Transcriptionally active chromatin loops contain both 'active' and 'inactive' histone modifications that exhibit exclusivity at the level of nucleosome clusters.

Chromatin state is thought to impart regulatory function to the underlying DNA sequence. This can be established through histone modifications and chromatin organisation, but exactly how these factors relate to one another to regulate gene expression is unclear. In this study, we have used super-resolution microscopy to image the Y loops of Drosophila melanogaster primary spermatocytes, which are enormous transcriptionally active chromatin fibres, each representing single transcription units that are individually resolvable in the nuclear interior. We previously found that the Y loops consist of regular clusters of nucleosomes, with an estimated median of 54 nucleosomes per cluster with wide variation.In this study, we report that the histone modifications H3K4me3, H3K27me3, and H3K36me3 are also clustered along the Y loops, with H3K4me3 more associated with diffuse chromatin compared to H3K27me3. These histone modifications form domains that can be stretches of Y loop chromatin micrometres long, or can be in short alternating domains. The different histone modifications are associated with different sizes of chromatin clusters and unique morphologies. Strikingly, a single chromatin cluster almost always only contains only one type of the histone modifications that were labelled, suggesting exclusivity, and therefore regulation at the level of individual chromatin clusters. The active mark H3K36me3 is more associated with actively elongating RNA polymerase II than H3K27me3, with polymerase often appearing on what are assumed to be looping regions on the periphery of chromatin clusters.These results provide a foundation for understanding the relationship between chromatin state, chromatin organisation, and transcription regulation - with potential implications for pause-release dynamics, splicing complex organisation and chromatin dynamics during polymerase progression along a gene.

Selective Occupation by E2F and RB of Loci Expressed by RNA Polymerase III.

In all cases tested, TFIIIB is responsible for recruiting pol III to its genetic templates. In mammalian cells, RB binds TFIIIB and prevents its interactions with both promoter DNA and pol III, thereby suppressing transcription. As TFIIIB is not recruited to its target genes when bound by RB, the mechanism predicts that pol III-dependent templates will not be occupied by RB; this contrasts with the situation at most genes controlled by RB, where it can be tethered by promoter-bound sequence-specific DNA-binding factors such as E2F. Contrary to this prediction, however, ChIP-seq data reveal the presence of RB in multiple cell types and the related protein p130 at many loci that rely on pol III for their expression, including RMRP, RN7SL, and a variety of tRNA genes. The sets of genes targeted varies according to cell type and growth state. In such cases, recruitment of RB and p130 can be explained by binding of E2F1, E2F4 and/or E2F5. Genes transcribed by pol III had not previously been identified as common targets of E2F family members. The data provide evidence that E2F may allow for the selective regulation of specific non-coding RNAs by RB, in addition to its influence on overall pol III output through its interaction with TFIIIB.

Variability in Practice and Implementation of Oxygen Target Saturation Policies in United States' Neonatal Intensive Care Units.

OBJECTIVE: This study aimed to describe target oxygen saturation (SpO2) ranges used for premature infants in United States' neonatal intensive care units (NICUs) and to describe if these target SpO2 ranges have changed in recent years. STUDY DESIGN: A 29-question survey focused on target SpO2 practices and policies was distributed via the NICU medical directors listservs for the American Academy of Pediatrics Section of Neonatal-Perinatal Medicine and Pediatrix Medical Group between August and October of 2021. Results were collected via Research Electronic Data Capture (REDCap). RESULTS: We received responses representing 170 unique, levels 2, 3, and 4 NICUs from 36 states. Most NICUs (130, 78%) have recently changed their SpO2 targets in response to target SpO2 clinical trials. Over time, the most commonly reported target SpO2 range has shifted from 88-92% to 90-95%. Of NICUs that changed limits, the most common lower SpO2 limits increased from 88 to 90% and the upper SpO2 limits changed from 92 to 95%. The interquartile range for lower SpO2 limit shifted from 85-88% to 88-90% and the IQR for upper SpO2 limit decreased from 92-95% to 94-95%. Most NICUs had designated conditions that would allow for deviations from standard target SpO2 ranges. These most commonly include pulmonary hypertension (152, 95%), severe bronchopulmonary dysplasia (81, 51%), and retinopathy of prematurity (51, 32%). CONCLUSION: Oxygen saturation limits have changed over time with an overall increase in targeted SpO2. However, there remains considerable interunit variation in SpO2 policies. There is a need to achieve consensus to optimize clinical outcomes. KEY POINTS: · What are the SpO2 ranges in United States' NICUs?. · There is a shift in SpO2 ranges for preterm infants in NICUs across United States.. · Variability still persists in SpO2 ranges for preterm infants in United States' NICUs..

Epstein-Barr virus protein EBNA-LP engages YY1 through leucine-rich motifs to promote naïve B cell transformation.

Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naïve but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knock Out (LPKO) virus-infected cells express EBNA2-activated genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naïve B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naïve B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naïve B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naïve and cord blood B cells. Because cellular PPAR-γ coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging cellular transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data and confirmed their biochemical association in LCLs by endogenous co-immunoprecipitation. Moreover, we found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. Finally, we used Cas9 to knockout YY1 in primary total B cells and naïve B cells prior to EBV infection and found YY1 to be essential for EBV-mediated transformation. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells.

Couplet Care-The Next Frontier of Care in the Newborn Intensive Care Unit.

Couplet care of mother and newborn intensive care unit (NICU) baby in the same room is a new, rapidly evolving option for the care of NICU babies. This change has structural and operational challenges that require careful planning but its successful implementation is likely to drive enhanced family participation in the care of their baby throughout the NICU stay as well as improve collaboration between obstetric and neonatal providers.

Improved outcomes for spinal versus general anesthesia for hip fracture surgery: a retrospective cohort study of the National Surgical Quality Improvement Program.

BACKGROUND: There is a lack of consensus in the literature as to whether anesthetic modality influences perioperative complications in hip fracture surgery. The aim of the present study was to assess the effect of spinal anesthesia compared with general anesthesia on postoperative morbidity and mortality in patients who underwent hip fracture surgery using data from the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP). METHODS: We used the ACS NSQIP to identify patients aged 50 and older who received either spinal or general anesthesia for hip fracture surgery from 2016 to 2019. Propensity-score matching was performed to control for clinically relevant covariates. The primary outcome of interest was the combined incidence of stroke, myocardial infarction (MI) or death within 30 days. Secondary outcomes included 30-day mortality, hospital length of stay and operative time. RESULTS: Among the 40 527 patients aged 50 and over who received either spinal or general anesthesia for hip fracture surgery from 2016 to 2019, 7358 spinal anesthesia cases were matched to general anesthesia cases. General anesthesia was associated with a higher incidence of combined 30-day stroke, MI or death compared with spinal anesthesia (OR 1.219 (95% CI 1.076 to 1.381); p=0.002). General anesthesia was also associated with a higher frequency of 30-day mortality (OR 1.276 (95% CI 1.099 to 1.481); p=0.001) and longer operative time (64.73 vs 60.28 min; p<0.001). Spinal anesthesia had a longer average hospital length of stay (6.29 vs 5.73 days; p=0.001). CONCLUSION: Our propensity-matched analysis suggests that spinal anesthesia as compared with general anesthesia is associated with lower postoperative morbidity and mortality in patients undergoing hip fracture surgery.

Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells.

Intramolecular interactions and the neutral loss of ammonia from collisionally activated, protonated ω-aminoalkyl-3-hydroxyfurazans.

Gas phase fragmentation reactions of monoprotonated 4-(3-aminopropyl)- and 4-(4-aminobutyl)-3-hydroxyfurazan were investigated to examine potential interactions between functional groups. The two heterocyclic alkyl amines were ionized by electrospray ionization (ESI, positive mode) and fragmented using tandem mass spectrometry (MS/MS). The fragmentation pathways were characterized using pseudo MS3 experiments, precursor-ion scans, and density functional computations. For both heterocyclic ions, loss of ammonia was the only fragmentation process observed at low collision energies. Computational analysis indicated that the most feasible mechanism was intramolecular nucleophilic displacement of ammonia from the protonated ω-aminoalkyl side chain by N5 of the furazan ring. The alkylated nitrogen in the resulting bicyclic product ion facilitated N-O bond cleavage; subsequent neutral losses of nitric oxide (NO) and carbon monoxide (CO) occurred by homolytic bond cleavages. Next in the multistep sequence, neutral loss of ethylene from a radical cation was observed. A less favorable, competing fragmentation pathway of protonated 4-(3-aminopropyl)-3-hydroxyfurazan was consistent with cleavage of the 3-hydroxyfurazan ring and losses of NO and CO. Overall, the similar fragmentation behavior found for protonated 4-(3-aminopropyl)- and 4-(4-aminobutyl)-3-hydroxyfurazan differed from that previously characterized for furazan analogs with shorter alkyl chains. These observations demonstrate that a small change in the structure of multifunctional, heterocyclic alkyl amines may significantly influence interactions between distinct functional groups and the nature of the fragmentation process.

Dopamine Modulates Effective Connectivity in Frontal Cortex.

