Characterization of gut microbiota in patients with stage 3-4 chronic kidney disease: a retrospective cohort study.

PURPOSE: Multiple factors, such as dietary patterns, pharmaceutical interventions, and exposure to harmful substances, possess the capacity to influence gut microbiota composition. Gut microbiota dysbiosis has emerged as a significant contributor to the progression of chronic kidney disease (CKD) and its associated complications. By comprehending the intricacies of the intestinal microbiota, this research endeavor holds the potential to offer novel perspectives on potential strategies for mitigating CKD progression. METHODS: In this retrospective analysis, we assessed gut microbiota composition in CKD patients. Fecal samples were collected from a cohort of 44 patients with stage 3-4 CKD, alongside a control group consisting of 132 healthy volunteers. Subsequently, 16 s rDNA sequencing was conducted to examine the composition of the gut microbiota. RESULTS: Our findings revealed significant alterations in the diversity of intestinal microbiota in fecal samples between patients with stage 3-4 CKD and healthy subjects. Among the 475 bacterial genera, 164 were shared, while 242 dominant genera were exclusive to healthy subjects and 69 to CKD stages 3-4 samples. Notably, healthy volunteers exhibited a prevalence of intestinal Firmicutes and Bacteroidetes, whereas stage 3-4 CKD patients displayed higher abundance of Proteobacteria and Actinobacteria. The presence of uncultured Coprobacillus sp. notably contributed to distinguishing between the two groups. ROC curve analysis identified distinct microbiota with superior diagnostic efficacy for discriminating stage 3-4 CKD patients from healthy individuals. Metabolic pathway analysis revealed differing dominant pathways between the two groups-the NADH dehydrogenase pathway in healthy individuals and the phosphate acetyltransferase pathway in stage 3-4 CKD patients. Moreover, the CKD cohort displayed a higher proportion of Gram-negative bacteria and facultative anaerobes. CONCLUSIONS: In conclusion, our study underscores the profound influence of gut microbiota dysbiosis on CKD progression. The distinct microbial profiles observed in CKD patients highlight the potential efficacy of microbiota-based interventions in mitigating CKD advancement.

Identification of a novel prognostic signature correlated with epithelial-mesenchymal transition, N6-methyladenosine modification, and immune infiltration in colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a commonly diagnosed human malignancy worldwide. Both epithelial-mesenchymal transition (EMT) and N6-methyladenosine (m6A) modification play a crucial role in CRC development. This study aimed to construct a prognostic signature based on the genes related to EMT and m6A modification. METHOD: Firstly, the mRNA expression profiling of CRC tissues was analyzed using TCGA and GEO databases. The prognostic hub genes related to EMT and m6A modification were selected using weighted correlation network and cox regression analysis. The prognostic signature was constructed based on hub genes, followed by validation in three external cohorts. Finally, the expression of the representative hub gene was detected in clinical samples, and its biological role was investigated using assays in vivo and in vitro. RESULTS: A prognostic signature was constructed using the following genes: YAP1, FAM3C, NUBPL, GLO1, JARID2, NFKB1, CDKN1B, HOOK1, and GIPC2. The signature effectively stratified the clinical outcome of CRC patients in the training cohort and two validation cohorts. The subgroup analysis demonstrated the signature could identify high-risk population from CRC patients within stage I-II or III-IV, female, male and elder patients. The signature was correlated with the infiltration of some immune cells (such as macrophage and regulatory T cells) and gene mutation counts. Finally, the hub gene GIPC2 was found to be downregulated in CRC tissues and most CRC cells lines. GIPC2 overexpression inhibited the malignant characteristics of CRC cells in vitro and in vivo through upregulating E-cadherin and downregulating N-cadherin, Vimentin, and Snail, while the opposite results were observed for GIPC2 knockdown in CRC cells. CONCLUSION: Our present study for the first time constructed a novel prognostic signature related to EMT, m6A modification, and immune infiltration for CRC risk stratification. In addition, GIPC2 is identified as a promising clinical biomarker or therapeutical target for CRC.

