Accurate molecular identification of different meat adulterations without carryover contaminations on a microarray chip PCR-directed microfluidic lateral flow strip device.

Meat adulteration-based food fraud has recently become one of the global major economical, illegal, religious, and public health concerns. In this work, we developed a microarray chip polymerase chain reaction (PCR)-directed microfluidic lateral flow strip (LFS) device that facilitates the accurate and simultaneous identification of beef adulterated with chicken, duck, and pork, especially in processed beef products. To realize this goal, four pairs of amplification primers were designed and applied for specifically amplifying genomic DNA extracted from mixed meat powders in microarray chip. With the prominent advantage of this device lies in the flexible combination and integration of sample loading, detection, and reporting in microstructures, all the DNA amplicons can be individually visualized on the LFS unit, leading to the appearance of test lines (TC line, TD line, TP line, or TB line) as well as the control line (C line) for the species identification and quantification in beef products. Based on this new method, the adulterants were successfully distinguished and identified in mixtures down to 0.01% (wt.%) while the carryover aerogel contamination in routine molecular diagnostic laboratories was effectively avoided. The practicability, accuracy, and reliability of the device were further confirmed by using real-time PCR as a gold standard control on the successful identification of 50 processed ground meat samples sourced from local markets. The method and device proposed herein could be a useful tool for on-site identification of food authentication.

Investigation of the Pathogenic Mechanism of Ciprofloxacin in Aortic Aneurysm and Dissection by an Integrated Proteomics and Network Pharmacology Strategy.

Aortic aneurysm and dissection (AAD) is a life-threatening disease worldwide. Recently, fluoroquinolones have been reported to significantly increase the risk of AAD. This study aimed to investigate the potential functional mechanism and molecular targets of fluoroquinolones in relation to AAD by an integrated proteomic and network pharmacology strategy. A total of 1351 differentially expressed proteins were identified in human aortic vascular smooth muscle cells (VSMCs) after ciprofloxacin (CIP) stimulation. The functional analysis emphasized the important roles of metabolism, extracellular matrix homeostasis, mitochondrial damage, focal adhesion, and apoptosis in CIP-stimulated VSMCs. CIP targets were predicted with online databases and verified by molecular docking. Protein-protein interaction (PPI) analysis and module construction of the 34 potential CIP targets and 37 selected hub molecules after CIP stimulation identified four critical target proteins in the module: PARP1, RAC1, IGF1R and MKI67. Functional analysis of the PPI module showed that the MAPK signalling pathway, focal adhesion, apoptosis, regulation of actin cytoskeleton, and PI3K-Akt signalling pathway were significantly enriched. Our results will provide novel insights into the pathogenic mechanism of fluoroquinolones in aortic diseases.

Identification of Novel Plasma Biomarkers for Abdominal Aortic Aneurysm by Protein Array Analysis.

Abdominal aortic aneurysm (AAA) is a potentially life-threatening disease that is common in the aging population. Currently, there are no approved diagnostic biomarkers or therapeutic drugs for AAA. We aimed to identify novel plasma biomarkers or potential therapeutic targets for AAA using a high-throughput protein array-based method. Proteomics expression profiles were investigated in plasma from AAA patients and healthy controls (HC) using 440-cytokine protein array analysis. Several promising biomarkers were further validated in independent cohorts using enzyme-linked immunosorbent assay (ELISA). Thirty-nine differentially expressed plasma proteins were identified between AAA and HC. Legumain (LGMN) was significantly higher in AAA patients and was validated in another large cohort. Additionally, "AAA without diabetes" (AAN) patients and "AAA complicated with type 2 diabetes mellitus" (AAM) patients had different cytokine expression patterns in their plasma, and nine plasma proteins were differentially expressed among the AAN, AAM, and HC subjects. Delta-like protein 1 (DLL1), receptor tyrosine-protein kinase erbB-3 (ERBB3), and dipeptidyl peptidase 4 (DPPIV) were significantly higher in AAM than in AAN. This study identified several promising plasma biomarkers of AAA. Their role as therapeutic targets for AAA warrants further investigation.

Accurate and Rapid Genetic Tracing the Authenticity of Floral Originated Honey with the Molecular Lateral Flow Strip.

