Search

Background and Aims: With the advent and implementation of high-sensitivity cardiac troponin assays, differentiation of patients with distinct types of myocardial injuries, including acute thrombotic myocardial infarction (TMI), acute non-thrombotic myocardial injury (nTMi), and chronic coronary atherosclerotic disease (cCAD), is of pressing clinical importance. Thermal liquid biopsy (TLB) emerges as a valuable diagnostic tool, relying on identifying thermally induced conformational changes of biomolecules in blood plasma. While TLB has proven useful in detecting and monitoring several cancers and autoimmune diseases, its application in cardiovascular diseases remains unexplored. In this proof-of-concept study, we sought to determine and characterize TLB profiles in patients with TMI, nTMi, and cCAD at multiple acute-phase time points (T 0 h, T 2 h, T 4 h, T 24 h, T 48 h) as well as a follow-up time point (Tfu) when the patient was in a stable state., Methods: TLB profiles were collected for 115 patients (60 with TMI, 35 with nTMi, and 20 with cCAD) who underwent coronary angiography at the event presentation and had subsequent follow-up. Medical history, physical, electrocardiographic, histological, biochemical, and angiographic data were gathered through medical records, standardized patient interviews, and core laboratory measurements., Results: Distinctive signatures were noted in the median TLB profiles across the three patient types. TLB profiles for TMI and nTMi patients exhibited gradual changes from T0 to Tfu, with significant differences during the acute and quiescent phases. During the quiescent phase, all three patient types demonstrated similar TLB signatures. An unsupervised clustering analysis revealed a unique TLB signature for the patients with TMI. TLB metrics generated from specific features of TLB profiles were tested for differences between patient groups. The first moment temperature ( T FM ) metric distinguished all three groups at time of presentation (T0). In addition, 13 other TLB-derived metrics were shown to have distinct distributions between patients with TMI and those with cCAD., Conclusion: Our findings demonstrated the use of TLB as a sensitive and data-rich technique to be explored in cardiovascular diseases, thus providing valuable insight into acute myocardial injury events., Competing Interests: ADF and NG are co-inventors of a patent assigned to and owned by the University of Louisville describing the use of DSC to differentially diagnose myocardial infarction types (US Patent No. 11,835,529). During study completion and analysis, ADF and NG were founders and had an equity interest in a start-up company, DSC Technologies LLC, which was involved in the development of DSC technologies; NG was a consultant for the calorimetry instrument supplier TA Instruments, Inc. involved in education for microcalorimetry applications and the characterization of microcalorimetry instrument performance. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Lidani, Buscaglia, Trainor, Tomar, Kaliappan, DeFilippis and Garbett.)

Background and Aims: Although several biomarkers have been studied in thromboembolic stroke, measuring the balance between thrombus formation and thrombolysis and data on its role in predicting stroke and atrial fibrillation (AF)-related stroke is limited. We sought to assess atherothrombotic biomarkers grouped into composite factors that reflect thrombotic and thrombolytic potential, and the balance between these factors as it relates to incident stroke or transient ischemic attack (TIA) and stroke/TIA in AF., Methods: A Thrombotic Factor, derived from fibrinogen, plasmin-antiplasmin complex, factor VIII, D-dimer, and lipoprotein(a); and a Thrombolytic Factor, derived from plasminogen and oxidized phospholipids on plasminogen, were evaluated at baseline in 5,764 Multi-Ethnic Study of Atherosclerosis (MESA) participants. We evaluated the association between these two factors representative of thrombotic and thrombolytic potential and incident stroke/TIA (n = 402), and AF-related stroke/TIA (n = 82) over a median of 13.9 and 3.7 years, respectively. Cox proportional hazard models adjusted for medication use, cardiovascular risk factors and CHA 2 DS 2 -VASc score were utilized. Harrell's C-index was estimated to evaluate model performance., Results: In models including both factors, Thrombotic Factor was positively while Thrombolytic Factor was inversely associated with incident stroke/TIA and AF-related stroke/TIA. Incorporating these factors along with the CHA 2 DS 2 -VASc in adjusted models resulted in a small improvement in risk prediction of incident stroke/TIA and AF-related stroke/TIA compared to models without the factors (C-index from 0.697 to 0.704, and from 0.657 to 0.675, respectively)., Conclusions: Composite biomarker factors, representative of the balance between thrombotic and thrombolytic propensity, provided an improvement in predicting stroke/TIA beyond CHA 2 DS 2 -VASc score., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Phytophthora blight, caused by Phytophthora capsici , is a serious disease of many vegetable crops worldwide. In New Mexico, U.S.A., the disease affects chile pepper ( Capsicum annuum L.), a major crop in the state. There is no single tool that effectively controls the disease. Continuous research is needed in identifying combination of tools that can reduce the impact of Phytophthora blight. We explored the potential of combining cover crops and biocontrol agents to reduce soilborne diseases. This study aimed to evaluate the effects of Indian mustard ( Brassica juncea L.) cover crop on the antagonistic ability of Trichoderma harzianum against P. capsici in vitro and to quantify the impacts of combining soil amendment with residues from B. juncea and barley ( Hordeum vulgare L.) cover crops and plastic covering on infection of chile pepper seedlings by P. capsici under greenhouse conditions. Volatiles from macerated tissue of B. juncea significantly reduced P. capsici and T. harzianum growth in the absence of soil by 89.0 and 79.0%, respectively. When incorporated in soils, volatiles from macerated tissue of B. juncea significantly reduced P. capsici and T. harzianum by 33.4 and 7.8%, respectively. T. harzianum was more resilient to B. juncea biofumigation than P. capsici. Significant reduction in disease incidence was observed with B. juncea -fumigated soil, while no disease suppression was observed with soil incorporation of H. vulgare residues. Covering soil with plastic was necessary for increasing the efficacy of B. juncea biofumigation., Competing Interests: The author(s) declare no conflict of interest.