There is increasing evidence that the left lateral frontal cortex is hierarchically organized such that higher-order regions have an asymmetric top-down influence over lower order regions. However, questions remain about the underlying neuroarchitecture of this hierarchical control organization. Within the frontal cortex, dopamine plays an important role in cognitive control functions, and we hypothesized that dopamine may preferentially influence top-down connections within the lateral frontal hierarchy. Using a randomized, double-blind, within-subject design, we analyzed resting-state fMRI data of 66 healthy young participants who were scanned once each after administration of bromocriptine (a dopamine agonist with preferential affinity for D2 receptor), tolcapone (an inhibitor of catechol-O-methyltransferase), and placebo, to determine whether dopaminergic stimulation modulated effective functional connectivity between hierarchically organized frontal regions in the left hemisphere. We found that dopaminergic drugs modulated connections from the caudal middle frontal gyrus and the inferior frontal sulcus to both rostral and caudal frontal areas. In dorsal frontal regions, effectivity connectivity strength was increased, whereas in ventral frontal regions, effective connectivity strength was decreased. These findings suggest that connections within frontal cortex are differentially modulated by dopamine, which may bias the influence that frontal regions exert over each other.

Reimagining the NICU: a human-centered design approach to healthcare innovation.

Design charettes have been utilized in architectural and design practice to generate innovative ideas. The Reimagining Workshop is a version that combines practical and blue-sky thinking to improve healthcare facility design. The workshop engages diverse stakeholders who follow a human-centered design framework. The Reimagining the Neonatal Intensive Care Unit workshop sought to generate ideas for the future, optimal NICU without specific site or client constraints. Key themes include family-centered care, technology-enabled care, neighborhood and village design and investing in the care team. Recommendations include a supportive physical environment, celebrating milestones, complementary and alternative medicine, enhancing the transition of care, aiding the transition period, and leveraging technology. The workshop showcased the potential for transformative change in NICU design and provided a roadmap for future advancements. These findings can inform regulatory standards for NICU design and drive improvements in family-centered care, patient experiences, and outcomes within the NICU environment.

Precise temperature control and rapid heating/cooling of infrared spectroscopy samples with a two-stage thermoelectric device.

The design and performance of an apparatus for heating and cooling samples during variable temperature infrared spectroscopy studies are described. The apparatus incorporates two thermoelectric device modules in a stacked configuration. The cascaded devices are powered in parallel and contained within a metal enclosure that maintains their alignment and applies clamping pressure between them to maximize thermal conductivity. By using this apparatus, sample temperatures can be increased or decreased at 2 °C s-1 rates and isothermal temperatures can be maintained precisely (±0.1 °C). The rapid heating and cooling capabilities of the apparatus facilitate programmed temperature step heating/cooling profiles with isothermal infrared spectrum measurements at pre-selected temperatures. Using linear heating and cooling temperature ramps, subtle temperature-dependent poly(styrene) infrared spectrum changes are elucidated and correlated with sample temperature. Results obtained by using a temperature step sample heating profile are compared with those obtained by using linear temperature ramp heating and cooling to characterize silica gel dehydration and re-hydration processes. By comparing infrared spectra acquired at different temperatures while heating and cooling the sample, silica gel spectrum changes associated with water desorption/adsorption and the thermal expansion/contraction of the Si-O-Si network are differentiated.

Racial and Ethnic Disparities in Delivery In-Hospital Mortality or Maternal End-Organ Injury: A Multistate Analysis, 2007-2020.

Background: In the United States, Black maternal mortality is 2-4 × higher than that of White maternal mortality, with differences also present in severe maternal morbidity and other measures. However, limited research has comprehensively studied multilevel social determinants of health, and their confounding and effect modification on obstetrical outcomes. Materials and Methods: We performed a retrospective multistate analysis of adult inpatient delivery hospitalizations (Florida, Kentucky, Maryland, New Jersey, New York, North Carolina, and Washington) between 2007 and 2020. Multilevel multivariable models were used to test the confounder-adjusted association for race/ethnicity and the binary outcomes (1) in-hospital mortality or maternal end-organ injury and (2) in-hospital mortality only. Stratified analyses were performed to test effect modification. Results: The confounder-adjusted odds ratio showed that Black (1.33, 95% confidence interval [CI]: 1.30-1.36) and Hispanic (1.14, 95% CI: 1.11-1.18) as compared with White patients were more likely to die in-hospital or experience maternal end-organ injury. For Black and Hispanic patients, stratified analysis showed that findings remained significant in almost all homogeneous strata. After statistical adjustment, Black as compared with White patients were more likely to die in-hospital (1.49, 95% CI: 1.21-1.82). Conclusions: Black and Hispanic patients had higher adjusted odds of in-patient mortality and end-organ damage after birth than White patients. Race and ethnicity serve as strong predictors of health care inequality, and differences in outcomes may reflect broader structural racism and individual implicit bias. Proposed solutions require immense and multifaceted active efforts to restructure how obstetrical care is provided on the societal, hospital, and patient level.