Honey is a natural product and is heavily consumed for its well-known nutritional functions. Honeys with different floral origins possess distinctive flavors, tastes, functions and economic values. It is vital to establish an effective strategy for identifying the authenticity of honey. The intrinsic genetic materials of pollen were adopted as target analytes for the effective identification of honey with floral origins. With an optimized protocol for the rapid gene extraction from honey, target genetic templates were amplified on-site with a portable device. Conveniently, all on-site amplified functional products were easily judged by the designed lateral flow strip (LFS), which was defined as the molecular LFS in this research. Additionally, the entire on-site genetic authentication of honey was completed in less than 2 h by visual observation. Commercial honey products have been successfully identified with excellent accuracy. This low-cost, high-efficiency and easy-operational strategy will greatly benefit the quality guarantee of foods with specific functions and geographical markers.

DNA methylation alternation in Stanford- A acute aortic dissection.

BACKGROUND: Acute aortic dissection (AAD) is a life-threatening cardiovascular disease. Recent studies have shown that DNA methylation may be associated with the pathological mechanism of AAD, but the panorama of DNA methylation needs to be explored. METHODS: DNA methylation patterns were screened using Infinium Human Methylation 450 K BeadChip in the aortic tissues from 4 patients with Stanford-A AAD and 4 controls. Gene enrichment was analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology (GO). DNA methylation levels of candidate genes were determined by pyrosequencing in the replication cohort including 16 patients with AAD and 7 controls. Protein expression level of candidate gene was assessed by Western blot. RESULTS: A total of 589 differentially methylated positions including 315 hypomethylated and 274 hypermethylated positions were found in AAD group. KEGG analysis demonstrated that differentially methylated position-associated genes were enriched in MAPK signaling pathway, TNF signaling pathway and apoptosis pathway, et al. GO analysis demonstrated that differentially methylated position-associated genes were enriched in protein binding, angiogenesis and heart development et al. The differential DNA methylation in five key genes, including Fas, ANGPT2, DUSP6, FARP1 and CARD6, was authenticated in the independent replication cohort. The protein expression level of the Fas was increased by 1.78 times, indicating the possible role of DNA methylation in regulation of gene expression. CONCLUSION: DNA methylation was markedly changed in the aortic tissues of Stanford-A AAD and associated with gene dysregulation, involved in AAD progression.

Genetic variants in Chinese patients with sporadic Stanford type A aortic dissection.

BACKGROUND: Genetic disorders are strongly associated with aortic disease. However, the identities of genetic mutations in sporadic Stanford type A aortic dissection (STAAD) are not clear. The present study analysed the possible genetic mutations of the known pathogenic genes of aortic disease and the clinical characteristics in patients with sporadic STAAD. METHODS: We analysed genetic mutations in 26 genes that underlie aortic aneurysms and dissections in 100 sporadic STAAD patients and 568 healthy controls after whole-genome sequencing (WGS). Clinical features and in-hospital death were determined in all STAAD patients. RESULTS: In total, 60 suspicious pathogenic mutations (56 novel and 4 previously reported) in 19 genes were identified in 50% (50/100) of patients, and 14 patients had more than 1 mutation. The ascending aortic diameter was extended in patients with mutations (49.1±12.3 vs. 43.7±11.2 mm, P=0.023), and the DeBakey type I phenotype was more common in patients with mutations in genes that coded extracellular matrix (ECM) components than in patients with mutations in other genes (96.6% vs. 66.7%, P=0.007). Patients with fibrillin-1 (FBN1) mutations were younger than patients without FBN1 mutations (44.7±11.0 vs. 53.5±12.1, P=0.030). Subgroup analyses revealed an increased risk of in-hospital mortality in mutation carriers (44.4% vs. 10.5%, P=0.029) but only in patients who received conservative treatment. CONCLUSIONS: Half of Chinese patients with a sporadic form of STAAD may carry mutations in known pathogenic genes of aortic disease, and these patients may exhibit distinct clinical features and poor clinical outcomes with the use of conservative treatment.

Proteomics applications in biomarker discovery and pathogenesis for abdominal aortic aneurysm.

