUSIONS: Medium and high concentration of LBP can improve insulin resistance of HepG2 cell, its mechanisns may be associated with decreasing the level of oxidative stress and increasing the protein expressions of insulin signaling pathway.

[Effect of arecoline on proliferation and apoptosis of MCF-7 human breast cancer cells].

OBJECTIVE: To observe the effects of arecoline on proliferation and apoptosis of MCF-7 human breast cancer cells and to ex-plore its possible mechanism. METHODS: Human breast cancer MCF-7 cells were treated with arecoline at the concentrations of 0,10,30,50, 100,300,500µmol/L, the cell proliferation were detected by MTT assay, cell apoptosis were analyzed by Hoechst 33342 staining and flow cy-tometry, the protein expression of Bax,Bcl-2 and P53 were detected by Western blot. RESULTS: Low concentration(0,10,30, 50 µmol/L) arecoline had no effect on the proliferation and apoptosis of MCF-7. However, high concentration(100,300,500µmol/L) arecoline inhibited proliferation and induced apoptosis of MCF-7 cells in a concentration-dependent manner, arecoline also significantly increased P53 and Bax protein expression and decreased Bcl-2 protein expression. CONCLUSIONS: High concentration arecoline inhibited the proliferation and induced the apoptosis of MCF-7 cells, the mechanism was probably corrected with increasing P53 and Bax protein expression and decreasing Bcl-2 pro-tein expression.

Association between TRAIL expression on peripheral blood lymphocytes and liver damage in chronic hepatitis B.

AIM: To explore a novel mechanism for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), upregulation of CD4(+) and CD8(+) T lymphocytes participating in the patho-physiological process of chronic hepatitis B (CHB). METHODS: The levels of serum soluble TRAIL (sTRAIL), serum IFN-gamma and membrane-bound TRAIL expression on peripheral leucocytes from 58 CHB patients were examined by ELISA and flow cytometry respectively. The levels of TRAIL were compared with the baseline levels of 17 healthy controls, and correlation analysis was performed between ALT, TBIL, PT, morphological change in hepatic tissues, and serum IFN-gamma. RESULTS: The results showed that TRAIL levels on membranes of CD4(+), CD8(+) T cells in CHB patients were much higher than those in healthy controls (P<0.001), and were correlated with serum TBIL (r = 0.354, P = 0.008 for CD4(+) and r = 0.522, P = 0.000 for CD8(+), respectively), ALT (r = 0.393, P = 0.003 for CD8(+)), PT (r = 0.385, P = 0.004 for CD8(+)) and serum IFN-gamma level (r = 0.302, P = 0.011 for CD4(+) and r = 0.307, P = 0.009 for CD8(+)). On the contrary to membrane-bound TRAIL expression, serum level of sTRAIL was not correlated with that of TBIL and PT, though it was higher than that of the normal population and was positively correlated with serum HBeAg expression (r = 0.695, P = 0.001). CONCLUSION: The expression level of TRAIL on the membrane of lymphocytes was upregulated and associated with the liver injury in CHB patients. These findings suggest that upregulation of TRAIL expression may be induced by virus antigen and inflammatory cytokine IFN-gamma.

Akt­mediated phosphorylation of Oct4 is associated with the proliferation of stem­like cancer cells.

Oct4 protein encoded by POU5F1 plays a pivotal role in maintaining the self­renewal of pluripotent stem cells; however, its presence in cancer cells remains controversial. In the present study, we provided evidence that the transcripts of authentic OCT4 gene (OCT4A) and its multiple pseudogenes were detected in a variety of cancer cell lines. A few major bands were also detected by western blotting using an anti­Oct4A monoclonal antibody. Moreover, an anti­Oct4­pT235 antibody was used to identify a band in the majority of the tested cancer cell lines that coincided with one of the anti­Oct4A bands which was decreasable by a specific shRNA. The Oct4­pT235 signals were also detected in human glioblastoma and liver cancer specimens by immunofluorescence microscopy and immunohistochemistry. U87 glioblastoma cells were cultured in a neural stem cell medium to induce the formation of neurospheres rich in stem­like cancer cells. The levels of Oct4­pT235 in the sphere cells were markedly increased compared to their monolayer parental cells, a result that was accompanied by upregulation of the PI3K­Akt pathway. Akti­1/2, a specific inhibitor of Akt, effectively reduced the level of Oct4­pT235 and attenuated the proliferation of U87 sphere cells. ITE, an agonist of the aryl hydrocarbon receptor, also significantly attenuated the Akt­mediated phosphorylation of Oct4 in glioblastoma and liver cancer cells, and reduced their tumorigenic potential in a xenograft tumor model. Taken together, we concluded that the Akt­mediated phosphorylation of Oct4A or its homolog protein was associated with the proliferation of stem­like cancer cells that may serve as a novel biomarker and drug target for certain types of cancer.