Background: Despite different prevalence, pathobiology, and prognosis between etiologically distinct myocardial infarction (MI) subtypes, prospective study of risk factor for MI in large NHLBI-sponsored cardiovascular cohorts is limited to acute MI as a singular entity. Therefore, we sought to utilize the Multi-Ethnic Study of Atherosclerosis (MESA), a large prospective primary prevention cardiovascular study, to define the incidence and risk factor profile of individual myocardial injury subtypes., Methods: We describe the rationale and design of re-adjudicating 4,080 events that occurred over the first 14 years of follow-up in MESA for the presence and subtype of myocardial injury as defined by the Fourth Universal Definition of MI: MI type 1 to 5, acute non-ischemic myocardial injury, and chronic myocardial injury. The project utilizes a 2-physician adjudication process via examination of medical records, abstracted data collection forms, cardiac biomarker results, and electrocardiograms of all relevant clinical events. Comparison of the magnitude and direction of associations between baseline traditional and novel cardiovascular risk factors with incident and recurrent acute MI subtypes and acute non-ischemic myocardial injury events will be made., Conclusions: This project will result in one of the first large prospective cardiovascular cohort with modern classification of acute MI subtypes, as well as a full accounting of non-ischemic myocardial injury events, with implications for numerous ongoing and future studies in MESA. By creating precise MI phenotypes, and defining their epidemiology, this project will allow for discovery of novel pathobiology-specific risk factors, allow for development of more accurate risk prediction, and suggest more targeted preventive strategies., Competing Interests: Conflict of interest None reported., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Angiotensinogen is the proximal precursor of the angiotensin peptide hormones of the renin-angiotensin-aldosterone system (RAAS). Clinical trials are ongoing targeting angiotensinogen for the treatment of hypertension and heart failure. The epidemiology of angiotensinogen is not well defined, particularly its relationship to ethnicity, sex, and blood pressure (BP)/hypertension., Objectives: The authors sought to determine the relationship of circulating angiotensinogen levels to ethnicity, sex, BP, incident hypertension, and prevalent hypertension in a modern sex-balanced ethnically diverse cohort., Methods: Plasma angiotensinogen levels were measured in 5,786 participants from the MESA (Multi-Ethnic Study of Atherosclerosis). Linear, logistic, and Cox proportional hazards models were utilized to examine the associations of angiotensinogen with BP, prevalent hypertension, and incident hypertension, respectively., Results: Angiotensinogen levels were significantly higher in females than males and differed across self-reported ethnicities with the ordering (from highest to lowest): White, Black, Hispanic, and Chinese adults. Higher levels were associated with higher BP and odds of prevalent hypertension, after adjusting for other risk factors. Equivalent relative differences in angiotensinogen were associated with greater differences in BP in males vs females. In males not taking RAAS-blocking medications, a standard deviation increment in log-angiotensinogen was associated with 2.61 mm Hg higher systolic BP (95% CI: 1.49-3.80), while in females the same increment in angiotensinogen was associated with 0.97 mm Hg higher systolic BP (95% CI: 0.30-1.65)., Conclusions: Significant differences in angiotensinogen levels are present between sexes and ethnicities. A positive association is present between levels and prevalent hypertension and BP, which differs between sexes., Competing Interests: Funding Support and Author Disclosures This research was supported by an Institutional Development Award from the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under grant no. P20GM103451 and a Research Enhancement Award to Dr Trainor from NIGMS under grant no. SC1GM139730. Additional support for this research, including sample processing and angiotensinogen measurements, was provided by Ionis Pharmaceuticals. MESA was supported by contracts HHSN268201500003I, N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, and N01-HC-95169 from the National Heart, Lung, and Blood Institute and by grants UL1-TR-000040, UL1-TR-001079, and UL1-TR-001420 from the National Center for Advancing Translational Sciences. The authors thank the other investigators, the staff, and the participants of MESA for their valuable contributions. A full list of participating MESA investigators and institutions can be found at http://www.mesa-nhlbi.org. Drs Brambatti, Mullick, and Morgan are employees of Ionis Pharmaceuticals. Although these relationships have been identified for conflict-of-interest management based on the overall scope of the project, the research findings included in this particular publication may not necessarily relate to the interests of the above companies. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict-of-interest policies. Dr Tsimikas is a co-inventor on and receives royalties from patents owned by University of California San Diego (UCSD); is a co-founder and has an equity interest in Oxitope and its affiliates Kleanthi Diagnostics and Covicept Therapeutics; and has a dual appointment at UCSD and Ionis Pharmaceuticals. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Aims: Traditional atherosclerotic cardiovascular disease (ASCVD) risk factors fail to address the full spectrum of the complex interplay of atherosclerotic and atherothrombotic factors integral to ASCVD events. This study sought to examine the association between atherothrombotic biomarkers and ASCVD events., Methods and Results: The association between atherothrombotic biomarkers and 877 ASCVD events with and without adjustment for traditional risk factors was evaluated via Cox proportional hazards models and factor analysis in 5789 Multi-Ethnic Study of Atherosclerosis participants over a median follow-up of 14.7 years. Factor analysis accounted for multidimensional relationship and shared variance among study biomarkers, which identified two new variables: a thrombotic factor (Factor 1), principally defined by shared variance in fibrinogen, plasmin-antiplasmin complex, factor VIII, D-dimer, and lipoprotein(a), and a fibrinolytic factor (Factor 2), principally defined by shared variance of plasminogen and oxidized phospholipids on plasminogen. In a model including both factors, the thrombotic factor was associated with the higher risk of ASCVD events [hazard ratio (HR) 1.57, 95% confidence interval (CI) 1.45, 1.70], while the fibrinolytic factor was associated with the lower risk of ASCVD events (HR 0.76, 95% CI 0.70, 0.82), with estimated ASCVD free survival highest for low atherothrombotic Factor 1 and high atherothrombotic Factor 2., Conclusion: Two atherothrombotic factors, one representative of thrombotic propensity and the other representative of fibrinolytic propensity, were significantly and complementarily associated with incident ASCVD events, remained significantly associated with incident ASCVD after controlling for traditional risk factors, and have promise for identifying patients at high ASCVD event risk specifically due to their atherothrombotic profile., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Many cancers, including breast cancer, have shown differential expression of human arylamine N -acetyltransferase 1 (NAT1). The exact effect this differential expression has on disease risk and progression remains unclear. While NAT1 is classically defined as a xenobiotic metabolizing enzyme, other functions and roles in endogenous metabolism have recently been described providing additional impetus for investigating the effects of varying levels of NAT1 on global gene expression. Our objective is to further evaluate the role of NAT1 in breast cancer by determining the effect of NAT1 overexpression, knockdown, and knockout on global gene expression in MDA-MB-231 cell lines. RNA-seq was utilized to interrogate differential gene expression (genes correlated with NAT1 activity) across three biological replicates of previously constructed and characterized MDA-MB-231 breast cancer cell lines expressing parental ( Scrambled ), increased ( Up ), decreased ( Down, CRISPR 2-12 ), or knockout ( CRISPR 2-19, CRISPR 5-50 ) levels of NAT1. 3,889 genes were significantly associated with the NAT1 N -acetylation activity of the cell lines ( adjusted p ≤ 0.05); of those 3,889 genes, 1,756 were positively associated with NAT1 N -acetylation activity and 2,133 were negatively associated with NAT1 N -acetylation activity. An enrichment of genes involved in cell adhesion was observed. Additionally, human arylamine N -acetyltransferase 2 (NAT2) transcripts were observed in the complete NAT1 knockout cell lines ( CRISPR 2-19 and CRISPR 5-50 ). This study provides further evidence that NAT1 functions as more than just a drug metabolizing enzyme given the observation that differences in NAT1 activity have significant impacts on global gene expression. Additionally, our data suggests the knockout of NAT1 results in transcription of its isozyme NAT2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Carlisle, Trainor, Doll and Hein.)

