Activatable Self-Dissociation of Watson-Crick Structures with Fluorescent Nucleotides for Sensing Multiple Human Glycosylases at Single-Cell Level.

Nucleobase oxidation and alkylation can destroy Watson-Crick base-pairing to challenge the genomic integrity. Human 8-oxoguanine glycosylase 1 (hOGG1) and alkyladenine glycosylase (hAAG) are evolved to counter these two cytotoxic lesions through base-excision repair, and their deregulations are implicated with multifactorial diseases and cancers. Herein, we demonstrate activatable self-dissociation of Watson-Crick structures with fluorescent nucleotides for sensing multiple human glycosylases at single-cell level. The presence of hOGG1 and hAAG catalyzes 8-oxoG and deoxyinosine removal in functional probe 1 to release two trigger probes (1 and 2). Then, trigger probes hybridize with functional probe 2 to activate the autocatalytic degradation of functional probes 2 (Cycle I) and 3 (Cycle II), replicating abundant trigger probes (1-4) and releasing two fluorophores (2-aminopurine (2-AP) and pyrrolo-dC (P-dC)). New trigger probes (1, 2) and (3, 4), in turn, hybridize with free functional probes 2 and 3, repeating Cycles I and II turnovers. Through multicycle self-dissociation of Watson-Crick structures, 2-AP and P-dC are exponentially accumulated for the simultaneous quantification of hOGG1 and hAAG. This nanodevice exhibits high sensitivity with a detection limit of 2.9 × 10-3 U/mL for hOOG1 and 1.5 × 10-3 U/mL for hAAG, and it can measure enzymatic kinetics, identify potential inhibitors, discriminate glycosylases between cancer and normal cell lines, and even quantify glycosylase activities in a single HeLa cell. Moreover, this assay may be rapidly and isothermally performed in one tube with only one tool enzyme in a quencher-free manner, promising a simple and powerful platform for multiple human glycosylase detection.

Toxin-Antitoxin Systems Alter Adaptation of Mycobacterium smegmatis to Environmental Stress.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.

Effects of Bedaquiline on Antimicrobial Activity and Cytokine Secretion of Macrophages Infected with Multidrug-Resistant Mycobacterium tuberculosis Strains.

Background: Bedaquiline (Bdq) exerts bactericidal effects against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains, including multidrug-resistant M. tuberculosis strains (MDR-MTBs). However, few reported investigations exist regarding Bdq effects on MDR-MTBs-infected macrophages activities and cytokine secretion. Here, Bdq bactericidal activities against MDR-MTBs and related cellular immune mechanisms were explored. Methods: Macrophages infected with MDR-MTBs or H37Rv received Bdq treatments (4 h/8 h/24 h/48 h) at 1 × the minimum inhibitory concentration (1 × MIC), 10 × MIC and 20 × MIC. Intracellular colony-forming units (CFUs) and culture supernatant IL-12/23 p40, TNF-α, IL-6, and IL-10 were determined using the Luminex® 200TM system. Normally distributed continuous data (mean ± standard deviation) were analyzed using t-test or F-test (SPSS 25.0, P < 0.05 deemed statistically significant). Results: (1) 100% of Bdq-treated macrophages (all doses applied over 4-48 h) survived with 0% inhibition of proliferation observed. (2) Intracellular CFUs of Bdq-treated MDR-MTBs-infected macrophages decreased over 4-48 h of treatment, were lower than preadministration and control CFUs, decreased with increasing Bdq dose, and resembled H37Rv-infected group CFUs (48 h). (3) For MDR-MTBs-infected macrophages (various Bdq doses), IL-12/23 p40 levels resembled preadministration group levels and exceeded controls (4 h); TNF-α levels exceeded preadministration group levels (24 h/48 h) and controls (24 h); IL-12/23 p40 and TNF-α levels resembled H37Rv-infected group levels (4 h/8 h/24 h/48 h); IL-6 levels exceeded preadministration and H37Rv-infected group levels (24 h/48 h) and controls (24 h); IL-10 levels resembled preadministration and H37Rv-infected group levels (4 h/8 h/24 h/48 h) and were lower than controls (24 h/48 h); IL-12/23 p40 and IL-10 levels remained unchanged as intracellular CFUs changed, with IL-12/23 p40 levels exceeding controls (4 h) and IL-10 levels remaining lower than controls (24 h/48 h); TNF-α and IL-6 levels increased as intracellular CFUs decreased (24 h/48 h) and exceed controls (24 h). Conclusion: Bdq was strongly bactericidal against intracellular MDR-MTBs and H37Rv in a time-dependent, concentration-dependent manner. Bdq potentially exerted immunomodulatory effects by inducing high-level Th1 cytokine expression (IL-12/23 p40, TNF-α) and low-level Th2 cytokine expression (IL-10).

