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Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman–Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6–60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

After more than a decade since the introduction of Statistical Engineering by Roger Hoerl and Ronald Snee, a group of leading applied statisticians from academia, industry, and government were invited to discuss their perspectives on progress made, the current status of this important movement, and what future Statistical Engineering holds on the path forward in a series of two panel discussion papers. In this first article, the invited panelists focus their discussion on the past and present of Statistical Engineering. They discuss notable advances and current obstacles to progress. They also consider the unique value added by Statistical Engineering, and the possible addition of decision making to the body of knowledge. The format of the article consists of the posed questions from the moderators, a summary of key ideas from all the panelists, and then the individual detailed answers. The goal of this series of articles is to inspire statisticians to consider their possible role to advance the adoption of Statistical Engineering to solve important problems.

In the second of two panel discussion articles focused on the evolution of statistical engineering (SE) as introduced by Roger Hoerl and Ronald Snee, a group of leading applied statisticians from academia, industry, and government present their perspectives on what the future might hold for this important movement. The invited panelists discuss the challenges and opportunities presented by the emergence of data science and the abundance of large amounts of data. They also consider the possible paths forward for SE, and the roles for statisticians in academia, industry, and government. The final question addresses what additional skills would be helpful to increase the effectiveness of the practice and advance SE. As with the first article, the format of the article follows the order of a posed question, a summary of key ideas, and then the detailed individual panelist answers. The article seeks to inspire statisticians to consider their possible role to leverage the potential of SE to solve important problems.

To describe the implementation of the age-friendly health systems (AFHS) 4Ms Framework, an evidence-based framework to assess and act on "What Matters, Medication, Mentation and Mobility to deliver Age-Friendly health care for patients 65 and older", to achieve the Institute for Health care Improvement (IHI) Committed to Care Excellence recognition in a convenient care health system and test two novel implementation strategies.The study was conducted in over 1100 convenient care clinics in 35 states and DC. MinuteClinics are located in community-based retail pharmacies in rural, suburban, and urban areas and staffed with approximately 3300 nurse practitioners and physician associates.In Year 1, the project used a quality improvement design, and in Year 2, a quasi-experimental implementation research design to pilot two strategies at the provider level (Virtual Clinic and Plan-Do-Study-Act (PDSA)). Statistical process control charts were used to assess changes in 4Ms documentation over time. Mixed-effects Poisson regression was used to assess the effectiveness of the pilot studies.The electronic health record (EHR) was enhanced to capture documentation of the AFHS 4Ms assessments and actions. A learning platform was created to teach and evaluate provider 4Ms competency, and the two data sources were merged into a registry. A formative evaluation was conducted using Tableau and reporting dashboards.After 18 months and the implementation of 20 strategies to improve the uptake of the 4Ms, MinuteClinic achieved the IHI Committed to Care Excellence recognition. A significant increase over time in the reliable delivery of all 4Ms and each M component individually was found. For the research, there were significant improvements in the mean number of Ms delivered per visit (M-Score) in the Virtual Clinic (Incident Rate Ratio [IRR]: 2.47, p = 0.001) and PDSA (IRR: 3.08, p = 0.002) strategy intervention groups when compared to controls.Application of quality improvement and implementation methodologies contributed to the success of implementing age-friendly 4Ms evidence-based practice.

Purpose: Evidence-based practice (EBP) integrates the best evidence from research, clinician expertise, and patient preferences and values to deliver the highest quality of care to improve patient outcomes. Occupational therapy (OT), physical therapy (PT), and rehabilitation students gain exposure to EBP through both didactic and experiential learning. The differences in cultural, educational systems, and student learning styles between the United States and China may lead to different students’ perceptions and attitudes towards EBP. The purpose of the study was to characterize and compare the perceptions of and attitudes towards EBP between the U.S. and Chinese OT and PT students. Methods: A cross-sectional survey of the Evidence-Based Practice Process Assessment Scale (EBPPAS) was sent to professional students enrolled in Doctor of Occupational Therapy (OTD), Doctor of Physical Therapy (DPT), and Master of Occupational Therapy (MOT) programs at three universities in the U.S. (n=1,062) and OT, PT, and rehabilitation students of four-year bachelor programs at four universities in China (n=1,017). Students’ perception of the overall and individual domain of EBP was compared between the U.S. and China with independent samples t-test. Results: In general, all students showed a positive attitude towards EBP across the five domains. The U.S. DPT students had the highest mean score of 3.90 in the domain of “attitude about EBP” followed by the U.S. MOT students (mean=3.88), and the U.S. OTD students (mean=3.84). On average, the U.S. students scored 0.44 (13.8%) higher than Chinese students in all domains combined. Responses from both countries showed the highest scores in the domain of “attitude about EBP” followed by “familiarity with EBP” and “intention to engage in EBP”. In addition, the overall mean score increased non-significantly by 0.07 for the U.S. students from 1st year to 3rd year while it increased significantly by 0.15 (pnd year to 4th year. Conclusion: Few research studies have compared professional students’ attitudes towards EBP between the U.S. and China. This study demonstrated that the U.S. students were more positive overall and in all five domains. Future studies may focus on novice ways to promote EBP in didactic teaching and in clinical practice.

