A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens.

Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.

Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data.

Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.

Disparities in Access to Outpatient Surgery Related to Removal of Procedures from Medicare's Inpatient Only List.

OBJECTIVE: We assessed the shift from inpatient to outpatient surgical care related to changes to the Inpatient Only List in 2020 and 2021 compared to 2019. SUMMARY BACKGROUND DATA: The extent to which procedures shift from the inpatient to outpatient setting following removal from Medicare's Inpatient Only List is unknown. Many health systems also encouraged a shift from inpatient to outpatient surgery during the COVID-19 pandemic. Assessing the relative change in outpatient surgical utilization for procedures removed from the Inpatient Only List during COVID-19 would provide empirical data on whether reimbursement policy changes or inpatient capacity needs during the pandemic were more likely to shift care from the inpatient to outpatient setting. METHODS: We used administrative data from the PINC AI Healthcare Database across 723 hospitals to determine the within-facility relative change in outpatient vs inpatient procedural volume in 2020 and 2021 compared to 2019 using a multivariable conditional fixed-effects Poisson regression model. We also assessed whether outpatient surgical utilization varied by race and ethnicity. Using a multivariable linear probability model, we assessed the absolute change in risk-adjusted 30-day complication, readmission, and mortality rates for inpatient and outpatient surgical procedures. RESULTS: In 2020 and 2021 compared to 2019 respectively, there was a 5.3% (95% CI, 1.4% to 9.5%) and 41.3% (95% CI 33.1% to 50.0%) relative increase in outpatient elective procedural volume. Outpatient procedural volume increased most significantly for hip replacement which was removed from the Inpatient Only List in 2020 (increase in outpatient surgical utilization of 589.3% (95% CI, 524.9% to 660.3%)). The shift to outpatient hip replacement procedures was concentrated among White patients; in 2021, hip replacement procedural volume increased by 271.1% (95% CI, 241.2% and 303.7%) for White patients and 29.5% (95% CI, 24.4% and 34.9%) for Black patients compared to 2019 levels. There were no consistent or large changes in 30-day complication, readmission, or mortality risk in 2020 and 2021 compared to 2019. CONCLUSION: There was a modest increase in elective outpatient surgeries and a pronounced increase in outpatient orthopedic surgeries which were removed from the Inpatient Only List during the COVID-19 pandemic. Utilization of outpatient surgical procedures was concentrated among White patients.

The microtubule targeting agent ST-401 triggers cell death in interphase and prevents the formation of polyploid giant cancer cells.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.

Predicting cellular responses to complex perturbations in high-throughput screens.

Recent advances in multiplexed single-cell transcriptomics experiments facilitate the high-throughput study of drug and genetic perturbations. However, an exhaustive exploration of the combinatorial perturbation space is experimentally unfeasible. Therefore, computational methods are needed to predict, interpret, and prioritize perturbations. Here, we present the compositional perturbation autoencoder (CPA), which combines the interpretability of linear models with the flexibility of deep-learning approaches for single-cell response modeling. CPA learns to in silico predict transcriptional perturbation response at the single-cell level for unseen dosages, cell types, time points, and species. Using newly generated single-cell drug combination data, we validate that CPA can predict unseen drug combinations while outperforming baseline models. Additionally, the architecture's modularity enables incorporating the chemical representation of the drugs, allowing the prediction of cellular response to completely unseen drugs. Furthermore, CPA is also applicable to genetic combinatorial screens. We demonstrate this by imputing in silico 5,329 missing combinations (97.6% of all possibilities) in a single-cell Perturb-seq experiment with diverse genetic interactions. We envision CPA will facilitate efficient experimental design and hypothesis generation by enabling in silico response prediction at the single-cell level and thus accelerate therapeutic applications using single-cell technologies.

Antiretroviral Therapy Use and Disparities Among Medicare Beneficiaries with HIV.