[Involvement of TRAIL up-regulation of CD4+, CD8+ T cells in liver injury in chronic hepatitis B].

OBJECTIVE: To describe a novel mechanism for TRAIL up-regulation of CD4+, CD8+ T cells to participate in the pathophysiological process in patients with chronic hepatitis B (CHB). METHODS: The serum levels of soluble TRAIL (sTRAIL), IFN-gamma and membrane bound TRAIL expression on peripheral leucocytes from 58 CHB patients were examined by ELISA and flow cytometry respectively. The levels of TRAIL were compared with the baseline levels of 15 healthy controls, and correlation analysis were performed between ALT, TBil and PT, morphological change in hepatic tissues. RESULTS: The results showed that TRAIL levels on membranes of CD4+, CD8+ T cells in CHB patients were much higher than the healthy controls (P < 0.001), which of CD4+ T cells positively correlated with serum TBil (r=0.354, P = 0.008), Serum IFN-gamma level (r=0.302, P = 0.011) and which of CD8+ T cells positively correlated with serum TBil (r=0.522, P = 0.000), ALT (r=0.393, P = 0.003), PT (r=0.385, P = 0.004), serum IFN-gamma level (r=0.307, P = 0.009). The serum levels of soluble TRAIL only correlated with serum HBeAg expression (r=0.695, P = 0.001). CONCLUSION: These findings suggest that the expression of TRAIL on the membranes of lymphocytes was up-regulated, which may take part in the immunopathogenesis in CHB patients. TRAIL expression can be induced either by virus-specific protein expression or by inflammation cytokine IFN-gamma

[The role of arecoline on hepatic insulin resistance in type 2 diabetes rats].

OBJECTIVE: To explore the effects of arecoline on hepatic insulin resistance in type 2 diabetes rats and to elucidate its possible mechanism. METHODS: Forty five Wistar rats were fed with high fructose diet for 12 weeks to induce type 2 diabetic rat model. rats were randomly divided into 5 groups (n = 8): control group, model group and model group were treated with different dose (0, 0.5, 1, 5 mg/kg) of arecoline. After 4 weeks, the fasting blood glucose, blood lipid and insulin level measured , mRNA expression of liver constitutive androstane receptor (CAR), pregnane X receptor (PXR), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected by reverse transcription polymerase chain reaction (RT-PCR), the protein expression of p-AKT and glucose transporter4 (GLUT4) were detected by Western blot. RESULTS: 1.5 mg/kg arecoline could significantly decrease the level of fasting blood glucose, blood lipid, blood insulin level and liver G6Pase, PEPCK, IL-6, TNF-alpha mRNA level in type 2 diabetes rats. 1.5 mg/kg arecoline also could significantly increase CAR, PXR mRNA level and p-AKT and GLUT4 protein expression. CONCLUSION: Arecoline improved hepatic insulin resistance in type 2 diabetes rats by increasing the mRNA levels of CAR and PXR leading to the creased glucose metabolism and inflammation related genes expression.

Hydrogen sulfide prevents formaldehyde-induced neurotoxicity to PC12 cells by attenuation of mitochondrial dysfunction and pro-apoptotic potential.

Hydrogen sulfide (H(2)S) has been shown to act as a neuroprotectant and antioxidant. Numerous studies have demonstrated that exposure to formaldehyde (FA) causes neuronal damage and that oxidative stress is one of the most critical effects of FA exposure. Accumulation of FA is involved in the pathogenesis of Alzheimer's disease (AD). The aim of present study is to explore the inhibitory effects of H(2)S on FA-induced cytotoxicity and apoptosis and the molecular mechanisms underlying in PC12 cells. We show that sodium hydrosulfide (NaHS), a H(2)S donor, protects PC12 cells against FA-mediated cytotoxicity and apoptosis and that NaHS preserves the function of mitochondria by preventing FA-induced loss of mitochondrial membrane potential and release of cytochrome c in PC12 cells. Furthermore, NaHS blocks FA-exerted accumulation of intracellular reactive oxygen species (ROS), down-regulation of Bcl-2 expression, and up-regulation of Bax expression. These results indicate that H(2)S protects neuronal cells against neurotoxicity of FA by preserving mitochondrial function through attenuation of ROS accumulation, up-regulation of Bcl-2 level, and down-regulation of Bax expression. Our study suggests a promising future of H(2)S-based preventions and therapies for neuronal damage after FA exposure.

Inhibition of hydrogen sulfide generation contributes to 1-methy-4-phenylpyridinium ion-induced neurotoxicity.