While there is an effective vaccine for Human Hepatitis B Virus (HBV), 257 million people have chronic infections for which there is no cure. The assembly process for the viral capsid is a potential therapeutic target. In order to understand the capsid assembly process, we investigated the dimeric building blocks of the capsid. To understand what blocks assembly, we took advantage of an assembly incompetent mutant dimer, Cp149-Y132A, located in the interdimer interface. This mutation leads to changes in protein dynamics throughout the structure of the dimer as measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS). To further understand how the HBV capsid assembles, the homologue woodchuck HBV (WHV) capsid protein dimer (Cp) was used. WHV is more stable than HBV in HDX-MS and native mass spectrometry experiments. Because the WHV Cp assembles more rapidly into viral capsids than HBV, it was suspected that an increase in stability of the intradimer interface and/or in the contact region leads to increased assembly rates. The differences in dynamics when comparing HBV and human Cp149-Y132A as well as the differences in dynamics when comparing the HBV and WHV Cps allowed us to map an allosteric network within the HBV dimer. Through a careful comparison of structure, stability, and dynamics using four different capsid protein dimers, we conclude that protein subunit dynamics regulate HBV capsid assembly.

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins (RIPs). When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B virus (HBV) core protein (Cp) is involved in most stages of the viral life cycle; it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g., Impβ) via Cp’s C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp allosteric modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Impβ and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Impβ synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Impβ. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV life cycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to “flip” to the capsid exterior. Core protein-directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has the potential to disrupt the virus life cycle and activate the innate immune system.

Human trafficking is a global problem with significant impacts on victims' physical and emotional health. Many health care professionals lack human trafficking knowledge, leading to missed opportunities for intervention. This cross-sectional study used evaluation data from a short course on human trafficking to evaluate the course's perceived impact on students. Closed-ended questions were analyzed descriptively while open-ended questions were analyzed using qualitative content analysis. A total of 241 students across eight professions/disciplines completed the evaluation. The vast majority indicated course content was valuable, applicable to their future practice, and recognized interprofessional teamwork is needed to address human trafficking. Despite course effectiveness, there remains a need to continue expanding interprofessional engagement and examining the longitudinal impact of this educational effort.

BackgroundAfter hospital discharge, patients can experience symptoms prompting them to seek acute medical attention. Early evaluation of patients’ post-discharge symptoms by healthcare providers may improve appropriate healthcare utilization and patient safety. Post-discharge follow-up phone calls, which are used for routine transitional care in U.S. hospitals, serve as an important channel for provider-patient communication about symptoms. This study aimed to assess the facilitators and barriers to evaluating and triaging pain symptoms in cardiovascular patients through follow-up phone calls after their discharge from a large healthcare system in Central Massachusetts. We also discuss strategies that may help address the identified barriers.MethodsGuided by the Practical, Robust, Implementation and Sustainability Model (PRISM), we completed semi-structured interviews with 7 nurses and 16 patients in 2020. Selected nurses conducted (or supervised) post-discharge follow-up calls on behalf of 5 clinical teams (2 primary care; 3 cardiology). We used thematic analysis to identify themes from interviews and mapped them to the domains of the PRISM model.ResultsParticipants described common facilitators and barriers related to the four domains of PRISM: Intervention (I), Recipients (R), Implementation and Sustainability Infrastructure (ISI), and External Environment (EE). Facilitators include: (1) patients being willing to receive provider follow-up (R); (2) nurses experienced in symptom assessment (R); (3) good care coordination within individual clinical teams (R); (4) electronic health record system and call templates to support follow-up calls (ISI); and (5) national and institutional policies to support post-discharge follow-up (EE). Barriers include: (1) limitations of conducting symptom assessment by provider-initiated follow-up calls (I); (2) difficulty connecting patients and providers in a timely manner (R); (3) suboptimal coordination for transitional care among primary care and cardiology providers (R); and (4) lack of emphasis on post-discharge follow-up call reimbursement among cardiology clinics (EE). Specific barriers for pain assessment include: (1) concerns with pain medication misuse (R); and (2) no standardized pain assessment and triage protocol (ISI).ConclusionsStrategies to empower patients, facilitate timely patient-provider communication, and support care coordination regarding pain evaluation and treatment may reduce the barriers and improve processes and outcomes of pain assessment and triage.

