Changes in Patient-Reported Outcomes Associated with Receiving Whole Health in the Veteran Health Administration (VHA)'s National Demonstration Project.

BACKGROUND: Whole Health (WH) is a patient-centered model of care being implemented by the Veterans Health Administration. Little is known about how use of WH services impacts patients' health and well-being. OBJECTIVE: We sought to assess the association of WH utilization with pain and other patient-reported outcomes (PRO) over 6 months. DESIGN: A longitudinal observational cohort evaluation, comparing changes in PRO surveys for WH users and Conventional Care (CC) users. Inverse probability of treatment weighting was used to balance the two groups on observed demographic and clinical characteristics. PARTICIPANTS: A total of 9689 veterans receiving outpatient care at 18 VA medical centers piloting WH. INTERVENTIONS: WH services included goal-setting clinical encounters, Whole Health coaching, personal health planning, and well-being services. MAIN OUTCOME MEASURES: The primary outcome was change in pain intensity and interference at 6 months using the 3-item PEG. Secondary outcomes included satisfaction, experiences of care, patient engagement in healthcare, and well-being. KEY RESULTS: By 6 months,1053 veterans had utilized WH and 3139 utilized only CC. Baseline pain PEG scores were 6.2 (2.5) for WH users and 6.4 (2.3) for CC users (difference p = 0.028), improving by - 2.4% (p = 0.006) and - 2.3% (p < 0.001), respectively. In adjusted analyses, WH use was unassociated with greater improvement in PEG scores compared to CC - 1.0% (- 2.9%, 1.2%). Positive trends were observed for 8 of 15 exploratory outcomes for WH compared to CC. WH use was associated with greater improvements at 6 months in likelihood to recommend VA 2.0% (0.9%, 3.3%); discussions of goals 11.8% (8.2%, 15.5%); perceptions of healthcare interactions 2.5% (0.4%, 4.6%); and engagement in health behaviors 2.2% (0.3%, 3.9%). CONCLUSION: This study provides early evidence supporting the delivery of WH patient-centered care services to improve veterans' experiences of and engagement in care. These are important first-line impacts towards the goals of better overall health and well-being outcomes for Veterans.

Racial and Ethnic Variation in Complementary and Integrative Health Therapy Use Among US Veterans.

Importance: White individuals are the greatest users of complementary and integrative health (CIH) therapies in the general population, but this might partially be due to differences in age, health condition, and location. Identifying the nuances in racial and ethnic differences in care is one important step to addressing them. Objective: To evaluate racial and ethnic differences in Veterans Affairs (VA)-covered CIH therapy use in a more nuanced manner by examining the association of 5 demographic characteristics, health conditions, and medical facility locations with those differences. Design, Setting, and Participants: Retrospective cross-sectional observational study of VA health care system users, using electronic health record and administrative data at all VA medical facilities and community-based clinics. Participants included veterans with nonmissing race and ethnicity data using VA-funded health care between October 2018 and September 2019. Data were analyzed from June 2022 to April 2023. Main Outcome and Measure: Any use of VA-covered acupuncture, chiropractic care, massage therapy, yoga, or meditation/mindfulness. Results: The sample consisted of 5 260 807 veterans with a mean (SD) age of 62.3 (16.4) years and was 91% male (4 788 267 veterans), 67% non-Hispanic White (3 547 140 veterans), 6% Hispanic (328 396 veterans), and 17% Black (903 699 veterans). Chiropractic care was the most used CIH therapy among non-Hispanic White veterans, Hispanic veterans, and veterans of other races and ethnicities, while acupuncture was the most commonly used therapy among Black veterans. When not accounting for the location of the VA medical facilities in which veterans used health care, Black veterans appeared more likely to use yoga and meditation than non-Hispanic White veterans and far less likely to use chiropractic care, while those of Hispanic or other race and ethnicity appeared more likely to use massage than non-Hispanic White veterans. However, those differences mostly disappeared once controlling for medical facility location, with few exceptions-after adjustment Black veterans were less likely than non-Hispanic White veterans to use yoga and more likely to use chiropractic care. Conclusions and Relevance: This large-scale, cross-sectional study found racial and ethnic differences in use of 4 of 5 CIH therapies among VA health care system users when not considering their medical facility location. Given those differences mostly disappeared once medical facilities were accounted for, the results demonstrated the importance of considering facilities and residential locations when examining racial differences in CIH therapy use. Medical facilities could be a proxy for the racial and ethnic composition of their patients, CIH therapy availability, regional patient or clinician attitudes, or therapy availability.

