Quantitative profiling of adaptation to cyclin E overproduction.

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Despite the toxicity of cyclin E overproduction in mammalian cells, the cyclin E gene is overexpressed in some cancers. To further understand how cells can tolerate high cyclin E, we characterized non-transformed epithelial cells subjected to chronic cyclin E overproduction. Cells overproducing cyclin E, but not cyclins D or A, briefly experienced truncated G1 phases followed by a transient period of DNA replication origin underlicensing, replication stress, and impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite high cyclin E-associated activity. Transcriptome analysis revealed adapted cells down-regulated a cohort of G1-regulated genes. Withdrawing cyclin E from adapted cells only partially reversed underlicensing indicating that adaptation is at least partly non-genetic. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing indirectly through premature S phase onset and provides mechanistic insight into the relationship between CDKs and licensing. It serves as an example of oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence.

To maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is minichromosome maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading before S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. Consequently, the first S phase cells are hypersensitive to replication stress. This underlicensing results from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher-risk first cell cycle that likely promotes genome instability.

Measuring psychological flexibility in medical students and residents: a psychometric analysis.

PURPOSE: Psychological flexibility involves mindful awareness of our thoughts and feelings without allowing them to prohibit acting consistently with our values and may have important implications for patient-centered clinical care. Although psychological flexibility appears quite relevant to the training and development of health care providers, prior research has not evaluated measures of psychological flexibility in medical learners. Therefore, we investigated the validity of our learners' responses to three measures related to psychological flexibility. METHODS: Fourth-year medical students and residents (n=275) completed three measures of overlapping aspects of psychological flexibility: (1) Acceptance and Action Questionnaire-II (AAQ-II); (2) Cognitive Fusion Questionnaire (CFQ); and (3) Mindful Attention and Awareness Questionnaire (MAAS). We evaluated five aspects of construct validity: content, response process, internal structure, relationship with other variables, and consequences. RESULTS: We found good internal consistency for responses on the AAQ (α=0.93), MAAS (α=0.92), and CFQ (α=0.95). Factor analyses demonstrated a reasonable fit to previously published factor structures. As expected, scores on all three measures were moderately correlated with one another and with a measure of life satisfaction (p<0.01). CONCLUSION: Our findings provide preliminary evidence supporting validity of the psychological flexibility construct in a medical education sample. As psychological flexibility is a central concept underlying self-awareness, this work may have important implications for clinical training and practice.

Strategic, value-based delivery in global health care: innovations at Harvard University and Brigham and Women's Hospital.

Investments in global health have more than doubled over the past decade, generating a cadre of new institutions. To date, most of the funded research in global health has focused on discovery, and, more recently, on the development of new tools, which has tightened the implementation bottleneck. This article introduces the concept of global health delivery and the need to catalog and analyze current implementation efforts to bridge gaps in delivery. Global health delivery is complex and context-dependent and requires an interdisciplinary effort, including the application of strategic principles. Furthermore, delivery is necessary to ensure that the investments in research, discovery, and development generate value for patients and populations. This article discusses the application of value-based delivery to global health. It provides some examples of approaches to aggregating implicit knowledge to inform practice. With global health delivery, the aim is to transform global health scale-up from a series of well-intentioned but often disconnected efforts to a value-based movement based upon 21st-century technology, standards, and efficiency.

Regulation of alternative splicing: more than just the ABCs.

Alternative pre-mRNA splicing, the differential inclusion or exclusion of portions of a nascent transcript into the final protein-coding mRNA, is widely recognized to be a ubiquitous mechanism for controlling protein expression. Thus, understanding the molecular basis of alternative splicing is essential for deciphering post-transcriptional control of the genome. Pre-mRNA splicing in general is catalyzed by a large dynamic macromolecular machine known as the spliceosome. Notably, the recognition of the intron substrate by spliceosomal components and the assembly of these components to form a catalytic spliceosome occur through a network of highly combinatorial molecular interactions. Many, if not all, of these interactions are subject to regulation, forming the basis of alternative splicing. This minireview focuses on recent advances in our understanding of the diversity of mechanisms by which the spliceosome can be regulated so as to achieve precise control of alternative splicing under a range of cellular conditions.

An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition.

Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosome's dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4-U6-U5 tri-snRNP-containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway.

Increasing the usability of cognitive processing therapy for survivors of child sexual abuse.

There is an ongoing need for empirically based treatments for child sexual abuse (CSA) that are time-efficient and cost-effective. This article describes a modification of cognitive processing therapy for child sexual abuse (CPT-SA) that increases the therapy's usability by reducing the number of individual therapy sessions required. The modifications are based on the developing literature on stage-based approaches to the treatment of CSA and incorporate dialectical behavior therapy skills training into the treatment protocol. Initial pilot data (N = 6) on modified CPT-SA suggests the therapy may be effective for the treatment of posttraumatic stress disorder (PTSD) and depression.

HnRNP L represses exon splicing via a regulated exonic splicing silencer.

Skipping of mammalian exons during pre-mRNA splicing is commonly mediated by the activity of exonic splicing silencers (ESSs). We have recently identified a regulated ESS within variable exon 4 of the CD45 gene, named ESS1, that is necessary and sufficient for partial exon repression in resting T cells and has additional silencing activity upon T-cell activation. In this study, we identify three heterogeneous nuclear ribonucleoproteins (hnRNPs) that bind specifically to ESS1. The binding of one of these proteins, hnRNP-L, is significantly decreased by mutations that disrupt both the basal and induced activities of ESS1. Recombinant hnRNP-L functions to repress exon inclusion in vitro in an ESS1-dependent manner. Moreover, depletion of hnRNP-L, either in vitro or in vivo, leads to increased exon inclusion. In contrast, the other ESS1-binding proteins, PTB and hnRNP E2, do not discriminate between wild-type and mutant ESS1 in binding studies, and do not specifically alter ESS1-dependent splicing in vitro. Together, these studies demonstrate that hnRNP-L is the primary protein through which CD45 exon 4 silencing is mediated by the regulatory sequence ESS1.

The moderating role of parental warmth on the effects of exposure to family violence.

Previous research has shown parental warmth to have mixed effects on individuals in violent families. While positively associated with psychological health in some victims, parental warmth has also been positively associated with measures of psychological distress in other victims. The current study examined two models (the "buffering" and "inconsistency" theories) to clarify the effects of parental warmth. The current study also sought to clarify the role of parental warmth within the context of exposure to different types of family violence (i.e., witnessing versus victimization). Results differed depending on the type of violence exposure. Both mother and father warmth were negatively associated with secure attachment and self-esteem in combined victims and witnesses of violence, whereas, mother warmth was positively associated with self-esteem in witnesses of violence. Father warmth did not significantly impact either outcome for witnesses. Parental warmth did not influence either outcome for those who had only experienced victimization.