Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development.

Tissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naive B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and "corrupted" developmental processes including cancer.

Changes in Physical Inactivity Among US Adults Overall and by Sociodemographic Characteristics, Behavioral Risk Factor Surveillance System, 2020 Versus 2018.

The COVID-19 pandemic may have disrupted people's work-life patterns and access to places to be physically active. Behavioral Risk Factor Surveillance System data were analyzed to assess changes in self-reported leisure-time physical inactivity. The results showed that prevalence of inactivity among US adults decreased 0.7 percentage points (95% CI: -1.2 to -0.3), from 24.5% in 2018 to 23.8% in 2020, and the greatest decreases were observed among rural-dwelling women, rural-dwelling men, and non-Hispanic White women. These findings highlight a need to understand and address factors that lead to differential changes in leisure-time physical inactivity across subpopulations during public health emergencies.

Attitudes Toward COVID-19 Illness and COVID-19 Vaccination among Pregnant Women: A Cross-Sectional Multicenter Study during August-December 2020.

OBJECTIVE: The aim of the study was to evaluate pregnant women's attitudes toward COVID-19 illness and vaccination and identify factors associated with vaccine acceptability. STUDY DESIGN: This was a cross-sectional survey among pregnant women enrolled in a prospective COVID-19 cohort study in Salt Lake City, UT, Birmingham, AL, and New York, NY, from August 9 to December 10, 2020. Women were eligible if they were 18 to 50 years old and <28 weeks of gestation. Upon enrollment, women completed surveys regarding concerns about COVID-19 illness and likelihood of getting COVID-19 vaccine if one were available during pregnancy. Vaccine acceptability was defined as a response of "very likely" or "somewhat likely" on a 4-point Likert scale. Factors associated with vaccine acceptability were assessed with multivariable logistic regression. RESULTS: Of 939 pregnant women eligible for the main cohort study, 915 (97%) consented to participate. Among these 915 women, 39% self-identified as White, 23% Black, 33% Hispanic, and 4% Other. Sixty-two percent received an influenza vaccine last season. Seventy-two percent worried about getting sick with COVID-19. If they were to get sick, 92% worried about harm to their pregnancy and 80% about harm to themselves. Only 41% reported they would get a vaccine. Of women who were unlikely to get vaccinated, the most frequently cited concern was vaccine safety for their pregnancy (82%). Non-Hispanic Black and Hispanic women had lower odds of accepting a vaccine compared with non-Hispanic White women (adjusted odds ratios [aOR] 0.4, 95% CI 0.2-0.6 for both). Receipt of influenza vaccine during the previous season was associated with higher odds of vaccine acceptability (aOR 2.1, 95% CI 1.5-3.0). CONCLUSION: Although most pregnant women worried about COVID-19 illness, <50% were willing to get vaccinated during pregnancy. Racial and ethnic disparities in plans to accept COVID-19 vaccine highlight the need to prioritize strategies to address perceived barriers among groups at high risk for COVID-19. KEY POINTS: · Less than half of pregnant patients stated they would get a COVID-19 vaccine.. · Protecting their baby was the most common reason for acceptance and refusal of the COVID-19 vaccine.. · Patients of minority race/ethnicity and those without prior influenza vaccination were less likely to accept the COVID-19 vaccine..

Cross-sectional study of changes in physical activity behavior during the COVID-19 pandemic among US adults.

BACKGROUND: Physical activity (PA) provides numerous health benefits relevant to the COVID-19 pandemic. However, concerns exist that PA levels may have decreased during the pandemic thus exacerbating health disparities. This study aims to determine changes in and locations for PA and reasons for decreased PA during the pandemic. METHODS: Reported percentage of changes in and locations for PA and reasons for decreased PA were examined in 3829 US adults who completed the 2020 SummerStyles survey. RESULTS: Overall, 30% reported less PA, and 50% reported no change or no activity during the pandemic; percentages varied across subgroups. Adults who were non-Hispanic Black (Black) or Hispanic (vs. non-Hispanic White, (White)) reported less PA. Fewer Black adults (vs. White) reported doing most PA in their neighborhood. Concern about exposure to the virus (39%) was the most common reason adults were less active. CONCLUSIONS: In June 2020, nearly one-third of US adults reported decreased PA; 20% reported increased PA. Decreased activity was higher among Black and Hispanic compared to White adults; these two groups have experienced disproportionate COVID-19 impacts. Continued efforts are needed to ensure everyone has access to supports that allow them to participate in PA while still following guidance to prevent COVID-19 transmission.

