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

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.

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.

Quality of Life Differences in Children and Adolescents With 0, 1 to 2, or 3+ Persistent Postconcussion Symptoms.

CONTEXT: Persistent postconcussion symptoms (PPCSs) are associated with lower health-related quality of life (HRQoL) in children and adolescents. Despite commonly cited criteria for PPCSs involving 3 or more complaints, many individuals experience just 1 or 2 symptoms that may still negatively affect HRQoL. OBJECTIVE: To determine differences in HRQoL between children and adolescents with 0, 1 to 2, or 3+ parent-reported persistent symptoms at 1 month postconcussion. DESIGN: Prospective cohort study. SETTING: Community practice clinics. PATIENTS OR OTHER PARTICIPANTS: Individuals aged 8 to 18 years presented for the initial visit within 3 days of a sport- or recreation-related concussion. One month later, parents or guardians reported persistent symptoms using the Rivermead Post Concussion Symptoms Questionnaire (RPQ). Individuals with complete symptom data were analyzed (n = 236/245, n = 97 females, age = 14.3 ± 2.1 years). Participants were grouped by the number of discrete RPQ symptoms reported as worse than preinjury (0, 1-2, or 3+). MAIN OUTCOME MEASURE(S): Total summary and subscale scores on the Pediatric Quality of Life Inventory (PedsQL) 23-item HRQoL inventory and 18-item Multidimensional Fatigue Scale (MDFS). RESULTS: Kruskal-Wallis rank sum tests highlighted differences in PedsQL HRQoL and MDFS total scores across symptom groups (PedsQL HRQoL: χ22 = 85.53, P < .001; MDFS: χ22 = 93.15, P < .001). Dunn post hoc analyses indicated all 3 groups were statistically significantly different from each other (P < .001). The median (interquartile range) values for the Peds QL Inventory HRQoL totals were 93.5 (84.2-98.8) for those with 0 symptoms; 84.8 (73.9-92.4) for those with 1 to 2 symptoms; and 70.7 (58.7-78.0) for those with 3+ symptoms. The median (interquartile range) values for the MDFS totals were 92.4 (76.4-98.6) for those with 0 symptoms; 78.5 (65.6-88.9) for those with 1 to 2 symptoms; and 54.2 (46.2-65.3) for those with 3+ symptoms. Similar group differences were observed for each PedsQL HRQoL and MDFS subscale score. CONCLUSIONS: Children and adolescents whose parents reported 1 to 2 PPCSs had lower HRQoL and more fatigue than those with 0 symptoms. Across all 3 groups, those with 3+ persistent symptoms had the lowest HRQoL and most fatigue. These findings indicate the continued need for intervention in this age group to prevent and address PPCSs.

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

Like other intestinal bacteria, the facultative pathogen Vibrio cholerae adapts to a wide range of osmotic environments. Under drastic osmotic down-shifts, Vibrio avoids mechanical rupture by rapidly releasing excessive metabolites through mechanosensitive (MS) channels that belong to two major types, low-threshold MscS and high-threshold MscL. To investigate each channel individual contribution to V. cholerae osmotic permeability response, we generated individual ΔmscS, ∆mscL, and double ΔmscL ΔmscS mutants in V. cholerae O395 and characterized their tension-dependent activation in patch-clamp experiments, as well as their millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models reflecting the kinetic competition of osmolyte release with water influx. Both mutants lacking MscS exhibited delayed osmolyte release kinetics and decreased osmotic survival rates compared to WT. The ΔmscL mutant showed comparable release kinetics to WT, but a higher osmotic survival, while ΔmscS had low survival, comparable to the double ΔmscL ΔmscS mutant. By analyzing release kinetics following rapid medium dilution, we illustrate the sequence of events and define the set of parameters that characterize discrete phases of the osmotic response. Osmotic survival rates are directly correlated to the extent and duration of cell swelling, the rate of osmolyte release and the onset time, and the completeness of the post-shock membrane resealing. Not only do the two channels interact functionally during the resealing phase, but there is also a compensatory up-regulation of MscS in the ΔmscL strain suggesting some transcriptional crosstalk. The data reveal the advantage of the low-threshold MscS channel in curbing tension surges, without which MscL becomes toxic, and the role of MscS in the proper termination of the osmotic permeability response in Vibrio.

Structural features of the Notch ankyrin domain-Deltex WWE2 domain heterodimer determined by NMR spectroscopy and functional implications.

