G-quadruplexes on chromosomal DNA negatively regulates topoisomerase 1 activity.

Human DNA topoisomerase 1 (Top1) is a crucial enzyme responsible for alleviating torsional stress on DNA during transcription and replication, thereby maintaining genome stability. Previous researches had found that non-working Top1 interacted extensively with chromosomal DNA in human cells. However, the reason for its retention on chromosomal DNA remained unclear. In this study, we discovered a close association between Top1 and chromosomal DNA, specifically linked to the presence of G-quadruplex (G4) structures. G4 structures, formed during transcription, trap Top1 and hinder its ability to relax neighboring DNAs. Disruption of the Top1-G4 interaction using G4 ligand relieved the inhibitory effect of G4 on Top1 activity, resulting in a further reduction of R-loop levels in cells. Additionally, the activation of Top1 through the use of a G4 ligand enhanced the toxicity of Top1 inhibitors towards cancer cells. Our study uncovers a negative regulation mechanism of human Top1 and highlights a novel pathway for activating Top1.

Baicalin attenuate diet-induced metabolic syndrome by improving abnormal metabolism and gut microbiota.

In this work, we examined whether baicalin (BC), a bioactive flavonoid in Scutellaria baicalensis Georgi, can reduce high-fat diet (HFD)-induced metabolic syndrome (MetS) in mice. The UPLC-QTOF/MS was used for metabolome profiles analysis, and an analysis of bacterial 16S rDNA in feces was used to examine the effects of BC on gut microbiota composition. Our results showed that BC (400 mg/kg) could reduce the body weight gain, decrease hepatic fat accumulation and abnormal blood lipids, and increase insulin sensitivity after 8 weeks of treatment. BC could reverse the alteration of 7 metabolites induced by HFD and the metabolic pathways responsive to BC intervention including citrate cycle, alanine, aspartate and glutamate metabolism, glycerophospholipid metabolism, and aminoacyl-tRNA biosynthesis. 16S rDNA analysis demonstrated that BC altered the composition and function of gut microbiota in MetS mice. Notably, we found that the change in succinic acid was negatively associated with the changes in Bacteroides and Sutterella, and positively associated with the change in Mucispirillum. Moreover, we confirmed that succinic acid displayed a metabolic protective effect on MetS mice. The antibiotic treatment verified that BC exerts metabolic protection through gut microbiota. Our findings suggested BC may be a potential therapeutic drug to ameliorate diet induced MetS and gut microbiome may be a novel mechanistic target of BC for treatment of MetS.

Spectrum-Effect Relationships Between High-Performance Liquid Chromatography Fingerprints and Hepatoprotective Activities of Cuscutae Semen.

BACKGROUND: Cuscutae Semen (CS) is a commonly used hepatoprotective traditional Chinese medicine, but the chemical components responsible for its hepatoprotective activity are unclear. OBJECTIVE: The purpose of this study was to evaluate the spectrum-effect relationships between HPLC fingerprints and hepatoprotective effects of CS, and to identify its bioactive components. METHODS: Phytochemical isolation of CS extracts was first carried out and 14 potential bioactive compounds were obtained. Chemical fingerprinting was performed on 27 batches of CS from different sources by HPLC, and further analyzed by similarity analysis (SA) and hierarchical clustering analysis (HCA). Pharmacodynamic testing was performed in a CCl4-induced, acute liver injury cell model to assess the hepatoprotective activity of CS by measuring the cell viability and levels of alanine transaminase (ALT) and aspartate aminotransferase (AST). Bivariate correlations analysis (BCA) and orthogonal projections to latent structures (OPLS) were used to analyze the spectrum-effect relationships of CS. RESULTS: The results showed that the chemical fingerprints of CS were closely correlated with its hepatoprotective activity. Peaks 1, 10, 18, 19, 21, 22, and 24 might be potential hepatoprotective compounds in CS, and the validation experiments of isolated compounds indicated that chlorogenic acid (P10), hyperoside (P21), isoquercitrin (P22), and astragalin (P24) were the main hepatoprotective components. CONCLUSION: By combining chemical fingerprints with hepatoprotective evaluation, the present study provides important guidance for QC and clinical use of CS. HIGHLIGHTS: (1) Ten potential bioactive compounds were isolated from CS; (2) The spectrum-effect relationship of CS was molded by HPLC and analysed by OPLS and BCA. (3) Four compounds including chlorogenic acid were the main hepatoprotective components.

