Quercetin-induced degradation of RhoC suppresses hepatocellular carcinoma invasion and metastasis.

BACKGROUND: Tumor metastasis and recurrence are major causes of mortality in patients with hepatocellular carcinoma (HCC) that is still lack of effective therapeutic targets and drugs. Previous reports implied that ras homolog family member C (RhoC) plays a toxic role on metastasis and proliferation of cancer. METHODS: In this research, the correlation between RhoC and metastasis ability was confirmed by in vitro experiments and TCGA database. We explored whether quercetin could inhibit cell migration or invasion by transwell assay. Real-time PCR, overexpression and ubiquitination assay, etc. were applied in mechanism study. Primary HCC cells and animal models including patient-derived xenografts (PDXs) were employed to evaluate the anti-metastasis effects of quercetin. RESULTS: Clinical relevance and in vitro experiments further confirmed the level of RhoC was positively correlated with invasion and metastasis ability of HCC. Then we uncovered that quercetin could attenuate invasion and metastasis of HCC by downregulating RhoC's level in vitro, in vivo and PDXs. Furthermore, mechanistic investigations displayed quercetin hindered the E3 ligase expression of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) leading to enhancement of RhoC's ubiquitination and proteasomal degradation. CONCLUSIONS: Our research has revealed the novel mechanisms quercetin regulates degradation of RhoC level by targeting SMURF2 and identified quercetin may be a potential compound for HCC therapy.

Comparison of oriented and non-oriented antibody conjugation with AIE fluorescence microsphere for the immunochromatographic detection of enrofloxacin.

Antibodies and labels were typically non-oriented conjugated in conventional immunochromatographic assays (ICAs). In this work, a C-terminal cysteine-tagged recombinant protein A (rPA) was conjugated in an oriented manner onto aggregation-induced emission fluorescence microsphere (AIEFM). The Fc fragment of anti-enrofloxacin monoclonal antibody (anti-ENR mAb) was then conjugated onto the rPA. The resulting oriented mAb-AIEFM probe was used in an ENR-ICA for the rapid detection of ENR, a widely abused animal drug. The ENR-ICA with the oriented probe saved 66.7% of anti-ENR mAb and 25% of ENR-bovine serum albumin, and had a limit of detection of 0.035 ng/mL, compared with 0.079 ng/mL for the non-oriented probe. The corresponding linear ranges of the ENR-ICA based on the oriented and non-oriented probes were 0.25-10 ng/mL and 0.1-2.5 ng/mL, respectively. This novel ICA based on the oriented probe has the potential to be used for sensitive and rapid detection in food safety.

DNA methylation mediates the genetic variants on ticagrelor major metabolite elimination and platelet function recovery after ticagrelor discontinuation.

Aim: To investigate the influence of DNA methylation on ticagrelor major metabolite M8 elimination and platelet function recovery after ticagrelor discontinuation. Materials & methods: Among healthy Chinese subjects, a causal inference test was conducted to identify CpG sites located on absorption, distribution, metabolism and excretion genes that mediate genetic variants on M8 elimination. Colocalization analysis was used to identify the CpG sites that shared causal variants with platelet function recovery. Results: cg05300248 (CHST9), cg05640674 (SLC22A5) and cg00846580 (DHRS7) mediated genetic variants on the M8 elimination. cg06338150 (NOTCH1) and cg17456097 (RPS6KA1) were demonstrated to have strong evidence of colocalization with platelet function recovery. Conclusion: The results provide new biological insights into the impact of DNA methylation on M8 elimination and platelet function recovery after ticagrelor discontinuation. Clinical trial registration: clinicaltrials.gov, identifier: NCT03092076.

Comprehensive Metabolomics Identified the Prominent Role of Glycerophospholipid Metabolism in Coronary Artery Disease Progression.

