Estimating RNA Secondary Structure Folding Free Energy Changes with efn2.

A number of analyses require estimates of the folding free energy changes of specific RNA secondary structures. These predictions are often based on a set of nearest neighbor parameters that models the folding stability of a RNA secondary structure as the sum of folding stabilities of the structural elements that comprise the secondary structure. In the software suite RNAstructure, the free energy change calculation is implemented in the program efn2. The efn2 program estimates the folding free energy change and the experimental uncertainty in the folding free energy change. It can be run through the graphical user interface for RNAstructure, from the command line, or a web server. This chapter provides detailed protocols for using efn2.

Genome-Wide DNA Changes Acquired by Candida albicans Caspofungin-Adapted Mutants.

Drugs from the echinocandin (ECN) class are now recommended 'front-line' treatments of infections caused by a prevailing fungal pathogen, C. albicans. However, the increased use of ECNs is associated with a rising resistance to ECNs. As the acquisition of ECN resistance in C. albicans is viewed as a multistep evolution, determining factors that are associated with the decreased ECN susceptibility is of importance. We have recently identified two cohorts of genes that are either up- or downregulated in concert in order to control remodeling of cell wall, an organelle targeted by ECNs, in laboratory mutants with decreased ECN susceptibility. Here, we profiled the global DNA sequence of four of these adapted mutants in search of DNA changes that are associated with decreased ECN susceptibility. We find a limited number of 112 unique mutations representing two alternative mutational pathways. Approximately half of the mutations occurred as hotspots. Approximately half of mutations and hotspots were shared by ECN-adapted mutants despite the mutants arising as independent events and differing in some of their phenotypes, as well as in condition of chromosome 5. A total of 88 mutations are associated with 43 open reading frames (ORFs) and occurred inside of an ORF or within 1 kb of an ORF, predominantly as single-nucleotide substitution. Mutations occurred more often in the 5'-UTR than in the 3'-UTR by a 1.67:1 ratio. A total of 16 mutations mapped to eight genomic features that were not ORFs: Tca4-4 retrotransposon; Tca2-7 retrotransposon; lambda-4a long terminal repeat; mu-Ra long terminal repeat; MRS-7b Major Repeat Sequence; MRS-R Major Repeat Sequence; RB2-5a repeat sequence; and tL (CAA) leucine tRNA. Finally, eight mutations are not associated with any ORF or other genomic feature. Repeated occurrence of single-nucleotide substitutions in non-related drug-adapted mutants strongly indicates that these DNA changes are accompanying drug adaptation and could possibly influence ECN susceptibility, thus serving as factors facilitating evolution of ECN drug resistance due to classical mutations in FKS1.

Caring again: Support for parent caregivers of wounded, ill, and/or injured adult children veterans.

BACKGROUND: Parents often provide care to adult children veterans with polytrauma, traumatic brain injury, and/or post-traumatic stress disorder. OBJECTIVE: This two-arm randomized clinical trial compared interventions to help parent caregivers improve their depression, anxiety, and burden and manage care by decreasing troubling and concerning behaviors. METHODS: Interventions were six one-hour structured one-on-one behavioral sessions (REACH) or six 30-minute prerecorded online educational webinars. Both focused on knowledge, strategies for care, and coping, but REACH sessions were targeted, interactive, and skills-based. Quantitative and qualitative data were collected by telephone. Quantitative analyses included chi-squared test or independent samples t-test and repeated measures mixed linear modeling, with theme development for qualitative data. RESULTS: There were 163 parent caregivers, mostly mothers. During six months, participants in both arms improved significantly in depression, anxiety, burden, and reported veteran troubling and concerning behaviors. REACH caregivers showed a group by time improvement in concerning behaviors. Benefits included resources, self-reflection, not feeling alone, new skills, improved self-efficacy, and helping others. Specific concerns include exclusion from military and veteran care briefings and concern for the future. CONCLUSION: The positive response to both interventions provides opportunities for organizations with varying resources to provide support for parent caregivers. Interventions need to be targeted to parents' particular concerns and needs.

Pandemic Shift: Virtual Self-Care Courses for Caregivers of Veterans.

Self-care improves health and well-being, yet many caregivers neglect it. During COVID-19, self-care courses for caregivers of veterans transitioned from in-person to virtual videoconferencing. The format remained the same with caregiver groups and a trainer. This observational study examined in-person and virtual caregivers' satisfaction with courses. Caregivers (1120 in-person, 962 virtual) could attend five courses before and following March 2020 transition to virtual. Evaluations (N = 1665) examined demographics, satisfaction, and utility. Characteristics were compared between in-person and virtual participants using chi-squared tests. Qualitative caregiver comments were compared. Half of the caregivers were over 60 years old; 49% had been caregivers at least 6 years. Caregivers were primarily women (91%) and spouses (75%), with more spouses virtually (p = 0.006) and more men in-person (p < 0.001). Both groups endorsed learning new information, planning to use it in caregiving and for themselves, increasing knowledge and skills, and having needs met. Caregiver comments revealed six types of benefits: new information, information review, positive effects, interaction, plans to act, and instructor qualities. Caregivers in virtual groups more often mentioned learning more information, being reminded of information, and planning to take further action; in-person caregivers more often mentioned interaction as a benefit. Caregivers were satisfied with and found benefit from in-person and virtual self-care courses. Although in-person courses allow for more social connection with others, virtual courses offer decreased travel costs for instructors and increased convenience and access for caregivers.

