Bromodomain protein BRD4 directs mitotic cell division of mouse fibroblasts by inhibiting DNA damage.

Bromodomain protein BRD4 binds to acetylated histones to regulate transcription. BRD4 also drives cancer cell proliferation. However, the role of BRD4 in normal cell growth has remained unclear. Here, we investigated this question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells; they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. In summary, BRD4 epigenetically marks above genes and serves as a master regulator of normal cell growth.

An integrated toolkit for human microglia functional genomics.

BACKGROUND: Microglia, the brain's resident immune cells, play vital roles in brain development, and disorders like Alzheimer's disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However, existing iMG generation protocols face challenges, such as prolonged differentiation time, lack of detailed characterization, and limited gene function investigation via CRISPR-Cas9. METHODS: Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step, 20-day process, producing iMG with a normal karyotype. We confirmed the iMG's authenticity and quality through single-cell RNA sequencing, chromatin accessibility profiles (ATAC-Seq), proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further, we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ . RESULTS: Our method generates iMG that closely align with human primary microglia in terms of transcriptomic, proteomic, and chromatin accessibility profiles. Functionally, these iMG exhibit Ca2 + transients, cytokine driven migration, immune responses to inflammatory signals, and active phagocytosis of CNS related substrates including synaptosomes, amyloid beta and myelin. Significantly, the toolkit facilitates repeated iMG harvesting, essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia, positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions. CONCLUSIONS: Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform, our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism, the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement.

Most of the photons that reionized the Universe came from dwarf galaxies.

The identification of sources driving cosmic reionization, a major phase transition from neutral hydrogen to ionized plasma around 600-800 Myr after the Big Bang1-3, has been a matter of debate4. Some models suggest that high ionizing emissivity and escape fractions (fesc) from quasars support their role in driving cosmic reionization5,6. Others propose that the high fesc values from bright galaxies generate sufficient ionizing radiation to drive this process7. Finally, a few studies suggest that the number density of faint galaxies, when combined with a stellar-mass-dependent model of ionizing efficiency and fesc, can effectively dominate cosmic reionization8,9. However, so far, comprehensive spectroscopic studies of low-mass galaxies have not been done because of their extreme faintness. Here we report an analysis of eight ultra-faint galaxies (in a very small field) during the epoch of reionization with absolute magnitudes between MUV ≈ -17 mag and -15 mag (down to 0.005L⋆ (refs. 10,11)). We find that faint galaxies during the first thousand million years of the Universe produce ionizing photons with log[ξion (Hz erg-1)] = 25.80 ± 0.14, a factor of 4 higher than commonly assumed values12. If this field is representative of the large-scale distribution of faint galaxies, the rate of ionizing photons exceeds that needed for reionization, even for escape fractions of the order of 5%.

A high black-hole-to-host mass ratio in a lensed AGN in the early Universe.

Early JWST observations have uncovered a population of red sources that might represent a previously overlooked phase of supermassive black hole growth1-3. One of the most intriguing examples is an extremely red, point-like object that was found to be triply imaged by the strong lensing cluster Abell 2744 (ref. 4). Here we present deep JWST/NIRSpec observations of this object, Abell2744-QSO1. The spectroscopy confirms that the three images are of the same object, and that it is a highly reddened (AV ≃ 3) broad emission line active galactic nucleus at a redshift of zspec = 7.0451 ± 0.0005. From the width of Hß (full width at half-maximum = 2,800 ± 250 km s-1), we derive a black hole mass of M BH = 4 - 1 + 2 × 1 0 7 M ⊙ . We infer a very high ratio of black-hole-to-galaxy mass of at least 3%, an order of magnitude more than that seen in local galaxies5 and possibly as high as 100%. The lack of strong metal lines in the spectrum together with the high bolometric luminosity (Lbol = (1.1 ± 0.3) × 1045 erg s-1) indicate that we are seeing the black hole in a phase of rapid growth, accreting at 30% of the Eddington limit. The rapid growth and high black-hole-to-galaxy mass ratio of Abell2744-QSO1 suggest that it may represent the missing link between black hole seeds6 and one of the first luminous quasars7.

IRF8 configures enhancer landscape in postnatal microglia and directs microglia specific transcriptional programs.

Microglia are innate immune cells in the brain. Transcription factor IRF8 is highly expressed in microglia. However, its role in postnatal microglia development and the mechanism of action is unknown. We demonstrate here that IRF8 binds to enhancer regions of postnatal microglia in a stepwise fashion reaching a maximum after day 14, which coincided with the initiation of microglia function. Constitutive Irf8 deletion led to the loss of microglia identity gene expression and aberrant induction of Alzheimer's disease and neurodegeneration associated genes. Conditional Irf8 deletion in adult microglia showed similar transcriptome profiles, revealing the requirement of continuous IRF8 expression. Additional genome-wide analyses showed IRF8 is critical for setting microglia-specific chromatin accessibility and DNA methylation patterns. Lastly, in the 5xFAD mouse AD model, Irf8 deletion lessened the formation and spread of amyloidß plaques, thereby reducing neuronal loss. Together, IRF8 sets the microglia epigenome landscape, required for eliciting microglia identity and function.