BACKGROUND: Antiretroviral therapy (ART) is recommended for all people with HIV. Understanding ART use among Medicare beneficiaries with HIV is therefore critically important for improving quality and equity of care among the growing population of older adults with HIV. However, a comprehensive national evaluation of filled ART prescriptions among Medicare beneficiaries is lacking. OBJECTIVE: To examine trends in ART use among Medicare beneficiaries with HIV from 2013 to 2019 and to evaluate whether racial and ethnic disparities in ART use are narrowing over time. DESIGN: Retrospective observational study. SUBJECTS: Traditional Medicare beneficiaries with Part D living with HIV in 2013-2019. MAIN MEASURES: Months of filled ART prescriptions each year. KEY RESULTS: Compared with beneficiaries not on ART, beneficiaries on ART were younger, less likely to be Black (41.6% vs. 47.0%), and more likely to be Hispanic (13.1% vs. 9.7%). While the share of beneficiaries who filled ART prescriptions for 10 + months/year improved (+ 0.48 percentage points/year [p.p.y.], 95% CI 0.34-0.63, p < 0.001), 25.8% of beneficiaries did not fill ART for 10 + months in 2019. Between 2013 and 2019, the proportion of beneficiaries who filled ART for 10 + months improved for Black beneficiaries (65.8 to 70.3%, + 0.66 p.p.y., 95% CI 0.43-0.89, p < 0.001) and White beneficiaries (74.8 to 77.4%, + 0.38 p.p.y.; 95% CI 0.19-0.58, p < 0.001), while remaining stable for Hispanic beneficiaries (74.5 to 75.0%, + 0.12 p.p.y., 95% CI - 0.24-0.49, p = 0.51). Although Black-White disparities in ART use narrowed over time, the share of beneficiaries who filled ART prescriptions for 10 + months/year was significantly lower among Black beneficiaries relative to White beneficiaries each year. CONCLUSIONS: ART use improved from 2013 to 2019 among Medicare beneficiaries with HIV. However, about 25% of beneficiaries did not consistently fill ART prescriptions within a given year. Despite declining differences between Black and White beneficiaries, concerning disparities in ART use persist.

Effect of Medicare Advantage on Hospital Readmission and Mortality Rankings.

BACKGROUND: Medicare links hospital performance on readmissions and mortality to payment solely on the basis of outcomes among fee-for-service (FFS) beneficiaries. Whether including Medicare Advantage (MA) beneficiaries, who account for nearly half of all Medicare beneficiaries, in the evaluation of hospital performance affects rankings is unknown. OBJECTIVE: To determine if the inclusion of MA beneficiaries in readmission and mortality measures reclassifies hospital performance rankings compared with current measures. DESIGN: Cross-sectional. SETTING: Population-based. PARTICIPANTS: Hospitals participating in the Hospital Readmissions Reduction Program or Hospital Value-Based Purchasing Program. MEASUREMENTS: Using the 100% Medicare files for FFS and MA claims, the authors calculated 30-day risk-adjusted readmissions and mortality for acute myocardial infarction, heart failure, chronic obstructive pulmonary disease, and pneumonia on the basis of only FFS beneficiaries and then both FFS and MA beneficiaries. Hospitals were divided into quintiles of performance based on FFS beneficiaries only, and the proportion of hospitals that were reclassified to a different performance group with the inclusion of MA beneficiaries was calculated. RESULTS: Of the hospitals in the top-performing quintile for readmissions and mortality based on FFS beneficiaries, between 21.6% and 30.2% were reclassified to a lower-performing quintile with the inclusion of MA beneficiaries. Similar proportions of hospitals were reclassified from the bottom performance quintile to a higher one across all measures and conditions. Hospitals with a higher proportion of MA beneficiaries were more likely to improve in performance rankings. LIMITATION: Hospital performance measurement and risk adjustment differed slightly from those used by Medicare. CONCLUSION: Approximately 1 in 4 top-performing hospitals is reclassified to a lower performance group when MA beneficiaries are included in the evaluation of hospital readmissions and mortality. These findings suggest that Medicare's current value-based programs provide an incomplete picture of hospital performance. PRIMARY FUNDING SOURCE: Laura and John Arnold Foundation.