Generation of Functional Human Cardiac Progenitor Cells by High-Efficiency Protein Transduction.

UNLABELLED: The reprogramming of fibroblasts to induced pluripotent stem cells raises the possibility that somatic cells could be directly reprogrammed to cardiac progenitor cells (CPCs). The present study aimed to assess highly efficient protein-based approaches to reduce or eliminate the genetic manipulations to generate CPCs for cardiac regeneration therapy. A combination of QQ-reagent-modified Gata4, Hand2, Mef2c, and Tbx5 and three cytokines rapidly and efficiently reprogrammed human dermal fibroblasts (HDFs) into CPCs. This reprogramming process enriched trimethylated histone H3 lysine 4, monoacetylated histone H3 lysine 9, and Baf60c at the Nkx2.5 cardiac enhancer region by the chromatin immunoprecipitation quantitative polymerase chain reaction assay. Protein-induced CPCs transplanted into rat hearts after myocardial infarction improved cardiac function, and this was related to differentiation into cardiomyocyte-like cells. These findings demonstrate that the highly efficient protein-transduction method can directly reprogram HDFs into CPCs. This protein reprogramming strategy lays the foundation for future refinements both in vitro and in vivo and might provide a source of CPCs for regenerative approaches. SIGNIFICANCE: The findings from the present study have demonstrated an efficient protein-transduction method of directly reprogramming fibroblasts into cardiac progenitor cells. These results have great potential in cell-based therapy for cardiovascular diseases.

The caspase-8 shRNA-modified mesenchymal stem cells improve the function of infarcted heart.

The beneficial effects of mesenchymal stem cells (MSCs) in cardiac cell therapy are greatly limited due to poor survival after transplantation into ischemic hearts. Here, we investigated whether caspase 8 small hairpin RNA (shRNA) modification enhance human MSCs (hMSCs) survival and improve infarcted heart function. Recombinant adenovirus encoding pre-miRNA-155-designed caspase 8 shRNA was prepared to inhibit caspase 8 expression in hMSCs. The effect of caspase 8 shRNA modification on protecting hMSCs from apoptosis under the conditions of serum deprivation and hypoxia was tested by Annexin V/PI staining and caspase 8 activity assay. The caspase 8 shRNA-modified and superparamagnetic iron oxide (SPIO)-labeled hMSCs were injected into the border zone of the infarcted region of rat heart. Echocardiography and Masson trichrome staining were performed to assess heart function and cardiac fibrosis. Our results showed that adenovirus-mediated caspase 8 shRNA could efficiently inhibit caspase 8 expression in hMSCs. Knock-down of caspase 8 expression lead to inhibition of hMSCs apoptosis, reduction of caspase 8 activity and up-regulations of HGF, IGF-1 and Bcl-2. Transplantation of caspase 8 shRNA-modified hMSCs could significantly improve infracted heart function, attenuate cardiac fibrosis. Consistently, the rate of cardiomyocyte apoptosis and caspase 8 activity were significantly decreased, and the survival rate of transplanted hMSCs was markedly elevated in the myocardium receiving caspase 8 shRNA-modified hMSCs transplantation. Together, our findings implicated the therapeutic potential of caspase 8 shRNA-modified hMSCs in improving the infarcted heart function.

The learning effect of a training programme on the diagnosis of oesophageal lesions by narrow band imaging magnification among endoscopists of varying experience.