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum. The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD(+), we propose a catalytic cycle involving formation of an intermediary NAD(+)-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP(+) oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD(+), the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B Virus (HBV) core protein (Cp) is involved in most stages of the viral lifecycle: it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g. Impβ) via Cp’s C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp Allosteric Modulators (CpAMs), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV lifecycle is poorly understood. We investigated how HAP12 influenced the interactions between empty or RNA-filled capsids with Impβ and trypsin in vitro. We showed that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrated that Impβ synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resisted the destabilizing effects of HAP12 and Impβ. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV lifecycle and also cause the virus to become vulnerable to host innate immune responses.IMPORTANCEThe HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to “flip” to the capsid exterior. Core-protein directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has potential to disrupt the virus lifecycle and activate the innate immune system.

BACKGROUND: There is increasing recognition of the importance of supporting patients in their health-related goals. Patient-provider discussions and health-related mobile applications (apps) can support patients to pursue health goals; however, their impact on patient goal setting and achievement is not well understood. OBJECTIVE: To examine the relationships between the following: (1) patient demographics, patient-provider discussions, and health-related goal setting and achievement, and (2) patient mobile health app use and goal achievement. DESIGN: Cross-sectional survey. PARTICIPANTS: Veterans who receive Veterans Health Administration (VA) healthcare and are users of VA patient-facing technology. MAIN MEASURES: Veteran demographics, goal-related behaviors, and goal achievement. METHODS: Veterans were invited to participate in a telephone survey. VA administrative data were linked to survey data for additional health and demographic information. Logistic regression models were run to identify factors that predict health-related goal setting and achievement. KEY RESULTS: Among respondents (n=2552), 75% of patients indicated having set health goals in the preceding 6 months and approximately 42% reported achieving their goal. Men (vs. women) had lower odds of setting goals (OR: 0.71; CI95: 0.53–0.97), as did individuals with worse (vs. better) health (OR: 0.18; CI95: 0.04–0.88). Individuals with advanced education—some college/college degrees, and post-college degrees (vs. no college education)—demonstrated higher odds of setting goals (OR: 1.35; CI95: 1.01–1.79; OR: 1.71; CI95: 1.28–2.28, respectively). Those who reported having discussed their goals with their providers were more likely to set goals (OR: 3.60; CI95: 2.97–4.35). Patient mobile health app use was not statistically associated with goal achievement. CONCLUSIONS: Efforts to further promote patient-led goal setting should leverage the influence of patient-provider conversations. Use of patient-facing technologies, specifically mobile health apps, may facilitate goal-oriented care, but further work is needed to examine the potential benefits of apps to support patient goals, particularly if providers discuss and endorse use of those apps with patients.

Replication protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes. RPA binds ssDNA utilizing multiple oligosaccharide/oligonucleotide binding domains and based on their individual DNA binding affinities are classified as high versus low-affinity DNA-binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen–deuterium exchange mass spectrometry (HDX-MS), we assessed the ssDNA-driven dynamics of the individual domains of human RPA. As expected, ssDNA binding shows HDX changes in DBDs A, B, C, D and E. However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. Slower allosteric changes transpire in the protein–protein interaction domains and linker regions, and thus do not directly interact with ssDNA. Within a dynamics-based model for RPA, we propose that DBD-A and -B act as the dynamic half and DBD-C, -D and -E function as the less-dynamic half. Thus, segments of ssDNA buried under the dynamic half are likely more readily accessible to RPA-interacting proteins., Graphical Abstract Graphical abstractRPA binds to DNA as two dynamic halves.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]–containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen–deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