Background: Coronary stenosis severity determines ischemic symptoms and adverse outcomes. The metabolomic analysis of human fluids can provide an insight into the pathogenesis of complex disease. Thus, this study aims to investigate the metabolomic and lipidomic biomarkers of coronary artery disease (CAD) severity and to develop diagnostic models for distinguishing individuals at an increased risk of atherosclerotic burden and plaque instability. Methods: Widely targeted metabolomic and lipidomic analyses of plasma in 1,435 CAD patients from three independent centers were performed. These patients were classified as stable coronary artery disease (SCAD), unstable angina (UA), and myocardial infarction (MI). Associations between CAD stages and metabolic conditions were assessed by multivariable-adjusted logistic regression. Furthermore, the least absolute shrinkage and selection operator logistic-based classifiers were used to identify biomarkers and to develop prediagnostic models for discriminating the diverse CAD stages. Results: On the basis of weighted correlation network analysis, 10 co-clustering metabolite modules significantly (p < 0.05) changed at different CAD stages and showed apparent correlation with CAD severity indicators. Moreover, cross-comparisons within CAD patients characterized that a total of 72 and 88 metabolites/lipid species significantly associated with UA (vs. SCAD) and MI (vs. UA), respectively. The disturbed pathways included glycerophospholipid metabolism, and cysteine and methionine metabolism. Furthermore, models incorporating metabolic and lipidomic profiles with traditional risk factors were constructed. The combined model that incorporated 11 metabolites/lipid species and four traditional risk factors represented better discrimination of UA and MI (C-statistic = 0.823, 95% CI, 0.783-0.863) compared with the model involving risk factors alone (C-statistic = 0.758, 95% CI, 0.712-0.810). The combined model was successfully used in discriminating UA and MI patients (p < 0.001) in a three-center validation cohort. Conclusion: Differences in metabolic profiles of diverse CAD subtypes provided a new approach for the risk stratification of unstable plaque and the pathogenesis decipherment of CAD progression.

Glucose oxidase-induced colorimetric immunoassay for qualitative detection of danofloxacin based on iron (â ¡) chelation reaction with phenanthroline.

In this study, we developed a competitive colorimetric immunoassay for qualitative detection of DAN based on oxidation of iron (Ⅱ) (Fe2+) in the presence of glucose oxidase (GOx) and color change induced by Fe2+-phenanthroline (Phen) chromogenic system. Streptavidin (SA) acted as a linker between biotinylated anti-DAN-monoantibody (bio-mAb) and biotinylated GOx (bio-GOx) to form the immunocomplexes bio-mAb-SA-bio-GOx. In the absence of DAN, the immunocomplexes bio-mAb-SA-bio-GOx combining with coated DAN-ovalbumin (DAN-OVA) will be immobilized and catalyze glucose to produce H2O2. Fe2+ is oxidized to Fe3+ by H2O2, giving rise to a colorless result. In the presence of DAN, Fe2+ produces a chelation reaction with Phen, leading to orange-red color. Under optimal conditions, the detection limit (LOD) by naked eyes was 2.5 ng mL-1 in milk, chicken, beef, and pork samples. Low LOD, no matrix effect, and no signal reader requirement make it possibly applied to quickly screen DAN on site.

Supramolecular Recognition-Mediated Layer-by-Layer Self-Assembled Gold Nanoparticles for Customized Sensitivity in Paper-Based Strip Nanobiosensors.

Herein, a smart supramolecular self-assembly-mediated signal amplification strategy is developed on a paper-based nanobiosensor to achieve the sensitive and customized detection of biomarkers. The host-guest recognition between ß-cyclodextrin-coated gold nanoparticles (AuNPs) and 1-adamantane acetic acid or tetrakis(4-carboxyphenyl)porphyrin is designed and applied to the layer-by-layer self-assembly of AuNPs at the test area of the strip. Thus, the amplified platform exhibits a high sensitivity with a detection limit at subattogram levels (approximately dozens of molecules per strip) and a wide dynamic range of concentration over seven orders of magnitude. The applicability and universality of this sensitive platform are demonstrated in clinically significant ranges to measure carcinoembryonic antigen and HIV-1 capsid p24 antigen in spiked serum and clinical samples. The customized biomarker detection ability for the on-demand needs of clinicians is further verified through cycle incubation-mediated controllable self-assembly. Collectively, the supramolecular self-assembly amplification method is suitable as a universal point-of-care diagnostic tool and can be readily adapted as a platform technology for the sensitive assay of many different target analytes.