We Built it, But Did They Come: Veterans' Use of VA Healthcare System-Provided Complementary and Integrative Health Approaches.

BACKGROUND: Interest in complementary and integrative health (CIH) approaches, such as meditation, yoga, and acupuncture, continues to grow. The evidence of effectiveness for some CIH approaches has increased in the last decade, especially for pain, with many being recommended in varying degrees in national guidelines. To offer nonpharmacological health management options and meet patient demand, the nation's largest integrated healthcare system, the Veterans Health Administration (VA), greatly expanded their provision of CIH approaches recently. OBJECTIVE: This paper addressed the questions of how many VA patients might use CIH approaches and chiropractic care if they were available at modest to no fee, and would patients with some health conditions or characteristics be more likely than others to use these therapies. DESIGN: Using electronic medical records, we conducted a national, three-year, retrospective analysis of VA patients' use of eleven VA-covered therapies: chiropractic care, acupuncture, Battlefield Acupuncture, biofeedback, clinical hypnosis, guided imagery, massage therapy, meditation, Tai Chi/Qigong, and yoga. PARTICIPANTS: We created a national cohort of veterans using VA healthcare from October 2016-September 2019. KEY RESULTS: Veterans' use of these approaches increased 70% in three years. By 2019, use was 5.7% among all VA patients, but highest among patients with chronic musculoskeletal pain (13.9%), post-traumatic stress disorder (PTSD; 10.6%), depression (10.4%), anxiety (10.2%), or obesity (7.8%). The approach used varied by age and race/ethnicity, with women being uniformly more likely than men to use each approach. Patients having chronic musculoskeletal pain, obesity, anxiety, depression, or PTSD were more likely than others to use each of the approaches. CONCLUSIONS: Veterans' use of some approaches rapidly grew recently and was robust, especially among patients most in need. This information might help shape federal/state health policy on the provision of evidence-based CIH approaches and guide other healthcare institutions considering providing them.

Effectiveness of a whole health model of care emphasizing complementary and integrative health on reducing opioid use among patients with chronic pain.

BACKGROUND: The opioid crisis has necessitated new approaches to managing chronic pain. The Veterans Health Administration (VHA) Whole Health model of care, with its focus on patient empowerment and emphasis on nonpharmacological approaches to pain management, is a promising strategy for reducing patients' use of opioids. We aim to assess whether the VHA's Whole Health pilot program impacted longitudinal patterns of opioid utilization among patients with chronic musculoskeletal pain. METHODS: A cohort of 4,869 Veterans with chronic pain engaging in Whole Health services was compared with a cohort of 118,888 Veterans receiving conventional care. All patients were continuously enrolled in VHA care from 10/2017 through 3/2019 at the 18 VHA medical centers participating in the pilot program. Inverse probability of treatment weighting and multivariate analyses were used to adjust for observable differences in patient characteristics between exposures and conventional care. Patients exposed to Whole Health services were offered nine complementary and integrative health therapies alone or in combination with novel Whole Health services including goal-setting clinical encounters, Whole Health coaching, and personal health planning. MAIN MEASURES: The main measure was change over an 18-month period in prescribed opioid doses starting from the six-month period prior to qualifying exposure. RESULTS: Prescribed opioid doses decreased by -12.0% in one year among Veterans who began complementary and integrative health therapies compared to similar Veterans who used conventional care; -4.4% among Veterans who used only Whole Health services such as goal setting and coaching compared to conventional care, and -8.5% among Veterans who used both complementary and integrative health therapies combined with Whole Health services compared to conventional care. CONCLUSIONS: VHA's Whole Health national pilot program was associated with greater reductions in prescribed opioid doses compared to secular trends associated with conventional care, especially when Veterans were connected with complementary and integrative health therapies.

Association Between Exposure to Complementary and Integrative Therapies and Opioid Analgesic Daily Dose Among Patients on Long-term Opioid Therapy.