U.S. Older Adults' Participation in Balance Activities.

The Physical Activity Guidelines for Americans, second edition recommends that older adults do multicomponent physical activity, which includes balance training in addition to aerobic and muscle-strengthening activities. The authors estimated the prevalence of U.S. older adults (age ≥65 years) who do balance activities and meet the aerobic and muscle-strengthening physical activity guidelines. The authors analyzed data on 1,012 respondents to the 2019 FallStyles survey, a nationwide web-based panel survey. Approximately four in 10 respondents (40.7%) reported doing balance activities on ≥1 day/week, 34.0% on ≥2 days/week, and 25.3% on ≥3 days/week. Prevalence differed by sex, education level, income level, census region, body mass index category, and meeting the aerobic and/or muscle-strengthening guidelines. The combined prevalence of participation in balance activities and meeting aerobic and muscle-strengthening guidelines ranged from 12.0% for ≥3 days/week to 15.8% for ≥1 day/week. Opportunities exist to introduce and increase participation in balance and multicomponent activities by older adults.

Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.

Cytometry is used extensively in clinical and laboratory settings to diagnose and track cell subsets in blood and tissue. High-throughput, single-cell approaches leveraging cytometry are developed and applied in the computational and systems biology communities by researchers, who seek to improve the diagnosis of human diseases, map the structures of cell signaling networks, and identify new cell types. Data analysis and management present a bottleneck in the flow of knowledge from bench to clinic. Multi-parameter flow and mass cytometry enable identification of signaling profiles of patient cell samples. Currently, this process is manual, requiring hours of work to summarize multi-dimensional data and translate these data for input into other analysis programs. In addition, the increase in the number and size of collaborative cytometry studies as well as the computational complexity of analytical tools require the ability to assemble sufficient and appropriately configured computing capacity on demand. There is a critical need for platforms that can be used by both clinical and basic researchers who routinely rely on cytometry. Recent advances provide a unique opportunity to facilitate collaboration and analysis and management of cytometry data. Specifically, advances in cloud computing and virtualization are enabling efficient use of large computing resources for analysis and backup. An example is Cytobank, a platform that allows researchers to annotate, analyze, and share results along with the underlying single-cell data.

Time Playing Outdoors Among Children Aged 3-5 Years: National Survey of Children's Health, 2021.

INTRODUCTION: Federal guidelines recommend physical activity throughout the day for preschool-aged children. Time playing outdoors can support physical activity participation, health, and development. Estimates of time playing outdoors among U.S. children aged 3-5 years have not been published. METHODS: Parent/caregiver-reported data on children aged 3-5 years from the 2021 National Survey of Children's Health were analyzed in 2022-23. Chi-square tests were used to identify differences in time playing outdoors by sociodemographic and neighborhood characteristics. Multiple logistic regression analyses were conducted with significant characteristics for weekdays and weekend days. RESULTS: Among 11,743 children aged 3-5 years, 37% played outdoors for ≤1 hour on weekdays, and 24% played outdoors for ≤1 hour on weekend days. In 9 states, ≥40% of children played outdoors for ≤1 hour on weekdays. Adjusted models for weekdays and weekend days showed a greater likelihood of ≤1 hour playing outdoors among those in all racial/ethnic groups compared to non-Hispanic White, those who lived in metropolitan statistical areas, those who did not participate in child care, and those whose adult proxy disagreed with "we watch out for each other's children in this neighborhood." The weekday model showed additional differences by sex, with girls more likely to have ≤1 hour of time playing outdoors. CONCLUSIONS: Nearly 40% of preschool-aged children play outdoors for ≤1 hour per day on weekdays, with differences by sociodemographic and neighborhood characteristics. Further study and interventions focused on building supportive, equitable communities might increase the amount of time preschool-aged children spend playing outdoors.