A genome-wide association study on lipoprotein (a) levels and coronary artery disease severity in a Chinese population.

Lipoprotein (a) [Lp(a)] is a genetically determined risk factor of coronary artery disease (CAD). Previous genome-wide association studies (GWASs), which were mostly carried out in Caucasians, have identified many Lp(a)-associated SNPs. Here, we performed a GWAS on Lp(a) levels and further explored the relationships between Lp(a)-associated SNPs and CAD severity in 1,403 Han Chinese subjects. We observed that elevated Lp(a) levels were significantly associated with the increased synergy between percutaneous coronary intervention with TAXUS and cardiac surgery (SYNTAX) score and the counts of heavily calcified lesions and long-range lesions (LRLs; P < 0.05), which are defined as lesions spanning >20 mm. Moreover, we identified four independent SNPs, namely, rs7770628, rs73596816, and rs6926458 in LPA, and rs144217738 in SLC22A2, that were significantly associated with Lp(a) levels. We also found that rs7770628 was associated with high SYNTAX scores [odds ratio (OR) (95% CI): 1.37 (1.05-1.80), P = 0.0213, false discovery rate (FDR) = 0.0852], and that rs7770628 and rs73596816 were associated with high risk of harboring LRLs [OR (95% CI): 1.53 (1.17-2.01), P = 0.0018, FDR = 0.0072 and 1.72 (1.19-2.49), P = 0.0040, FDR = 0.0080, respectively]. Our study was a large-scale GWAS to identify Lp(a)-associated variants in the Han Chinese population. Our findings highlight the importance and potential of Lp(a) intervention and expand our understanding of CAD prevention and treatment.

SLCO1B1 521T > C polymorphism associated with rosuvastatin-induced myotoxicity in Chinese coronary artery disease patients: a nested case-control study.

PURPOSE: This nested case-control study aimed to evaluate the association of candidate genetic variants with statin-induced myotoxicity in Chinese patients with coronary artery disease (CAD). METHODS: One hundred forty-eight Chinese patients experiencing statin-induced myotoxicity were included in our study, and 255 patients without muscular side effects served as controls. Five SNPs in CYP3A5, SLCO1B1, and APOE were genotyped. The effect of genetic variants on statin-induced myotoxicity was assessed. RESULTS: Patients who carried at least one SLCO1B1 521C allele had a higher risk for myotoxicity (OR = 1.69, 95%CI = 1.07-2.67, P = 0.024). Significant association was found between SLCO1B1 521C mutant allele mutation and risk of myotoxicity in individuals that received rosuvastatin (OR = 3.67, 95%CI = 1.42-9.47, P = 0.007). However, non-significant association was observed between 521C mutant allele and risk of myotoxicity (P > 0.5) in patients that received atorvastatin and simvastatin. The other four single nucleotide polymorphisms (SNPs), namely rs776746, rs2306283, rs7412, and rs429358, showed no significant association with any statin induced myotoxicity (P > 0.5). CONCLUSIONS: SLCO1B1 (rs4149056, 521T > C) is associated with statin-induced myotoxicity in Chinese patients with coronary artery disease. In addition, SLCO1B1 521C mutant allele increased the risk of rosuvastatin-associated myotoxicity.

Size dependent biodistribution and toxicokinetics of iron oxide magnetic nanoparticles in mice.

In spite of the immense benefits from iron oxide magnetic nanoparticles (IOMNs), there is scanty information regarding their metabolic activities and toxicity in vivo. In this study, we investigated the size dependent in vivo biodistribution, toxicokinetics, and toxicity and gene expression changes of various sizes of carboxyl coated IOMNs (diameters of 10, 20, 30, and 40 nm). Our findings demonstrated that the various sizes of IOMNs accumulated primarily in the liver and spleen on the first day post-injection. Interestingly, size dependent biodistribution and transport were observed: the smallest IOMNs (10 nm) showed the highest uptake by the liver, whereas the largest IOMNs (40 nm) showed the highest uptake by the spleen. Moreover, the IOMNs with the smallest size (10 nm) were cleared faster from the liver and kidneys, but more readily entered the brain and the uterus. IOMNs with the largest size (40 nm) accumulated more readily but were easily eliminated in the spleen. However, the level of iron in the heart decreased in all IOMN exposed groups. In addition, blood biochemistry, hematological analyses and histological examination demonstrated that there was no apparent acute toxicity caused by IOMNs in mice. However, smaller IOMNs (10 nm and 20 nm) more effectively changed the expression level of sensitive genes related to oxidant stress, iron transport, metabolic process, apoptosis, and others.

