GET: a foundation model of transcription across human cell types.

Transcriptional regulation, involving the complex interplay between regulatory sequences and proteins, directs all biological processes. Computational models of transcription lack generalizability to accurately extrapolate in unseen cell types and conditions. Here, we introduce GET, an interpretable foundation model designed to uncover regulatory grammars across 213 human fetal and adult cell types. Relying exclusively on chromatin accessibility data and sequence information, GET achieves experimental-level accuracy in predicting gene expression even in previously unseen cell types. GET showcases remarkable adaptability across new sequencing platforms and assays, enabling regulatory inference across a broad range of cell types and conditions, and uncovering universal and cell type specific transcription factor interaction networks. We evaluated its performance on prediction of regulatory activity, inference of regulatory elements and regulators, and identification of physical interactions between transcription factors. Specifically, we show GET outperforms current models in predicting lentivirus-based massive parallel reporter assay readout with reduced input data. In fetal erythroblasts, we identify distal (>1Mbp) regulatory regions that were missed by previous models. In B cells, we identified a lymphocyte-specific transcription factor-transcription factor interaction that explains the functional significance of a leukemia-risk predisposing germline mutation. In sum, we provide a generalizable and accurate model for transcription together with catalogs of gene regulation and transcription factor interactions, all with cell type specificity.

Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis.

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of S-phase proliferative tip cells relative to stalk cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip and stalk cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest and dynamics at the front. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.

Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

BACKGROUND: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. METHODS: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. RESULTS: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. CONCLUSIONS: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis.

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of proliferative tip cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.

Endothelial cell flow-mediated quiescence is temporally regulated and utilizes the cell cycle inhibitor p27.

Background: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. Methods: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time to cell cycle re-entry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA seq analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. Results: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous-flow exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. Conclusions: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence mis-regulation that leads to vascular dysfunction and disease.

Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development.

BMP signaling is crucial to blood vessel formation and function, but how pathway components regulate vascular development is not well-understood. Here, we find that inhibitory SMAD6 functions in endothelial cells to negatively regulate ALK1-mediated responses, and it is required to prevent vessel dysmorphogenesis and hemorrhage in the embryonic liver vasculature. Reduced Alk1 gene dosage rescued embryonic hepatic hemorrhage and microvascular capillarization induced by Smad6 deletion in endothelial cells in vivo. At the cellular level, co-depletion of Smad6 and Alk1 rescued the destabilized junctions and impaired barrier function of endothelial cells depleted for SMAD6 alone. Mechanistically, blockade of actomyosin contractility or increased PI3K signaling rescued endothelial junction defects induced by SMAD6 loss. Thus, SMAD6 normally modulates ALK1 function in endothelial cells to regulate PI3K signaling and contractility, and SMAD6 loss increases signaling through ALK1 that disrupts endothelial cell junctions. ALK1 loss-of-function also disrupts vascular development and function, indicating that balanced ALK1 signaling is crucial for proper vascular development and identifying ALK1 as a 'Goldilocks' pathway in vascular biology that requires a certain signaling amplitude, regulated by SMAD6, to function properly.

Endothelial Cell SMAD6 Balances ACVRL1/Alk1 Function to Regulate Adherens Junctions and Hepatic Vascular Development.

BMP signaling is critical to blood vessel formation and function, but how pathway components regulate vascular development is not well-understood. Here we find that inhibitory SMAD6 functions in endothelial cells to negatively regulate ALK1/ACVRL1-mediated responses, and it is required to prevent vessel dysmorphogenesis and hemorrhage in the embryonic liver vasculature. Reduced Alk1 gene dosage rescued embryonic hepatic hemorrhage and microvascular capillarization induced by Smad6 deletion in endothelial cells in vivo . At the cellular level, co-depletion of Smad6 and Alk1 rescued the destabilized junctions and impaired barrier function of endothelial cells depleted for SMAD6 alone. At the mechanistic level, blockade of actomyosin contractility or increased PI3K signaling rescued endothelial junction defects induced by SMAD6 loss. Thus, SMAD6 normally modulates ALK1 function in endothelial cells to regulate PI3K signaling and contractility, and SMAD6 loss increases signaling through ALK1 that disrupts endothelial junctions. ALK1 loss-of-function also disrupts vascular development and function, indicating that balanced ALK1 signaling is crucial for proper vascular development and identifying ALK1 as a "Goldilocks" pathway in vascular biology regulated by SMAD6.

Innovations in user-defined analysis: dynamic grouping and customized user datasets in VistaPHw.

Flexible, ready access to community health assessment data is a feature of innovative Web-based data query systems. An example is VistaPHw, which provides access to Washington state data and statistics used in community health assessment. Because of its flexible analysis options, VistaPHw customizes local, population-based results to be relevant to public health decision-making. The advantages of two innovations, dynamic grouping and the Custom Data Module, are described. Dynamic grouping permits the creation of user-defined aggregations of geographic areas, age groups, race categories, and years. Standard VistaPHw measures such as rates, confidence intervals, and other statistics may then be calculated for the new groups. Dynamic grouping has provided data for major, successful grant proposals, building partnerships with local governments and organizations, and informing program planning for community organizations. The Custom Data Module allows users to prepare virtually any dataset so it may be analyzed in VistaPHw. Uses for this module may include datasets too sensitive to be placed on a Web server or datasets that are not standardized across the state. Limitations and other system needs are also discussed.

HIV/AIDS knowledge and attitudes in Chinese medical professionals and students before and after an informational lecture on HIV/AIDS.

The rapid increase in human immunodeficiency virus (HIV) prevalence and incidence requires that Chinese health professionals mobilize for HIV prevention. The article describes an HIV/acquired immune deficiency syndrome knowledge and attitude assessment administered before and after a lecture. HIV transmission knowledge was good, with 90 percent to 100 percent of participants correctly identifying means of transmission. Prior to and after the lecture, 43 percent and 21 percent, respectively, were unwilling to sit or work with an HIV-infected person. To increase the likelihood of prevention activities' success, HIV knowledge and attitudes of health care personnel in China must be monitored and improved.

Case-reporting of acute hepatitis B and C among injection drug users.

Although public health surveillance system data are widely used to describe the epidemiology of communicable disease, occurrence of hepatitis B and C virus (HBV and HCV, respectively) infections may be misrepresented by under-reporting in injection drug users (IDUs). This study was carried out to examine the relationship between HBV and HCV incidence and case-reporting of hepatitis B and C in Seattle IDUs. Names of participants in a Seattle IDU cohort study who acquired HBV or HCV infection over a 12-month follow-up period were compared to a database of persons with acute hepatitis B and C reported to the health department surveillance unit over the same period. Of 2,208 IDUs enrolled in the cohort who completed a follow-up visit, 63/759 acquired HBV infection, 53/317 acquired HCV infection, and 3 subjects acquired both HBV and HCV. Of 113 cohort subjects who acquired HBV or HCV, only 2 (1.5%) cases were reported; both had acute hepatitis B. The upper 95% confidence limit for case-reporting of hepatitis C in the cohort was 5.7%, and for hepatitis B, it was 7.5%. In this study, reporting of acute hepatitis in IDUs was extremely low, raising questions regarding the use of community surveillance data to estimate underlying incidence in that population group.