The Activities and Secretion of Cytokines Caused by Delamanid on Macrophages Infected by Multidrug-Resistant Mycobacterium tuberculosis Strains.

Background: Delamanid (Dlm) is an effective drug against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains, including Multidrug-resistant Mycobacterium tuberculosis (MDR-MTB). There are few reports on the activity and secretion of cytokines caused by Dlm on macrophages infected by MDR-MTB strains. Therefore, this article aims to observe the bactericidal activity and secretion of cytokines of the macrophages infected by MDR-MTB strains after Dlm was administered, so as to provide a basis for further perfecting the mechanism of Dlm. Methods: Samples were respectively collected to count the intracellular colony-forming unit (CFU) of macrophages infected by MDR-MTB or H37Rv strains at 4, 8, 24, and 48 h after Dlm at MIC, 10MIC, and 20MIC were administered. Samples were respectively collected to detect the level of IL-12/23 p40, TNF-α, IL-6, and IL-10 in the culture supernatant of macrophages infected by MDR-MTB or H37Rv strains at 4, 24, and 48 h after Dlm at MIC were administered. The levels of four cytokines in the culture supernatant were measured using the Luminex® 200™ (Luminex, USA) according to the manufacturer's instructions. Data were analyzed by SPSS 25.0 software. The continuous data in normal distribution were expressed as mean ± standard deviation ( x¯ ± s) and analyzed by t or F test. P<0.05 was considered statistically significant. Results: (1) After Dlm was applied to macrophages infected by MDR-MTB strains:(A) The intracellular CFU gradually decreased, reached the lowest value at 48 h, and was lower than that of Dlm before administration and infection group (P<0.05). (B) The intracellular CFU was further reduced after increasing Dlm dose to 10MIC and 20MIC, and the latter was lower than that of the former (P<0.05). (C) The intracellular CFU of MDR-MTB group was higher than that of H37Rv group at 4~48 h after administration (P<0.05). (2) After Dlm at MIC dose was applied to macrophages infected by MDR-MTB strains: (A) The level of IL-12/23 p40 at any time didn't change compared with that of Dlm before administration (P>0.05), while the level of IL-12/23 p40 at 4 h was higher than that of the infection group (P<0.05). The levels of TNF-α at 24 and 48 h were higher than that of Dlm before administration (P<0.05), but were similar to that of the infection group (P>0.05). In addition, the levels of IL-12/23 p40 and TNF-α at any time were similar to that of the H37Rv group after administration (P>0.05). (B) The levels of IL-6 at 24 and 48 h were higher than that of Dlm before administration (P<0.05), but were similar to that of H37Rv group (P>0.05) and were lower than that of infection group (P<0.05). The level of IL-10 at any time didn't change compared with that of Dlm before administration (P>0.05), but was lower than that of the infection group at 4~48 h and was lower than that of the H37Rv group at 24 h (P<0.05). (C) The level of IL-12/23 p40 and IL-10 didn't change with the change of intracellular CFU (P<0.05), while the level of TNF-α and IL-6 increased with the intracellular CFU decreasing, and the increase level of TNF-α was lower than that of the infection group (P<0.05). Conclusions: Dlm had strong bactericidal activity against intracellular MDR-MTB, which was time-dependent and concentration-dependent. Its bactericidal activity against intracellular MDR-MTB strains was weaker than that against drug-susceptible tuberculosis strains. Dlm might have immunomodulatory effect, inducing low expression of Th2 cytokines IL-6 and IL-10 at different times after administration.