Introduction: Abdominal aortic aneurysm (AAA) is a common, complex, and life-threatening disease. Currently, the pathogenesis of AAA is not well understood. No biomarkers or specific drugs are available for AAA in clinical applications. Proteomics is a powerful tool in biomarker discovery, exploration of pathogenesis, and drug target identification.Areas covered: We review the application of mass spectrometry-based proteome analysis in AAA patients within the last ten years. Differentially expressed proteins associated with AAA were identified in multiple sample sources, including vascular tissue, intraluminal thrombus, tissue secretome, blood, and cells. Some potential disease biomarkers, pathogenic mechanisms, or therapeutic targets for AAA were discovered using proteome analysis. The challenges and prospects of proteomics applied to AAA are also discussed.Expert opinion: Since most of the previous proteomic studies used relatively small sample sizes, some promising biomarkers need to be validated in multicenter cohorts to accelerate their clinical application. With the rapid development of mass spectrometry technology, modification-specific proteomics and multi-omics research in the future will enhance our understanding of the pathogenesis of AAA and promote biomarker discovery and drug development for clinical translation.

Angiopoietin 2 as a Novel Potential Biomarker for Acute Aortic Dissection.

Biomarker-assisted diagnosis of acute aortic dissection (AAD) is important for initiation of treatment and improved survival. However, identification of biomarkers for AAD in blood is a challenging task. The present study aims to find the potential AAD biomarkers using a transcriptomic strategy. Arrays based genome-wide gene expression profiling were performed using ascending aortic tissues which were collected from AAD patients and healthy donors. The differentially expressed genes were validated using quantitative reverse transcriptase PCR (qRT-PCR) and western blot. The plasma levels of a potential biomarker, angiopoietin 2 (ANGPT2) were determined in case-control cohort (77 AAD patients and 82 healthy controls) by enzyme linked immunosorbent assay. Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic power of ANGPT2 for AAD. Transcriptome data demonstrated that a total of 18 genes were significantly up-regulated and 28 genes were significantly down-regulated among AAD tissues (foldchange>3.0, p < 0.01). By bioinformatic analysis, we identified ANGPT2 as a candidate biomarker for blood-based detection of AAD. The qRT-PCR and protein expression demonstrated that ANGPT2 increased 2.4- and 4.2 folds, respectively in aortic tissue of AAD patients. Immunohistochemical staining demonstrated that ANGPT2 was markedly increased in intima of the aortic wall in AAD. Furthermore, ANGPT2 was significantly elevated in AAD patients as compared with controls (median 1625 vs. 383 pg/ml, p < 1E-6). ROC curve analysis showed that ANGPT2 was highly predictive of a diagnosis of type A AAD (area under curve 0.93, p < 1E-6). Sensitivity and specificity were 81 and 90%, respectively at the cutoff value of 833 pg/ml. In conclusion, ANGPT2 could be a promising biomarker for diagnosis of AAD; however, more studies are still needed to verify its specificity in diagnosing of AAD.

Integrin α9 is involved in the pathopoiesis of acute aortic dissection via mediating phenotype switch of vascular smooth muscle cell.

Acute aortic dissection (AAD) is a devastating disease with high mortality; however, the pathogenic mechanisms of AAD remain poorly understood. Our present study aimed to identify genes associated with AAD and explore the molecular function of candidate genes in the pathogenesis of AAD. We used a whole-genome transcriptional microarray to identify putative AAD genes using ascending aortic tissues from four patients with AAD and four healthy organ donors. The differentially expressed genes were further validated in eight patients with AAD and eight healthy organ donors. Functional assessments were conducted to analyze the effects of the identified AAD genes on the phenotype of aortic vascular smooth muscle cells (VSMCs). The whole-genome transcriptional microarray analysis found 129 dysregulated genes in the ascending aortic tissues of AAD (fold change≥2), which were mainly associated with the focal adhesion pathway and actin cytoskeleton regulation pathway. Among these genes, integrin α9 (ITGA9) was identified to be involved in both pathways and downregulated by 50% in AAD patients. The association of ITGA9 with AAD was confirmed by Western blotting analysis (P = 0.003). Functional studies showed that knocking down ITGA9 in VSMCs resulted in a decrease in contractile markers (SM22α and α-SMA) and an increase in synthetic markers (OPN and SMemb), suggesting that the VSMCs switched from a contractile to a synthetic phenotype. After overexpression of ITGA9 by a recombinant adenovirus vector in VSMCs, SM22α and α-SMA were upregulated, while SMemb was downregulated, indicating a phenotypic switch from the synthetic to contractile phenotype of VSMCs. In conclusion, our study identified ITGA9 as a novel AAD gene. This gene is downregulated in patients with AAD and is involved in the regulation of the phenotypic switch of VSMCs from a contractile to a synthetic phenotype.