BRD4 directs mitotic cell division by inhibiting DNA damage.

BRD4 binds to acetylated histones to regulate transcription and drive cancer cell proliferation. However, the role of BRD4 in normal cell growth remains to be elucidated. Here we investigated the question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. We suggest that BRD4 epigenetically marks those genes and serves as a master regulator of normal cell growth.

BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses.

BRD4 is a BET family protein that binds acetylated histones and regulates transcription. BET/BRD4 inhibitors block blood cancer growth and inflammation and serve as a new therapeutic strategy. However, the biological role of BRD4 in normal hematopoiesis and inflammation is not fully understood. Analysis of Brd4 conditional knockout (KO) mice showed that BRD4 is required for hematopoietic stem cell expansion and progenitor development. Nevertheless, BRD4 played limited roles in macrophage development and inflammatory response to LPS ChIP-seq analysis showed that despite its limited importance, BRD4 broadly occupied the macrophage genome and participated in super-enhancer (SE) formation. Although BRD4 is critical for SE formation in cancer, BRD4 was not required for macrophage SEs, as KO macrophages created alternate, BRD4-less SEs that compensated BRD4 loss. This and additional mechanisms led to the retention of inflammatory responses in macrophages. Our results illustrate a context-dependent role of BRD4 and plasticity of epigenetic regulation.

Leveraging Evidence-Based Public Policy and Advocacy to Advance Newborn Screening in California.

In 2016, the EveryLife Foundation for Rare Diseases, in partnership with Dr Pan, who is a pediatrician and state senator in California, launched legislation to advance and expand newborn screening. Researchers have shown that newborn screening can be cost-effective and can greatly improve health outcomes for patients with rare diseases. However, adding additional diseases in newborn screening is a long process, requiring legislative approval in addition to new state funding. Such process delays can lead to protracted diagnostic odysseys for patients, especially those with rare diseases. These delays can result in irreversible morbidity and, in some cases, early mortality for patients. To improve this process, legislation known as Senate Bill 1095 was introduced to require California to adhere to the latest federal recommendations for newborn screening within 2 years. We provide insight and describe the process of advancing state legislation, coalition building, and managing opposition. Senate Bill 1095 would become law in 2016, requiring California to screen for 2 new rare diseases by August 2018: mucopolysaccharidosis type I and Pompe disease. This case study can serve as a model for advocates looking to expand state newborn-screening programs.

Evolving Federal and State Health Care Policy: Toward a More Integrated and Comprehensive Care-Delivery System for Children With Medical Complexity.

Irrespective of any future changes in federal health policy, the momentum to shift from fee-for-service to value-based payment systems is likely to persist. Public and private payers continue to move toward alternative payment models that promote novel care-delivery systems and greater accountability for health outcomes. With a focus on population health, patient-centered medical homes, and care coordination, alternative payment models hold the potential to promote care-delivery systems that address the unique needs of children with medical complexity (CMC), including nonmedical needs and the social determinants of health. Notwithstanding, the implementation of care systems with meaningful quality measures for CMC poses unique and substantive challenges. Stakeholders must view policy options for CMC in the context of transformation within the overall health system to understand how broader health system changes impact care delivery for CMC.

Residency exposures and anticipated future involvement in community settings.

OBJECTIVE: To assess how exposures to community activities in residency impact anticipated future involvement in community child health settings. METHODS: Prospective cohort study of pediatric residents from 10 programs (12 sites) who completed training between 2003 and 2009. Residents reported annual participation for ≥ 8 days in each of 7 community activities (eg, community settings, child health advocacy) in the prior year. At the start and end of residency, residents reported anticipated involvement in 10 years in 8 community settings (eg, school, shelter). Anticipated involvement was dichotomized: moderate/substantial ("high") versus none/limited ("low"). Logistic regression modeled whether residency exposures independently influenced anticipated future involvement at the end of residency. RESULTS: A total of 683 residents completed surveys at the start and end of residency (66.8% participation). More than half of trainees reported ≥ 8 days' of involvement in community settings (65.6%) or child health advocacy (53.6%) in residency. Fewer anticipated high involvement in at least 1 community setting at the end of residency than at the start (65.5% vs 85.6%, P < .001). Participation in each community activity mediated but did not moderate relations between anticipated involvement at the start and end of residency. In multivariate models, exposure to community settings in residency was associated with anticipated involvement at end of residency (adjusted odds ratio 1.5; 95% confidence interval 1.2, 2.0). No other residency exposures were associated. CONCLUSIONS: Residents who anticipate high involvement in community pediatrics at the start of residency participate in related opportunities in training. Exposure to community settings during residency may encourage community involvement after training.

Attributes affecting the medical school primary care experience.