Platelet activation and subsequent aggregation is a vital component of atherothrombosis resulting in acute myocardial infarction. Therefore, quantifying platelet aggregation is a valuable measure for elucidating the pathogenesis of acute coronary syndromes (ACS). Circulating platelet-monocyte conjugates (PMC) as determined by flow cytometry (FCM) are an important measure of in vivo platelet aggregation. However, the influence of sample handling on FCM measurement of PMC is not well-studied. The changes in FCM measurement of PMC with variation in sample handling techniques were evaluated. The stability of PMC concentrations over time with changes in fixation and immunolabeling intervals was assessed. The effect of Time-to-Fix and Time-to-Stain on FCM PMC measurements was investigated in five healthy volunteers. Time-to-Fix (i.e., interval between phlebotomy and sample fixation) was performed at 3, 30, and 60 min. Time-to-Stain (i.e., time of fixed sample storage to staining) was performed at 1, 24, and 48 h. Increasing Time-to-Stain from 1 to 24 or 48 h resulted in lower PMC measures (p < 0.0001). A statistically significant difference in PMC measurement with increasing Time-to-Fix was not observed (p < 0.41). Postponement of sample staining has deleterious effects on the measurement of PMC via FCM. Delays in immunolabeling of fixed samples compromised measurement of PMC by 30% over the first 24 h. Staining/FCM should be completed within an hour of collection.