A homogeneous immunosensor for AFB1 detection based on FRET between different-sized quantum dots.

Mycotoxins are fatal threats in food safety due to their strong carcinogenesis and toxicity, thus requiring highly sensitive detections. Herein, different-sized quantum dots (QDs) were used to construct a Förster resonance energy transfer (FRET) based immunosensor for sensitive detection of aflatoxin B1 (AFB1) in rice grains. To avoid irregular aggregation between two kinds of QDs, monovalent monoclonal antibody (mAb)-labeled red QDs (~0.84 anti-AFB1 mAbs per QD) and multivalent hapten-labeled green QDs (~6.8 AFB1 per QD) were designed as acceptor and donor, respectively. The anti-AFB1 mAbs and AFB1 interactions promoted one or more acceptors bound with a multivalent AFB1-labeled donor, resulting in energy transfer from the green QDs to the red QDs. Various parameters that influence the immunoassay including reactant ratio of donor to acceptor, buffer pH value, buffer ionic strength and immunoreaction time were systematically investigated and optimized. With optimal conditions, the obtained energy transfer efficiency is proportional to the logarithm of AFB1 concentration in a range over 0.19-16 pM (0.06-5 ng/mL), while offering a limit of detection of 0.13 pM (0.04 ng/mL) in rice extracts. The recovery rates of the intra-assay for spiked samples at AFB1 concentrations of 0.1, 1.0, and 5.0 ng/mL were 83.27% ± 3.27%, 97.36% ± 4.55% and 83.04% ± 4.94%, respectively, and those for the inter-assay were 81.28% ± 6.11%, 95.97% ± 7.07%, and 82.78% ± 5.99%, respectively. Statistical analysis using t-test had no significant difference between the proposed FRET-based immunoassay and the commercial enzyme-linked immunosorbent assay kit.

Identification of an outer membrane protein of Salmonella enterica serovar Typhimurium as a potential vaccine candidate for Salmonellosis in mice.

We report our investigation of the functions of PagN in Salmonella pathogenesis and its potential as a vaccine candidate. Further investigation conducted in this study indicates that the outer membrane protein PagN is important for Salmonella adhesion/invasion of epithelial cells as well as bacterial virulence. When pagN was deleted from Salmonella enterica serovar Typhimurium (S. Typhimurium), the adhesion and invasion of HT-29 epithelial cells was significantly decreased compared with the wild type strain. Mice infected with the pagN mutant strain exhibited less pathological signs in the intestine and survived longer than the wild-type-infected mice. PagN is widely distributed and conserved among clinical isolates of different Salmonella serovars, making PagN a potential vaccine candidate for Salmonella infection. To elucidate the potential of PagN as a vaccine, we expressed and purified recombinant PagN (rPagN). When rPagN was tested in mice, it provided significant protection against Salmonella infection in vivo. In vitro, anti-PagN serum enhanced clearance of Salmonella, indicating a contribution of PagN-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with rPagN. Our preliminary results indicate more functions of PagN in S. Typhimurium virulence as well as its potential as a protective vaccine.

Development of a lateral-flow assay for rapid screening of the performance-enhancing sympathomimetic drug clenbuterol used in animal production; food safety assessments.

A lateral-flow assay that could provide visual evidence of the presence of clenbuterol in swine urine was developed. Colloidal gold was prepared and conjugated with anti-clenbuterol monoclonal antibody. Immunochromatographic test strips were produced, and then, 210 samples were tested on these strips. Analysis was completed in 10 min. Detection limit was 3 ppb of clenbuterol. Parallel GC-MS data indicated that clenbuterol rapid detection strip had no false negative. The false positive rate was 4.4%. Immunochromatographic strip has great applied value in the food safety field because it possesses benefits of sensitivity, stability, reproducibility, ease of use and inexpensive.