BACKGROUND: Magnifying narrow-band imaging using intra-epithelial papillary capillary loop analysis has been confirmed as a promising diagnostic modality for oesophageal lesions. Little is known about its learning curve. AIM: To evaluate the effect of a training programme on the diagnosis of oesophageal lesions by different modalities among endoscopists of varying experience. METHODS: We divided endoscopists into three groups based on their experience. A 2-h training programme on magnifying narrow-band imaging and intra-epithelial papillary capillary loop patterns was provided to trainees. They evaluated the test images and suggested diagnoses both before and after training. Diagnostic accuracy and interobserver agreement of three modalities were analysed. RESULTS: The diagnostic accuracies of magnifying narrow-band imaging for differentiating oesophageal neoplastic lesions and predicting lesion depth were significantly improved in less-experienced (92.8% vs. 55.9%, 63.8% vs. 17.5%) and non-experienced endoscopist groups (84.2% vs. 47.4%, 50% vs. 10%), and kappa (κ) values for both groups achieved good agreement after training (0.76 vs. 0.43, 0.68 vs. 0.24, respectively), all P<0.05. CONCLUSION: Magnifying narrow-band imaging could be learnt easily and rapidly by beginners. For diagnosis of oesophageal neoplastic lesions, our training programme improved the diagnostic skill of less-experienced endoscopists to the level of highly experienced endoscopists.

[Desmoplakin expression silencing affects cardiac voltage-gated sodium channel Nav1.5 in HL-1 cells].

OBJECTIVE: To investigate the association of desmoplakin with the distribution and function of Nav1.5 by RNA silencing technology in HL-1 cells. METHODS: HL-1 cells with desmoplakin expression suppression by RNA silencing were examined for desmoplakin and Nav1.5 protein expressions by Western blotting, and the distribution and co-location of desmoplakin and Nav1.5 protein were detected by immunofluorescence staining. Patch-clamp recording was applied to analyze the changes in whole-cell sodium current after desmoplakin silencing. RESULTS: Compared with the untreated group and negative control group, the cells with desmoplakin silencing showed obviously reduced expressions of desmoplakin and Nav1.5 proteins. Co-localization of desmoplakin and Nav1.5 was detected at cell-cell contact in untreated and control conditions, and desmoplakin expression silencing induced a drastic redistribution of Nav1.5 with decreased peak current density (156.3∓6.2 vs 41.8∓3.1, n=6, P<0.05), a shift in voltage dependence of steady-state inactivation (-42 mV vs -61 mV, n=5, P<0.05), and prolonged time of recovery from inactivation. CONCLUSION: Desmoplakin silencing caused redistribution of Nav1.5 protein and also changes in its electrophysiological properties in HL-1 cells.

Silencing of desmoplakin decreases connexin43/Nav1.5 expression and sodium current in HL­1 cardiomyocytes.

Desmosomes and gap junctions are situated in the intercalated disks of cardiac muscle and maintain the integrity of mechanical coupling and electrical impulse conduction between cells. The desmosomal plakin protein, desmoplakin (DSP), also plays a crucial role in the stability of these interconnected components as well as gap junction connexin proteins. In addition to cell­to­cell junctions, other molecules, including voltage­gated sodium channels (Nav1.5) are present in the intercalated disk and support the contraction of cardiac muscle. Mutations in genes encoding desmosome proteins may result in fatal arrhythmias, including arrhythmogenic right ventricular cardiomyopathy (ARVC). Therefore, the aim of the present study was to determine whether the presence of DSP is necessary for the normal function and localization of gap junction protein connexin43 (Cx43) and Nav1.5. To examine this hypothesis, RNA interference was utilized to knock down the expression of DSP in HL­1 cells and the content, distribution and function of Cx43 and Nav1.5 was assessed. Western blotting and flow cytometry experiments revealed that Cx43 and Nav1.5 expression decreased following DSP silencing. In addition, immunofluorescence studies demonstrated that a loss of DSP expression led to an abnormal distribution of Cx43 and Nav1.5, while scrape­loading dye/transfer revealed a decrease in dye transfer in DSP siRNA­treated cells. The sodium current was also recorded by the whole­cell patch clamp technique. The results indicated that DSP suppression decreased sodium current and slowed conduction velocity in cultured cells. The present study indicates that impaired mechanical coupling largely affects electrical synchrony, further uncovering the pathogenesis of ARVC.

[The potential pathogenesis of gastrointestinal vascular malformation and the potential mechanism of thalidomide in the treatment of gastrointestinal vascular malformation].

