Comprehensive analysis of DNA replication timing across 184 cell lines suggests a role for MCM10 in replication timing regulation.

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing.

Developmental stress has sex-specific effects on contextual and cued fear conditioning in adulthood.

Stress-induced deviations in central nervous system development has long-term effects on adult mental health. Previous research in humans demonstrates that prenatal or adolescent stress increases the risk for psychiatric disorders. Animal models investigating the effects of stress during prenatal or adolescent development produces behavioral outcomes analogous to those observed in humans. However, whether adolescent stress exposure potentiates the effects of prenatal stress is currently unknown. Thus, the current study tested whether adolescent stress increases the impact of prenatal stress on contextual and cued fear memory in adulthood. Male and female Sprague Dawley rats were exposed to a chronic variable stress schedule during the last week of gestation, during adolescence, or during both developmental periods before undergoing fear conditioning training in adulthood. Our hypothesis predicted that the combined effects of prenatal and adolescent stress on contextual and cued fear memory would be greater than the effects of stress during either time period alone. In contrast to our hypothesis, however, we found independent effects of prenatal and adolescent stress on contextual and cued fear memory in both sexes, with no additional combined impact of stress exposure during both developmental phases. In males, developmental stress increased freezing behavior during contextual and cued testing regardless of whether stress exposure was prenatal, adolescent, or combined prenatal and adolescent stress exposure. In contrast, the effects of developmental stress in females were both test- and ovarian hormone status-dependent. During cued testing, nonstressed female freezing behavior depended on estrous cycle phase, whereas freezing behavior in stressed females did not, suggesting that developmental stress interferes with hormone-dependent cued fear memory. No effects of developmental stress or estrous cycle phase were observed for contextual fear memory in females. The results of the current study suggest that the effects of prenatal and adolescent stress on contextual and cued fear memory are not cumulative, but the effects of developmental stress on associative memory differ between males and females.

Implementing AACN's Healthy Work Environment Framework in an Intensive Care Unit.

BACKGROUND: Bedside nurse turnover in the United States is 15.9%, representing a national challenge that has been attributed to poor work environments. Healthy work environments are associated with improved nurse satisfaction and retention as well as positive patient outcomes; unhealthy work environments have the opposite effects. OBJECTIVES: To implement the American Association of Critical-Care Nurses (AACN) healthy work environment (HWE) framework in an intensive care unit and to evaluate staff satisfaction, turnover, and tenure 2 years later. METHODS: A pre-post study design was used to evaluate implementation of the HWE framework in an intensive care unit in a large academic medical facility. Interventions for each of the 6 HWE standards were performed. The AACN HWE assessment survey was used to measure skilled communication, true collaboration, effective decision-making, appropriate staffing, meaningful recognition, and authentic leadership in 2017 and in 2019. RESULTS: Nurse cohorts (n = 165 in 2017; n = 176 in 2019) had a mean age of 31 (median, 27; range, 23-63) years, were predominantly female (76%), and had a mean of 5 (median, 3) years of intensive care unit nursing experience. Statistically significant improvements were found in all standards except the skilled communication and overall measures. Registered nurse turnover remained stable and tenure increased by 79 days in this 2-year period. CONCLUSIONS: Findings from this study suggest that interventions addressing the HWE standards are associated with improved staff satisfaction, turnover, and average tenure, further demonstrating the value of the HWE framework in improving retention.

A Modern Dilemma: How Experts Grapple with Ambiguous Genetic Test Results.

Objective. Clinicians regularly use panel genetic testing to identify hereditary breast cancer risk, but this practice increases the rate of receiving an ambiguous test result, the variant of uncertain significance (VUS). VUS results are a growing and long-term challenge for providers and have caused negative patient outcomes. The objective of this study was to elicit expert opinions about patients' decision making after receiving a VUS result to provide future guidance for VUS disclosure. Methods. Using an adapted mental models approach, experts (N = 25) completed an online survey and in-depth interview eliciting qualitative judgments of the factors relevant to informed patient decision making after receiving a VUS result. Content analysis of interview transcripts clarified the basis for these judgments. Results. Participants identified 11 decisions facing patients after receiving VUS results grouped into ambiguity management or risk management. The experts also identified 24 factors relevant to each decision, which reflected 2 themes: objective factors (e.g., clinical information, guidelines) and psychosocial factors (e.g., understanding or risk perception). Conclusion. This study presents an adaptation of the mental models approach for communication under conditions of ambiguity. Findings suggest providers who present VUS results from genetic testing for hereditary breast cancer should discuss decisions related to ambiguity management that focus on hope for future reclassification, and be directive when discussing risk management decisions. Objective and psychosocial factors should influence both ambiguity and risk management decisions, but especially risk management decisions.