PURPOSE: Favorable primary care (PC) experiences might encourage more medical students to pursue generalist careers, yet academicians know little about which attributes influence the medical school PC experience. The authors sought to identify such attributes and weight their importance. METHOD: Semistructured interviews with 16 academic generalist leaders of family medicine, general internal medicine, and general pediatrics led to the development of a Web-based survey, administered to a national sample of 126 generalist faculty. Survey respondents rated (on a nine-point Likert-like scale) the importance of each interview-generated PC medical school attribute and indicated (yes/no) whether outside experts' assessment of the attributes would be valid. The authors assessed interrater agreement. RESULTS: Interview thematic analysis generated 58 institutional attributes in four categories: informal curriculum (23), institutional infrastructure (6), educational/curricular infrastructure (6), and specific educational experiences (23). Of these 58, 31 (53%) had median importance ratings of >7 (highly important). For 14 of these (45%), more than two-thirds of respondents indicated external expert surveys would provide a valid assessment. Of the 23 informal curriculum attributes, 20 (87%) received highly important ratings; however, more than two-thirds of respondents believed that external expert survey ratings would be valid for only 4 (20%) of them. Strong agreement occurred among respondents across the generalist fields. CONCLUSIONS: Academic generalist educators identified several attributes as highly important in shaping the quality of the medical school PC experience. Informal curriculum attributes appeared particularly influential, but these attributes may not be validly assessed via expert surveys, suggesting the need for other measures.

Community-based advocacy training: applying asset-based community development in resident education.

Communities and Physicians Together (CPT) at University of California, Davis Health System provides a novel approach to teaching residents to be effective community advocates. Founded in 1999, CPT is a partnership between a pediatric residency program, five community collaboratives located in diverse neighborhoods, and a grassroots child advocacy organization. Using the principles of Asset-Based Community Development, the program emphasizes establishing partnerships with community members and organizations to improve child health and identifies community assets and building capacity. Community members function as the primary faculty for CPT.The authors describe the CPT curriculum, which teaches residents to build partnerships with their assigned community. Residents have three, two-week blocks each year for CPT activities and maintain a longitudinal relationship with their community. In the first year, collaborative coordinators from each community orient residents to their community. Residents identify community assets and perform activities designed to provide them with a community member's perspective. In the second and third years, residents partner with community members and organizations to implement a project to improve the health of children in that community. CPT also provides faculty development to community partners including a workshop on medical culture and resident life. A qualitative evaluation demonstrated residents' attitudes of their role as pediatricians in the community changed with CPT.CPT is unique because it provides a model of service learning that emphasizes identifying and utilizing strengths and building capacity. This approach differs from the traditional medical model, which emphasizes deficits and needs.

Financing graduate medical education to meet the needs of children and the future pediatrician workforce.

This policy statement articulates the positions of the American Academy of Pediatrics on graduate medical education and the associated costs and funding mechanisms. It reaffirms the policy of the American Academy of Pediatrics that graduate medical education is a public good and is an essential part of maintaining a high-quality physician workforce. The American Academy of Pediatrics advocates for lifelong learning across the continuum of medical education. This policy statement focuses on the financing of one component of this continuum, namely residency education. The statement calls on federal and state governments to continue their support of residency education and advocates for stable means of funding such as the establishment of an all-payer graduate medical education trust fund. It further proposes a portable authorization system that would allocate graduate medical education funds for direct medical education costs to accredited residency programs on the basis of the selection of the program by qualified student or residents. This system allows the funding to follow the residents to their program. Recognizing the critical workforce needs of many pediatric medical subspecialties, pediatric surgical specialties, and other pediatric specialty disciplines, this statement maintains that subspecialty fellowship training and general pediatrics research fellowship training should receive adequate support from the graduate medical education financing system, including funding from the National Institutes of Health and other federal agencies, as appropriate. Furthermore, residency education that is provided in freestanding children's hospitals should receive a level of support equivalent to that of other teaching hospitals. The financing of graduate medical education is an important and effective tool to ensure that the future pediatrician workforce can provide optimal heath care for infants, children, adolescents, and young adults.

A noninvasive parametric evaluation of stress effects on global cardiovascular function.

Stress has been recognized as an important contributing factor to many forms of cardiovascular diseases. Its quantification has been sought for decades, but to no avail. We have developed a wholly noninvasive approach to quantitatively assess mental and physical stress effects on parameters that are associated with global cardiovascular function. Blood pressure, electrocardiogram, respiration and pulse volume are recorded simultaneously in experimental subjects during imposed arithmetic mental stress and Valsalva maneuver. Results show that parameters related to heart rate variability, respiratory rate, T-wave amplitude and pulse transit time are significantly modified during stress. Changes in these parameters involved differing mechanisms, although complex, can be delineated with logical analysis of electrophysiological, hemodynamic and neurogenic origins. This noninvasive technique is useful for both psychological evaluation and for clinical stress management.