OBJECTIVE: To study the pathogenesis of gastrointestinal vascular malformation (GIVM) and the potential mechanism of thalidomide in the treatment of gastrointestinal bleeding due to GIVM. METHODS: We collected the surgical intestinal specimens from 10 patients who suffered from massive hemorrhage of gastrointestinal tract owning to GIVM and the normal intestinal mucosa around the lesions, as well as normal intestinal mucosa from healthy subjects. Immunohistochemical (IHC) staining was carried out to investigate the differences of angiopoietin 2 (Ang2), Notch1 and delta like ligand 4 (Dll4) in the above three intestinal mucosa to find the relationship with the pathogenesis of GIVM. Human umbilical vein endothelial cells (HUVECs) were cultured with 0, 25, 50, 100 and 200 mg/L thalidomide for 24 or 48 hours to observe their mRNA and protein expressions of Ang2, Notch1, Dll4 by real-time PCR and Western blot. RESULTS: By IHC staining, more expressions of Ang2, Notch1 and Dll4 in the lesions were detected than those in the normal intestinal mucosa around the lesions and the normal intestinal mucosa in healthy people. The expressions of Ang2, Notch1 and Dll4 were significantly correlated (P = 0.016, r = 0.732), and the expressions of Notch1 and Dll4 were absolutely correlated (P = 0.000, r = 1.000). Real-time PCR and Western blot showed that thalidomide could down-regulate the expressions of them, which were in a concentration-dependent manner. CONCLUSION: Ang2, Notch1 and Dll4 may correlate with the pathogenesis of GIVM, while thalidomide can concentration-dependently down-regulate the expression of Ang2, Notch1 and Dll4, which may be one of the mechanism that thalidomide play a therapeutic role in GIVM.

Association between polymorphisms of the renin-angiotensin system and coronary artery disease in Chinese patients with type 2 diabetes.

BACKGROUND: The renin-angiotensin system (RAS) is regarded as one of the most important regulatory systems for cardiovascular homeostasis. In this study, we investigated associations of the five genetic polymorphisms in the RAS and presence of coronary artery disease (CAD) in type 2 diabetes. METHODS: Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism analysis. We used the generalised multifactor dimensionality reduction (GMDR) method to identify gene-gene interactions. RESULTS: The D allele in the ACE gene was significantly more frequent in type 2 diabetic patients with CAD (p = 0.04). In multivariate logistic regression analysis, the DD genotype was associated with a significantly increased risk of CAD (p = 0.044). 1675G/A variant in the AT2R gene was found to be associated with CAD in female subjects with type 2 diabetes (p = 0.025). The three other polymorphisms of the RAS do not seem to influence the development of CAD in type 2 diabetes. No significant gene-gene interaction for any combinations of genotypes was found in the GMDR method. CONCLUSION: The DD variant of the ACE gene polymorphism is associated with increased risk of developing CAD in Chinese patients with type 2 diabetes. A slight impact of AT2R 1675G/A polymorphism on CAD was found only in female diabetic patients.

Integrating interacting drugs and genetic variations to improve the predictability of warfarin maintenance dose in Chinese patients.

OBJECTIVE: Compared with genetic factors, drug interactions are largely unexplored in pharmacogenetic studies. This study sought to systematically investigate the effects of VKORC1, STX4A, CYP2C9, CYP4F2, CYP3A4, and GGCX gene polymorphisms and interacting drugs on warfarin maintenance dose. METHODS: A retrospective study of 845 Chinese patients after heart valve replacement receiving long-term warfarin maintenance therapy was conducted. Thirteen polymorphisms in the six genes were genotyped, and 36 drugs that may interact with warfarin were investigated. RESULTS: Single-nucleotide polymorphism association analysis showed that VKORC1, CYP2C9 and CYP4F2 variations were highly associated with the warfarin maintenance dose. Among 36 drugs that may interact with warfarin, fluconazole, amiodarone, and omeprazole were associated with the requirement for 45.8, 16.7, and 16.7% lower median warfarin dose (all P<0.05 with a false discovery rate <0.05). The final pharmacogenetic equation explained 43.65% of interindividual variation of warfarin maintenance dose with age, body surface area, VKORC1 g.3588G>A, CYP2C9*3, CYP4F2 c.1297G>A, amiodarone, fluconazole, and diltiazem accounting for 1.97, 2.74, 24.12, 3.94, 1.64, 5.92, 2.47, and 0.84% of variation. CONCLUSION: The present study indicated that VKORC1, CYP4F2, and CYP2C9 genotypes and interacting drugs had a significant impact on the warfarin maintenance dose in Chinese patients with heart valve replacement and demonstrated that integrating interacting drugs can largely improve the predictability of the dose algorithm.