Background Widespread adoption, use, and integration of patient-facing technologies into the workflow of health care systems has been slow, thus limiting the realization of their potential. A growing body of work has focused on how best to promote adoption and use of these technologies and measure their impacts on processes of care and outcomes. This body of work currently suffers from limitations (eg, cross-sectional analyses, limited patient-generated data linked with clinical records) and would benefit from institutional infrastructure to enhance available data and integrate the voice of the patient into implementation and evaluation efforts. Objective The Veterans Health Administration (VHA) has launched an initiative called the Veterans Engagement with Technology Collaborative cohort to directly address these challenges. This paper reports the process by which the cohort was developed and describes the baseline data being collected from cohort members. The overarching goal of the Veterans Engagement with Technology Collaborative cohort is to directly engage veterans in the evaluation of new VHA patient-facing technologies and in so doing, to create new infrastructure to support related quality improvement and evaluation activities. Methods Inclusion criteria for veterans to be eligible for membership in the cohort included being an active user of VHA health care services, having a mobile phone, and being an established user of existing VHA patient-facing technologies as represented by use of the secure messaging feature of VHA’s patient portal. Between 2017 and 2018, we recruited veterans who met these criteria and administered a survey to them over the telephone. Results The majority of participants (N=2727) were male (2268/2727, 83.2%), White (2226/2727, 81.6%), living in their own apartment or house (2519/2696, 93.4%), and had completed some college (1176/2701, 43.5%) or an advanced degree (1178/2701, 43.6%). Cohort members were 59.9 years old, on average. The majority self-reported their health status as being good (1055/2725, 38.7%) or very good (524/2725, 19.2%). Most cohort members owned a personal computer (2609/2725, 95.7%), tablet computer (1616/2716, 59.5%), and/or smartphone (2438/2722, 89.6%). Conclusions The Veterans Engagement with Technology Collaborative cohort is an example of a VHA learning health care system initiative designed to support the data-driven implementation of patient-facing technologies into practice and measurement of their impacts. With this initiative, VHA is building capacity for future, rapid, rigorous evaluation and quality improvement efforts to enhance understanding of the adoption, use, and impact of patient-facing technologies.

BACKGROUND Widespread adoption, use, and integration of patient-facing technologies into the workflow of health care systems has been slow, thus limiting the realization of their potential. A growing body of work has focused on how best to promote adoption and use of these technologies and measure their impacts on processes of care and outcomes. This body of work currently suffers from limitations (eg, cross-sectional analyses, limited patient-generated data linked with clinical records) and would benefit from institutional infrastructure to enhance available data and integrate the voice of the patient into implementation and evaluation efforts. OBJECTIVE The Veterans Health Administration (VHA) has launched an initiative called the Veterans Engagement with Technology Collaborative cohort to directly address these challenges. This paper reports the process by which the cohort was developed and describes the baseline data being collected from cohort members. The overarching goal of the Veterans Engagement with Technology Collaborative cohort is to directly engage veterans in the evaluation of new VHA patient-facing technologies and in so doing, to create new infrastructure to support related quality improvement and evaluation activities. METHODS Inclusion criteria for veterans to be eligible for membership in the cohort included being an active user of VHA health care services, having a mobile phone, and being an established user of existing VHA patient-facing technologies as represented by use of the secure messaging feature of VHA’s patient portal. Between 2017 and 2018, we recruited veterans who met these criteria and administered a survey to them over the telephone. RESULTS The majority of participants (N=2727) were male (2268/2727, 83.2%), White (2226/2727, 81.6%), living in their own apartment or house (2519/2696, 93.4%), and had completed some college (1176/2701, 43.5%) or an advanced degree (1178/2701, 43.6%). Cohort members were 59.9 years old, on average. The majority self-reported their health status as being good (1055/2725, 38.7%) or very good (524/2725, 19.2%). Most cohort members owned a personal computer (2609/2725, 95.7%), tablet computer (1616/2716, 59.5%), and/or smartphone (2438/2722, 89.6%). CONCLUSIONS The Veterans Engagement with Technology Collaborative cohort is an example of a VHA learning health care system initiative designed to support the data-driven implementation of patient-facing technologies into practice and measurement of their impacts. With this initiative, VHA is building capacity for future, rapid, rigorous evaluation and quality improvement efforts to enhance understanding of the adoption, use, and impact of patient-facing technologies.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain–mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.This research was originally published in the Journal of Biological Chemistry. Cairns. Biophysical characterization of SARAH domain–mediated multimerization of Hippo pathway complexes in Drosophila. J. Biol. Chem. 2020; 295:<>. © the American Society for Biochemistry and Molecular Biology or © the Author(s). Deposited by shareyourpaper.org and openaccessbutton.org. We've taken reasonable steps to ensure this content doesn't violate copyright. However, if you think it does you can request a takedown by emailing help@openaccessbutton.org.