PHF23 negatively regulates the autophagy of chondrocytes in osteoarthritis.

AIM: Osteoarthritis (OA) is the main cause of disability and joint replacement surgery in the elderly. As a crucial cell survival mechanism, autophagy has been reported to decrease in OA. PHF23 is a new autophagy inhibitor which was first reported by us previously. This study aimed to explore the anti-autophagic mechanism of PHF23 to make it a possible therapeutic target of OA. MAIN METHOD: Lentiviral vectors specific to PHF23 were used on chondrocytes (C28/I2) to establish PHF23 overexpressed or knockdown stable cell strains. Interleukin (IL)-1ß (10 ng/mL) and chloroquine (CQ, 25 uM) were used as an inducer of OA and inhibitor of lysosome, respectively. Autophagy was evaluated by autophagosome formation using transmission electron microscopy (TEM) and western blot analysis of P62 and LC3B on different groups of cells. Effects of PHF23 on OA were evaluated by collagen II immunofluorescent staining and western blot analysis of OA-associated proteins MMP13 and ADAMTS5. Effects of PHF23 on AMPK and mTOR/S6K pathways and mitophagy were determined by western blot analysis. KEY FINDINGS: Knockdown of PHF23 enhanced IL-1ß-induced autophagy, while overexpression of PHF23 exerted the opposite effect. Knockdown of PHF23 protected chondrocytes against IL-1ß-induced OA by decreasing the levels of OA-associated proteins and increasing expression of Collagen II. Knockdown of PHF23 also increased mitophagy level and altered the phosphorylation levels of AMPK, mTOR, and S6K. SIGNIFICANCE: PHF23 downregulates autophagy, mitophagy in IL-1ß-induced OA-like chondrocytes and alters the activities of AMPK and mTOR/S6K, which suggests that PHF23 may be a possible therapeutic target for OA.

Inflammation responses in patients with pulmonary tuberculosis in an intensive care unit.

Pulmonary tuberculosis caused by Mycobacterium tuberculosis remains a global problem. Inflammatory responses are the primary characteristics of patients with pulmonary tuberculosis in intensive care units (ICU). The aim of the present study was to investigate the clinical importance of inflammatory cells and factors for patients with pulmonary tuberculosis in ICU. A total of 124 patients with pulmonary tuberculosis in ICU were recruited for the present study. The inflammatory responses in patients with pulmonary tuberculosis in ICU were examined by changes in inflammatory cells and factors in the serum. The results indicated that serum levels of lymphocytes, plasma cells, granulocytes and monocytes were increased in patients with pulmonary tuberculosis in ICU compared with healthy controls. The serum levels of inflammatory factors interleukin (IL)-1, IL-6, IL-10, IL-12, and IL-4 were upregulated in patients with pulmonary tuberculosis in ICU. Lower plasma concentrations of IL-2, IL-15 and interferon-γ were detected in patients with pulmonary tuberculosis compared with healthy controls. It was demonstrated that high mobility group box-1 protein expression levels were higher in the serum of patients with pulmonary tuberculosis compared with healthy controls. Notably, an imbalance of T-helper cell (Th)1/Th2 cytokines was observed in patients with pulmonary tuberculosis. Pulmonary tuberculosis caused by M. tuberculosis also upregulated expression of matrix metalloproteinase (MMP)-1 and MMP-9 in hPMCs. In conclusion, these outcomes demonstrated that inflammatory responses and inflammatory factors are associated with the progression of pulmonary tuberculosis, suggesting that inhibition of inflammatory responses and inflammatory factors may be beneficial for the treatment of patients with pulmonary tuberculosis in ICU.

Polymorphisms in IRG1 gene associated with immune responses to hepatitis B vaccination in a Chinese Han population and function to restrain the HBV life cycle.