High-capacity sample multiplexing for single cell chromatin accessibility profiling.

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

Association Between Emergency Physician's Age and Mortality of Medicare Patients Aged 65 to 89 Years After Emergency Department Visit.

STUDY OBJECTIVE: To determine the association between emergency physicians' ages and patient mortality after emergency department visits. METHODS: This observational study used a 20% random sample of Medicare fee-for-service beneficiaries aged 65 to 89 years treated by emergency physicians at EDs from 2016 to 2017. We investigated whether 7-day mortality after ED visits differed by the age of the emergency physician, adjusting for patient and physician characteristics and hospital fixed effects. RESULTS: We observed 2,629,464 ED visits treated by 32,570 emergency physicians (mean age 43.5). We found that patients treated by younger emergency physicians had lower mortality rates compared with those treated by older physicians. Adjusted 7-day mortality was 1.33% for patients treated by emergency physicians aged less than 40 years, 1.36% (adjusted difference, 0.03%; 95% confidence interval [CI], -0.001% to 0.06%) for physicians ages 40 to 49, 1.40% (0.08%; 95% CI 0.04% to 0.12%) for physicians ages 50 to 59, and 1.43% (0.11%; 95% CI 0.06% to 0.16%) for those with a physician age of 60 years and more. Similar patterns were observed when stratified by the patient's disposition (discharged vs admitted), and the association was more pronounced for patients with higher severity of illness. CONCLUSIONS: Medicare patients aged 65 to 89 years treated by emergency physicians aged under 40 years had lower 7-day mortality rates than those treated by physicians aged 50 to 59 years and 60 years or older within the same hospital. Potential mechanisms explaining the association between emergency physician age and patient mortality (eg, differences in training received and other unobservable patient/physician characteristics) are uncertain and require further study.

Estimating the cost of HIV services for key populations provided by the LINKAGES program in Kenya and Malawi.

BACKGROUND: Data remain scarce on the costs of HIV services for key populations (KPs). The objective of this study was to bridge this gap in the literature by estimating the unit costs of HIV services delivered to KPs in the LINKAGES program in Kenya and Malawi. We estimated the mean total unit costs of seven clinical services: post-exposure prophylaxis (PEP), pre-exposure prophylaxis (PrEP), HIV testing services (HTS), antiretroviral therapy (ART), sexually transmitted infection (STI) services, sexual and reproductive health (SRH) services, and management of sexual violence (MSV). These costs take into account the costs of non-clinical services delivered alongside clinical services and the pre-service and above-service program management integral to the LINKAGES program. METHODS: Data were collected at all implementation levels of the LINKAGES program including 30 drop-in-centers (DICs) in Kenya and 15 in Malawi. This study was conducted from the provider's perspective. We estimated economic costs for FY 2019 and cost estimates include start-up costs. Start-up and capital costs were annualized using a discount rate of 3%. We used a combination of top-down and bottom-up costing approaches. Top-down methods were used to estimate the costs of headquarters, country offices, and implementing partners. Bottom-up micro-costing methods were used to measure the quantities and prices of inputs used to produce services in DICs. Volume-weighted mean unit costs were calculated for each clinical service. Costs are presented in 2019 United States dollars (US$). RESULTS: The mean total unit costs per service ranged from US$18 (95% CI: 16, 21) for STI services to US$635 (95% CI: 484, 785) for PrEP in Kenya and from US$41 (95% CI: 37, 44) for STI services to US$1,240 (95% CI 1156, 1324) for MSV in Malawi. Clinical costs accounted for between 21 and 59% of total mean unit costs in Kenya, and between 25 and 38% in Malawi. Indirect costs-including start-up activities, the costs of KP interventions implemented alongside clinical services, and program management and data monitoring-made up the remaining costs incurred. CONCLUSIONS: A better understanding of the cost of HIV services is highly relevant for budgeting and planning purposes and for optimizing HIV services. When considering all service delivery costs of a comprehensive HIV service package for KPs, costs of services can be significantly higher than when considering direct clinical service costs alone. These estimates can inform investment cases, strategic plans and other budgeting exercises.