The Hippo pathway controls cell proliferation and differentiation through the precisely tuned activity of a core kinase cassette. The activity of Hippo kinase is modulated by interactions between its C-terminal coiled-coil, termed the SARAH domain, and the SARAH domains of either dRassF or Salvador. Here, we wanted to understand the molecular basis of SARAH domain–mediated interactions and their influence on Hippo kinase activity. We focused on Salvador, a positive effector of Hippo activity and the least well-characterized SARAH domain–containing protein. We determined the crystal structure of a complex between Salvador and Hippo SARAH domains from Drosophila. This structure provided insight into the organization of the Salvador SARAH domain including a folded N-terminal extension that expands the binding interface with Hippo SARAH domain. We also found that this extension improves the solubility of the Salvador SARAH domain, enhances binding to Hippo, and is unique to Salvador. We therefore suggest expanding the definition of the Salvador SARAH domain to include this extended region. The heterodimeric assembly observed in the crystal was confirmed by cross-linked MS and provided a structural basis for the mutually exclusive interactions of Hippo with either dRassF or Salvador. Of note, Salvador influenced the kinase activity of Mst2, the mammalian Hippo homolog. In co-transfected HEK293T cells, human Salvador increased the levels of Mst2 autophosphorylation and Mst2-mediated phosphorylation of select substrates, whereas Salvador SARAH domain inhibited Mst2 autophosphorylation in vitro. These results suggest Salvador enhances the effects of Hippo kinase activity at multiple points in the Hippo pathway.

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here we use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

The biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E(m)= −320 mV) coupled to reduction of flavodoxin semiquinone (E(m)= −460 mV) and reduction of coenzyme Q (E(m)= +10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another electron delivery route to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in electron transfer to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs on a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain–mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.

Background Smoking is the leading cause of preventable death and disease, yet implementation of smoking cessation in inpatient settings is inconsistent. The Technology Inpatient Program for Smokers (TIPS) is an implementation program designed to reach smokers with a mobile health (mHealth) intervention using stakeholder-supported strategies. Objective The purpose of this study was to determine the impact of the TIPS implementation strategies on smoker-level engagement of the mHealth intervention during care transition. Methods We examined varying intensities (passive motivational posters only and posters + active nurse-led facilitation) of TIPS strategies on four hospital units located in two sites. Unit-level and smoker-level adoption was monitored during active implementation (30 weeks) and sustainability follow-up (30 weeks). Process measures reflecting the reach, effectiveness, adoption, implementation, maintenance (RE-AIM) framework, stakeholder reported adaptations of strategies, and formative evaluation data were collected and analyzed. Results For our smoker-level reach, 103 smokers signed up for the mHealth intervention in-hospital, with minimal decline during sustainability follow-up. While posters + nurse facilitation did not lead to higher reach than posters alone during active implementation (27 vs 30 signed up), it did lead to higher engagement of smokers (85.2% vs 73.3% completion of the full 2-week intervention). TIPS strategy adoption and fidelity varied by unit, including adoption of motivational posters (range: weeks 1 and 5), fidelity of posters (0.4% to 16.2% of posters missing per unit weekly) and internal facilitation of nurse training sessions (average of 2 vs 7.5 by site). Variable maintenance costs of the program totaled US $6.63 (US $683.28/103) per smoker reached. Reported family-member facilitation of mHealth sign-up was an observation of unintended behavior. Conclusions TIPS is a feasible and low-cost implementation program that successfully engages smokers in an mHealth intervention and sustains engagement after discharge. Further testing of nurse facilitation and expanding reach to patient family and friends as an implementation strategy is needed.