OBJECTIVES: To describe the association between exposure to selected complementary and integrative health (CIH) modalities and the trajectory of prescribed opioid analgesic dose within a national cohort of patients receiving long-term opioid therapy (LTOT) in the Veterans Health Administration (VHA). MATERIALS AND METHODS: Using national data from VHA electronic health records between October 1, 2017 and September 30, 2019, CIH use was analyzed among 57,437 patients receiving LTOT within 18 VHA facilities serving as evaluation sites of VHA's Whole Health System of Care. Using linear mixed effects modeling controlling for covariates, opioid dose was modeled as a function of time, CIH exposure, and their interaction. RESULTS: Overall, 11.91% of patients on LTOT used any of the focus CIH therapies; 43.25% of those had 4 or more encounters. Patients used acupuncture, chiropractic care, and meditation modalities primarily. CIH use was associated with being female, Black, having a mental health diagnosis, obesity, pain intensity, and baseline morphine-equivalent daily dose. Mean baseline morphine-equivalent daily dose was 40.81 milligrams and dose decreased on average over time. Controlling for covariates, patients with any CIH exposure experienced 38% faster dose tapering, corresponding to a mean difference in 12-month reduction over patients not engaging in CIH of 2.88 milligrams or 7.06% of the mean starting dose. DISCUSSION: Results support the role of CIH modalities in opioid tapering. The study design precludes inference about the causal effects of CIH on tapering. Analyses did not consider the trend in opioid dose before cohort entry nor the use of other nonopioid treatments for pain. Future research should address these questions and consider tapering-associated adverse events.

From patient outcomes to system change: Evaluating the impact of VHA's implementation of the Whole Health System of Care.

OBJECTIVE: To describe how a partnered evaluation of the Whole Health (WH) system of care-comprised of the WH pathway, clinical care, and well-being programs-produced patient outcomes findings, which informed Veterans Health Administration (VA) policy and system change. DATA SOURCES: Electronic health records (EHR)-based cohort of 1,368,413 patients and a longitudinal survey of Veterans receiving care at 18 WH pilot medical centers. STUDY DESIGN: In partnership with VA operations, we focused the evaluation on the impact of WH services utilization on Veterans' (1) use of opioids and (2) care experiences, care engagement, and well-being. Outcomes were compared between Veterans who did and did not use WH services identified from the EHR. DATA COLLECTION: Pharmacy records and WH service data were obtained from the VA EHR, including WH coaching, peer-led groups, personal health planning, and complementary, integrative health therapies. We surveyed veterans at baseline and 6 months to measure patient-reported outcomes. PRINCIPAL FINDINGS: Opioid use decreased 23% (31.5-6.5) to 38% (60.3-14.4) among WH users depending on level of WH use compared to a secular 11% (12.0-9.9) decrease among Veterans using Conventional Care. Compared to Conventional Care users, WH users reported greater improvements in perceptions of care (SMD = 0.138), engagement in health care (SMD = 0.118) and self-care (SMD = 0.1), life meaning and purpose (SMD = 0.152), pain (SMD = 0.025), and perceived stress (SMD = 0.191). CONCLUSIONS: Evidence developed through this partnership yielded key VA policy changes to increase Veteran access to WH services. Findings formed the foundation of a congressionally mandated report in response to the Comprehensive Addiction and Recovery Act, highlighting the value of WH and complementary, integrative health and well-being programs for Veterans with pain. Findings subsequently informed issuance of an Executive Decision Memo mandating the integration of WH into mental health and primary care across VA, now one lane of modernization for VA.

Telehealth and Rural-Urban Differences in Receipt of Pain Care in the Veterans Health Administration.

OBJECTIVE: Examine changes in specialty pain utilization in the Veterans Health Administration (VHA) after establishing a virtual interdisciplinary pain team (TelePain). DESIGN: Retrospective cohort study. SETTING: A single VHA healthcare system, 2015-2019. SUBJECTS: 33,169 patients with chronic pain-related diagnoses. METHODS: We measured specialty pain utilization (in-person and telehealth) among patients with moderate to severe chronic pain. We used generalized estimating equations to test the association of time (pre- or post-TelePain) and rurality on receipt of specialty pain care. RESULTS: Among patients with moderate to severe chronic pain, the reach of specialty pain care increased from 11.1% to 16.2% in the pre- to post-TelePain periods (adjusted odds ratio [aOR]: 1.37, 95% confidence interval [CI]: 1.26-1.49). This was true of both urban patients (aOR: 1.62, 95% CI: 1.53-1.71) and rural patients (aOR: 1.16, 95% CI: 0.99-1.36), although the difference for rural patients was not statistically significant. Among rural patients who received specialty pain care, a high percentage of the visits were delivered by telehealth (nearly 12% in the post-TelePain period), much higher than among urban patients (3%). CONCLUSIONS: We observed increased use of specialty pain services among all patients with chronic pain. Although rural patients did not achieve the same degree of access and utilization overall as urban patients, their use of pain telehealth increased substantially and may have substituted for in-person visits. Targeted implementation efforts may be needed to further increase the reach of services to patients living in areas with limited specialty pain care options.