Vaccination against the hepatitis B virus (HBV) is extensively used as an effective method to prevent HBV infection. However, nearly 10% of healthy adults fail to produce a protective level of antibodies against the hepatitis B vaccine, and multiple genetic variants are known to affect the immune response to the hepatitis B vaccine. The aim of the present study was to investigate the association between polymorphisms in immunoresponsive gene 1 (IRG1) gene and the immune response to hepatitis B vaccination in a Chinese Han population. Four single nucleotide polymorphisms (SNPs) located in the IRG1 gene were genotyped in 1230 high-responders and 451 non-responders to hepatitis B vaccination. The SNPs rs17470171 and rs17385627 were associated with the immune response to hepatitis B vaccination (P = 0.014 and 0.029, respectively). In addition, the haplotypes G-A-A-A (rs614171-rs17470171-rs9530614-rs17385627, P = 0.0042, OR = 0.68) and A-A (rs17470171-rs17385627, P = 0.0065, OR = 0.72) exerted a protective role in the immune response to hepatitis B vaccination. Allele 'A' of rs17470171 and allele 'A' of rs17385627 show higher levels of expression for the IRG1 gene compared with allele 'C' of rs17470171 and allele 'T' of rs17385627 as demonstrated by luciferase reporter and overexpression assays. In addition, we observed that IRG1 inhibited the HBV life cycle and that IRG1 rs17385627 allele 'A' was more effective than rs17385627 allele 'T' at eliminating HBV in HepG2.2.15 cells. These findings suggest that polymorphisms in the IRG1 gene are associated with the immune response to hepatitis B vaccination. The antiviral effect of IRG1 was confirmed using HBV infection cell models.

Behavioral intervention reduces unhealthy eating behaviors in preschool children via a behavior card approach.

Many eating behaviors form in childhood, and some unhealthy behaviors may persist into adulthood and have potential impacts on people's health. This study evaluated the effectiveness of behavioral intervention in reducing consumption of Western fast food, sweetened beverages, fried food in preschool children, and changing parents' rewarding behaviors that encourage the consumption of the unhealthy foods. The research was a cluster randomized trial of seven kindergartens, involving 1138 children aged 3-6 years and their parents in Beijing, China. Parents and children allocated to the intervention group received two lectures and printed resources, including behavior cards, educational sheets. Children's behavior cards, applied with behavior-changing techniques, were used to intervene, and monitor behavior changes over time. Children in the control group just followed their usual health education curriculum in kindergartens. Intervention effects on food consumption behaviors were assessed by examining pre- and post-questionnaires. Of the 1138 children screened at baseline, 880 (77.3%) were measured at the end of the intervention period. The intervention lasted from March to June in 2010. The results showed that consumption of Western fast food, sweetened beverages, and fried food was decreased among the intervention group (P<0.001). Proportions of parents using Western fast food as rewards for their children were decreased (P=0.002). From March to June 2010, the frequency of each target behavior in children tended to decrease over the intervention period (P<0.001). Most parents favored regularly-delivered behavior cards or materials for behavioral intervention. In conclusion, the behavioral intervention encourages the healthier eating behaviors of children and reduces the parents' practice of using unhealthy foods as reward.

Analysis of plasma microRNA expression profiles in male textile workers with noise-induced hearing loss.