BACKGROUND Smoking is the leading cause of preventable death and disease, yet implementation of smoking cessation in inpatient settings is inconsistent. The Technology Inpatient Program for Smokers (TIPS) is an implementation program designed to reach smokers with a mobile health (mHealth) intervention using stakeholder-supported strategies. OBJECTIVE The purpose of this study was to determine the impact of the TIPS implementation strategies on smoker-level engagement of the mHealth intervention during care transition. METHODS We examined varying intensities (passive motivational posters only and posters + active nurse-led facilitation) of TIPS strategies on four hospital units located in two sites. Unit-level and smoker-level adoption was monitored during active implementation (30 weeks) and sustainability follow-up (30 weeks). Process measures reflecting the reach, effectiveness, adoption, implementation, maintenance (RE-AIM) framework, stakeholder reported adaptations of strategies, and formative evaluation data were collected and analyzed. RESULTS For our smoker-level reach, 103 smokers signed up for the mHealth intervention in-hospital, with minimal decline during sustainability follow-up. While posters + nurse facilitation did not lead to higher reach than posters alone during active implementation (27 vs 30 signed up), it did lead to higher engagement of smokers (85.2% vs 73.3% completion of the full 2-week intervention). TIPS strategy adoption and fidelity varied by unit, including adoption of motivational posters (range: weeks 1 and 5), fidelity of posters (0.4% to 16.2% of posters missing per unit weekly) and internal facilitation of nurse training sessions (average of 2 vs 7.5 by site). Variable maintenance costs of the program totaled US $6.63 (US $683.28/103) per smoker reached. Reported family-member facilitation of mHealth sign-up was an observation of unintended behavior. CONCLUSIONS TIPS is a feasible and low-cost implementation program that successfully engages smokers in an mHealth intervention and sustains engagement after discharge. Further testing of nurse facilitation and expanding reach to patient family and friends as an implementation strategy is needed.

Adaptive prokaryotic immune systems rely on clustered regularly interspaced short palindromic repeats and their associated genes to provide the components necessary to clear infection by foreign genetic elements. These immune systems are based on highly specific nucleases that bind DNA or RNA and, upon sequence recognition, degrade the bound nucleic acid. Because of their specificity, CRISPR-Cas systems are being co-opted to edit genes in eukaryotic cells. While the general function of these systems is well understood, an understanding of mechanistic details to facilitate engineering and application to this new arena remains a topic of intense study. Here, we present two methods that have been successfully used to study the structure and mechanism of the Type IE CRISPR system, Cascade, from Escherichia coli. We provide the protocol for a typical native mass spectrometry experiment which, because it allows for analysis of a protein complex without disruption of the noncovalent interactions within the complex, can be used to determine complex composition, architecture, and relative affinity between subunits. We, also, provide the protocol for intact protein hydrogen-deuterium exchange mass spectrometry, which provides insight into the overall conformational stability of the complex and changes in complex stability based on conditions such as substrate binding. Investigating the solution-phase structure, stability, and dynamics of these complexes improves the overall understanding of the mechanism facilitating engineered adjustments to function or utility.

Adaptive prokaryotic immune systems rely on clustered regularly interspaced short palindromic repeats and their associated genes to provide the components necessary to clear infection by foreign genetic elements. These immune systems are based on highly specific nucleases that bind DNA or RNA and, upon sequence recognition, degrade the bound nucleic acid. Because of their specificity, CRISPR-Cas systems are being co-opted to edit genes in eukaryotic cells. While the general function of these systems is well understood, an understanding of mechanistic details to facilitate engineering and application to this new arena remains a topic of intense study. Here, we present two methods that have been successfully used to study the structure and mechanism of the Type IE CRISPR system, Cascade, from Escherichia coli. We provide the protocol for a typical native mass spectrometry experiment which, because it allows for analysis of a protein complex without disruption of the noncovalent interactions within the complex, can be used to determine complex composition, architecture, and relative affinity between subunits. We, also, provide the protocol for intact protein hydrogen-deuterium exchange mass spectrometry, which provides insight into the overall conformational stability of the complex and changes in complex stability based on conditions such as substrate binding. Investigating the solution-phase structure, stability, and dynamics of these complexes improves the overall understanding of the mechanism facilitating engineered adjustments to function or utility.