Geospatial Distribution of Prenatally and Postnatally Diagnosed Congenital Heart Disease: Implications for Equitable Care from a Fetal Heart Society Research Collaborative Study.

OBJECTIVE: To characterize patterns in the geospatial distribution of pre- and postnatally diagnosed congenital heart disease (CHD) across 6 surgical centers. STUDY DESIGN: A retrospective, multicenter case series from the Fetal Heart Society identified patients at 6 centers from 2012 through 2016 with prenatally (PrND) or postnatally (PoND) diagnosed hypoplastic left heart syndrome (HLHS) or d-transposition of the great arteries (TGA). Geospatial analysis for clustering was done by the average nearest neighbor (ANN) tool or optimized hot spot tool, depending on spatial unit and data type. Both point location and county case rate per 10 000 live births were assessed for geographic clustering or dispersion. RESULTS: Of the 453 CHD cases, 26% were PoND (n = 117), and 74% were PrND (n = 336). PrND cases, in all but one center, displayed significant geographic clustering by the ANN. Conversely, PoND cases tended toward geographic dispersion. Dispersion of PoND HLHS occurred in 2 centers (ANN = 1.59, P < .001; and 1.47, P = .016), and PoND TGA occurred in 2 centers (ANN = 1.22, P < .05; and ANN = 1.73, P < .001). Hot spot analysis of all CHD cases (TGA and HLHS combined) revealed clustering near areas of high population density and the tertiary surgical center. Hot spot analysis of county-level case rate, accounting for population density, found variable clustering patterns. CONCLUSION: Geographic dispersion among postnatally detected CHD highlights the need for a wider reach of prenatal cardiac diagnosis tailored to the specific needs of a community. Geospatial analysis can support centers in improving the equitable delivery of prenatal care.

Shifting the narrative from living at risk to living with risk: validating and pilot-testing a clinical decision support tool: a mixed methods study.

BACKGROUND: When there are safety concerns, healthcare professionals (HCPs) may disregard older adults' wishes to return or remain at home. A paradigm shift is needed for HCPs to move from labelling older adults as living at risk to helping them live with risk. The Living with Risk: Decision Support Tool (LwR:DST) was developed to support older adults and HCPs with difficult decision-making regarding living with risk. The study objectives were to: (1) validate, and (2) pilot-test the LwR:DST in hospital and community settings. METHODS: The study was conducted across Canada during the pandemic. The LwR:DST's content was validated with quantitative and qualitative data by: (1) 71 HCPs from hospital and community settings using the Delphi method, and (2) 17 older adults and caregivers using focus groups. HCPs provided feedback on the LwR:DST's content, format and instruction manual while older adults provided feedback on the LwR:DST's communication step. The revised LwR:DST was pilot-tested by 14 HCPs in one hospital and one community setting, and 17 older adults and caregivers described their experience of HCPs using this approach with them. Descriptive and thematic analysis were performed. RESULTS: The LwR:DST underwent two iterations incorporating qualitative and quantitative data provided by HCPs, older adults and caregivers. The quantitative Delphi method data validated the content and the process of the LwR:DST, while the qualitative data provided practical improvements. The pilot-testing results suggest that using the LwR:DST broadens HCPs' clinical thinking, structures their decision-making, improves their communication and increases their competence and comfort with risk assessment and management. Our findings also suggest that the LwR:DST improves older adults' healthcare experience by feeling heard, understood and involved. CONCLUSIONS: This revised LwR:DST should help HCPs systematically identify frail older adults' risks when they remain at or return home and find acceptable ways to mitigate these risks. The LwR:DST induces a paradigm shift by acknowledging that risks are inherent in everyday living and that risk-taking has positive and negative consequences. The challenges involved in integrating the LwR:DST into practice, i.e., when, how and with whom to use it, will be addressed in future research.

Geographic Distribution of Congenital Heart Disease: A Single Surgical Center Experience.

OBJECTIVE: To determine presence of spatial clustering or dispersion of pre and postnatally detected hypoplastic left heart syndrome (HLHS) and d-transposition of the great arteries (TGA) cases. STUDY DESIGN: This retrospective study examined all patients with a prenatal or postnatal diagnosis of HLHS or TGA who had an initial visit or hospitalization at our tertiary care center over a 5-year period from 2012 to 2016 (n = 105). Using geographic information systems software, the nearest neighbor ratio (NNR) tool was used to determine whether statistically significant clustering or dispersion occurred. RESULTS: Geographic clustering was observed among prenatally diagnosed pooled cases of HLHS and TGA and all total cases (NNR = 0.73 and 0.66, respectively), but not postnatally detected cases (NNR = 1.08). Notably, there was significant dispersion of postnatally detected TGA cases (NNR = 1.22) There was no pattern for prenatally detected TGA or HLHS when analyzed individually. CONCLUSIONS: The spatial distribution of HLHS and TGA is not random; these conditions occur in geographic clusters. Clustering of all patients in the study population and dispersion of postnatal diagnosis of TGA represent opportunities for improved delivery of fetal cardiac care.