Regulation of PDGFR-ß gene expression by targeting the G-vacancy bearing G-quadruplex in promoter.

G-quadruplex is an essential element in gene transcription that serves as a promising drug target. Guanine-vacancy-bearing G-quadruplex (GVBQ) is a newly identified G-quadruplex that has distinct structural features from the canonical G-quadruplex. Potential GVBQ-forming motifs are widely distributed in gene promoter regions. However, whether GVBQ can form in genomic DNA and be an effective target for manipulating gene expression is unknown. Using photo-crosslinking, dimethyl sulfate footprinting, exonuclease digestion and in vitro transcription, we demonstrated the formation of a GVBQ in the G-rich nuclease hypersensitivity element within the human PDGFR-ß gene promoter region in both single-stranded and double-stranded DNA. The formation of GVBQ in dsDNA could be induced by negative supercoiling created by downstream transcription. We also found that the PDGFR-ß GVBQ was specifically recognized and stabilized by a new synthetic porphyrin guanine conjugate (mPG). Targeting the PDGFR-ß GVBQ in human cancer cells using the mPG could specifically alter PDGFR-ß gene expression. Our work illustrates that targeting GVBQ with mPG in human cells can regulate the expression level of a specific gene, thus indicating a novel strategy for drug development.

Urinary Metabolite Signatures for Predicting Elderly Stroke Survivors with Depression.

BACKGROUND: Post-stroke depression (PSD) is a major complication in stroke survivors, especially in elderly stroke survivors. But there are still no objective methods to diagnose depression in elderly stroke survivors. Thus, this study was conducted to identify potential biomarkers for diagnosing elderly PSD subjects. METHODS: Elderly (60 years or older) stroke survivors with depression were assigned into the PSD group, and elderly stroke survivors without depression and elderly healthy controls (HCs) were assigned into the non-depressed group. Urinary metabolite signatures obtained from gas chromatography-mass spectrometry (GC-MS)-based metabolomic platform were collected. Both univariate and multivariate statistical analysis were used to find the differential urinary metabolites between the two groups. RESULTS: The 78 elderly HCs, 122 elderly stroke survivors without depression and 124 elderly PSD subjects were included. A set of 13 differential urinary metabolites responsible for distinguishing PSD subjects from non-depressed subjects were found. The Phenylalanine, tyrosine and tryptophan biosynthesis, Phenylalanine metabolism and Galactose metabolism were found to be significantly changed in elderly PSD subjects. The phenylalanine was significantly negatively correlated with age and depressive symptoms. Meanwhile, a biomarker panel consisting of 3-hydroxyphenylacetic acid, tyrosine, phenylalanine, sucrose, palmitic acid, glyceric acid, azelaic acid and α-aminobutyric acid was identified. CONCLUSION: These results provided candidate molecules for developing objective methods to diagnose depression in elderly stroke survivors, suggested that taking supplements of phenylalanine might be an effective method to prevent depression in elderly stroke survivors, and would be helpful for future revealing the pathophysiological mechanism of PSD.

Diabetic nephropathy and risk factors for peripheral artery disease in Chinese with type 2 diabetes mellitus.

The risk for peripheral arterial disease (PAD) is increased in patients with chronic kidney disease. We investigated the effects of renal function on PAD in Chinese with type 2 diabetes mellitus. This study enrolled a total of 2983 (1342 men and 1641 women) Chinese adults with diabetes. The mean age was 63.2 +/- 11.9 years. Peripheral arterial disease was diagnosed by an ankle-brachial index less than 0.9. Renal function was evaluated by serum creatinine (SCr), estimated glomerular filtration rate, and urinary albumin-creatinine ratio (ACR). Risk factors for PAD were evaluated using multiple logistic regression analysis. Age, cholesterol, and high-density lipoprotein cholesterol (HDL-C) (inverse association) were significant risk factors in men, whereas age, body mass index (inverse association), low-density lipoprotein cholesterol, and HDL-C (inverse association) were significant risk factors for diabetic women. After adjustment for age, body mass index, blood pressure, glycosylated hemoglobin, cholesterol, HDL-C, low-density lipoprotein cholesterol, and triglyceride levels, we found that SCr levels greater than 1.5 mg/dL, estimated glomerular filtration rate less than 60 mL/min, and urinary ACR greater than 30 mg/g were independent risk factors for PAD in diabetic men and that SCr levels greater than 1.4 mg/dL and urinary ACR greater than 30 mg/g were independently associated with PAD in diabetic women. The risk factors for PAD are somewhat different between men and women with diabetes in Chinese population in Taiwan. Diabetic nephropathy is significantly associated with PAD in this patient population.