Human arylamine N-acetyltransferase 1 (NAT1), present in all tissues, is classically described as a phase-II xenobiotic metabolizing enzyme but can also catalyze the hydrolysis of acetyl-Coenzyme A (acetyl-CoA) in the absence of an arylamine substrate using folate as a cofactor. NAT1 activity varies inter-individually and has been shown to be overexpressed in estrogen receptor-positive (ER+) breast cancers. NAT1 has also been implicated in breast cancer progression however the exact role of NAT1 remains unknown. The objective of this study was to evaluate the effect of varying levels of NAT1 N-acetylation activity in MDA-MB-231 breast cancer cells on global cellular metabolism and to probe for unknown endogenous NAT1 substrates. Global, untargeted metabolomics was conducted via ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on MDA-MB-231 breast cancer cell lines constructed with siRNA and CRISPR/Cas9 technologies to vary only in NAT1 N-acetylation activity. Many metabolites were differentially abundant in NAT1-modified cell lines compared to the Scrambled parental cell line. N-acetylasparagine and N-acetylputrescine abundances were strongly positively correlated (r = 0.986 and r = 0.944, respectively) with NAT1 N-acetylation activity whereas saccharopine abundance was strongly inversely correlated (r = -0.876). Two of the most striking observations were a reduction in de novo pyrimidine biosynthesis and defective β-oxidation of fatty acids in the absence of NAT1. We have shown that NAT1 expression differentially affects cellular metabolism dependent on the level of expression. Our results support the hypothesis that NAT1 is not just a xenobiotic metabolizing enzyme and may have a role in endogenous cellular metabolism.

Elevated measures of matrix metalloproteinases (MMPs) are associated with acute myocardial infarction (MI), but it is not known how long these changes persist post-MI or if these measures differ between atherothrombotic versus non-atherothrombotic MI. MMPs-2, 3, and 9 were measured in 80 subjects with acute MI (atherothrombotic and non-atherothrombotic MI) or stable coronary artery disease (CAD). Measurements were made at, the time of acute MI, and > 3-month following acute MI (quiescent phase). Outcome measures were compared between groups and between time of acute MI and quiescent post-MI follow-up using Wilcoxon's and repeated measures analysis of variance. Forty-nine subjects met the criteria for acute MI with clearly defined atherothrombotic (n = 22) and non-atherothrombotic (n = 12) subsets. Fifteen subjects met criteria for stable CAD. MMP-3 was higher in acute MI versus stable CAD subjects at the time of acute MI: (453 vs. 217 pg/mL, p = 0.010) but not at quiescent phase follow-up (p > 0.05). MMP-9 was higher in acute MI versus stable CAD subjects at the time of acute MI: (412 vs. 168 pg/mL, p = 0.002) but not at the quiescent phase follow-up (p > 0.05). MMP-9 was higher at the time of acute MI versus quiescent phase follow-up in acute MI (412 vs. 213 pg/mL, p = 0.001) and atherothrombotic MI specifically (458 vs. 212 pg/mL, p = 0.001). No difference in MMP-2, 3, or 9 was observed between atherothrombotic versus non-atherothrombotic MI subgroups. MMPs-3 and 9 are significantly elevated in acute MI verses stable CAD subjects at time of acute MI but not different at quiescent phase follow-up. MMP-9 is elevated at the time of acute MI and specifically in acute atherothrombotic MI at time of MI versus quiescent phase follow-up.

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can also hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. Expression of NAT1 varies between individuals and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. To date, however, the exact mechanism by which NAT1 expression affects mitochondrial bioenergetics in breast cancer cells has not been described. To further evaluate the role of NAT1 in energy metabolism MDA-MB-231 breast cancer cells with parental, increased, and knockout levels of NAT1 activity were compared for bioenergetics profile. Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured followed by programmed sequential injection of Oligomycin (ATP synthase inhibitor), FCCP (ETC uncoupler), Antimycin A (Complex III inhibitor), and Rotenone (Complex I inhibitor) to evaluate mitochondrial bioenergetics. Compared to the cell lines with parental NAT1 activity, NAT1 knockout MDA-MB-231 cell lines exhibited significant differences in bioenergetics profile, while those with increased NAT1 did not. Significant increases in reserve capacity, maximum mitochondrial capacity, and glycolytic reserve capacity were observed in NAT1 knockout MDA-MB-231 cell lines compared to those with parental and increased NAT1 activity. These data indicate that NAT1 knockout in MDA-MB-231 breast cancer cells may enhance adaptation to stress by increasing plasticity in response to energy demand., (© 2018 Wiley Periodicals, Inc.)

This study evaluated the integration and methlyation of human papillomavirus type 16 (HPV16) in head and neck squamous cell carcinoma (HNSCC) and its oral precursor, high-grade oral epithelial dysplasia (hgOED). Archival samples of HPV16-positive hgOED ( N = 19) and HNSCC ( N = 15) were evaluated, along with three HNSCC (UMSCC-1, -47 and -104) and two cervical cancer (SiHa and CaSki) cell lines. HgOED cases were stratified into three groups with increasing degrees of cytologic changes (mitosis, karyorrhexis and apoptosis). The viral load was higher and the E2/E6 ratio lower (indicating a greater tendency toward viral integration) in group 3 than in groups 1 or 2 ( p = 0.002, 0.03). Methylation was not observed in hgOED cases and occurred variably in only three HNSCC cases (26.67%, 60.0% and 93.3%). In HNSCC cell lines, lower E7 expression correlated with higher levels of methylation. HgOED with increased cytologic change, now termed HPV-associated oral epithelial dysplasia (HPV-OED), exhibited an increased viral load and a tendency toward DNA integration, suggesting a potentially increased risk for malignant transformation. More detailed characterization and clinical follow-up of HPV-OED patients is needed to determine whether HPV-OED is a true precursor to HPV-associated HNSCC and to clarify the involvement of HPV in HNSCC carcinogenesis., Competing Interests: CONFLICTS OF INTEREST All authors have no conflicts of interest.