Assessing the impact of the COVID-19 pandemic on pragmatic clinical trial participants.

Characterizing the impacts of disruption attributable to the COVID-19 pandemic on clinical research is important, especially in pain research where psychological, social, and economic stressors attributable to the COVID-19 pandemic may greatly impact treatment effects. The National Institutes of Health - Department of Defense - Department of Veterans Affairs Pain Management Collaboratory (PMC) is a collective effort supporting 11 pragmatic clinical trials studying nonpharmacological approaches and innovative integrated care models for pain management in veteran and military health systems. The PMC rapidly developed a brief pandemic impacts measure for use across its pragmatic trials studying pain while remaining broadly applicable to other areas of clinical research. Through open discussion and consensus building by the PMC's Phenotypes and Outcomes Work Group, the PMC Coronavirus Pandemic (COVID-19) Measure was iteratively developed. The measure assesses the following domains (one item/domain): access to healthcare, social support, finances, ability to meet basic needs, and mental or emotional health. Two additional items assess infection status (personal and household) and hospitalization. The measure uses structured responses with a three-point scale for COVID-19 infection status and four-point ordinal rank response for all other domains. We recommend individualized adaptation as appropriate by clinical research teams using this measure to survey the effects of the COVID-19 pandemic on study participants. This can also help maintain utility of the measure beyond the COVID-19 pandemic to characterize impacts during future public health emergencies that may require mitigation strategies such as periods of quarantine and isolation.

Bringing chronic-pain care to rural veterans: A telehealth pilot program description.

Opioid-related harms disproportionately affect rural communities. Recent research-based policy changes have called for reductions in opioid prescribing and substitution of safe and effective alternatives to opioids for treating chronic pain, but such alternatives are often difficult to access in rural areas. Telehealth services can help address this disparity by bringing evidence-based, biopsychosocial chronic-pain services to rural and underserved patients with chronic pain. This article describes a 2-year pilot project for delivering chronic-pain care by pain specialists from central hubs at Veterans Health Administration (VA) medical centers to spokes at VA community-based outpatient clinics (CBOCs). The VA Puget Sound Pain Telehealth pilot program offered pain education classes, cognitive-behavioral therapy groups, opioid-safety education, and acupuncture education. The program delivered 501 encounters to patients from 1 hub to 4 CBOC spoke sites from 2016 to 2018, and supported training, administration, equipment acquisition, and grant-writing. The quality-improvement project was rolled out using existing local resources. We present initial findings about the patients who utilized Pain Telehealth, share lessons learned, and discuss future directions for expansion. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Assessing the Relative Effectiveness of Combining Self-Care with Practitioner-Delivered Complementary and Integrative Health Therapies to Improve Pain in a Pragmatic Trial.

BACKGROUND: Many health care systems are beginning to encourage patients to use complementary and integrative health (CIH) therapies for pain management. Many clinicians have anecdotally reported that patients combining self-care CIH therapies with practitioner-delivered therapies report larger health improvements than do patients using practitioner-delivered or self-care CIH therapies alone. However, we are unaware of any trials in this area. DESIGN: The APPROACH Study (Assessing Pain, Patient-Reported Outcomes and Complementary and Integrative Health) assesses the value of veterans participating in practitioner-delivered CIH therapies alone or self-care CIH therapies alone compared with the combination of self-care and practitioner-delivered care. The study is being conducted in 18 Veterans Health Administration sites that received funding as part of the Comprehensive Addiction and Recovery Act to expand availability of CIH therapies. Practitioner-delivered therapies under study include chiropractic care, acupuncture, and therapeutic massage, and self-care therapies include tai chi/qi gong, yoga, and meditation. The primary outcome will be improvement on the Brief Pain Inventory 6 months after initiation of CIH as compared with baseline scores. Patients will enter treatment groups on the basis of the care they receive because randomizing patients to specific CIH therapies would require withholding therapies routinely offered at VA. We will address selection bias and confounding by using sites' variations in business practices and other encouragements to receive different types of CIH therapies as a surrogate for direct randomization by using instrumental variable econometrics methods. SUMMARY: Real-world evidence about the value of combining self-care and practitioner-delivered CIH therapies from this pragmatic trial will help guide the VA and other health care systems in offering specific nonpharmacological approaches to manage patients' chronic pain.

Fibroscan liver stiffness after anti-viral treatment for hepatitis C is independently associated with adverse outcomes.