The Notch signaling pathway, an important cell fate determination pathway, is modulated by the ubiquitin ligase Deltex. Here, we investigate the structural basis for Deltex-Notch interaction. We used nuclear magnetic resonance (NMR) spectroscopy to assign the backbone of the Drosophila Deltex WWE2 domain and mapped the binding site of the Notch ankyrin (ANK) domain to the N-terminal WWEA motif. Using cultured Drosophila S2R+ cells, we find that point substitutions within the ANK-binding surface of Deltex disrupt Deltex-mediated enhancement of Notch transcriptional activation and disrupt ANK binding in cells and in vitro. Likewise, ANK substitutions that disrupt Notch-Deltex heterodimer formation in vitro block disrupt Deltex-mediated stimulation of Notch transcription activation and diminish interaction with full-length Deltex in cells. Surprisingly, the Deltex-Notch intracellular domain (NICD) interaction is not disrupted by deletion of the Deltex WWE2 domain, suggesting a secondary Notch-Deltex interaction. These results show the importance of the WWEA:ANK interaction in enhancing Notch signaling.

Individual Symptom Report Prevalence in Children and Adolescents With One, Two, and Three or More Persistent Symptoms After Concussion: A Brief Report.

OBJECTIVE: To examine differences in individual symptom report prevalence between children and adolescents experiencing 1 to 2 versus ≥3 persistent postconcussion symptoms. DESIGN: A prospective cohort study. SETTING: Three community practice concussion clinics within a family practice network. PATIENTS: Children and adolescents aged 8 to 18 years presenting to clinics within 72 hours of a sport-related or recreation-related concussion. Those with complete symptom data from a 1-month follow-up time point (n = 236) were included in analyses. INDEPENDENT VARIABLES: One hundred thirty-six patients (n = 136) reported 0 symptoms as worse than preinjury at the 1-month time point. Participants reporting 1+ symptoms as worse than preinjury at 1 month were assigned to groups based on the number of symptoms endorsed: those with 1 to 2 (n = 38) compared with those experiencing ≥3 (n = 62). MAIN OUTCOME MEASURES: Fisher exact tests were used to compare symptom report prevalence for each item of the Rivermead Postconcussion Symptoms Questionnaire. This research question was formulated and examined after completion of data collection. RESULTS: Across both groups, headache and fatigue were the most commonly reported persistent postconcussion symptoms. Several emotional symptoms (eg, irritability, depression) were primarily or only present in those reporting ≥3 persistent symptoms. CONCLUSIONS: Findings provide detail regarding the clinical manifestation of experiencing fewer versus more persistent postconcussion symptoms, underscoring the importance of developing individualized, multifaceted rehabilitation programs.

Presence of Persistent Parent Reported Emotional and Behavioral-Related Concussion Symptoms Is Associated with Lower Health-Related Quality of Life in Adolescent Athletes.

Persistent concussion symptoms in adolescents are associated with lower health-related quality of life (HRQOL). The association between persistent emotional and behavioral-related concussion symptoms (EBS) and HRQOL is unknown, however. This study was a prospective cohort of adolescent athletes presenting to a concussion clinic within three days post-concussion and completing a one-month follow-up. The independent variable in these analyses was parent reported EBS symptom presence grouped as: (1) no EBS; (2) EBS present at pre-concussion levels; and (3) EBS worse than pre-concussion. The EBS included the following concussion symptoms: feeling irritable, depressed, frustrated/impatient, restless, reduced tolerance to stress/emotion, poor concentration, and fear of permanent symptoms. Dependent variables were parent reported psychosocial, physical, and total HRQOL. Separate multi-variable linear regression models controlling for age, sex, and concussion history were used to assess the association between EBS and HRQOL. Estimated adjusted mean differences (MD) and 95% confidence intervals (CI) were used to assess associations; MDs with a 95%CI excluding 0.0 were considered statistically significant. Overall, n = 245 presented to the study clinic three days post-concussion and completed the one-month follow-up (Mage = 14.28 ± 2.09 years, 59.02% male, 90.64% Caucasian, 31.84% with concussion history). At one-month post-concussion, adolescents with pre-concussion EBS levels had significantly lower psychosocial, physical, and total HRQOL than those with no EBS. In addition, those with EBS worse than pre-concussion had significantly lower psychosocial, physical, and total HRQOL than those with no EBS and EBS at pre-concussion levels. These findings highlight the importance of HRQOL assessments and that targeted interventions may be needed for those with EBS at one-month post-concussion to improve HRQOL.

Exclusivity and Compensation in NFκB Dimer Distributions and IκB Inhibition.