Introduction: Heart disease remains a leading cause of global mortality. While acute myocardial infarction (colloquially: heart attack), has multiple proximate causes, proximate etiology cannot be determined by a blood-based diagnostic test. We enrolled a suitable patient cohort and conducted a non-targeted quantification of plasma metabolites by mass spectrometry for developing a test that can differentiate between thrombotic MI, non-thrombotic MI, and stable disease. A significant challenge in developing such a diagnostic test is solving the NP-hard problem of feature selection for constructing an optimal statistical classifier., Objective: We employed a Wisdom of Artificial Crowds (WoAC) strategy for solving the feature selection problem and evaluated the accuracy and parsimony of downstream classifiers in comparison with traditional feature selection techniques including the Lasso and selection using Random Forest variable importance criteria., Materials and Methods: Artificial Crowd Wisdom was generated via aggregation of the best solutions from independent and diverse genetic algorithm populations that were initialized with bootstrapping and a random subspaces constraint., Results/conclusions: Strong evidence was observed that a statistical classifier utilizing WoAC feature selection can discriminate between human subjects presenting with thrombotic MI, non-thrombotic MI, and stable Coronary Artery Disease given abundances of selected plasma metabolites. Utilizing the abundances of twenty selected metabolites, a leave-one-out cross-validation estimated misclassification rate of 2.6% was observed. However, the WoAC feature selection strategy did not perform better than the Lasso over the current study., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Platelet count has been shown to be lower and mean platelet volume (MPV) to be higher in acute myocardial infarction (MI). However, it is not known whether these changes persist post-MI or if these measures are able to distinguish between acute thrombotic and non-thrombotic MI. Platelet count and MPV were measured in 80 subjects with acute MI (thrombotic and non-thrombotic) and stable coronary artery disease (CAD) at cardiac catheterization (acute phase) and at >3-month follow-up (quiescent phase). Subjects were stratified using stringent clinical, biochemical, histological, and angiographic criteria. Outcome measures were compared between groups by analysis of variance. Forty-seven subjects met criteria for acute MI with clearly defined thrombotic (n = 22) and non-thrombotic (n = 12) subsets. Fourteen subjects met criteria for stable CAD. No significant difference was observed in platelet count between subjects with acute MI and stable CAD at the acute or quiescent phase. MPV was higher in acute MI (9.18 ± 1.21) compared to stable CAD (8.13 ± 0.66; P = 0.003) at the acute phase but not at the quiescent phase (8.48 ± 0.58 vs 8.94 ± 1.42; P = 0.19). No difference in platelet count or MPV was detected between thrombotic and non-thrombotic subsets at acute or quiescent phases. The power to detect differences in these measures between thrombotic and non-thrombotic subsets was 58%. Higher MPV at the time of acute MI is not observed by 3 months post-MI (quiescent phase). Platelet count and MPV do not differ in subjects with thrombotic versus non-thrombotic MI. Further investigation is warranted to evaluate the utility of these measures in the diagnosis of acute MI.

Background: Collagen is a major determinant of atherosclerotic plaque stability. Thus, identification of differences in enzymes that regulate collagen integrity could be useful for predicting susceptibility to atherothrombosis or for diagnosing plaque rupture. In this study, we sought to determine whether prolidase, the rate-limiting enzyme of collagen turnover, differs in human subjects with acute myocardial infarction (MI) versus those with stable coronary artery disease (CAD)., Methods: We measured serum prolidase activity in 15 patients with stable CAD and 49 patients with acute MI, of which a subset had clearly defined thrombotic MI ( n  = 22) or non-thrombotic MI ( n  = 12). Prolidase activity was compared across study time points (at cardiac catheterization, T0; 6 h after presentation, T6; and at a quiescent follow-up, Tf/u) in acute MI and stable CAD subjects. We performed subgroup analyses to evaluate prolidase activity in subjects presenting with acute thrombotic versus non-thrombotic MI., Results: Although prolidase activity was lower at T0 and T6 versus the quiescent phase in acute MI and stable CAD subjects ( p  < 0.0001), it was not significantly different between acute MI and stable CAD subjects at any time point (T0, T6, and Tf/u) or between thrombotic and non-thrombotic MI groups. Preliminary data from stratified analyses of a small number of diabetic subjects ( n  = 8) suggested lower prolidase activity in diabetic acute MI subjects compared with non-diabetic acute MI subjects ( p  = 0.02)., Conclusion: Circulating prolidase is not significantly different between patients with acute MI and stable CAD or between patients with thrombotic and non-thrombotic MI. Further studies are required to determine if diabetes significantly affects prolidase activity and how this might relate to the risk of MI.