Role of vascular endothelial growth factor in angiodysplasia: an interventional study with thalidomide.

BACKGROUND AND AIM: The pathogenesis of angiodysplasia is still not fully understood and effective therapy is not available. Thalidomide was reported to be effective in the treatment of angiodysplasia, but the mechanisms underlying its activity are, as yet, unknown. We aimed to investigate the expression of vascular endothelial growth factor (VEGF) in angiodysplasia tissues, and the role of hypoxia-inducible factor-1α (HIF-1α) and basic fibroblast growth factor (bFGF) on VEGF expression in human umbilical vein endothelial cells (HUVEC). Additionally, we aimed to study the role of thalidomide in these parameters. METHODS: Immunohistochemistry was performed to visualize VEGF in angiodysplasia lesions. HUVEC were incubated under hypoxic conditions or in the presence of bFGF. Effects of exposure to thalidomide were studied. Cell growth was assessed in methylthiazolyte-trazolium assays. Enzyme-linked immunosorbent assays and real-time polymerase chain reaction were performed to assess the expression of VEGF at protein and mRNA levels. Western blot was performed to detect the expression of HIF-1α under hypoxic conditions. RESULTS: VEGF was strongly expressed in 75% of patients with angiodysplasia lesions, as compared to expression in patients without angiodysplasia lesions. VEGF was also induced in HUVEC under hypoxic conditions (P < 0.05). bFGF was found to stimulate the proliferation of HUVEC and enhance the expression of VEGF. Thalidomide suppressed bFGF-induced proliferation significantly and decreased VEGF expression, both at the protein and mRNA levels. Thalidomide also inhibited HIF-1α in a dose-dependent manner (P < 0.05). CONCLUSIONS: VEGF may play an important role in the pathogenesis of angiodysplasia. Thalidomide can suppress VEGF, either induced by HIF-1α or bFGF.

[Distribution of variant alleles association with warfarin pharmacokinetics and pharmacodynamics in the Han population in China].

OBJECTIVE: To investigate distribution of CYP2C9, CYP3A4, VKORC1 and GGCX gene polymorphisms in the Han population of Guangdong. METHODS: The subjects included were 970 Chinese Han patients who received long-term warfarin anticoagulant therapy orally after valve replacement in Guangdong General Hospital between 2000 and 2008. By selecting and analyzing the 12 single nucleotide polymorphisms (SNPs) loci, rs12572351 G>A, rs9332146 G>A, rs4917639 G>T, rs1057910 A>C (CYP2C9*3), rs1934967 G>T, rs1934968 G>A, rs2242480 T>C, rs2246709 G>A, rs9923231 C>T (VKORC1-1639 G>A), rs2359612 G>A (VKORC1*2), rs10871454 C>T, and rs699664 T>C, in 4 genes including CYP2C9, CYP3A4, VKORC1 and GGCX that were possibly correlated with warfarin pharmacodynamics and pharmacokinetics through literature retrieval, the distribution of mutation frequencies of the 12 SNPs loci in Chinese Han population were obtained systematically. SNaPshot technique was used to detect gene SNPs, Hardy-Weinberg genetic equilibrium test was used to test population representativeness. RESULTS: The allelic mutation frequency at CYP2C9 gene rs12572351 G>A, rs9332146 G>A, rs4917639 C>A, rs1057910 A>C (*3), rs1934967 G>T and rs1934968 G>A loci was 32.53%, 2.16%, 8.25%, 3.61%, 19.18% and 37.37%, respectively; the allelic mutation frequency at CYP3A4 gene rs2242480 T>C and rs2246709 G>A loci was 29.07% and 40.41%, respectively; the allelic mutation frequency at VKORC1 gene rs9923231 C>T, rs2359612 G>A and rs10871454 C>T SNPs loci was 87.99%, 87.94% and 91.34%, respectively; the allelic mutation frequency at GGCX gene rs699664 T>C locus was 31.86%. CONCLUSION: It is of important clinical significance in individualized warfarin therapy to systematically study distribution of mutation frequencies at 12 polymorphisms loci in 4 genes including CYP2C9, CYP3A4 , VKORC1 and GGCX related to warfarin pharmacodynamics and pharmacokinetics in the Chinese Han population receiving valve replacement.