Physical Activity Behaviors and Negative Safety and Violence Experiences Among High School Students - Youth Risk Behavior Survey, United States, 2023.

Schools are in a unique position to offer opportunities for students to be physically active throughout the school day and promote health and well-being. However, experiences that threaten safety or perceptions of safety might affect students' physical activity behaviors. Using the 2023 national Youth Risk Behavior Survey, six physical activity behaviors and five negative safety and violence experiences were examined from a nationally representative sample of U.S. high school students. This report updates national estimates for physical activity behaviors overall and by sex, grade, race and ethnicity, and sexual identity. In addition, associations between negative experiences and physical activity behaviors were examined, stratified by sex, via unadjusted and adjusted prevalence ratios. Regardless of negative safety and violence experiences, male students had a higher prevalence of meeting aerobic, muscle-strengthening, and both aerobic and muscle-strengthening physical activity guidelines compared with female students. In adjusted models among female students, a positive association was observed between being threatened or injured with a weapon at school and meeting the aerobic guideline, meeting the muscle-strengthening guideline, and playing on ≥1 sports team. Among male students, positive associations were observed between witnessing neighborhood violence and meeting the aerobic guideline and the muscle-strengthening guideline. A negative association was observed between attending physical education classes on all 5 days and witnessing neighborhood violence among female students and being bullied electronically among male students. Physical activity might serve as a mechanism that students employ to cope with negative safety and violence experiences. Understanding current physical activity behaviors among students with these negative experiences will be useful for school leaders, teachers, and public health practitioners who influence physical activity infrastructure and programming in schools and work to support safe, supportive, and inclusive school environments for student health. Although future research is needed to further explore these associations, physical activity continues to be an important behavior to prioritize for adolescent health in the school setting.

Association between passively collected walking and bicycling data and purposefully collected active commuting survey data-United States, 2019.

Commercially-available location-based services (LBS) data derived primarily from mobile devices may provide an alternative to surveys for monitoring physically-active transportation. Using Spearman correlation, we compared county-level metrics of walking and bicycling from StreetLight with metrics of physically-active commuting among U.S. workers from the American Community Survey. Our strongest pair of metrics ranked counties (n = 298) similarly for walking (rho = 0.53 [95% CI: 0.44-0.61]) and bicycling (rho = 0.61 [0.53-0.67]). Correlations were higher for denser and more urban counties. LBS data may offer public health and transportation professionals timely information on walking and bicycling behavior at finer geographic scales than some existing surveys.

Awareness and Knowledge of the Physical Activity Guidelines for Americans, 2nd Edition.

BACKGROUND: Assessing awareness and knowledge of the Physical Activity Guidelines for Americans, 2nd edition (Guidelines), released in 2018, is important for monitoring factors that contribute to increasing physical activity. METHODS: We estimated prevalence of awareness and knowledge of the adult aerobic guideline (≥150 min/wk of moderate-intensity equivalent aerobic physical activity preferably spread out over a week) among adults (n = 3471) and of the youth aerobic guideline (≥60 min/d of mostly moderate- to vigorous-intensity aerobic physical activity) among a subset of parents (n = 744) from a nationwide sample of US adults in the 2019 FallStyles survey. We estimated odds ratios using logistic regression, adjusting for demographic and other characteristics. RESULTS: Approximately 1 in 10 US adults and parents reported being aware of the Guidelines. Only 3% of adults knew the correct adult aerobic guideline. The most common responses were "don't know/not sure" (44%) and "30 minutes a day, 5 or more days a week" (28%). Among parents, 15% knew the youth aerobic guideline. Awareness and knowledge tended to be lower with lower education and income. CONCLUSIONS: Limited awareness and knowledge of the Guidelines suggest communication about the Guidelines could be strengthened, especially among adults with low income or education.

Dietary and Physical Activity Behaviors in 2021 and Changes from 2019 to 2021 Among High School Students - Youth Risk Behavior Survey, United States, 2021.