Gender-dependent effect of ACE I/D and AGT M235T polymorphisms on the progression of urinary albumin excretion in Taiwanese with type 2 diabetes.

BACKGROUND: We investigated the gender differences in the effect of ACE I/D and AGT M235T polymorphisms on the prognosis of diabetic nephropathy (DN). METHODS: A total of 525 type 2 diabetics were enrolled to participate in this prospective observational study. ACE and AGT gene polymorphisms were analyzed by polymerase chain reaction. The progression of DN was defined as a shift to a higher stage of DN or a doubling of the baseline serum creatinine level by the end of the study. RESULTS: The baseline biophysical parameters show no gender differences in progression and non-progression of DN. The women who were ACE D allele carriers were found to be at an increased risk of DN progression compared to those with II genotypes (p = 0.024, OR 2.176). No such difference was seen in male patients (p = 0.619, OR 0.833). After adjusting for confounding factors (age, SBP, DBP, BMI, HbA1c, total cholesterol, TG, HDL-C, LDL-C, ACEI, and ARB) in our multiple regression analysis, these women were still found to be at increased risk of progressing to more severe DN (p = 0.008, OR 3.082) but not the men (p = 0.183, OR 0.586). Neither the AGT TT genotype nor the T allele were associated with the progression of DN in either sex after adjusting for confounding factors. CONCLUSION: Our follow-up study suggests that female diabetic carriers of the ACE D allele might be at an increased risk of DN progression.

The association of endothelial nitric oxide synthase G894T polymorphism with C-reactive protein level and metabolic syndrome in a Chinese study group.

Some studies have reported a possible relationship between endothelial nitric oxide synthase (eNOS) and metabolic syndrome (MS), which is associated with an increased risk for cardiovascular disease. A recent meta-analysis study found the eNOS G894T polymorphism to be associated with ischemic heart disease. Here, we examine the association of eNOS G894T polymorphism with MS in a Chinese population (n = 397). The eNOS T+ (TT and GT) genotypes (56.92% vs 38.86%; odds ratio, 2.08; 95% confidence interval, 1.21-3.56; P = .007) and T allele (33.08% vs 23.34%; odds ratio, 1.62; 95% confidence interval, 1.08-2.44; P = .019) were significantly more frequent in subjects who had MS. Furthermore, subjects with eNOS T+ genotypes had significantly higher plasma C-reactive protein levels as compared with GG subjects (P = .004). This study shows that, in a Chinese population, eNOS G894T polymorphism is associated with an elevated C-reactive protein level and MS.

Chronic kidney disease as a risk factor for coronary artery disease in Chinese with type 2 diabetes.

BACKGROUND: Chronic kidney disease (CKD) is associated with cardiovascular disease (CVD) in the general population. We investigated the effects of renal function on coronary artery disease (CAD) in Chinese with type 2 diabetes who have a high risk of developing diabetic nephropathy but who may have a low risk of developing CAD. METHODS: We recruited a total of 2,434 Chinese with type 2 diabetes (1,078 men and 1,356 women) and diagnosed CAD by history or with an abnormal electrocardiogram (coronary probable or possible by Minnesota codes). Renal function was evaluated by serum creatinine (SCr) levels, estimated glomerular filtration rate (eGFR) (calculated by the abbreviated Modification of Diet in Renal Disease Study Croup formula) and urinary albumin/creatinine ratio (ACR). RESULTS: We found that patients with CAD were older, had higher SCr levels and body mass index (BMI), and had lower serum high-density lipoprotein cholesterol (HDL-c) levels. After adjusting for age, BMI, blood pressure, glycosylated hemoglobin, cholesterol, LDL-c, HDL-c, and triglycerides, we found that SCr levels >1.5 mg/dl, eGFR <60 ml/min, and urinary ACR >30 mg/g were independent risk factors for CAD in diabetic men, and that SCr levels >1.4 mg/dl and eGFR <60 ml/min were independently associated with CAD in women. CONCLUSION: Our findings indicate that Chinese with type 2 diabetes and CKD are likely to have had CAD previously and CKD is 'CVD risk state' in diabetic Chinese.