BACKGROUND: Circulating microRNAs (miRNAs) have attracted interests as non-invasive biomarkers of physiological and pathological conditions, which may be applied in noise-induced hearing loss (NIHL). However, no epidemiology studies have yet examined the potential effects of NIHL or noise exposure on miRNA expression profiles. OBJECTIVES: We sought to identify permanent NIHL-related miRNAs and to predict the biological functions of the putative genes encoding the indicated miRNAs. METHODS: In the discovery stage, we used a microarray assay to detect the miRNA expression profiles between pooled plasma samples from 10 noise-exposed individuals with normal hearing and 10 NIHL patients. In addition, we conducted a preliminary validation of six candidate miRNAs in the same 20 workers. Subsequently, three miRNAs were selected for expanded validation in 23 non-exposed individuals with normal hearing and 46 noise-exposed textile workers which including 23 noise-exposed workers with normal hearing and 23 NIHL patients. Moreover, we predicted the biological functions of the putative target genes using a Gene Ontology (GO) function enrichment analysis. RESULTS: In the discovery stage, compared with the noise exposures with normal hearing, 73 miRNAs demonstrated at least a 1.5-fold differential expression in the NIHL patients. In the preliminary validation, compared with the noise exposures, the plasma levels of miR-16-5p, miR-24-3p, miR-185-5p and miR-451a were all upregulated (P < 0.001) in the NIHL patients. In the expanded validation stage, compared with the non-exposures, the plasma levels of miR-24, miR-185-5p and miR-451a were all significantly downregulated (P < 0.001) in the exposures. And compared with the noise exposures, the plasma levels of miR-185-5p and miR-451a were slightly elevated (P < 0.001) in the NIHL patients, which were consistent with the results of preliminary validation and microarray analysis. CONCLUSION: The two indicated plasma miRNAs may be biomarkers of indicating responses to noise exposure. However, further studies are necessary to prove the causal association between miRNAs changes and noise exposure, and to determine whether these two miRNAs are clear biomarkers to noise exposure.

The suppression of epileptiform discharges in cultured hippocampal neurons is regulated via alterations in full-length tropomyosin-related kinase type B receptors signalling activity.

Epilepsy is a common neurological disease. Understanding the mechanisms of epileptogenesis at the cellular and molecular levels may provide novel targets for preventing this disorder. Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase type B (TrkB) are believed to be critical for epileptogenesis. Previous studies have revealed possible changes in the expression of full-length TrkB receptors (TrkB.FL) and truncated TrkB receptors (TrkB.T) in neurodegenerative disorders. In this study, we investigated alterations in TrkB receptor expression and TrkB signalling activity in a rat hippocampal neuronal model of spontaneous recurrent epileptiform discharges (SREDs) and the effects on the epileptiform discharges. To induce epileptiform discharges, we established a model with Mg(2+) -free treatment. We found a dramatic upregulation of TrkB.T and a decrease in TrkB.FL in the SREDs model. Calpain contributed to the downregulation of TrkB.FL. The upregulation of TrkB.T required transcription and translation activity. Furthermore, BDNF induced the activation of TrkB.FL signalling. However, TrkB.FL signalling was inhibited in the SREDs model. Although calpain inhibitors prevented a decrease in TrkB.FL, they did not restrain the downregulation of TrkB.FL signalling activity in the model. However, a SREDs model with a translation inhibitor prevented the increase in TrkB.T and re-activated TrkB.FL signalling activity. Finally, we used electrophysiology to observe that a downregulation of TrkB.T could relieve the representative epileptiform discharges in the model. These results, taken together, demonstrate that alterations in TrkB.FL signalling may be regulated via TrkB.T receptors. Upregulation of TrkB.FL signalling suppresses epileptiform discharges in the SREDs model.

[Therapeutic effects of umbilical cord mesenchymal stem cells transplantation on systemic lupus erythematosus].

OBJECTIVE: To observe the efficacy of umbilical cord mesenchymal stem cells (UC-MSCs) transplantation for the patients with refractory systemic lupus erythematosus (SLE). METHODS: Thirty seven patients with SLE were enrolled in this study, and divided into conventional treatment group (control group, n = 20) and UC-MSCS adjuvant treatment group (treatment group, n= 17). All the patients in both two groups were treated with glucocorticoids and cyclophosphamide (CTX). In the UC-MSCs group, each patient additionally received the transplantation of 3 x 10(7) UC-MSCs infusion intravenously. The clinical manifestations and laboratory parameters of each patient were observed before the treatments and 2 weeks, 1 month, 2 months, 3 months, months,9 months and 12 months after the treatments. RESULTS: All the 37 patients were observed for 12 months. 24 h urinary protein excretion (U-Pro), anti nuclear antibody (ANA), erythrocyte sedimentation rate (ESR), high-sensitivity C-reactive protein (hs-CRP), systemic lupus erythematosus disease activity index (SLEDAI) of these two groups decreased significantly (P < 0.05). serum albumin (ALB), C3, and C4 of two groups were higher after the treatments (P < 0.05). ALB and C3 in treatment group exceeded the control group (P < 0.05). The positive rates of Anti-dsDNA in control and treatment group were 40% and 10% respectively, while the recurrence rates were 50% and 20% respectively, these difference between the two groups were statistically significant (P < 0.05). There were no transplantation related complications observed. CONCLUSION: UC-MSCs transplantation could be effective and safe for refractory SLE on basis of glucocorticoid and cyclophosphamide therapy.