BACKGROUND: Fibroscan-derived liver stiffness decreases after anti-viral treatment for hepatitis C virus (HCV) infection, which may affect the associations and interpretation of liver stiffness. AIMS: To assess whether liver stiffness pre- or post-anti-viral therapy is associated with the development of decompensated cirrhosis, hepatocellular carcinoma (HCC) or death. METHODS: In this retrospective cohort study, we identified US veterans who initiated HCV treatment and had at least one liver stiffness before (n = 492) or after (n = 877) HCV therapy. We used Cox proportional hazards regression (adjusting for age, race/ethnicity, history of cirrhosis, body mass index, diabetes, FIB-4 score, Charlson comorbidity index, alcohol use disorder, Model for end-stage liver disease score and sustained virological response status) to determine the associations between pre- or post-treatment liver stiffness values and the development of decompensated cirrhosis, HCC, death or liver transplant. RESULTS: In the post-treatment liver stiffness cohort, during a mean follow-up of 27.3 months, 21 (2.4%) developed decompensated cirrhosis, 26 (3.0%) developed HCC and 57 (6.5%) died or underwent liver transplant. Compared to patients with post-treatment liver stiffness ≤12.5 kPa, those with post-treatment liver stiffness >20 kPa, had higher rates of developing decompensated cirrhosis (adjusted HR 3.85, 95% CI 1.29-11.50) and the composite outcome of death, liver transplant, decompensated cirrhosis or HCC (adjusted HR 1.95, 95% CI: 1.07-3.56). There were no significant associations between pre-treatment liver stiffness and any outcomes on multivariable analysis. CONCLUSIONS: Post-treatment liver stiffness >20 kPa, but not pre-treatment liver stiffness, was independently associated with the development of decompensated cirrhosis and the composite outcome in multivariable analyses. Measuring liver stiffness should be considered after anti-viral treatment because it predicts adverse outcomes even beyond routinely available clinical predictors.

Does Offering Battlefield Acupuncture Lead to Subsequent Use of Traditional Acupuncture?

OBJECTIVES: Veterans Health Administration encourages auricular acupuncture (Battlefield Acupuncture/BFA) as a nonpharmacologic approach to pain management. Qualitative reports highlighted a "gateway hypothesis": providing BFA can lead to additional nonpharmacologic treatments. This analysis examines subsequent use of traditional acupuncture. RESEARCH DESIGN: Cohort study of Veterans treated with BFA and a propensity score matched comparison group with a 3-month follow-up period to identify subsequent use of traditional acupuncture. Matching variables included pain, comorbidity, and demographics, with further adjustment in multivariate regression analysis. SUBJECTS: We identified 41,234 patients who used BFA across 130 Veterans Health Administration medical facilities between October 1, 2016 and March 31, 2019. These patients were matched 2:1 on Veterans who used VA care but not BFA during the same period resulting in a population of 24,037 BFA users and a comparison cohort of 40,358 non-BFA users. Patients with prior use of traditional acupuncture were excluded. RESULTS: Among Veterans receiving BFA, 9.5% subsequently used traditional acupuncture compared with 0.9% of non-BFA users (P<0.001). In adjusted analysis, accounting for patient characteristics and regional availability of traditional acupuncture, patients who used BFA had 10.9 times greater odds (95% confidence interval, 8.67-12.24) of subsequent traditional acupuncture use. CONCLUSIONS: Providing BFA, which is easy to administer during a patient visit and does not require providers be formally certified, led to a substantial increase in use of traditional acupuncture. These findings suggest that the value of offering BFA may not only be its immediate potential for pain relief but also subsequent engagement in additional therapies.

The APPROACH trial: Assessing pain, patient-reported outcomes, and complementary and integrative health.