Reactive oxygen species (ROS) overproduction contributes to the neurotoxicity of 1-methy-4-phenylpyridinium ion (MPP(+)). Increasing studies have shown that hydrogen sulfide (H(2)S) is an endogenous antioxidant gas. We have hypothesized that MPP(+)-caused neurotoxicity may involve the imbalance of proportion to this endogenous protective antioxidant gas. The aim of this study is to evaluate whether MPP(+) disturbs H(2)S synthesis in PC12 cells, a clonal rat pheochromocytoma cell line, and whether disturbance of H(2)S generation induced by MPP(+) is an underlying mechanism of MPP(+)-induced neurotoxicity. We show that exposure of PC12 cells to MPP(+) causes a significant decrease in H(2)S generation and results in remarkable cell damage. We find that cystathionine-ß-synthetase (CBS) is catalyzed in PC12 cells to generate H(2)S, and that both expression and activity of CBS are inhibited by MPP(+) treatment. Exposure of sodium hydrosulfide (NaHS), a donor of H(2)S, extenuates MPP(+)-induced cytotoxicity and ROS accumulation in PC12 cells, while inhibition of CBS by amino-oxyacetate (AOAA) exacerbates the effects of MPP(+). These results indicate that MPP(+) neurotoxicity involves reduction of H(2)S production, which is caused by inhibition of CBS. This study provides novel insights into cell death observed in neurodegenerative disease such as Parkinson's disease.

[miRNA expression change of differentiation of mice marrow mesenchymal stem cells into adipocytes].

OBJECTIVE: To explore miRNA expression change of differentiation of mice marrow mesenchymal stem cells (MSCs) into adipocytes, which lay the foundation for further studies on molecular mechanism of miRNA regulating the differentiation of MSCs into adipocytes. METHODS: C57BL/6 mice MSCs were isolated, cultured through the whole bone marrow method, amplified by the differential adherent method. Cell growth was observed by morphology and the expression of superficial antigen CD29, CD44, CD34 were detected through immunohistochemistry. MSCs was induced to differentiation into adipocytes with adipocyte differentiation medium, and adipogenic differentiation of MSCs was analyzed by oil Red O staining. MicroRNA microarray was used to investigate the differentially expressed miRNAs in MSCs and adipocytes. RESULTS: (1) The fifth passage of MSCs had high purity under an inverted m icroscope. Immunohistochemistry staining showed that CD29, CD44 were positive and CD34 was negative in more than 90% MSCs. There were a large number of lipid droplets in cytoplasm after MSCs were induced with adipocyte differentiation medium, Oil O staining was positive. (2) The microarray experiment showed that 75 differentially expressed miRNAs were obtained in adipocytes compared with MSCs, 20 up-regulated and 55 down-regulated miRNAs were observed among them. CONCLUSION: There was a expression change of miRNA of differentiation of MSCs into adipocytes, some miRNAs might play important roles in MSCs adipogenic differentiation.

Rapid detection of clonal expansion of T-cell receptor-beta gene in patients with HBV using the real-time PCR with DNA melting curve analysis.

AIM: The gene melting spectral pattern (GMSP) of PCR products from 24 T-cell receptor beta chain variable (TCRBV) gene families was developed to determine sequence bias and feature of TCRBV CDR3 gene family. METHODS: The assay was based on reverse transcript quantitative polymerase chain reaction and their DNA melting curves. RESULTS: We discovered that the relatively conserved amino acid sequences X-Q and X-G are present in TCRBV CDR3 from patients with HBV. Further, the X of the X-Q motif is preferentially E (glutamic acid), P (proline) or T (threonine) when accompanied by the BJ2.7, BJ1.5, or BJ2.3, respectively. The frequency of sequence bias in the TCRBV gene family showed a positive correlation with the T cell receptor excision circles (TRECs) content, and an inverse correlation with the HBV DNA loading. CONCLUSION: These results suggest that the GMSP assay could be used to monitor the features of TCRBV gene distribution quickly, and facilitate the further study of HBV-specific T cell in patients with HBV.

Hydrogen sulfide antagonizes homocysteine-induced neurotoxicity in PC12 cells.

Hydrogen sulfide (H2S) has been shown to protect neurons against oxidative stress. Lower levels of H(2)S as well as accumulation of homocysteine (Hcy), a strong risk of Alzheimer's disease (AD), are reported in the brains of AD patients. The aim of present study is to explore the protection of H2S against Hcy-induced cytotoxicity and apoptosis and the molecular mechanisms underlying in PC12 cells. We show that sodium hydrosulfide (NaHS), a H2S donor, protects PC12 cells against Hcy-mediated cytotoxicity and apoptosis by preventing both the loss of mitochondrial membrane potential (MMP) and the increase in intracellular reactive oxygen species (ROS) induced by Hcy. NaHS not only promotes the expression of bcl-2, but also blocks the down-regulation of bcl-2 by Hcy. These results indicate that H2S protects neuronal cells against neurotoxicity of Hcy by preserving MMP and attenuating ROS accumulation through up-regulation of bcl-2 level. Our study suggests a promising future of H2S-based therapies for neurodegenerative diseases such as AD.