Efficacy of thalidomide for refractory gastrointestinal bleeding from vascular malformation.

BACKGROUND & AIMS: Patients with recurrent bleeding from gastrointestinal vascular malformations are a challenge to treat. We investigated the long-term efficacy and safety of thalidomide for refractory bleeding from gastrointestinal vascular malformations in an open-label, randomized study. METHODS: Eligible patients were randomly assigned to groups that were given either 100 mg thalidomide (n = 28) or 400 mg iron (n = 27, controls), daily for 4 months; patients were followed for at least 1 year (mean, 39 months). Bleeding was defined by a positive result from an immunoassay fecal occult blood test. The primary end point was the effective response rate, defined as the proportion of patients in whom bleeding episodes had decreased by ≥ 50% in the first year of the follow-up period. The secondary end points included the rates of cessation of bleeding, blood transfusion, overall hospitalization, and hospitalization for bleeding. We also quantified yearly bleeding episodes, bleeding duration, levels of hemoglobin, and yearly requirements for transfusions of red cells, numbers of hospitalizations for bleeding, and hospital stays. Plasma levels of vascular endothelial growth factor were measured in the group given thalidomide. RESULTS: Rates of response in the thalidomide and control groups were 71.4% and 3.7%, respectively (P < .001). All secondary end points differed significantly different between groups; thalidomide was more effective. No severe adverse effects were observed, although minor side effects were common among patients in the thalidomide group. Levels of vascular endothelial growth factor were significantly reduced by thalidomide (P < .001). CONCLUSIONS: Thalidomide is an effective and relatively safe treatment for patients with refractory bleeding from gastrointestinal vascular malformations. Mechanisms of thalidomide activity might involve vascular endothelial growth factor.

The influence of genetic polymorphisms and interacting drugs on initial response to warfarin in Chinese patients with heart valve replacement.

PURPOSE: Compared with genetic factors, drug interactions were largely unexplored in warfarin pharmacogenetic studies. This study sought to systematically investigate the effects of genetic polymorphisms of VKORC1, STX4A, CYP2C9, CYP3A4, and GGCX and interacting drugs on the initial responses to warfarin in Chinese patients with heart valve replacement (HVR). METHODS: A retrospective study was conducted in 809 patients starting warfarin therapy after HVR. The relationships between 12 polymorphisms plus 47 drugs and primary outcomes of the time to the first international normalized ratio (INR) ≥ 1.8 and the time to the first INR > 3.5 and the secondary outcomes of the proportion of time INR < 1.8, the proportion of time INR > 3.5, and the daily warfarin dose in the first 28 days after the initiation of warfarin treatment were analyzed. RESULTS: Genetic polymorphisms and interacting drugs could significantly affect the primary and secondary outcomes. The time to the first INR ≥ 1.8 was significantly influenced by the body surface area (BSA), VKORC1 g.3588G > A allele, and CYP2C9*3 allele, with hazard ratio (HR; 95% confidence interval [CI]) of 0.34 (0.17-0.66), 2.71 (2.2-3.35) and 1.43 (1.07-1.93) respectively. The time to the first INR > 3.5 was affected not only by BSA, VKORC1 g.3588G > A allele, and CYP2C9*3 allele with HR (95%CI) of 0.26 (0.07-0.99), 2.76 (1.61-4.72), and 3.09 (2.02-4.74) respectively, but also by age and interacting drugs, including fluconazole, amiodarone, and simvastatin with HR (95%CI) of 1.02 (1.01-1.04), 2.66 (1.16-6.08), 1.78 (1.17-2.73), and 5.33 (1.67-16.96) respectively. CONCLUSIONS: Not only VKORC1 and CYP2C9 genotypes, but also interacting drugs, had a significant impact on the variability of the initial response to warfarin.