Statistical classification is a critical component of utilizing metabolomics data for examining the molecular determinants of phenotypes. Despite this, a comprehensive and rigorous evaluation of the accuracy of classification techniques for phenotype discrimination given metabolomics data has not been conducted. We conducted such an evaluation using both simulated and real metabolomics datasets, comparing Partial Least Squares-Discriminant Analysis (PLS-DA), Sparse PLS-DA, Random Forests, Support Vector Machines (SVM), Artificial Neural Network, k -Nearest Neighbors ( k -NN), and Naïve Bayes classification techniques for discrimination. We evaluated the techniques on simulated data generated to mimic global untargeted metabolomics data by incorporating realistic block-wise correlation and partial correlation structures for mimicking the correlations and metabolite clustering generated by biological processes. Over the simulation studies, covariance structures, means, and effect sizes were stochastically varied to provide consistent estimates of classifier performance over a wide range of possible scenarios. The effects of the presence of non-normal error distributions, the introduction of biological and technical outliers, unbalanced phenotype allocation, missing values due to abundances below a limit of detection, and the effect of prior-significance filtering (dimension reduction) were evaluated via simulation. In each simulation, classifier parameters, such as the number of hidden nodes in a Neural Network, were optimized by cross-validation to minimize the probability of detecting spurious results due to poorly tuned classifiers. Classifier performance was then evaluated using real metabolomics datasets of varying sample medium, sample size, and experimental design. We report that in the most realistic simulation studies that incorporated non-normal error distributions, unbalanced phenotype allocation, outliers, missing values, and dimension reduction, classifier performance (least to greatest error) was ranked as follows: SVM, Random Forest, Naïve Bayes, sPLS-DA, Neural Networks, PLS-DA and k -NN classifiers. When non-normal error distributions were introduced, the performance of PLS-DA and k -NN classifiers deteriorated further relative to the remaining techniques. Over the real datasets, a trend of better performance of SVM and Random Forest classifier performance was observed., Competing Interests: The authors declare no conflict of interest.

Myocardial infarction (MI) is an acute event characterized by myocardial necrosis. Thrombotic MI is caused by spontaneous atherosclerotic plaque disruption that results in a coronary thrombus; non-thrombotic MI occurs secondary to oxygen supply-demand mismatch. We sought to characterize the differential metabolic perturbations associated with these subtypes utilizing a systems approach. Subjects presenting with thrombotic MI, non-thrombotic MI and stable coronary artery disease (CAD) were included. Whole blood was collected at two acute time-points and at a time-point representing the quiescent stable disease state. Plasma metabolites were analyzed by untargeted UPLC-MS/MS and GC-MS. A weighted network was constructed, and modules were determined from the resulting topology. To determine perturbed modules, an enrichment analysis for metabolites that demonstrated between-group differences in temporal change across the disease state transition was then conducted., Biological Significance: We report evidence of metabolic perturbations of acute MI and determine perturbations specific to thrombotic MI. Specifically, a module characterized by elevated glucocorticoid steroid metabolites following acute MI showed greatest perturbation following thrombotic MI. Modules characterized by elevated pregnenolone metabolites, monoacylglycerols, and acylcarnitines were perturbed following acute MI. A module characterized by a decrease in plasma amino acids following thrombotic MI was differentially perturbed between MI subtypes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objectives: Reported cytologic alterations associated with high-risk human papillomavirus (HR-HPV) in oral epithelial dysplasia (HPV-OED) need further characterization., Study Design: Archival cases of high-grade oral epithelial dysplasia (hgOED) (N = 38) were assigned a cytologic score (CS) based on the average number of mitotic, karyorrhectic, and apoptotic cells per high-power field. Three groups were then generated on the basis of increasing CS: Focal (group 1, N = 14), Intermediate (group 2, N = 12), and Diffuse (group 3, N = 12). Polymerase chain reaction-based HPV genotyping and p16 immunohistochemistry were performed., Results: HR-HPV was found significantly more in group 3 (83.3%) compared with groups 1 and 2 (group 1&2; 42.9% and 41.7%, respectively; P = .047). HPV16 predominated in HR-HPV-positive cases (90.5%). By location, the tongue or the floor of mouth was associated with all groups (P = .04). Increasing CS was associated with a slightly younger age (P = .04) and increased expression of p16 (P = .005). CS and p16 expression were not sensitive but were highly specific predictors for HR-HPV presence. Based on limited follow-up information, HPV-OED does not differ in clinical aggressiveness compared with conventional OED., Conclusions: Increased CS in hgOED is strongly associated with HR-HPV (mostly HPV16) and p16 expression. CS and p16 expression are specific predictors of HR-HPV presence. Further molecular study and long-term follow-up of HPV-OED are needed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aims: Current non-invasive diagnostics for acute myocardial infarction (MI) identify myocardial necrosis rather than the primary cause and therapeutic target-plaque disruption and resultant thrombosis. The aim of this study was to identify changes specific to plaque disruption and pathological thrombosis that are distinct from acute myocardial necrosis., Methods and Results: We quantified 1,032 plasma metabolites by mass spectrometry in 11 thrombotic MI, 12 non-thrombotic MI, and 15 stable coronary artery disease (CAD) subjects at two acute phase (time of catheterization [T0], six hours [T6]) and one quiescent (>3 months follow-up) time points. A statistical classifier was constructed utilizing baseline (T0) abundances of a parsimonious set of 17 qualifying metabolites. Qualifying metabolites were those that demonstrated a significant change between the quiescent phase and the acute phase and that were distinct from any change seen in non-thrombotic MI or stable CAD subjects. Classifier performance as estimated by 10-fold cross-validation was suggestive of high sensitivity and specificity for differentiating thrombotic from non-thrombotic MI and stable CAD subjects at presentation. Nineteen metabolites demonstrated an intra-subject change from time of acute thrombotic MI presentation to the quiescent state that was distinct from any change measured in both the non-thrombotic MI and stable CAD subjects undergoing cardiac catheterization over the same time course (false discovery rate <5%)., Conclusions: We have identified a candidate metabolic signature that differentiates acute thrombotic MI from quiescent state after MI, from acute non-thrombotic MI, and from stable CAD. Further validation of these metabolites is warranted given their potential as diagnostic biomarkers and novel therapeutic targets for the prevention or treatment of acute MI.