SIRT1 accelerates the progression of activity-based anorexia.

Food consumption is fundamental for life, and eating disorders often result in devastating or life-threatening conditions. Anorexia nervosa (AN) is characterized by a persistent restriction of energy intake, leading to lowered body weight, constant fear of gaining weight, and psychological disturbances of body perception. Herein, we demonstrate that SIRT1 inhibition, both genetically and pharmacologically, delays the onset and progression of AN behaviors in activity-based anorexia (ABA) models, while SIRT1 activation accelerates ABA phenotypes. Mechanistically, we suggest that SIRT1 promotes progression of ABA, in part through its interaction with NRF1, leading to suppression of a NMDA receptor subunit Grin2A. Our results suggest that AN may arise from pathological positive feedback loops: voluntary food restriction activates SIRT1, promoting anxiety, hyperactivity, and addiction to starvation, exacerbating the dieting and exercising, thus further activating SIRT1. We propose SIRT1 inhibition can break this cycle and provide a potential therapy for individuals suffering from AN.

Next-Generation Sequencing Enables Spatiotemporal Resolution of Human Centromere Replication Timing.

Centromeres serve a critical function in preserving genome integrity across sequential cell divisions, by mediating symmetric chromosome segregation. The repetitive, heterochromatic nature of centromeres is thought to be inhibitory to DNA replication, but has also led to their underrepresentation in human reference genome assemblies. Consequently, centromeres have been excluded from genomic replication timing analyses, leaving their time of replication unresolved. However, the most recent human reference genome, hg38, included models of centromere sequences. To establish the experimental requirements for achieving replication timing profiles for centromeres, we sequenced G1- and S-phase cells from five human cell lines, and aligned the sequence reads to hg38. We were able to infer DNA replication timing profiles for the centromeres in each of the five cell lines, which showed that centromere replication occurs in mid-to-late S phase. Furthermore, we found that replication timing was more variable between cell lines in the centromere regions than expected, given the distribution of variation in replication timing genome-wide. These results suggest the potential of these, and future, sequence models to enable high-resolution studies of replication in centromeres and other heterochromatic regions.

Dietary choline supplementation to dams during pregnancy and lactation mitigates the effects of in utero stress exposure on adult anxiety-related behaviors.

Brain cholinergic dysfunction is associated with neuropsychiatric illnesses such as depression, anxiety, and schizophrenia. Maternal stress exposure is associated with these same illnesses in adult offspring, yet the relationship between prenatal stress and brain cholinergic function is largely unexplored. Thus, using a rodent model, the current study implemented an intervention aimed at buffering the potential effects of prenatal stress on the developing brain cholinergic system. Specifically, control and stressed dams were fed choline-supplemented or control chow during pregnancy and lactation, and the anxiety-related behaviors of adult offspring were assessed in the open field, elevated zero maze and social interaction tests. In the open field test, choline supplementation significantly increased center investigation in both stressed and nonstressed female offspring, suggesting that choline-supplementation decreases female anxiety-related behavior irrespective of prenatal stress exposure. In the elevated zero maze, prenatal stress increased anxiety-related behaviors of female offspring fed a control diet (normal choline levels). However, prenatal stress failed to increase anxiety-related behaviors in female offspring receiving supplemental choline during gestation and lactation, suggesting that dietary choline supplementation ameliorated the effects of prenatal stress on anxiety-related behaviors. For male rats, neither prenatal stress nor diet impacted anxiety-related behaviors in the open field or elevated zero maze. In contrast, perinatal choline supplementation mitigated prenatal stress-induced social behavioral deficits in males, whereas neither prenatal stress nor choline supplementation influenced female social behaviors. Taken together, these data suggest that perinatal choline supplementation ameliorates the sex-specific effects of prenatal stress.