Hydrogen sulfide inhibits MPP(+)-induced apoptosis in PC12 cells.

AIMS: Hydrogen sulfide (H2S) is a well-known cytotoxic gas. Recently it has been shown to protect neurons against oxidative stress caused by glutamate, hypochlorous acid (HOCl), and beta-amyloid. The aim of the present study is to explore the cytoprotection of H2S against 1-methyl-4-phenylpyridinium ion (MPP(+))-induced apoptosis and the molecular mechanisms underlying in PC12 cells, a rat cell line derived from pheochromocytoma cells. MAIN METHODS: Cell viability was determined by the conventional 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. Apoptosis was assessed by Hoechst 33258 nuclear staining and flow cytometric (FCM) analysis after propidium iodide staining. The mitochondrial membrane potential (MMP) was measured by rhodamine 123 (Rh123) probe and reactive oxygen species (ROS) were measured by dihydrorhodamine probe using FCM analysis. KEY FINDINGS: MPP(+) reduced the cell viability and induced apoptosis of PC12 cells along with dissipation of MMP as well as overproduction of ROS. Sodium hydrosulfide (NaHS), a H2S donor, protected PC12 cells against MPP(+)-induced cytotoxicity and apoptosis not only by reducing the loss of MMP, but also by attenuating an increase in intracellular ROS. SIGNIFICANCE: H2S significantly protected PC12 cells against cytotoxicity and apoptosis induced by MPP(+), which was associated with the inhibition by H(2)S of MPP(+)-induced dissipation of MMP and overproduction of ROS. These findings can significantly advance therapeutic approaches to the neurodegenerative diseases which are associated with oxidative stress, such as Parkinson's disease.

[The emergence of candidate pathogenicity island 89K DNA sequence of Streptococcus suis isolated from sporadic patients in Zhejiang province].

OBJECTIVE: To identify the presence of candidate pathogenicity island 89K DNA sequence of Streptococcus suis serotype 2 (SS2) strains isolated from patient in Zhejiang province. METHODS: Genes and DNA fragments were amplified by PCR, using specific primers, and three amplified fragments of the 89K sequence were directly sequenced. The results were analyzed using software related to bioinformatics and epidemiology. RESULTS: 8 strains of SS2 all contained 89K sequence, cps2J and mrp virulent genes, and species-specific 16S rDNA. 3 amplified fragments of 89K candidate pathogenicity island of SS2 ZJ0501 were above 99% similar to SS2 strain identified from outbreaks in Jiangsu in 1998, and the gene fragment of coding DNA recombinant protein in the 89K sequence was highly homological with that of S. dysgalactiae and S.agalactiae. CONCLUSION: In recent years SS2 strains isolated from patients with clinical symptoms in Zhejiang province had been detected to have contained candidate pathogenic 89K DNA fragment.

[Effects of hyperthermia on brainstem auditory evoked potentials and middle latency response in rats].

AIM: To study the effects of hyperthermia on brainstem auditory evoked potentials (BAEP) and middle latency response (MLR) in rats. METHODS: BAEP and MLR were recorded at the skull surface of rats. The body temperature of anesthetized rats increased gradually with a physical method and was detected by a digital thermometer inserted into the rectum. The peak latency (PL), interpeak latency (IPL), wave amplitude and the critical body temperature at which BAEP and MLR completely lost had been observed. RESULTS: All PL and I - II, I - III and I -IV IPL of BAEP shortened more and more as the body temperature increased step by step from 37 degrees C to 41.5 degrees C. But all PL and I - II and I -IV IPL did not shortened further and prolonged a little contrary as the body temperature at 42 degrees C and over 42 degrees C. All PL and P1-P3 and P2-P3 IPL of MLR also shortened as the body temperature increased from 37 degrees C to 43 degrees C. The wave amplitudes of BAEP and MLR decreased as the body temperature increased, especially as the body temperature over 42 degrees C. BAEP and MLR lost completely and synchronously at the body temperature (43.1 +/- 0.5) degrees C, which was not reversed as the body temperature returning to normal by cooling. CONCLUSION: There were obvious effects of hyperthermia on both BAEP and MLR in rats, and irreversible impairments appeared at a critical body temperature.