Cholesteryl ester transfer protein B1B1 genotype as a predictor of coronary artery disease in Taiwanese with type 2 diabetes mellitus.

Diabetes is known to be a high-risk factor for coronary artery disease (CAD), and lipid abnormalities have been found to possibly contribute to CAD in diabetic patients. Cholesteryl ester transfer protein (CETP) gene TaqIB polymorphism is associated with lipid profile variability, and this polymorphism may be a risk factor for CAD in diabetic patients. To clarify the relationship between CETP TaqIB gene polymorphism and CAD, we enrolled in our study 365 Taiwanese with type 2 diabetes mellitus (101 with CAD and 264 without CAD). The genotype of the subjects for TaqIB polymorphism of CETP in intron 1 was analyzed by using polymerase chain reaction-restriction fragment length polymorphism. The CETP B1B1 genotype (18.8% vs 8.5%, P = .002) and B1 allele (42.1% vs 29.7%, P = .002) were significantly more frequent in diabetic patients with CAD than those without CAD. Logistic regression analysis revealed that the CETP B1B1 genotype was associated with CAD in patients with type 2 diabetes mellitus (odds ratio, 3.18; 95% confidence interval, 1.54-6.54; P = .002). Interestingly, in diabetic patients, serum creatinine levels higher than 1.4 mg/dL were also associated with increased risk for CAD (odds ratio, 2.09; 95% confidence interval, 1.12-3.91; P = .02). Our results suggest that the CETP B1B1 genotype is a strong genetic predictor of CAD in Taiwanese with type 2 diabetes mellitus.

Elevated serum anti-endothelial cell autoantibodies titer is associated with lupus nephritis in patients with systemic lupus erythematosus.

BACKGROUND AND PURPOSE: Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disease associated with endothelial dysfunction and the existence of multiple species of autoantibodies. However, the association between endothelial dysfunction and renal manifestations remains unclear in Taiwanese SLE patients. METHODS: Serum samples were collected from SLE patients with biopsy-proven lupus nephritis (n = 32), stable SLE patients (n = 32) and healthy controls (n = 32). The SLE Disease Activity Index (SLEDAI) of SLE patients was scored, and levels of anti-endothelial cell antibodies (AECA) and anti-endothelial activities in serum samples were measured by cell-enzyme-linked immunosorbent assay and crystal violet assay, respectively, using cultured human endothelial EA.hy926 cells. RESULTS: Significantly higher AECA (p<0.001) and anti-endothelial activities (p<0.001) were found in sera from patients with lupus nephritis compared with that from stable SLE patients or controls. Moreover, AECA titers (p<0.001) and anti-endothelial activities (p<0.001) were strongly correlated with SLEDAI scores in these patients. CONCLUSION: The strong correlations of AECA and anti-endothelial activity with lupus nephritis activity support an endothelial origin for renal complications in Taiwanese SLE patients.

Modulation of angiogenic processes in cultured endothelial cells by low density lipoproteins subfractions from patients with familial hypercholesterolemia.

OBJECTIVE: Electronegative low density lipoprotein (LDL) subfractions are cytotoxic to endothelial cells. To continue our study of homozygotic familial hypercholesterolemic (FH)-LDL, we report the effects of FH-LDL subfractions (FH-L1 to FH-L5) on the angiogenic processes in cultured endothelial cells. METHODS AND RESULTS: Subconfluent bovine aortic endothelial cells (BAEC) were treated with LDL subfractions (20 microg/ml), and the effects on angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, secretion of matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) were determined. The electronegative FH-L4 and FH-L5 inhibited cell proliferation while the other FH-LDL subfractions and LDL from normocholesterolemic subjects (N-LDL) had negligible effects. Like Cu2+ ox-LDL, FH-L5 strongly inhibited endothelial cell viability and FH-L4 had a milder effects. Similarly, FH-L4 and FH-L5 but not the other subfractions retarded cell migration, induced cell apoptosis, and perturbed tube formation by BAEC in matrigel. FH-L5 inhibited secretion of MMP-2 and MMP-9 by BAEC without affecting their endogenous levels. In contrast, FH-L5 increased the VEGF expression in endothelial cells. CONCLUSIONS: Our results show for the first time that FH-L5, a circulating LDL subfraction from hypercholesterolemic patients, modulates various angiogenic processes, thereby dysregulating endothelial function in a way that may be atherogenic.