(99m)Tc-DTPA renal dynamic imaging method may be unsuitable to be used as the reference method in investigating the validity of CDK-EPI equation for determining glomerular filtration rate.

OBJECTIVE: To compare the measurements of glomerular filtration rate (GFR) determined by (99m)Tc-diethylene triamine pentaacetic acid ((99m)Tc-DTPA) renal dynamic imaging with those estimated by Chronic Kidney Disease Epidemiology Collaboration (CDK-EPI) equation and to identify a more accurate measurement of GFR of chronic kidney disease (CKD) patients in clinical practice. METHODS: The GFR was determined simultaneously by 3 methods: (a) dual plasma sample clearance method (tGFR); (b) renal dynamic imaging method (dGFR); (c) CDK-EPI equation (eGFR). The tGFR was employed as the reference method. The correlation, regression, and limit of agreement of dGFR and eGFR were used to demonstrate the validity of the two methods. The comparison of bias, precision, and accuracy between dGFR and eGFR was analyzed to identify the most suitable method. The analysis of bias, precision and accuracy was repeated after stratifying patients by a measured tGFR cutpoint of 60 ml·min(-1)·(1.73 m(2))(-1). RESULTS: A total of 149 patients were enrolled. Both dGFR and eGFR correlated well with tGFR and the regression equation of dGFR and eGFR against tGFR was respectively Y = -4.289+0.962X (r = 0.919; RMSE = 14.323 ml.min(-1). (1.73 m(2))(-1); P<0.001) and Y = 2.462+0.914X (r = 0.909; RMSE = 15.123 ml.min(-1). (1.73 m(2))(-1); P<0.001). In addition, Bland-Altman analysis showed preferable agreement between the two methods and the reference method. The comparison revealed that eGFR, compared with dGFR, showed better performance on bias and 50% accuracy and similar performance on other indexes in the whole cohort and the lower-GFR subgroup, whereas in the higher-GFR subgroup the difference of the two methods was not significant in all parameters. CONCLUSIONS: Although both CDK-EPI equation and renal dynamic imaging can be used to determine the GFR of CKD patients, CDK-EPI equation is more accurate than renal dynamic imaging. As a result, (99m)Tc-DTPA renal dynamic imaging may be unsuitable to be used as the reference method in investigating the validity of CDK-EPI equation.

CDK-EPI equation may be the most proper formula based on creatinine in determining glomerular filtration rate in Chinese patients with chronic kidney disease.