Background: Coronary angiography is frequently employed to aid in the diagnosis of acute coronary thrombosis, but there is limited data to support its efficacy. The aim of the study was to evaluate sensitivity and specificity of five commonly used angiographic characteristics for diagnosis of acute coronary thrombosis: Ambrose complex lesion morphology; spherical, ovoid, or irregular filling defect; abrupt vessel cutoff; intraluminal staining; and any coronary filling defect., Methods: Coronary angiography of 80 acute myocardial infarction or stable coronary artery disease subjects were assessed in blinded fashion, for the presence or absence of five angiographic characteristics. Only lesions of ≥ 10% stenosis were included in the analysis. Presence or absence of each angiographic characteristic was compared between lesions with or without the following study defined outcomes: 1) histologically confirmed thrombus, 2) highly probable thrombus, and 3) highly unlikely thrombus., Results: A total of 323 lesions were evaluated. All studied angiographic characteristics were associated with histologically confirmed and highly probable thrombotic lesions vs. lesions not meeting criteria for these outcomes (p < 0.03), except for complex Ambrose morphology which was not associated with any of the study outcomes (p > 0.05). Specificity for identifying histologically confirmed or highly probable thrombotic lesion was high (92-100%), especially for spherical, ovoid, or irregular filling defect (99-100%) and intraluminal staining (99%). Sensitivity for identification of histologically confirmed or highly probable thrombotic lesions was low for all tested angiographic characteristics (17-60%)., Conclusions: The presence of spherical, ovoid, or irregular filling defect or intraluminal staining was highly suggestive of coronary thrombus. However, none of the evaluated angiographic characteristics were useful for ruling out the presence of coronary thrombus. If confirmed in an independent cohort, these angiographic characteristic will be of significant value in confirming the diagnosis of acute coronary thrombosis.

We demonstrate the rapid and label-free capture of breast cancer cells spiked in blood using nanotube-antibody micro-arrays. 76-element single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (anti-EpCAM), Anti-human epithelial growth factor receptor 2 (anti-Her2) and non-specific IgG antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester. Following device functionalization, blood spiked with SKBR3, MCF7 and MCF10A cells (100/1000 cells per 5 μl per device, 170 elements totaling 0.85 ml of whole blood) were adsorbed on to the nanotube device arrays. Electrical signatures were recorded from each device to screen the samples for differences in interaction (specific or non-specific) between samples and devices. A zone classification scheme enabled the classification of all 170 elements in a single map. A kernel-based statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping series to classify device electrical signals that corresponded to plain blood (control) or SKBR3 spiked blood (case) on anti-Her2 functionalized devices with ∼90% sensitivity, and 90% specificity in capture of 1000 SKBR3 breast cancer cells in blood using anti-Her2 functionalized devices. Screened devices that gave positive electrical signatures were confirmed using optical/confocal microscopy to hold spiked cancer cells. Confocal microscopic analysis of devices that were classified to hold spiked blood based on their electrical signatures confirmed the presence of cancer cells through staining for DAPI (nuclei), cytokeratin (cancer cells) and CD45 (hematologic cells) with single cell sensitivity. We report 55%-100% cancer cell capture yield depending on the active device area for blood adsorption with mean of 62% (∼12 500 captured off 20 000 spiked cells in 0.1 ml blood) in this first nanotube-CTC chip study.