Expanding Access to Fetal Telecardiology During the COVID-19 Pandemic.

Background: This study aims to describe one center's experience in expanding a fetal telecardiology program through collaborative work with maternal fetal medicine (MFM) clinics with the goal of safely reaching mothers during the COVID-19 pandemic. We sought to define the extent of fetal telehealth conversion at a large fetal cardiac care center and evaluate the diagnostic accuracy for studies performed. Methods: At our center, fetal telemedicine expanded from one MFM site before the pandemic to four additional sites by May 2020. A retrospective review of fetal telecardiology visits between March 15 and July 15, 2020, was performed. The chart was reviewed for confirmation of diagnosis postnatally. Results: With pandemic onset, there was a large increase in the number of telemedicine visits with a total of 122 mothers seen between five MFM clinics. Fourteen mothers (11.5%) had abnormal fetal echocardiograms requiring additional follow-up, and seven mothers (5.8%) had a fetal echocardiogram suspicious for a critical congenital heart disease (CCHD). All the fetal echocardiograms suspicious for CCHD were confirmed on postnatal echocardiogram. To our knowledge, none of the normal fetal echocardiograms were found to have congenital heart disease postnatally. Conclusions: In response to the COVID-19 pandemic, we rapidly transitioned to fetal telecardiology using a variety of formats. This has reduced potential infectious exposure for pregnant mothers and minimized contact between physicians without compromising diagnostic accuracy. In our experience, the expansion of a telemedicine program requires strong initial infrastructure, prior relationships with MFM providers, and appropriate training among obstetric sonographers.

Exploiting machine learning for end-to-end drug discovery and development.

A variety of machine learning methods such as naive Bayesian, support vector machines and more recently deep neural networks are demonstrating their utility for drug discovery and development. These leverage the generally bigger datasets created from high-throughput screening data and allow prediction of bioactivities for targets and molecular properties with increased levels of accuracy. We have only just begun to exploit the potential of these techniques but they may already be fundamentally changing the research process for identifying new molecules and/or repurposing old drugs. The integrated application of such machine learning models for end-to-end (E2E) application is broadly relevant and has considerable implications for developing future therapies and their targeting.

[Reduction of needlestick injuries by 48 % in 1 year : Effects of improvement of the safety concept according to the European Union Council directive 2010/32/EU at a large regional hospital]. / Reduktion von Nadelstichverletzungen um 48 % in einem Jahr : Auswirkungen einer Verbesserung des Sicherheitskonzeptes nach der EU-Direktive 2010/32/EU an einem großen regionalen Krankenhaus.

BACKGROUND: Needlestick injuries (NSI) are potentially infectious injuries from sharp or pointed medical instruments and through contact with blood on mucous membranes or nonintact skin. Although the European Union (EU) Council directive 2010/32/EU on the prevention of NSI was implemented in EU countries in 2013, information on the effectiveness of the measures is limited. OBJECTIVE: The aim of this study was to evaluate the effectiveness of a safety concept according to the EU Council Directive 2010/32/EU on prevention of NSI. MATERIAL AND METHODS: In 2016 the NSI safety concept at a large regional hospital was improved according to 2010/32/EU, specifically by an update of blood screening profiles and standard operating procedures (SOP), better dissemination of information to employees and complete conversion to safety cannulas and scalpels. The medical records of all NSIs from 2015-2017 were retrospectively anonymized and evaluated and a cost analysis was performed. RESULTS: The number of NSIs in 2017 was significantly reduced by 48.4% as compared to 2016 and NSIs with scalpels were completely prevented. The proportion of employees with NSIs who were adequately immunized against hepatitis B was significantly increased to 84.1% in 2017. Furthermore, identification of the index patient was significantly increased to 82.5% in 2017. The cost of avoiding NSIs increased by a total of 24.1% in 2017 as compared to 2015 before introduction of the safety concept. CONCLUSION: Implementation of the EU Council directive 2010/32/EU, resulted in an almost 50% reduction in NSIs over 1 year, including the complete prevention of NSIs due to scalpels. In addition, the anamnestic presence of immunization against hepatitis B and index patient identification were significantly increased.

Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly.