Hyperglycemic myocardial damage is mediated by proinflammatory cytokine: macrophage migration inhibitory factor.

BACKGROUND: Diabetes has been regarded as an inflammatory condition which is associated with left ventricular diastolic dysfunction (LVDD). The purpose of this study was to examine the expression levels of macrophage migration inhibitory factor (MIF) and G protein-coupled receptor kinase 2 (GRK2) in patients with early diabetic cardiomyopathy, and to investigate the mechanisms involved in MIF expression and GRK2 activation. METHODS: 83 patients in the age range of 30-64 years with type 2 diabetes and 30 matched healthy men were recruited. Left ventricular diastolic function was evaluated by cardiac Doppler echocardiography. Plasma MIF levels were determined by ELISA. To confirm the clinical observation, we also studied MIF expression in prediabetic rats with impaired glucose tolerance (IGT) and relationship between MIF and GRK2 expression in H9C2 cardiomyoblasts exposed to high glucose. RESULTS: Compared with healthy subjects, patients with diabetes have significantly increased levels of plasma MIF which was further increased in diabetic patients with Left ventricular diastolic dysfunction (LVDD). The increased plasma MIF levels in diabetic patients correlated with plasma glucose, glycosylated hemoglobin and urine albumin levels. We observed a significant number of TUNEL-positive cells in the myocardium of IGT-rats but not in the control rats. Moreover, we found higher MIF expression in the heart of IGT with cardiac dysfunction compared to that of the controls. In H9C2 cardiomyoblast cells, MIF and GRK2 expression was significantly increased in a glucose concentration-dependant manner. Furthermore, GRK2 expression was abolished by siRNA knockdown of MIF and by the inhibition of CXCR4 in H9C2 cells. CONCLUSIONS: Our findings indicate that hyperglycemia is a causal factor for increased levels of pro-inflammatory cytokine MIF which plays a role in the development of cardiomyopathy occurring in patients with type 2 diabetes. The elevated levels of MIF are associated with cardiac dysfunction in diabetic patients, and the MIF effects are mediated by GRK2.

[Impact of CYP2C9 and VKORC1 polymorphism on warfarin response during initiation of therapy].

OBJECTIVE: To investigate potential contributions of genetic variants of cytochrome P-450 2C9 (CYP2C9) and vitamin K expoxide reductase (VKORC1) to the anticoagulation response during the initiation of warfarin therapy in the Han Chinese population. METHODS: A total of 798 Han Chinese patients received long-term warfarin anticoagulant therapy orally after valve replacement in our hospital between 2000 and 2008 were included in this study. Nine single nucleotide polymorphism (SNP) loci [rs12572351 G > A, rs9332146 G > A, rs4917639 G > T, rs1057910 A > C (CYP2C9(*)3), rs1934967 G > T, rs1934968 G > A, rs9923231 C > T (VKORC1-1639 G > A), rs2359612 G > A and rs10871454 C > T] in 2 genes including CYP2C9 and VKORC1, which were possibly correlated with warfarin pharmacokinetics and pharmacodynamics through literature retrieval, were selected and analyzed. Warfarin steady-state dose requirement, time to the INR (the international normalized ratio) within the therapeutic range and percent of the INR of more than 3.5 were compared among genotype subgroups. SNaPshot technique was used to detect gene SNPs; Hardy-Weinberg genetic equilibrium test was used to test population representativeness. RESULTS: CYP2C9(*)3 genotype did not affect the required warfarin dose while it was associated with increased risk of bleeding when treated with routine dosage regimen during the initiation of treatment. The allelic mutation frequency at VKORC1 gene rs10871454G > A and VKORC1-1639G > A SNP loci was 92.04% and 88.03%, respectively and rs10871454 was in perfect linkage disequilibrium with-1639. Patients with VKORC1 rs10871454 genetic mutation required lower warfarin dose in the first 28 days of therapy. VKORC1-1639 genetic polymorphism was also associated with shorter time to the INR within the therapeutic range and increased risk of over-anticoagulation. CONCLUSION: Detecting genetic polymorphism of CYP2C9 and VKORC1 could guide clinical use of warfarin to reduce the risk of adverse reactions including bleeding in patients receiving chronic anticoagulation therapy.