Influence of Age on Clinical Presentation, Therapeutic Options, and Outcome in Chinese Patients with Acute Aortic Dissection.

It has been shown in previous studies that Chinese patients with acute aortic dissection (AD) were approximately 10 years younger than patients from western countries. However, there is a lack of studies concerning the age-related differences in clinical characteristics and outcomes in Chinese patients with acute AD. A total of 1,061 patients with AD (570 type A and 491 type B AD) were enrolled between 2006 and 2008. The clinical characteristics were compared between the patients in our study and those in the International Registry of Acute Aortic Dissection (IRAD). Compared with patients in the IRAD, those in our study were relatively younger, comprised more males, and had a higher proportion of Marfan syndrome but received fewer surgical interventions. When stratified by 10-year age, younger patients were more likely to have type A AD, familial AD, and Marfan syndrome, whereas older patients tended to comprise more females and type B AD. As age increased, the proportion of surgical intervention gradually decreased regardless of the type of AD. During a median follow-up of 2.2 years, 147 patients died, of whom 94 (63.9%) had type A AD and 53 (36.1%) had type B AD. Long-term mortality increased with increasing age, especially in patients above 70 years old. Furthermore, the recurrence rate of AD was higher in both the young and the older patients. In conclusion, compared with western patients with AD, Chinese patients have distinct characteristics and more attention should be paid to the young and older patients because of their high long-term mortality and recurrence rate.

Thyroid-stimulating hormone within the normal range and risk of major adverse cardiovascular events in nonischemic dilated cardiomyopathy patients with severe left ventricular dysfunction.

BACKGROUND: The association between thyroid-stimulating-hormone (TSH) and prognosis of nonischemic dilated cardiomyopathy (NIDCM) in patients with normal thyroid function remains unclear. HYPOTHESIS: Our aim was to investigate the association between TSH and major adverse cardiovascular events in euthyroid NIDCM patients. METHODS: The original cohort consisted of 216 consecutive euthyroid NIDCM patients, with left ventricular ejection fraction (LVEF) ≤35%, who were observed from 2010 to 2013. Patients with persistent ventricular arrhythmia (VA) histories, amiodarone taken for VA prevention, or on heart transplant list within 1 year were excluded. A follow-up evaluation was performed, and VA events, heart failure (HF) exacerbation/heart transplant, cardiac death, or death from any cause were separately evaluated. RESULTS: A total of 184 patients were enrolled, and 97.8% (180/184) ultimately received follow-up evaluations. During the median 4.6-year follow-up, 24 VA events, 28 cardiac deaths, 30 all-cause deaths, 40 HF exacerbations, and 11 heart transplant events occurred. Serum TSH levels showed good predictive efficacies for VA events (area under the curve [AUC] = 0.702, 95% confidence interval [CI]: 0.629-0.767), and the risk of VA events increased, according to serum TSH quarters, as determined by Kaplan-Meier analysis (2.2% vs 13.4% vs 21.0% vs 30.0%, Q1-Q4, P = 0.011). Multivariable Cox analysis showed that patients at the Q4 level of serum TSH (>2.67 mIU/L) suffered an increased risk of VA events, compared with those at the Q1 level of TSH (hazard ratio [HR] = 15.88, 95% CI: 2.01-65.15) or those at the other three quarters (HR = 3.17, 95% CI: 1.38-7.26). However, the Q4 TSH level was not associated with other adverse cardiac events. CONCLUSION: An association between TSH levels and the risk of VA events may exist in euthyroid NIDCM patients.

The effect of admission serum potassium levels on in-hospital and long-term mortality in type A acute aortic dissection.