The NEDD4-2 (neural precursor cell-expressed developmentally down-regulated 4-2) HECT ligase catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two functionally distinct E2∼ubiquitin-binding sites, analogous to the trimeric E6AP/UBE3A HECT ligase. This conserved catalytic mechanism suggests that NEDD4-2, and presumably all HECT ligases, requires oligomerization to catalyze polyubiquitin chain assembly. To explore this hypothesis, we examined the catalytic mechanism of NEDD4-2 through the use of biochemically defined kinetic assays examining rates of 125I-labeled polyubiquitin chain assembly and biophysical techniques. The results from gel filtration chromatography and dynamic light-scattering analyses demonstrate for the first time that active NEDD4-2 is a trimer. Homology modeling to E6AP revealed that the predicted intersubunit interface has an absolutely conserved Phe-823, substitution of which destabilized the trimer and resulted in a ≥104-fold decrease in kcat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (Ki = 8 ± 1.2 mm) of polyubiquitin chain elongation by destabilizing the active trimer, suggesting a mechanism for therapeutically targeting HECT ligases. Additional kinetic experiments indicated that monomeric NEDD4-2 catalyzes only HECT∼ubiquitin thioester formation and monoubiquitination, whereas polyubiquitin chain assembly requires NEDD4-2 oligomerization. These results provide evidence that the previously identified sites 1 and 2 of NEDD4-2 function in trans to support chain elongation, explicating the requirement for oligomerization. Finally, we identified a conserved catalytic ensemble comprising Glu-646 and Arg-604 that supports HECT-ubiquitin thioester exchange and isopeptide bond formation at the active-site Cys-922 of NEDD4-2.

Deviant Cyber-Sexual Activities in Young Adults: Exploring Prevalence and Predictions Using In-Person Sexual Activities and Social Learning Theory.

Technology has shifted some human interactions to the virtual world. For many young adults, sexual encounters now occur through virtual means, as social media, picture exchanges, sexually explicit Web sites, and video chatting have become popular alternative outlets for these activities to occur. This study used the self-report responses of 812 undergraduate students (282 men and 530 women), collected from an online survey. In addition to using 10 personal demographic control variables, this study used five sexual activity/relationship characteristics (number of sexual partners, relationship status, age to first use pornography, frequency of sexual activity/intercourse, and frequency of masturbation), and the four constructs of Akers' social learning theory (identified as differential association, differential reinforcement, imitation/modeling, and definitions favorable) to predict a seven-item count of deviant cyber-sexual activities, and two measures of "sexting" behaviors. Gender, self-esteem, sexual orientation, race, and religion were strongly significant predictors in the models, but Akers' four elements of social learning performed the strongest in predicting the two measures of sexting and the overall deviant cyber-sexual activities scale. This finding indicates that peer associations and peer reinforcements have a strong influence on individuals' willingness to engage in deviant cyber-sexual activities. This study explored different avenues for young adults' engagement in sexual deviancy and the results suggest that sexual behaviors performed in-person may not be the strongest predictors of online sexual behavior.

Effect of anaemia on the diagnosis of rheumatic heart disease using World Heart Federation criteria.

BACKGROUND: There is overlap between pathological mitral regurgitation seen in borderline rheumatic heart disease using World Heart Federation echocardiography criteria and physiologic regurgitation found in normal children. One possible contributing factor is higher rates of anaemia in endemic countries. OBJECTIVE: To investigate the contribution of anaemia as a potential confounder in the diagnosis of rheumatic heart disease detected in echocardiographic screening. METHOD/DESIGN: A novel Server 2012 data warehouse tool was used to incorporate haematology and echocardiography databases. The study included a convenience sample of patients from 5 to 18 years old without structural or functional heart disease that had a haemoglobin value within 1 month prior to an echocardiogram. Echocardiogram images were reviewed to determine presence or absence of World Heart Federation criteria for rheumatic heart disease. The rate of rheumatic heart disease among anaemic and non-anaemic children according to gender- and age-based norms groups was compared. RESULTS: Of the 935 patients who met the study inclusion criteria, 406 were classified as anaemic. There was no difference in the rate of echocardiograms meeting criteria for borderline rheumatic heart disease in anaemic (2.0%, 95% CI 0.6-3.3%) and non-anaemic children (1.3%, 95% CI 0.3-2.3%). However, there was a statistically significant increase in rates of mitral regurgitation of unclear significance among anaemic versus non-anaemic patients (8.6 versus 3.6%; p = 0.0012). CONCLUSION: Anaemia does not increase the likelihood of meeting echocardiographic criteria for borderline rheumatic heart disease. Future studies should evaluate for the correlation between anaemia and mitral regurgitation in endemic settings.