Nanopore Sequencing with GraphMap for Comprehensive Pathogen Detection in Potato Field Soil.

Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, such as soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests of three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs further experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.

Intoxicación infantil por plomo en México: otras fuentes de exposición más allá del barro vidriado (Ensanut 2022).

OBJETIVO: Describir y actualizar al año 2022 la prevalencia de intoxicación por plomo (Pb) en la población infantil de 1-4 años en México y describir fuentes de exposición ambiental, paraocupacional y uso de barro vidriado (BV). Material y métodos. Estudio en una muestra de menores que participaron en la Encuesta Nacional de Salud y Nutrición 2022 (Ensanut 2022). Se midió la concentración de plomo en sangre (PbS) capilar. Se aplicó un cuestionario para indagar sobre fuentes de exposición a Pb. RESULTADOS: Se midió PbS en 1 158 menores de 1-4 años. La prevalencia nacional de intoxicación por Pb (≥5.0 µg/dL) fue 16.8% (IC95%:13.6,20.5). La proporción de hogares que reportó exposición a una fuente exclusiva fue de 21.2, 17.7 y 7.4% para BV, exposición ambiental y paraocupacional, respectivamente. La prevalencia de intoxicación de los expuestos a una sola fuente fue 21.5%, y esta cifra aumenta a 24.2% entre quienes reportaron exposición combinada tres tipos de fuentes. Conclusión. La prevalencia nacional de intoxicación por plomo permaneció estable con respecto a lo reportado en la Ensanut 2018-19. El uso de BV es una fuente de exposición relevante, pero no es la única. Se identificó una contribución importante por exposición ambiental y, en menor medida, de exposición paraocupacional.

Contribución de diversas fuentes de exposición a la concentración de plomo en sangre en población infantil mexicana, Ensanut 2022.

OBJETIVO: Analizar la asociación entre fuentes de exposición al plomo (FEPb) y la concentración en sangre capilar (PbS) en menores de 1 a 4 años de edad a nivel nacional y regional, así como cuantificar la contribución relativa de las distintas FEPb. Material y métodos. Se utilizaron datos de la Encuesta Nacional de Salud y Nutrición (Ensanut 2022). Las FEPb consideradas fueron uso de loza de barro vidriada con plomo (LBVPb), residencia cercana a sitios contaminados y exposición paraocupacional. Se estimaron prevalencias de intoxicación (PbS ≥ 5.0 mg/dL) y medias geométricas de PbS. Se utilizaron modelos de regresión para PbS (escala logarítmica) y la descomposición Shapley-Owen de R2 para evaluar la contribución relativa de cada FEPb. RESULTADOS: Las FEPb estudiadas explican el 6% de la variabilidad de PbS a nivel nacional; de este, el 87.3% lo explica el uso de LBVPb, el 4.2% otras FEPb ambiental y 1.3% FEPb paraocupacionales. La contribución relativa del uso de LBVPb varía entre regiones, desde 38.1 a 76.8%. Algunas regiones destacan la FEPb ambiental, pero no paraocupacional. CONCLUSIONES: Los resultados confirman que el uso de LBVPb es la principal fuente de exposición reportada y sugieren que la población no identifica las principales FEPb documentadas hasta ahora.

Racial and Ethnic Disparities in Heart and Cerebrovascular Disease Deaths During the COVID-19 Pandemic in the United States.