The fall of 2021 was the first school semester to begin with widespread in-person learning since the COVID-19 pandemic began. Understanding dietary and physical activity behaviors of adolescents during this time can provide insight into potential health equity gaps and programmatic needs in schools and communities. This report uses data from the 2021 national Youth Risk Behavior Survey conducted among a nationally representative sample of U.S. public and private school students in grades 9-12 to update estimates of dietary and physical activity behaviors among U.S. high school students overall and by sex and race and ethnicity. In addition, 2-year comparisons (2019 versus 2021) of these behaviors were examined. In 2021, daily consumption of fruits, vegetables, and breakfast during the past 7 days remained low and decreased overall with specific disparities by sex and race and ethnicity from 2019 to 2021. The overall prevalence of students attending physical education classes daily, exercising to strengthen muscles on ≥3 days/week (i.e., met the guideline for muscle-strengthening activity), and playing on at least one sports team decreased from 2019 to 2021; whereas being physically active for ≥60 minutes/day on all 7 days (i.e., met the guideline for aerobic activity) and meeting both aerobic and muscle-strengthening guidelines remained low but did not change. These findings underscore the need for strategies to increase healthy dietary and physical activity behaviors both in the recovery phase of COVID-19 and longer term.

Traffic as a barrier to walking safely in the United States: Perceived reasons and potential mitigation strategies.

Motor vehicle traffic is commonly cited as a barrier to walking, but national level perceptions of traffic characteristics that negatively influence walking and potential traffic mitigation strategies remain unclear. The objectives of this study were to describe perceptions of (1) traffic characteristics that make walking unsafe in the United States and (2) potential mitigation strategies to address these concerns among those who report traffic as a barrier to walking. Data were from FallStyles, a nationwide internet panel survey conducted in October 2019 (n = 3,284 adults). Respondents reported if traffic makes walking unsafe where they live; those who answered yes were then asked about traffic characteristics that make walking unsafe (number of vehicles, speed of vehicles, distracted or impaired driving, types of vehicles, and other reasons) and potential mitigation strategies (new or improved sidewalks, crosswalks, pedestrian signals, street lighting, things that slow vehicles down, separating the sidewalk from the road, fewer vehicle lanes, and other). Prevalence of responses was assessed overall and by select sociodemographic and geographic characteristics, and by walking status. Nearly 25% of US adults reported that traffic is a barrier to walking where they live. Of these, 79% selected vehicle speed as a contributing traffic characteristic, and 57% indicated new or improved sidewalks as a potential mitigation strategy. These top responses were shared across all sociodemographic, geographic, and walking behavior subgroups. Speed reduction efforts and built environment enhancements such as sidewalks may alleviate pedestrian safety concerns. Promotion campaigns may be needed to bring awareness to such changes.

Sex-Stratified Trends in Meeting Physical Activity Guidelines, Participating in Sports, and Attending Physical Education Among US Adolescents, Youth Risk Behavior Survey 2009-2019.

BACKGROUND: During the past decade, guidelines for youth aerobic and muscle-strengthening physical activity remained unchanged. Active People, Healthy NationSM highlighted school and youth strategies (eg, sports and physical education [PE]) to increase physical activity. Sex, grade, and race/ethnicity disparities exist. This study examines sex-specific trends and differences by grade and race/ethnicity for the prevalence of 5 youth physical activity behaviors from 2009 to 2019. METHODS: The national Youth Risk Behavior Survey assesses adolescents (grades 9-12) meeting the aerobic, muscle-strengthening, and both guidelines (2011-2019) and sports participation and daily PE (2009-2019). Sex-stratified logistic regression assessed trends and 2009 or 2011-2019 differences by grade and racial/ethnic subgroups. RESULTS: Decreases in meeting the aerobic, muscle-strengthening, and both guidelines were observed for nearly all male subgroups by grade and race/ethnicity, whereas female subgroups exhibited declines or no change to low prevalence. Sports and PE participation remained mostly constant; select subgroups showed decreases (ie, Hispanic males [sports]; Black males and ninth-grade females [PE]). CONCLUSIONS: Past decade prevalence and patterns suggest that school-based and other strategies for all adolescents and tailored interventions for sex-specific subgroups may be needed to supplement sports and PE in promoting high school youth physical activity.

Trends in Aerobic and Muscle-Strengthening Physical Activity by Race/Ethnicity Across Income Levels Among US Adults, 1998-2018.