BACKGROUND: Mild fluctuations in serum potassium (K+) levels are related to the prognosis of cardiovascular disease. This study aimed to determine the effect of admission serum potassium levels on in-hospital and long-term mortality in patients with Stanford type A acute aortic dissection (AAD). MATERIALS AND METHODS: A total of 588 consecutive patients with type A AAD were enrolled, and they were grouped according to admission serum potassium level: <3.5, 3.5 to <4.0, 4.0 to <4.5, 4.5 to <5.0, and ≥5.0mmol/L. Clinical outcomes were in-hospital death and long-term all-cause mortality. RESULTS: The in-hospital and long-term all-cause mortality rates were 10.7% and 16.3%, respectively. A U-shaped relationship was observed between admission serum potassium levels and both in-hospital death and long-term mortality. Univariate Cox regression identified potassium levels outside the interval of <3.5 to 4.5mmol/L to be a risk factor for both in-hospital and long-term death. After adjusting for age, gender, surgery and other risk factors, potassium levels outside the interval of <3.5 to 4.5mmol/L still had a significant association with long-term death [hazard ratio (HR)=1.72, 95% confidence interval (95% CI): 1.07-2.74, P=0.024]. Surgical intervention was the main protective factor associated with both in-hospital (HR=0.01, 95% CI 0.01-0.06, P<0.001) and long-term survival (HR=0.06, 95% CI 0.03-0.12, P<0.001). CONCLUSIONS: Among patients with Stanford type A AAD, admission serum potassium levels other than 3.5 to 4.5mmol/L might be associated with an increased risk of in-hospital death and long-term mortality.

Onset seasons and clinical outcomes in patients with Stanford type A acute aortic dissection: an observational retrospective study.

OBJECTIVES: To evaluate the association of onset season with clinical outcome in type A acute aortic dissection (AAD). DESIGN: A single-centre, observational retrospective study. SETTING: The study was conducted in Fuwai Hospital, the National Centre for Cardiovascular Disease, Beijing, China. PARTICIPANTS: From 2008 to 2010, a set of consecutive patients with type A AAD, confirmed by CT scanning, were enrolled and divided into four groups according to onset season: winter (December, January and February), spring (March, April and May), summer (June, July and August) and autumn (September, October and November). The primary end points were in-hospital death and all-cause mortality during follow-up. RESULTS: Of the 492 cases in this study, 129 occurred in winter (26.2%), 147 in spring (29.9%), 92 in summer (18.7%), and 124 in autumn (25.2%). After a median follow-up of 20.4 months (IQR 9.7-38.9), the in-hospital mortality in cases occurring in autumn was higher than in the other three seasons (23.4% vs 8.4%, p<0.01). Long-term mortality was comparable among the four seasonal groups (p=0.63). After adjustment for age, gender and other risk factors, onset in autumn was still an independent factor associated with increased risk of in-hospital mortality (HR 2.05; 95% CI 1.15 to 3.64, p=0.02) in addition to surgical treatment. Further analysis showed that the seasonal effect on in-hospital mortality (autumn vs other seasons: 57.4% vs 27.3%, p<0.01) was only significant in patients who did not receive surgical treatment. No seasonal effect on long-term clinical outcomes was found in this cohort. CONCLUSIONS: Onset in autumn may be a factor that increases the risk of in-hospital death from type A AAD, especially in patients who receive conservative treatment. Immediate surgery improves the short-term and long-term outcomes regardless of onset season.

Admission white blood cell count predicts short-term clinical outcomes in patients with uncomplicated Stanford type B acute aortic dissection.

OBJECTIVES: Inflammation has been shown to be related with acute aortic dissection (AAD). The present study aimed to evaluate the association of white blood cell counts (WBCc) on admission with both in-hospital and long-term all-cause mortality in patients with uncomplicated Stanford type B AAD. METHODS: From 2008 to 2010, a total of 377 consecutive patients with uncomplicated type B AAD were enrolled and then followed up. Clinical data and WBCc on admission were collected. The primary end points were in-hospital death and long-term all-cause death. RESULTS: The in-hospital death rate was 4.2%, and the long-term all-cause mortality rate was 6.9% during a median follow-up of 18.9 months. WBCc on admission was identified as a risk factor for in-hospital death by univariate Cox regression analysis as both a continuous variable and a categorical variable using a cut off of 11.0 × 109 cell/L (all P < 0.05). After adjusting for age, sex and other risk factors, elevated admission WBCc was still a significant predictor for in-hospital death as both a continuous variable [hazard ratio (HR): 1.052, 95% CI: 1.024-1.336, P = 0.002] and a categorical variable using a cut off of 11.0 × 109 cell/L (HR: 2.056, 95% CI: 1.673-5.253, P = 0.034). No relationship was observed between WBCc on admission and long-term all-cause death. CONCLUSIONS: Our results indicate that elevated WBCc upon admission might be used as a predictor for increased risk of in-hospital death in uncomplicated type B AAD. There might be no predictive value of WBCc for the long-term survival of type B AAD.