BACKGROUND: Cardiovascular deaths increased during the early phase of the COVID-19 pandemic in the United States. However, it is unclear whether diverse racial/ethnic populations have experienced a disproportionate rise in heart disease and cerebrovascular disease deaths. METHODS: We used the National Center for Health Statistics to identify heart disease and cerebrovascular disease deaths for non-Hispanic White, non-Hispanic Black, non-Hispanic Asian, and Hispanic individuals from March to August 2020 (pandemic period), as well as for the corresponding months in 2019 (historical control). We determined the age- and sex-standardized deaths per million by race/ethnicity for each year. We then fit a modified Poisson model with robust SEs to compare change in deaths by race/ethnicity for each condition in 2020 versus 2019. RESULTS: There were a total of 339 076 heart disease and 76 767 cerebrovascular disease deaths from March through August 2020, compared with 321 218 and 72 190 deaths during the same months in 2019. Heart disease deaths increased during the pandemic in 2020, compared with the corresponding period in 2019, for non-Hispanic White (age-sex standardized deaths per million, 1234.2 versus 1208.7; risk ratio for death [RR], 1.02 [95% CI, 1.02-1.03]), non-Hispanic Black (1783.7 versus 1503.8; RR, 1.19 [95% CI, 1.17-1.20]), non-Hispanic Asian (685.7 versus 577.4; RR, 1.19 [95% CI, 1.15-1.22]), and Hispanic (968.5 versus 820.4; RR, 1.18 [95% CI, 1.16-1.20]) populations. Cerebrovascular disease deaths also increased for non-Hispanic White (268.7 versus 258.2; RR, 1.04 [95% CI, 1.03-1.05]), non-Hispanic Black (430.7 versus 379.7; RR, 1.13 [95% CI, 1.10-1.17]), non-Hispanic Asian (236.5 versus 207.4; RR, 1.15 [95% CI, 1.09-1.21]), and Hispanic (264.4 versus 235.9; RR, 1.12 [95% CI, 1.08-1.16]) populations. For both heart disease and cerebrovascular disease deaths, Black, Asian, and Hispanic populations experienced a larger relative increase in deaths than the non-Hispanic White population (interaction term, P<0.001). CONCLUSIONS: During the COVID-19 pandemic in the United States, Black, Hispanic, and Asian populations experienced a disproportionate rise in deaths caused by heart disease and cerebrovascular disease, suggesting that these groups have been most impacted by the indirect effects of the pandemic. Public health and policy strategies are needed to mitigate the short- and long-term adverse effects of the pandemic on the cardiovascular health of diverse populations.

Spending outcomes among patients with cancer in accountable care organizations 4 years after implementation.

BACKGROUND: The long-term impact of affordable care organizations (ACOs) on cancer spending remains unknown. The authors examined whether practices that became ACOs altered their spending for patients with cancer in the first 4 years after ACO implementation. METHODS: By using national Medicare data from 2011 to 2017, a random sample of 20% of fee-for-service Medicare beneficiaries aged 65 years and older with cancer was obtained (n = 866,532), and each patient was assigned to a practice. Practices that became ACOs in the Medicare Shared Savings Program were matched to non-ACO practices. Total, cancer-specific, and service category-specific yearly spending per patient was calculated. A difference-in-differences model was used to determine spending changes associated with ACO status for patients with cancer in the 4 years after ACO implementation. RESULTS: The introduction of ACOs did not have a significant impact on overall spending for patients with cancer in the 2 years after ACO implementation (difference, -$38; 95% CI, -$268, $191; P = .74). Changes in spending also did not differ between ACO and non-ACO patients within service categories or among the 11 cancer types examined. The lack of difference in spending for patients with cancer in ACO and non-ACO practices persisted in the third and fourth years after ACO implementation (difference, -$120; 95% CI, -$284, $525; P = .56). CONCLUSIONS: ACOs did not significantly change spending for patients with cancer in the first 4 years after their implementation compared with non-ACOs. This prompts a reevaluation of the current efficacy of ACOs in reducing spending for cancer care and may encourage policymakers to reconsider the incentive structures of ACOs. LAY SUMMARY: Accountable care organizations (ACOs) were developed to curtail health care spending and improve quality, but their effects on cancer spending in their first 2 years have been minimal. The long-term impact of ACOs on cancer spending remains unknown. By using data from 866,532 Medicare beneficiaries with cancer, the authors observed that the association of a practice with an ACO did not significantly change total yearly spending per patient in the first 4 years after ACO implementation. This finding prompts a reevaluation of the current efficacy of ACOs in reducing spending for cancer care.

Neural G0: a quiescent-like state found in neuroepithelial-derived cells and glioma.

