Unraveling paralog-specific Notch signaling through thermodynamics of ternary complex formation and transcriptional activation of chimeric receptors.

Notch signaling in humans is mediated by four paralogous receptors that share conserved architectures and possess overlapping, yet non-redundant functions. The receptors share a canonical activation pathway wherein upon extracellular ligand binding, the Notch intracellular domain (NICD) is cleaved from the membrane and translocates to the nucleus where its N-terminal RBP-j-associated molecule (RAM) region and ankyrin repeat (ANK) domain bind transcription factor CSL and recruit co-activator Mastermind-like-1 (MAML1) to activate transcription. However, different paralogs can lead to distinct outcomes. To better understand paralog-specific differences in Notch signaling, we performed a thermodynamic analysis of the Notch transcriptional activation complexes for all four Notch paralogs using isothermal titration calorimetry. Using chimeric constructs, we find that the RAM region is the primary determinant of stability of binary RAMANK:CSL complexes, and that the ANK regions are largely the determinants of MAML1 binding to pre-formed RAMANK:CSL complexes. Free energies of these binding reactions (ΔGRA and ΔGMAML) vary among the four Notch paralogs, although variations for Notch2, 3, and 4 offset in the free energy of the ternary complex (ΔGTC, where ΔGTC = ΔGRA + ΔGMAML). To probe how these affinity differences affect Notch signaling, we performed transcriptional activation assays with the paralogous and chimeric NICDs, and analyzed the results with an independent multiplicative model that quantifies contributions of the paralogous RAM, ANK, and C-terminal regions (CTR) to activation. This analysis shows that transcription activation correlates with ΔGTC, but that activation is further modified by CTR identity in a paralog-specific way.

Unit-Based Nurses' Development of a Couplet Care Acuity Scoring Tool.

OBJECTIVE: To evaluate content validity (CV) and interrater reliability (IRR) of an acuity scoring tool developed for the couplet care/postpartum/nursery patient population and to determine if there was agreement between supervisor or director scoring and staff scoring. DESIGN: A scoring tool to assess the acuity of the couplet care/postpartum/nursery patients was developed. SETTING: Two hospitals: one Level 2 hospital, one Level 3 hospital. Unit-based patient care councils participated in the development, and all couplet care nurses participated in scoring patients for testing. MEASUREMENTS: The final tool was evaluated for CV and IRR using expert review, universal agreement scores, and discriminant content validation. RESULTS: Regarding CV for the Couplet Care Acuity Scoring Tool, the average of the number of experts in agreement divided by the total number of experts across all items was 1.00. Regarding IRR, the intraclass correlation coefficient was 0.85, indicating that the tool is valid and reliable for the study sample. CONCLUSION: The tool was reliable and valid in this study. Future testing is needed with larger samples and different health care facilities.

The dynamic hypoosmotic response of Vibrio cholerae relies on the mechanosensitive channel MscS.

Vibrio cholerae adapts to osmotic down-shifts by releasing metabolites through two mechanosensitive (MS) channels, low-threshold MscS and high-threshold MscL. To investigate each channel's contribution to the osmotic response, we generated ΔmscS, ΔmscL, and double ΔmscL ΔmscS mutants in V. cholerae O395. We characterized their tension-dependent activation in patch-clamp, and the millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models describing osmolyte and water fluxes. We illustrate the sequence of events and define the parameters that characterize discrete phases of the osmotic response. Survival is correlated to the extent of cell swelling, the rate of osmolyte release, and the completeness of post-shock membrane resealing. Not only do the two channels interact functionally, but there is also an up-regulation of MscS in the ΔmscL strain, suggesting transcriptional crosstalk. The data reveal the role of MscS in the termination of the osmotic permeability response in V. cholerae.

A study protocol for risk stratification in children with concussion (RSiCC): Theoretical framework, design, and methods.

Research shows that one in five children will experience a concussion by age 16. Compared to adults, children experience longer and more severe postconcussive symptoms (PCS), with severity and duration varying considerably among children and complicating management of these patients. Persistent PCS can result in increased school absenteeism, social isolation, and psychological distress. Although early PCS diagnosis and access to evidence-based interventions are strongly linked to positive health and academic outcomes, symptom severity and duration are not fully explained by acute post-injury symptoms. Prior research has focused on the role of neuroinflammation in mediating PCS and associated fatigue; however relationship between inflammatory biomarkers and PCS severity, has not examined longitudinally. To identify which children are at high risk for persistent PCS and poor health, academic, and social outcomes, research tracking PCS trajectories and describing school-based impacts across the entire first year postinjury is critically needed. This study will 1) define novel PCS trajectory typologies in a racially/ethnically diverse population of 500 children with concussion (11-17 years, near equal distribution by sex), 2) identify associations between these typologies and patterns of inflammatory biomarkers and genetic variants, 3) develop a risk stratification model to identify children at risk for persistent PCS; and 4) gain unique insights and describe PCS impact, including fatigue, on longer-term academic and social outcomes. We will be the first to use NIH's symptom science model and patient-reported outcomes to explore the patterns of fatigue and other physical, cognitive, psychological, emotional and academic responses to concussion in children over a full year. Our model will enable clinicians and educators to identify children most at risk for poor long-term health, social, and academic outcomes after concussion. This work is critical to meeting our long-term goal of developing personalized concussion symptom-management strategies to improve outcomes and reduce disparities in the health and quality of life of children.