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Abstract Circulating tumor cell clusters (CTCCs) are rare cellular events found in the blood stream of metastatic tumor patients. Despite their scarcity, they represent an increased risk for metastasis. Label-free detection methods of these events remain primarily limited to in vitro microfluidic platforms. Here, we expand on the use of confocal backscatter and fluorescence flow cytometry (BSFC) for label-free detection of CTCCs in whole blood using machine learning for peak detection/classification. BSFC uses a custom-built flow cytometer with three excitation wavelengths (405 nm, 488 nm, and 633 nm) and five detectors to detect CTCCs in whole blood based on corresponding scattering and fluorescence signals. In this study, detection of CTCC-associated GFP fluorescence is used as the ground truth to assess the accuracy of endogenous back-scattered light-based CTCC detection in whole blood. Using a machine learning model for peak detection/classification, we demonstrated that the combined use of backscattered signals at the three wavelengths enable detection of ~ 93% of all CTCCs larger than two cells with a purity of > 82% and an overall accuracy of > 95%. The high level of performance established through BSFC and machine learning demonstrates the potential for label-free detection and monitoring of CTCCs in whole blood. Further developments of label-free BSFC to enhance throughput could lead to important applications in the isolation of CTCCs in whole blood with minimal disruption and ultimately their detection in vivo. 

Abstract Background We investigate the relationships among political preferences, risk for COVID-19 complications, and complying with preventative behaviors, such as social distancing, quarantine, and vaccination, as they remain incompletely understood. Since those with underlying health conditions have the highest mortality risk, prevention strategies targeting them and their caretakers effectively can save lives. Understanding caretakers’ adherence is also crucial as their behavior affects the probability of transmission and quality of care, but is understudied. Examining the degree to which adherence to prevention measures within these populations is affected by their health status vs. voting preference, a key predictor of preventative behavior in the U. S, is imperative to improve targeted public health messaging. Knowledge of these associations could inform targeted COVID-19 campaigns to improve adherence for those at risk for severe consequences. Methods We conducted a nationally-representative online survey of U.S. adults between May–June 2020 assessing: 1) attempts to socially-distance; 2) willingness/ability to self-quarantine; and 3) intention of COVID-19 vaccination. We estimated the relationships between 1) political preferences 2) underlying health status, and 3) being a caretaker to someone with high-risk conditions and each dependent variable. Sensitivity analyses examined the associations between political preference and dependent variables among participants with high-risk conditions and/or obesity. Results Among 908 participants, 75.2% engaged in social-distancing, 94.4% were willing/able to self-quarantine, and 60.1% intended to get vaccinated. Compared to participants intending to vote for Biden, participants who intended to vote for Trump were significantly less likely to have tried to socially-distance, self-quarantine, or intend to be vaccinated. We observed the same trends in analyses restricted to participants with underlying health conditions and their caretakers Underlying health status was independently associated with social distancing among individuals with obesity and another high-risk condition, but not other outcomes. Conclusion Engagement in preventative behavior is associated with political voting preference and not individual risk of severe COVID-19 or being a caretaker of a high-risk individual. Community based strategies and public health messaging should be tailored to individuals based on political preferences especially for those with obesity and other high-risk conditions. Efforts must be accompanied by broader public policy. 

Abstract Hypertrophic cardiomyopathy (HCM) is considered a primary disorder of the sarcomere resulting in unexplained left ventricular hypertrophy but the paradoxical association of nonmyocyte phenotypes such as fibrosis, mitral valve anomalies and microvascular occlusion is unexplained. To understand the interplay between cardiomyocyte and nonmyocyte cell types in human HCM, single nuclei RNA-sequencing was performed on myectomy specimens from HCM patients with left ventricular outflow tract obstruction and control samples from donor hearts free of cardiovascular disease. Clustering analysis based on gene expression patterns identified a total of 34 distinct cell populations, which were classified into 10 different cell types based on marker gene expression. Differential gene expression analysis comparing HCM to Normal datasets revealed differences in sarcomere and extracellular matrix gene expression. Analysis of expressed ligand-receptor pairs across multiple cell types indicated profound alteration in HCM intercellular communication, particularly between cardiomyocytes and fibroblasts, fibroblasts and lymphocytes and involving integrin β1 and its multiple extracellular matrix (ECM) cognate ligands. These findings provide a paradigm for how sarcomere dysfunction is associated with reduced cardiomyocyte secretion of ECM ligands, altered fibroblast ligand-receptor interactions with other cell types and increased fibroblast to lymphocyte signaling, which can further alter the ECM composition and promote nonmyocyte phenotypes. 