Electronic health record data can be used in multiple ways to facilitate real-world pragmatic studies. Electronic health record data can provide detailed information about utilization of treatment options to help identify appropriate comparison groups, access historical clinical characteristics of participants, and facilitate measuring longitudinal outcomes for the treatments being studied. An additional novel use of electronic health record data is to assess and understand referral pathways and other business practices that encourage or discourage patients from using different types of care. We describe an ongoing study utilizing access to real-time electronic health record data about changing patterns of complementary and integrative health services to demonstrate how electronic health record data can provide the foundation for a pragmatic study when randomization is not feasible. Conducting explanatory trials of the value of emerging therapies within a healthcare system poses ethical and pragmatic challenges, such as withholding access to specific services that are becoming widely available to patients. We describe how prospective examination of real-time electronic health record data can be used to construct and understand business practices as potential surrogates for direct randomization through an instrumental variables analytic approach. In this context, an example of a business practice is the internal hiring of acupuncturists who also provide yoga or Tai Chi classes and can offer these classes without additional cost compared to community acupuncturists. Here, the business practice of hiring internal acupuncturists is likely to encourage much higher rates of combined complementary and integrative health use compared to community referrals. We highlight the tradeoff in efficiency of this pragmatic approach and describe use of simulations to estimate the potential sample sizes needed for a variety of instrument strengths. While real-time monitoring of business practices from electronic health records provides insights into the validity of key independence assumptions associated with the instrumental variable approaches, we note that there may be some residual confounding by indication or selection bias and describe how alternative sources of electronic health record data can be used to assess the robustness of instrumental variable assumptions to address these challenges. Finally, we also highlight that while some clinical outcomes can be obtained directly from the electronic health record, such as longitudinal opioid utilization and pain intensity levels for the study of the value of complementary and integrative health, it is often critical to supplement clinical electronic health record-based measures with patient-reported outcomes. The experience of this example in evaluating complementary and integrative health demonstrates the use of electronic health record data in several novel ways that may be of use for designing future pragmatic trials.

Recommendations for the collection and use of multiplexed functional data for clinical variant interpretation.

Variants of uncertain significance represent a massive challenge to medical genetics. Multiplexed functional assays, in which the functional effects of thousands of genomic variants are assessed simultaneously, are increasingly generating data that can be used as additional evidence for or against variant pathogenicity. Such assays have the potential to resolve variants of uncertain significance, thereby increasing the clinical utility of genomic testing. Existing standards from the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) and new guidelines from the Clinical Genome Resource (ClinGen) establish the role of functional data in variant interpretation, but do not address the specific challenges or advantages of using functional data derived from multiplexed assays. Here, we build on these existing guidelines to provide recommendations to experimentalists for the production and reporting of multiplexed functional data and to clinicians for the evaluation and use of such data. By following these recommendations, experimentalists can produce transparent, complete, and well-validated datasets that are primed for clinical uptake. Our recommendations to clinicians and diagnostic labs on how to evaluate the quality of multiplexed functional datasets, and how different datasets could be incorporated into the ACMG/AMP variant-interpretation framework, will hopefully clarify whether and how such data should be used. The recommendations that we provide are designed to enhance the quality and utility of multiplexed functional data, and to promote their judicious use.

Heat shock-induced chaperoning by Hsp70 is enabled in-cell.

Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard ß-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a "preemptive holdase" mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function.

A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Correction to: A statistical framework for analyzing deep mutational scanning data.

CORRECTION: After publication of our article [1] it was brought to our attention that a line of code was missing from our program to combine the within-replicate variance and between-replicate variance. This led to an overestimation of the standard errors calculated using the Enrich2 random-effects model.

A statistical framework for analyzing deep mutational scanning data.

Deep mutational scanning is a widely used method for multiplex measurement of functional consequences of protein variants. We developed a new deep mutational scanning statistical model that generates error estimates for each measurement, capturing both sampling error and consistency between replicates. We apply our model to one novel and five published datasets comprising 243,732 variants and demonstrate its superiority in removing noisy variants and conducting hypothesis testing. Simulations show our model applies to scans based on cell growth or binding and handles common experimental errors. We implemented our model in Enrich2, software that can empower researchers analyzing deep mutational scanning data.

The Effect of Fluorescent Protein Tags on Phosphoglycerate Kinase Stability Is Nonadditive.

It is frequently assumed that fluorescent protein tags used in biological imaging experiments are minimally perturbing to their host protein. As in-cell experiments become more quantitative and measure rates and equilibrium constants, rather than just "on-off" activity or the presence of a protein, it becomes more important to understand such perturbations. One criterion for a protein modification to be a perturbation is additivity of two perturbations (a linear effect on the protein free energy). Here we show that adding fluorescent protein tags to a host protein in vitro has a large nonadditive effect on its folding free energy. We compare an unlabeled, three singly labeled, and a doubly labeled enzyme (phosphoglycerate kinase). We propose two mechanisms for nonadditivity. In the "quinary interaction" mechanism, two tags interact transiently with one another, relieving the host protein from unfavorable tag-protein interactions. In the "crowding" mechanism, adding two tags provides the minimal crowding necessary to overcome destabilizing interactions of individual tags with the host protein. Both of these mechanisms affect protein stability in cells; we show here that they must also be considered for tagged proteins used for reference in vitro.