Single-cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA-seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent-like state in neuroepithelial-derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non-dividing neural progenitors. Putative glioblastoma stem-like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down-regulation of quiescence-associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent-like state found in neuroepithelial-derived cells and gliomas.

Dynamics of Gene Expression in Single Root Cells of Arabidopsis thaliana.

Single cell RNA sequencing can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach to Arabidopsis (Arabidopsis thaliana) root cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat-shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.

ACO Spending and Utilization Among Medicare Patients at the End of Life: an Observational Study.

BACKGROUND: End-of-life (EOL) costs constitute a substantial portion of healthcare spending in the USA and have been increasing. ACOs may offer an opportunity to improve quality and curtail EOL spending. OBJECTIVE: To examine whether practices that became ACOs altered spending and utilization at the EOL. DESIGN: Retrospective analysis of Medicare claims. PATIENTS: We assigned patients who died in 2012 and 2015 to an ACO or non-ACO practice. Practices that converted to ACOs in 2013 or 2014 were matched to non-ACOs in the same region. A total of 23,643 ACO patients were matched to 23,643 non-ACO patients. MAIN MEASURES: Using a difference-in-differences model, we examined changes in EOL spending and care utilization after ACO implementation. KEY RESULTS: The introduction of ACOs did not significantly impact overall spending for patients in the last 6 months of life (difference-in-difference (DID) = $192, 95%CI -$841 to $1125, P = 0.72). Changes in spending did not differ between ACO and non-ACO patients across spending categories (inpatient, outpatient, physician services, skilled nursing, home health, hospice). No differences were seen between ACO and non-ACO patients in rates of ED visits, inpatient admissions, ICU admission, mean healthy days at home, and mean hospice days at 180 and 30 days prior to death. However, non-ACO patients had a significantly greater increase in hospice utilization compared to ACO patients at 180 days (DID P-value = 0.02) and 30 days (DID P-value = 0.01) prior to death. CONCLUSIONS: With the exception of hospice care utilization, spending and utilization were not different between ACOs and non-ACO patients at the EOL. Longer follow-up may be necessary to evaluate the impact of ACOs on EOL spending and care.

Changes in Employment Status and Access to Care During COVID-19 Pandemic Among Low-Income Adults in 4 Southern States.

BACKGROUND: While the impact of the COVID-19 recession on the economy is clear, there is limited evidence on how the COVID-19 pandemic-related job losses among low-income people may have affected their access to health care. OBJECTIVE: To determine the association of job loss during the pandemic with insurance coverage and access to and affordability of health care among low-income adults. DESIGN: Using a random digit dialing telephone survey from October 2020 to December 2020 of low-income adults in 4 states-Arkansas, Kentucky, Louisiana, and Texas-we conducted a series of multivariable logistic regression analyses, adjusting for demographics, chronic conditions, and state of residence. PARTICIPANTS: US citizens aged 19-64 with a family income less than 138% of the federal poverty line who became newly unemployed during pandemic, remained employed during pandemic, or were chronically unemployed before and during the pandemic. MAIN MEASURES: Rates of insurance, type of insurance coverage, measures of access to/affordability of care, and food/housing security KEY RESULTS: Of 1,794 respondents, 14.5% were newly unemployed, 49.6% were chronically unemployed, and 35.7% were employed. The newly unemployed were slightly younger and more likely Black or Latino. The newly unemployed were more likely to report uninsurance compared to the employed (+16.4 percentage points, 95% CI 6.0-26.9), and the chronically unemployed (+26.4 percentage points, 95% CI 16.2-36.6), mostly driven by Texas' populations. The newly unemployed also reported lower rates of access to care and higher rates of financial barriers to care. They were also more likely to report food and housing insecurity compared to others. CONCLUSIONS: In a survey of 4 Southern States during pandemic, the newly unemployed had higher rates of uninsurance and worse access to care-largely due to financial barriers-and reported more housing and food insecurity than other groups. Our study highlights the vulnerability of low-income populations who experienced a job loss, especially in Texas, which did not expand Medicaid.