Abstract Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context. 

ABSTRACT The rapid expansion of food and nutrition information requires new ways of data sharing and dissemination. Interactive platforms integrating data portals and visualization dashboards have been effectively utilized to describe, monitor, and track information related to food and nutrition; however, a comprehensive evaluation of emerging interactive systems is lacking. We conducted a systematic review on publicly available dashboards using a set of 48 evaluation metrics for data integrity, completeness, granularity, visualization quality, and interactivity based on 4 major principles: evidence, efficiency, emphasis, and ethics. We evaluated 13 dashboards, summarized their characteristics, strengths, and limitations, and provided guidelines for developing nutrition dashboards. We applied mixed effects models to summarize evaluation results adjusted for interrater variability. The proposed metrics and evaluation principles help to improve data standardization and harmonization, dashboard performance and usability, broaden information and knowledge sharing among researchers, practitioners, and decision makers in the field of food and nutrition, and accelerate data literacy and communication. 

Public health agencies routinely collect time-referenced records to describe and compare foodborne outbreak characteristics. Few studies provide comprehensive metadata to inform researchers of data limitations prior to conducting statistical modeling. We described the completeness of 103 variables for 22,792 outbreaks publicly reported by the United States Centers for Disease Control and Prevention’s (US CDC’s) electronic Foodborne Outbreak Reporting System (eFORS) and National Outbreak Reporting System (NORS). We compared monthly trends of completeness during eFORS (1998–2008) and NORS (2009–2019) reporting periods using segmented time series analyses adjusted for seasonality. We quantified the overall, annual, and monthly completeness as the percentage of outbreaks with blank records per our study period, calendar year, and study month, respectively. We found that outbreaks of unknown genus (n = 7401), Norovirus (n = 6414), Salmonella (n = 2872), Clostridium (n = 944), and multiple genera (n = 779) accounted for 80.77% of all outbreaks. However, crude completeness ranged from 46.06% to 60.19% across the 103 variables assessed. Variables with the lowest crude completeness (ranging 3.32–6.98%) included pathogen, specimen etiological testing, and secondary transmission traceback information. Variables with low (<35%) average monthly completeness during eFORS increased by 0.33–0.40%/month after transitioning to NORS, most likely due to the expansion of surveillance capacity and coverage within the new reporting system. Examining completeness metrics in outbreak surveillance systems provides essential information on the availability of data for public reuse. These metadata offer important insights for public health statisticians and modelers to precisely monitor and track the geographic spread, event duration, and illness intensity of foodborne outbreaks. 

Hypertrophic Cardiomyopathy (HCM) is a common inherited disorder characterized by unexplained left ventricular hypertrophy with or without left ventricular outflow tract (LVOT) obstruction. Single-nuclei RNA-sequencing (snRNA-seq) of both obstructive and nonobstructive HCM patient samples has revealed alterations in communication between various cell types, but no direct and integrated comparison between the two HCM phenotypes has been reported. We performed a bioinformatic analysis of HCM snRNA-seq datasets from obstructive and nonobstructive patient samples to identify differentially expressed genes and distinctive patterns of intercellular communication. Differential gene expression analysis revealed 37 differentially expressed genes, predominantly in cardiomyocytes but also in other cell types, relevant to aging, muscle contraction, cell motility, and the extracellular matrix. Intercellular communication was generally reduced in HCM, affecting the extracellular matrix, growth factor binding, integrin binding, PDGF binding, and SMAD binding, but with increases in adenylate cyclase binding, calcium channel inhibitor activity, and serine-threonine kinase activity in nonobstructive HCM. Increases in neuron to leukocyte and dendritic cell communication, in fibroblast to leukocyte and dendritic cell communication, and in endothelial cell communication to other cell types, largely through changes in the expression of integrin-β1 and its cognate ligands, were also noted. These findings indicate both common and distinct physiological mechanisms affecting the pathogenesis of obstructive and nonobstructive HCM and provide opportunities for the personalized management of different HCM phenotypes.