PURPOSE: To compare the performance of the Chronic Kidney Disease Epidemiology Collaboration (CDK-EPI) equation, the 24-h creatinine clearance rate (24hCCr), Cockroft-Gault (C-G) formula, the abbreviated Modification of Diet in Renal Disease (aMDRD) equation, the modified Modification of Diet in Renal Disease (mMDRD) equation in determining glomerular filtration rate (GFR) in Chinese patients with chronic kidney disease (CKD) and detect the most proper method to measure GFR in clinical practice. METHODS: One hundred and fifty-four patients with CKD were enrolled in the present study. (99m)Tc-diethylene triamine pentaacetic acid ((99m)Tc-DTPA) plasma clearance method measured by dual plasma sampling method (rGFR) was considered as the reference standard. GFR was estimated simultaneously using five methods: (1) CDK-EPI equation (eGFR1); (2) 24hCCr (eGFR2); (3) C-G formula (eGFR3); (4) abbreviated MDRD equation (eGFR4); (5) mMDRD equation (eGFR5). The comparison of correlation, regression, bias, precision, accuracy, limit of agreement, and receiver-operating characteristics (ROC) for detecting CKD (with a GFR cutoff of 60 mL min(-1) 1.73 m(-2)) among the methods was analyzed to identify the most suitable method. RESULTS: All the equations correlated well with rGFR, and the correlation coefficient of CDK-EPI equation was the highest (reGFR1 = 0.922, P < 0.001). The Bland-Altman analysis showed that the limit of agreement for CDK-EPI equation was -25.5 to 30.3 mL min(-1) 1.73 m(-2), which was the least range among the tested equations. The CDK-EPI equation represented the best capability in precision (14.24 mL min(-1) 1.73 m(-2)). The ROC curve showed the best performance in detecting CKD. The accuracy within ±30 % of CDK-EPI equation, 24hCCr, and mMDRD equation was 72.08, 69.48, and 70.13 %, respectively, and no statistical significant difference was found (P > 0.05). However, CDK-EPI equation had the highest accuracy when compared with the other two equations (P < 0.05). And its performance on bias showed no statistically significant difference compared with other four equations. CONCLUSIONS: Although its bias and accuracy did not overmatch the other four equations in our patient group, the CDK-EPI equation outperformed the other equations based on creatinine in correlation, precision, limit of agreement, and detecting CKD, and it is very simple, time-saving, and cost-effective. So we recommend intensely that the CDK-EPI equation is the most suitable method in determining GFR in Chinese patients with CKD and can be applied generally in clinical practice.

CD3Z genetic polymorphism in immune response to hepatitis B vaccination in two independent Chinese populations.

Vaccination against hepatitis B virus is an effective and routine practice that can prevent infection. However, vaccine-induced immunity to hepatitis B varies among individuals. CD4(+) T helper cells, which play an important role in both cellular and humoral immunity, are involved in the immune response elicited by vaccination. Polymorphisms in the genes involved in stimulating the activation and proliferation of CD4(+) T helper cells may influence the immune response to hepatitis B vaccination. In the first stage of the present study, a total of 111 single nucleotide polymorphisms (SNPs) in 17 genes were analyzed, using the iPLEX MassARRAY system, among 214 high responders and 107 low responders to hepatitis B vaccination. Three SNPs (rs12133337 and rs10918706 in CD3Z, rs10912564 in OX40L) were associated significantly with the immune response to hepatitis B vaccination (P = 0.008, 0.041, and 0.019, respectively). The three SNPs were analyzed further with the TaqMan-MGB or TaqMan-BHQ probe-based real-time polymerase chain reaction in another independent population, which included 1090 high responders and 636 low responders. The minor allele 'C' of rs12133337 continued to show an association with a lower response to hepatitis B vaccination (P = 0.033, odds radio = 1.28, 95% confidence interval = 1.01-1.61). Furthermore, in the stratified analysis for both the first and second populations, the association of the minor allele 'C' of rs12133337 with a lower response to hepatitis B vaccination was more prominent after individuals who were overweight or obese (body mass index ≥25 kg/m(2)) were excluded (1(st) stage: P = 0.003, 2(nd) stage: P = 0.002, P-combined = 9.47e-5). These findings suggest that the rs12133337 polymorphism in the CD3Z gene might affect the immune response to hepatitis B vaccination, and that a lower BMI might increase the contribution of the polymorphism to immunity to hepatitis B vaccination.

Cloning a peanut resveratrol synthase gene and its expression in purple sweet potato.