BACKGROUND: Although disparities in leisure-time physical activity (LTPA) participation by race/ethnicity and income are known, the combined association of these characteristics with LTPA participation is less understood. This study aims to describe trends and determine whether racial/ethnic differences in adult physical activity by income level have changed over the past 2 decades. METHODS: The authors estimated LTPA participation (outcomes: any aerobic activity, meeting the aerobic activity guideline, meeting the muscle-strengthening guideline, and meeting the combined aerobic and muscle-strengthening guidelines) among adults ≥18 years by race/ethnicity across income levels using 1998-2018 National Health Interview Survey data in 3-year aggregates. They also tested for trends, prevalence differences, and difference in differences using logistic regression. RESULTS: LTPA participation increased from 1998-2000 to 2016-2018 for all outcomes for non-Hispanic white, non-Hispanic black, and Hispanic adults at all income levels. Disparities narrowed for some groups but persisted between white and racial/ethnic minority groups across income levels for engaging in any aerobic activity and meeting the aerobic guideline (0.2-8.8 percentage point difference in differences). Disparities in meeting the muscle-strengthening and combined guidelines were less common. CONCLUSIONS: Opportunities exist to ensure that adults, particularly members of lower income racial/ethnic minority groups, have support to help them participate in LTPA.

Surveillance of Physical Activity and Sedentary Behavior Among Youth and Adults in the United States: History and Opportunities.

BACKGROUND: Surveillance is a core function of public health, and approaches to national surveillance of physical activity and sedentary behavior have evolved over the past 2 decades. The purpose of this paper is to provide an overview of surveillance of physical activity and sedentary behavior in the United States over the past 2 decades, along with related challenges and emerging opportunities. METHODS: The authors reviewed key national surveillance systems for the assessment of physical activity and sedentary behavior among youth and adults in the United States between 2000 and 2019. RESULTS: Over the past 20 years, 8 surveillance systems have assessed physical activity, and 5 of those have assessed sedentary behavior. Three of the 8 originated in nonpublic health agencies. Most systems have assessed physical activity and sedentary behavior via surveys. However, survey questions varied over time within and also across systems, resulting in a wide array of available data. CONCLUSION: The evolving nature of physical activity surveillance in the United States has resulted in both broad challenges (eg, balancing content with survey space; providing data at the national, state, and local level; adapting traditional physical activity measures and survey designs; and addressing variation across surveillance systems) and related opportunities.

A New Decade of Healthy People: Considerations for Comparing Youth Physical Activity Across 2 Surveillance Systems.

BACKGROUND: Healthy People 2030 includes objectives to increase meeting the aerobic physical activity guideline for ages 6-13 years (of ages 6-17 y, monitored by National Survey of Children's Health [NSCH]) and grades 9 to 12 (mostly aged 14-18+ y, monitored by Youth Risk Behavior Survey [YRBS]). This study compares methodologies, prevalence, and patterns of meeting the guideline, particularly for overlapping ages 14-17 years. METHODS: Nationally representative surveys, 2016-2017 NSCH (adult proxy report, 6-17 y) and 2015 and 2017 YRBS (self-report, grades 9-12), assess meeting the guideline of ≥60 minutes of daily moderate to vigorous physical activity. Prevalence and odds ratios were estimated by age group and demographics. RESULTS: For youth aged 14-17 years, 17.4% (95% confidence interval [CI], 16.1-18.7; NSCH) and 27.0% (95% CI, 25.6-28.5; YRBS) met the guideline. 25.9% (95% CI, 24.8-27.2) aged 6-13 years (NSCH) and 26.6% (95% CI, 25.3-28.0) in grades 9 to 12 (YRBS) met the guideline. Across surveys, fewer females (P < .001) and Asian youth (P < .001 except among NSCH 14-17 y) met the guideline. CONCLUSIONS: Neither methodology nor estimates for meeting the aerobic guideline are similar across surveys, so age continuity between juxtaposed estimates should not be assumed by magnitude nor age for separate Healthy People 2030 youth physical activity objectives.

Dietary and Physical Activity Behaviors Among High School Students - Youth Risk Behavior Survey, United States, 2019.