The NFκB transcription factor family members RelA, p50, and cRel form homo- and heterodimers that are inhibited by IκBα, IκBß, and IκBε. These NFκB family members have diverse biological functions, and their expression profiles differ, leading to different concentrations in different tissue types. Here we present definitive biophysical measurements of the NFκB dimer affinities and inhibitor affinities to better understand dimer exchange and how the presence of inhibitors may alter the equilibrium concentrations of NFκB dimers in the cellular context. Fluorescence anisotropy binding experiments were performed at low concentrations to mimic intracellular concentrations. We report binding affinities much stronger than those that had been previously reported by non-equilibrium gel shift and analytical ultracentrifugation assays. The results reveal a wide range of NFκB dimer affinities and a strong preference of each IκB for a small subset of NFκB dimers. Once the preferred IκB is bound, dimer exchange no longer occurs over a period of days. A mathematical model of the cellular distribution of these canonical NFκB transcription factors based on the revised binding affinities recapitulates intracellular observations and provides simple, precise explanations for observed cellular phenomena.

RNAs interact with BRD4 to promote enhanced chromatin engagement and transcription activation.

The bromodomain and extra-terminal motif (BET) protein BRD4 binds to acetylated histones at enhancers and promoters via its bromodomains (BDs) to regulate transcriptional elongation. In human colorectal cancer cells, we found that BRD4 was recruited to enhancers that were co-occupied by mutant p53 and supported the synthesis of enhancer-directed transcripts (eRNAs) in response to chronic immune signaling. BRD4 selectively associated with eRNAs that were produced from BRD4-bound enhancers. Using biochemical and biophysical methods, we found that BRD4 BDs function cooperatively as docking sites for eRNAs and that the BDs of BRD2, BRD3, BRDT, BRG1, and BRD7 directly interact with eRNAs. BRD4-eRNA interactions increased BRD4 binding to acetylated histones in vitro and augmented BRD4 enhancer recruitment and transcriptional cofactor activities. Our results suggest a mechanism by which eRNAs are directly involved in gene regulation by modulating enhancer interactions and transcriptional functions of BRD4.

Prediction of the presence of a seventh ankyrin repeat in IκBε from homology modeling combined with hydrogen-deuterium exchange mass spectrometry (HDX-MS).

The ankyrin repeat (AR) structure is a common protein-protein interaction motif and ankyrin repeat proteins comprise a vast family across a large array of different taxa. Natural AR proteins adopt a conserved fold comprised of several repeats with the N- and C-terminal repeats generally being of more divergent sequences. Obtaining experimental crystal structures for natural ankyrin repeat domains (ARD) can be difficult and often requires complexation with a binding partner. Homology modeling is an attractive method for creating a model of AR proteins due to the highly conserved fold; however, modeling the divergent N- and C-terminal "capping" repeats remains a challenge. We show here that amide hydrogen/deuterium exchange mass spectrometry (HDX-MS), which reports on the presence of secondary structural elements and "foldedness," can aid in the refinement and selection of AR protein homology models when multiple templates are identified with variations between them localizing to these terminal repeats. We report a homology model for the AR protein IκBε from three different templates and use HDX-MS to establish the presence of a seventh AR at the C-terminus identified by only one of the three templates used for modeling.

DNA and IκBα Both Induce Long-Range Conformational Changes in NFκB.

We recently discovered that IκBα enhances the rate of release of nuclear factor kappa B (NFκB) from DNA target sites in a process we have termed molecular stripping. Coarse-grained molecular dynamics simulations of the stripping pathway revealed two mechanisms for the enhanced release rate: the negatively charged PEST region of IκBα electrostatically repels the DNA, and the binding of IκBα appears to twist the NFκB heterodimer so that the DNA can no longer bind. Here, we report amide hydrogen/deuterium exchange data that reveal long-range allosteric changes in the NFκB (RelA-p50) heterodimer induced by DNA or IκBα binding. The data suggest that the two Ig-like subdomains of each Rel-homology region, which are connected by a flexible linker in the heterodimer, communicate in such a way that when DNA binds to the N-terminal DNA-binding domains, the nuclear localization signal becomes more highly exchanging. Conversely, when IκBα binds to the dimerization domains, amide exchange throughout the DNA-binding domains is decreased as if the entire domain is becoming globally stabilized. The results help understand how the subtle mechanism of molecular stripping actually occurs.

Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα.

Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of IκBα, the temporally regulated inhibitor of canonical NFκB signaling. Under native conditions, most of the IκBα molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NFκB(RelA/p50)-IκBα complex. Similar high FRET efficiencies were found when the IκBα molecules were either free or in complex with NFκB(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound IκBα molecules (0.67) than for the free IκBα molecules (0.74), apparently indicating that binding of NFκB(RelA/p50) stretches the ARD of IκBα. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NFκB(RelA/p50) and IκBα encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on IκBα as well as IκBα bound to dimerization-domain-only constructs or full-length NFκB(RelA/p50). Although we expected the stretched IκBα to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NFκB NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the IκBα ARD induced by interactions of the IκBα proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

Dual allosteric activation mechanisms in monomeric human glucokinase.

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In ß-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.