In silico modeling of the cryptic E2∼ubiquitin-binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.

To understand the mechanism for assembly of Lys48-linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys820∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations. To understand substrate inhibition, we exploited the PatchDock algorithm to model in silico UbcH7∼ubiquitin bound to Site 1, validated by chain assembly kinetics of selected point mutants. The predicted structure buries an extensive solvent-excluded surface bringing the UbcH7∼ubiquitin thioester bond within 6 Šof the Cys820 nucleophile. Modeling onto the active E6AP trimer suggests that substrate inhibition arises from steric hindrance between Sites 1 and 2 of adjacent subunits. Confirmation that Sites 1 and 2 function in trans was demonstrated by examining the effect of E6APC820A on wild-type activity and single-turnover pulse-chase kinetics. A cyclic proximal indexation model proposes that Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys820 before stochastic en bloc transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys820-linked 125I-polyubiquitin thioester intermediate. Other studies suggest that Glu550 serves as a general base to generate the Cys820 thiolate within the low dielectric binding interface and Arg506 functions to orient Glu550 and to stabilize the incipient anionic transition state during thioester exchange.

The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly.

The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of 125I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys63-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free 125I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration (Km = 44 ± 6 nm; kcat = 0.020 ± 0.007 s-1) and substrate inhibition above 0.5 µm (Ki = 2.5 ± 1.3 µm) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin-binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 (Ki = 2.0 ± 0.5 µm), consistent with the presence of the second E2-binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high-affinity Site 1 (Ki = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 (Kact = 750 ± 260 nm). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrate that the cryptic Site 1 is associated with thioester formation, whereas binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca2+-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin-binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family.

Unique methionine-aromatic interactions govern the calmodulin redox sensor.

Calmodulin contains multiple redox sensitive methionines whose oxidation alters the regulation of numerous targets. Molecular dynamics simulations were used to define the molecular principles that govern how calmodulin is structurally poised to detect and respond to methionine oxidation. We found that calmodulin's open and closed states were preferentially stabilized by unique, redox sensitive, methionine-aromatic interactions. Key methionine-aromatic interactions were coupled to reorientation of EF hand helices. Methionine to glutamine substitutions designed to mimic methionine oxidation strongly altered conformational transitions by modulating the strength of methionine-aromatic interactions. Together, these results suggest a broadly applicable redox sensing mechanism though which methionine oxidation by cellular oxidants alters the strength of methionine-aromatic interactions critical for functional protein dynamics.

Patient experiences of a lifestyle program for metabolic syndrome offered in family medicine clinics: a mixed methods study.

BACKGROUND: Patient perspectives on new programs to manage metabolic syndrome (MetS) are critical to evaluate for possible implementation in the primary healthcare system. Participants' perspectives were sought for the Canadian Health Advanced by Nutrition and Graded Exercise (CHANGE) study, which enrolled 293 participants, and demonstrated 19% reversal of MetS after 1 year. The main purpose of this study was to examine participants' perceptions of their experiences with the CHANGE program, enablers and barriers to change. METHODS: A convergent parallel mixed methods design combined patients' perspectives collected by questionnaires (n = 164), with insights from focus groups (n = 41) from three sites across Canada. Qualitative data were thematically analyzed using interpretative description. Insights were organized within a socio-ecologic framework. RESULTS: Key aspects identified by participants included intra-individual factors (personal agency, increased time availability), inter-individual factors (trust, social aspects) and organizational factors (increased mental health support, tailored programs). CONCLUSION: Results revealed participants' overall support for the CHANGE program, especially the importance of an extended program under the guidance of a family physician along with a skilled and supportive team. Team delivery of a lifestyle program in primary care or family medicine clinics is a complex intervention and use of a mixed methods design was helpful for exploring patient experiences and key issues on enablers and barriers to health behavior change.

The active form of E6-associated protein (E6AP)/UBE3A ubiquitin ligase is an oligomer.

Employing 125I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508Å2 and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe(727) and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases kcat 62-fold but fails to affect E2 ubiquitin binding or subsequent formation of the Hect domain Cys(820) ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe(727)-dependent trimer formation and catalytic activity (Ki12 mM), as does a conserved-helical peptide corresponding to residues 474­490 of E6A Pisoform 1 (Ki22M) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe(727) intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (Kactivation 1.5 nM) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the kcat defect of the Phe(727) mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity.

Impact of methionine oxidation on calmodulin structural dynamics.

We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron-electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM's structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4nm (closed) and another at ∼6nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each population ranging from 1 to 3nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM's functional interaction with its target proteins by perturbing this Ca-dependent structural shift.