Establishing healthy dietary and physical activity patterns among youths is an important public health strategy for improving health and preventing chronic diseases; however, few adolescents meet U.S. government recommendations for dietary or physical activity behaviors, and disparities by sex and race/ethnicity exist. CDC analyzed data from the 2019 Youth Risk Behavior Survey to update estimates of dietary and physical activity behaviors among U.S. high school students overall and by sex and race/ethnicity. In addition, 2-year comparisons (2017 and 2019) and trends in prevalence of these behaviors during 2009-2019 were examined. In 2019, overall, during the 7 days before the survey, 41.8% of students had eaten fruit or drunk 100% fruit juices <1 time/day; 40.7% had eaten vegetables <1 time/day; and 16.7% had not eaten breakfast on all 7 days. Moreover, although 57.4% of students had played on ≥1 sports team during the 12 months before the survey, less than half of students had been physically active for ≥60 minutes/day on all 7 days (23.2%), had exercised to strengthen or tone their muscles on ≥3 days/week (49.5%), had met both aerobic and muscle-strengthening physical activity guidelines (16.5%), or had attended physical education classes on all 5 days in an average school week (25.9%). Trend data indicate limited progress in shifting dietary and physical activity behaviors. That is, with the exception of decreases in the percentage of students who had consumed soda ≥1 time/day (2009: 29.2%; 2019: 15.1%), sports drinks ≥1 time/day (2015: 13.8%; 2019: 10.6%), and <3 glasses/day of plain water (2015: 50.5%; 2019: 44.6%), high school students' dietary and physical activity behaviors have not improved and, in certain cases, have worsened. These findings support the need for multicomponent approaches, including policy and environmental changes, and opportunities for adolescents to learn about and practice making healthy choices.

Integrating metabolic, transcriptional regulatory and signal transduction models in Escherichia coli.

MOTIVATION: The effort to build a whole-cell model requires the development of new modeling approaches, and in particular, the integration of models for different types of processes, each of which may be best described using different representation. Flux-balance analysis (FBA) has been useful for large-scale analysis of metabolic networks, and methods have been developed to incorporate transcriptional regulation (regulatory FBA, or rFBA). Of current interest is the integration of these approaches with detailed models based on ordinary differential equations (ODEs). RESULTS: We developed an approach to modeling the dynamic behavior of metabolic, regulatory and signaling networks by combining FBA with regulatory Boolean logic, and ordinary differential equations. We use this approach (called integrated FBA, or iFBA) to create an integrated model of Escherichia coli which combines a flux-balance-based, central carbon metabolic and transcriptional regulatory model with an ODE-based, detailed model of carbohydrate uptake control. We compare the predicted Escherichia coli wild-type and single gene perturbation phenotypes for diauxic growth on glucose/lactose and glucose/glucose-6-phosphate with that of the individual models. We find that iFBA encapsulates the dynamics of three internal metabolites and three transporters inadequately predicted by rFBA. Furthermore, we find that iFBA predicts different and more accurate phenotypes than the ODE model for 85 of 334 single gene perturbation simulations, as well for the wild-type simulations. We conclude that iFBA is a significant improvement over the individual rFBA and ODE modeling paradigms. AVAILABILITY: All MATLAB files used in this study are available at http://www.simtk.org/home/ifba/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Commonly Occurring Cell Subsets in High-Grade Serous Ovarian Tumors Identified by Single-Cell Mass Cytometry.

We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a focus for therapeutic intervention and disease monitoring.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators.

Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on human samples at single-cell resolution, but instruments process only one sample at a time. Here we describe mass-tag cellular barcoding (MCB), which increases mass cytometry throughput by using n metal ion tags to multiplex up to 2n samples. We used seven tags to multiplex an entire 96-well plate, and applied MCB to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics and cell-to-cell communication, signaling variability between PBMCs from eight human donors, and the effects of 27 inhibitors on this system. For each inhibitor, we measured 14 phosphorylation sites in 14 PBMC types at 96 conditions, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional, systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors and revealed off-target effects. High-content, high-throughput screening with MCB should be useful for drug discovery, preclinical testing and mechanistic investigation of human disease.