[Clinical application of anticoagulation treatment with warfarin after prosthetic heart valve replacement: a single center-based survey].

OBJECTIVE: To investigate the clinical application of anticoagulation treatment with warfarin after prosthetic heart valve replacement and compare the effect and safety of different anticoagulant intensities. METHODS: A total of 845 Chinese patients receiving oral warfarin for anticoagulant treatment after prosthetic heart valve replacement in Guangdong General Hospital between 2000 and 2008 were enrolled in this survey. The general data, clinical data, medications, international normalized ratio (INR) and results of echocardiogram of these patients were followed up to observe the incidence of complication of thrombo-embolism and such adverse effect as hemorrhage. RESULTS: All the patients were of Han nationality, and Cantonese accounted for 88.04%. The daily mean maintenance dose of warfarin was 2.92∓0.88 mg in these patients with a median INR of 2.09∓0.39. Of these patients, 44.62% received low-intensity anticoagulant treatment with warfarin with the INR maintained between 1.5 and 2.0, and 56.45% had standard anticoagulant intensity with the INR maintained between 2.0 and 3.0. The total incidence of thrombo-embolism was 4.14%. Severe hemorrhage occurred in 14 cases (1.66%), most frequently in the alimentary tract. The events of hemorrhage were correlated to the type of prosthetic heart valve replacement, occurring more frequently in patients with mechanical prosthetic heart valve replacement than in those with biological ones. No significant difference was found in the incidence of thrombo-embolism and server hemorrhage between the two groups receiving low and standard intensity therapy anticoagulant. CONCLUSION: The effect and safety of low-intensity anticoagulant treatment are comparable to that of standard intensity treatment in Chinese Han patients, and anticoagulation treatment with warfarin is effective and safe to maintain the INR between 1.8-3.0.

[High glucose up-regulates GRK2 gene expression in H9C2 cardiomyoblasts in vitro].

OBJECTIVE: To study the effect of high glucose on GRK2 gene expression in H9C2 cardiomyoblasts in vitro. METHODS: H9C2 cardiomyoblasts were cultured for 72 h in the presence of 0, 5.5, 12.5, 25 or 33 mmol/L glucose (with the osmotic pressure adjusted with monnitol). Semi-quantitative detection of GRK2 gene expression in H9C2 cardiomyoblasts was carried out using RT-PCR and phosph-Akt (Ser473) protein level was measured by Western blotting. RESULTS: Glucose in the culture medium (5.5 to 33 mmol/L) concentration-dependently increased the mRNA expression of GRK2 concentration and decreased phosphorylation Akt (ser473) level in in H9C2 cardiomyoblasts. CONCLUSION: Increased GRK2 gene expression may play an important role in cardiac dysfunction in diabetes.

[Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and CD74 using a yeast two-hybrid system].

OBJECTIVE: To investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system. METHODS: By using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media. RESULTS: The results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1. CONCLUSION: MIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).

[An efficient method for screening effective siRNAs using dual-luciferase reporter assay system].

OBJECTIVE: To establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system. METHODS: Based on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR. RESULTS: In the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01). CONCLUSION: The dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.