A resveratrol synthase gene was cloned from the peanut plant (Arachis hypogaea) by RT-PCR and was transformed into purple sweet potato (Ipomoea batatas) by Agrobacterium-mediated transformation. Stem sections were infected with bacterial solution of OD(600) = 0.4 for 20 min and then cocultured for 2 days. Infected explants were cultured on MS media containing 50 mg/l kanamycin, 0.02 mg/l NAA and 1 mg/l 6-BA for bud induction or containing 75 mg/l kanamycin, 1.0 mg/l NAA and 0.1 mg/l 6-BA for root formation. The bud and root induction rates were 37.5 and 25.0%, respectively. 105 regenerated plants were obtained, with 11 positive plants by PCR and Southern blotting analyses. A high level of resveratrol glucoside (340 µg/g dry weight), but no resveratrol, was detected in the transformed plants by HPLC. This study also provides a stable genetic transformation and plant regeneration method for metabolic modification of purple sweet potato.

Occurrence, function and potential medicinal applications of the phytohormone abscisic acid in animals and humans.

Abscisic acid (ABA) is an important phytohormone that regulates plant growth, development, dormancy and stress responses. Recently, it was discovered that ABA is produced by a wide range of animals including sponges (Axinella polypoides), hydroids (Eudendrium racemosum), human parasites (Toxoplasma gondii), and by various mammalian tissues and cells (leukocytes, pancreatic cells, and mesenchymal stem cells). ABA is a universal signaling molecule that stimulates diverse functions in animals through a signaling pathway that is remarkably similar to that used by plants; this pathway involves the sequential binding of ABA to a membrane receptor and the activation of ADP-ribose cyclase, which results in the overproduction of the intracellular cyclic ADP-ribose and an increase in intracellular Ca²âº concentrations. ABA stimulates the stress response (heat and light) in animal cells, immune responses in leukocytes, insulin release from pancreatic ß cells, and the expansion of mesenchymal and colon stem cells. ABA also inhibits the growth and induces the differentiation of cancer cells. Unlike some drugs that act as cell killers, ABA, when functioning as a growth regulator, does not have significant toxic side effects on animal cells. Research indicated that ABA is an endogenous immune regulator in animals and has potential medicinal applications for several human diseases. This article summarizes recent advances involving the discovery, signaling pathways and functions of ABA in animals.

Cloning, microbial expression and structure-activity relationship of polyphenol oxidases from Camellia sinensis.

Polyphenol oxidase (PPO) can be used for organic synthesis and degradation of wastes and dyes in industries. Lack of enzyme sources is a major barrier for its application. A PPO gene, with a full length of 1.8kb without introns, was cloned by PCR from genomic DNA of five common cultivars of Camellia sinensis. They had a 98.2-99.9% degree of identity in nucleotides and 94.7-96.1% in amino acids and encoded a polypeptide of 599 amino acids with a signal peptide targeting the chloroplast and three Cu-binding domains. The mature PPO showed high expression and enzyme activity after refolding the inclusion bodies in Escherichia coli BL21 (DE3) using pET30c expression vector, but low expression in Pichia pastoris GS115 using both the secretory and non-secretory vectors pPICZalphaA and pPICZA. The expression of PPO mutants demonstrated that the signal sequences prevented recombinant gene expression in E. coli. PPO activity was not affected by the C-terminus and was slightly inhibited by the CuC domain. Other domains were important for its activity. A 3.1-fold increase in PPO activity over non-recombinant controls was obtained by expressing the PPO fragment without signal sequences and the CuC domain in E. coli BL21 (DE3) using the pET30c vector.

[Effect of Panax notoginseng saponins on human kidney fibroblast].

OBJECTIVE: To study the effect of Panax notoginseng saponins (PNS) on cell proliferation, type I collagen secretion and integrin beta 1 expression in human kidney fibroblast (KFB). METHODS: KFB were cultured and stimulated by PNS in vitro. The cell proliferation, type I collagen secretion and integrin beta 1 expression in KFB were measured by methyl thiazolyl tetrazolium (MTT), enzyme-linked immunoabsorbent assay (ELISA) and flowcytometry respectively. RESULTS: Within its optimal concentration range and effective time range, PNS could obviously inhibit the proliferation, type I collagen secretion and integrin beta 1 expression (all P < 0.05) in human KFB. CONCLUSION: PNS would possibly be an effective drug for renal interstitial fibrosis prevention and treatment.