Conformationally trapping the actin-binding cleft of myosin with a bifunctional spin label.

We have trapped the catalytic domain of Dictyostelium (Dicty) myosin II in a weak actin-binding conformation by chemically crosslinking two engineered cysteines across the actin-binding cleft, using a bifunctional spin label (BSL). By connecting the lower and upper 50 kDa domains of myosin, the crosslink restricts the conformation of the actin-binding cleft. Crosslinking has no effect on the basal ATPase activity of isolated myosin, but it impairs rigor actin binding and actin-activation of myosin ATPase. EPR spectra of BSL provide insight into actomyosin structural dynamics. BSL is highly immobilized within the actin-binding cleft and is thus exquisitely sensitive to the global orientation and rotational motions of the myosin head. Conventional EPR shows that myosin heads bound to oriented actin filaments are highly disordered with respect to the actin filament axis, in contrast to the nearly crystalline order of myosin heads in rigor. This disorder is similar to that of weakly bound heads induced by ATP, but saturation transfer EPR shows that the disorder of crosslinked myosin is at least 100 times slower. Thus this cleft-crosslinked myosin is remarkably similar, in both actin affinity and rotational dynamics, to SH1-SH2 crosslinked BSL-myosin S1. We conclude that, whether myosin is trapped at the actin-myosin interface or in the force-generating region between the active site and lever arm, the structural state of myosin is intermediate between the weak-binding state preceding phosphate release and the strong-binding state that succeeds it. We propose that it represents the threshold of force generation.

E6AP/UBE3A ubiquitin ligase harbors two E2~ubiquitin binding sites.

By exploiting (125)I-polyubiquitin chain formation as a functional readout of enzyme activity, we have quantitatively examined the mechanism of human E6AP/UBE3A for the first time. Initial rate studies identify UbcH7 as the cognate E2 carrier protein for E6AP, although related Ubc5 isoforms and the ISG15-specific UbcH8 paralog also support E6AP with reduced efficacy due to impaired binding and catalytic competence. Initial rates of polyubiquitin chain formation displayed hyperbolic kinetics with respect to UbcH7 concentration (K(m) = 57.6 ± 5.7 nM and kcat = 0.032 ± 0.001 s(-1)) and substrate inhibition above 2 µM. Competitive inhibition by an isosteric UbcH7C86S-ubiquitin oxyester substrate analog (K(i) = 64 ± 18 nM) demonstrates that Km reflects intrinsic substrate affinity. In contrast, noncompetitive inhibition by a UbcH7C86A product analog (K(i) = 7 ± 0.7 µM) and substrate inhibition at high concentrations require two functionally distinct E2∼ubiquitin substrate binding sites. The kinetics of polyubiquitin chain formation reflect binding at a cryptic Site 1 not previously recognized that catalyzes E6AP∼ubiquitin thioester formation. Subsequent binding of E2∼ubiquitin at the canonical Site 2 present in the extant crystal structure is responsible for polyubiquitin chain elongation. Other rate studies show that the conserved -4 Phe(849) residue is required for polyubiquitin chain formation rather than target protein conjugation as originally suggested. The present studies unambiguously preclude earlier models for the mechanism of Hect domain-catalyzed conjugation through the canonical binding site suggested by the crystal structure and define a novel two-step mechanism for formation of the polyubiquitin degradation signal.

Redox-sensitive residue in the actin-binding interface of myosin.

We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress.

Electron paramagnetic resonance resolves effects of oxidative stress on muscle proteins.

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to explore the effects of oxidation on muscle function, with particular focus on the actin-myosin interaction. EPR measurements show that aging or oxidative modification causes a decrease in the fraction of myosins in the strong-binding state, which can be traced to the actin-binding cleft of the myosin catalytic domain.

Managing constipation: implementing a protocol in a geriatric rehabilitation setting.

This study examined the effect of implementing a constipation management protocol (CMP) within a geriatric rehabilitation setting. A convergent mixed-methods research design was used. Quantitative data on bowel activity, laxative use, opiate drug use, and nursing documentation regarding bowel care were gathered through a review of health records for 305 patients admitted to three geriatric rehabilitation units before (n = 137) and after (n = 168) protocol implementation over two 3-month periods. Focus groups were conducted examining nursing staff's experiences with such a protocol. Findings revealed that although implementation of the CMP did not reduce constipation rates among older patients, the average number of incidences of constipation per patient was reduced after implementation of the protocol. More importantly, it resulted in more due diligence by staff regarding patients' bowel patterns as well as improved bowel care documentation. Findings and recommendations extend current literature and have practical implications for nurses interested in improving management of patients' bowel care.