Introduction: Human arylamine N -acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. Expression of NAT1 is elevated in several cancers including breast cancer. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear., Objective: This study explored polar metabolome differences between MDA-MB-231 breast cancer cells expressing varying levels of NAT1 activity using an untargeted approach., Methods: Three MDA-MB-231 breast adenocarcinoma cell lines that stably express wild-type, increased, and decreased levels of human NAT1 were investigated for differences in polar metabolic profile using a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) system., Results: Increased levels of human NAT1 in the transformed cell lines resulted in a statistically significant decreased abundance of the metabolite palmitoleic acid (q = 0.0006), when compared to normal and decreased levels of human NAT1. The fatty acid synthesis pathway utilizes acetyl coenzyme A (acetyl-CoA) in the first two reactions of the pathway and eventually leads to the synthesis of palmitoleic acid., Conclusion: These data suggest a link between increased levels of NAT1 activity and decreased flux of acetyl-CoA through this portion of the fatty acid synthesis pathway., Competing Interests: The authors declare that they have no conflict of interest.

Oxidized phospholipids (OxPL) are abundant in atherosclerotic plaques. They are also bound to circulating plasminogen after myocardial infarction (MI), and their binding to plasminogen may accentuate fibrinolysis. We sought to assess whether circulating levels of plasminogen and OxPL bound to plasminogen (OxPL-PLG) increase following acute MI and whether this increase differs between atherothrombotic (Type 1) and non-atherothrombotic (Type 2) MI. We measured circulating levels of plasminogen and OxPL-PLG at 0, 6, 24, 48 h, and >3 months (stable state) following acute MI and following an angiogram for stable coronary artery disease (CAD). Forty-nine subjects met the criteria for acute MI, of whom 34 had clearly defined atherothrombotic (n = 22) or non-atherothrombotic (n = 12) MI; 15 patients met the criteria for stable CAD. Mean baseline levels of plasminogen and OxPL-PLG were lower in the acute MI group than in the stable CAD group (9.75 vs 20.2, p < 0.0001 for plasminogen and 165.5 vs 275.1, p = 0.0002 for OxPL-PLG) and did not change over time or between time points, including the 3-month follow-up. Mean baseline levels of plasminogen and OxPL-PLG were also lower in atherothrombotic (Type 1) than in non-atherothrombotic (Type 2) MI subjects (8.65 vs 12.1, p < 0.03 for plasminogen and 164.5 vs 245.7, p = 0.02 for OxPL-PLG), and this relationship did not change over time or between time points. Plasminogen and OxPL-PLG were lower in patients presenting with an acute MI than in those with stable CAD and also in those with atherothrombotic MI (Type 1) vs. those with non-atherothrombotic MI (Type 2). These findings persisted at a median follow-up of 3 months post-MI. The association of plasminogen and OxPL-PLG with acute MI, particularly atherothrombotic MI (Type 1), could reflect a reduced fibrinolytic capacity, associated with an increased risk of atherothrombotic events differentiating stable CAD from unstable CAD and atherothrombotic MI (Type 1) from non-atherothrombotic MI (Type 2). Additional study with a larger sample size is warranted.

The development of biosensors that produce time series data will facilitate improvements in biomedical diagnostics and in personalized medicine. The time series produced by these devices often contains characteristic features arising from biochemical interactions between the sample and the sensor. To use such characteristic features for determining sample class, similarity-based classifiers can be utilized. However, the construction of such classifiers is complicated by the variability in the time domains of such series that renders the traditional distance metrics such as Euclidean distance ineffective in distinguishing between biological variance and time domain variance. The dynamic time warping (DTW) algorithm is a sequence alignment algorithm that can be used to align two or more series to facilitate quantifying similarity. In this article, we evaluated the performance of DTW distance-based similarity classifiers for classifying time series that mimics electrical signals produced by nanotube biosensors. Simulation studies demonstrated the positive performance of such classifiers in discriminating between time series containing characteristic features that are obscured by noise in the intensity and time domains. We then applied a DTW distance-based k -nearest neighbors classifier to distinguish the presence/absence of mesenchymal biomarker in cancer cells in buffy coats in a blinded test. Using a train-test approach, we find that the classifier had high sensitivity (90.9%) and specificity (81.8%) in differentiating between EpCAM-positive MCF7 cells spiked in buffy coats and those in plain buffy coats., Competing Interests: The authors report no conflicts of interest in this work.

Objective: Human papillomavirus (HPV) typing of oral lesions microscopically consistent with multifocal epithelial hyperplasia (MEH) was performed to identify potential novel clinical presentations., Study Design: MEH (N = 22 lesions, 17 patients) and squamous papilloma control samples (N = 9 lesions, 9 patients) were compared by using polymerase chain reaction-based HPV genotyping. Student's t tests were used to compare continuous characteristics., Results: Of the study cases, 86.4% of MEH and only 11% of controls were positive for HPV (P = .0002). In MEH lesions, 45.5% contained HPV32, 36.4% HPV6, and 4.5% HPV40. MEH lesions were mostly multifocal (50%) and occurred in HIV-negative patients (81.3%). They predominated on the labial/buccal mucosa (63.3%), and there were significant differences between groups by anatomic site (P < .0001). HPV32, but not HPV6, was detected in known HIV-positive patients., Conclusions: A novel clinical presentation of MEH associated with HPV32 in HIV-negative, middle-aged to older adults is reported here. One case with HPV40 is the first to be reported. Future detection protocols should include HPV32, as it may be currently overlooked., (Copyright © 2015 Elsevier Inc. All rights reserved.)