Nearest neighbor rules for RNA helix folding thermodynamics: improved end effects.

Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated 5'GGUC3' paired to 3'CUGG5' separately from other 5'GU3'/3'UG5' stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37°C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.

Identification of variant HIV envelope proteins with enhanced affinities for precursors to anti-gp41 broadly neutralizing antibodies.

HIV envelope protein (Env) is the sole target of broadly neutralizing antibodies (BNAbs) that are capable of neutralizing diverse strains of HIV. While BNAbs develop spontaneously in a subset of HIV-infected patients, efforts to design an envelope protein-based immunogen to elicit broadly neutralizing antibody responses have so far been unsuccessful. It is hypothesized that a primary barrier to eliciting BNAbs is the fact that HIV envelope proteins bind poorly to the germline-encoded unmutated common ancestor (UCA) precursors to BNAbs. To identify variant forms of Env with increased affinities for the UCA forms of BNAbs 4E10 and 10E8, which target the Membrane Proximal External Region (MPER) of Env, libraries of randomly mutated Env variants were expressed in a yeast surface display system and screened using fluorescence activated cell sorting for cells displaying variants with enhanced abilities to bind the UCA antibodies. Based on analyses of individual clones obtained from the screen and on next-generation sequencing of sorted libraries, distinct but partially overlapping sets of amino acid substitutions conferring enhanced UCA antibody binding were identified. These were particularly enriched in substitutions of arginine for highly conserved tryptophan residues. The UCA-binding variants also generally exhibited enhanced binding to the mature forms of anti-MPER antibodies. Mapping of the identified substitutions into available structures of Env suggest that they may act by destabilizing both the initial pre-fusion conformation and the six-helix bundle involved in fusion of the viral and cell membranes, as well as providing new or expanded epitopes with increased accessibility for the UCA antibodies.

Metformin Associated With Increased Survival in Type 2 Diabetes Patients With Pancreatic Cancer and Lymphoma.

BACKGROUND: The biguanide drug metformin is one of the most commonly used medications for the treatment of type 2 diabetes mellitus. Diabetics are at an increased risk for cancer. Previous studies have demonstrated improved outcomes in patients taking metformin suffering from prostate, colon, lung, thyroid, and esophageal cancers. Metformin's main antineoplastic mechanism of action is thought to be mediated through inhibition of mammalian target of rapamycin, inhibition of hypoxia-inducible factor 1 (HIF-1) alpha, and activation of p53. We investigated the overall survival of type 2 diabetic patients on metformin with pancreatic cancer and lymphoma using the Computerized Patient Record System at the Veterans Affairs Medical Center, Memphis TN. METHODS: Lymphoma and pancreatic cancer patients with type 2 diabetes were sorted into an experimental (metformin) group and a control (nonmetformin) group. Patients were compared on baseline characteristics including race, body mass index, and age. Cancer outcomes including overall survival, metastasis, recurrences, and incidence of new malignancies were recorded. Hemoglobin A1C, creatinine and cancer treatment modalities were recorded and compared. Statistical analyses used included unpaired t tests and Chi-squared tests. RESULTS: There was significantly greater overall long-term survival in the metformin group compared to the nonmetformin group for lymphoma (5.89 vs 1.29 years, P < 0.001) and for pancreatic cancer (0.68 vs 0.22 years, P = 0.016). Cancer treatment modalities in both groups were comparable. CONCLUSIONS: Metformin is associated with a significant, positive effect of increased overall survival in type 2 diabetes patients with pancreatic cancer and lymphoma. These results are encouraging, and prospective studies should be done to further investigate metformin's effects in cancer.

Estimating uncertainty in predicted folding free energy changes of RNA secondary structures.

Nearest neighbor parameters for estimating the folding stability of RNA are commonly used in secondary structure prediction, for generating folding ensembles of structures, and for analyzing RNA function. Previously, we demonstrated that we could quantify the uncertainties in each nearest neighbor parameter by perturbing the underlying optical melting data within experimental error and rederiving the parameters, which accounts for the substantial correlations that exist between the parameters. In this contribution, we describe a method to estimate uncertainty in the estimated folding stabilities of RNA structures, accounting for correlations in the nearest neighbor parameters. This method is incorporated in the RNA structure software package.

Vaginal Anatomy on MRI: New Information Obtained Using Distention.

OBJECTIVES: To demonstrate and confirm the presence of three anatomic zones of the vagina (a superficial sphincteric zone; a central wedge shaped transition zone; and a deep, expanded forniceal zone) using pelvic magnetic resonance imaging with contrast distention of the vagina. METHODS: A total of 107 consecutive female pelvic magnetic resonance imaging scans using vaginal contrast distention were retrospectively reviewed. The images were observed for the three-zone configuration. Anteroposterior and transverse diameter measurements were taken in the proximal, mid, and distal sphincteric, transition, and forniceal zones. Means and standard deviations were calculated at each site. Adjacent sites were compared using paired t tests. RESULTS: The three-zone configuration was observed in all of the cases but one. Statistically significant increases and decreases of mean anteroposterior diameters occurred at all levels expected by visual observation. CONCLUSIONS: The three-zone configuration of the distended vagina was confirmed by this study. The configuration of the vagina is more complex than has been reported previously. This configuration may facilitate parturition and may be useful in the design of intravaginal devices.

Analysis of RNA nearest neighbor parameters reveals interdependencies and quantifies the uncertainty in RNA secondary structure prediction.

RNA secondary structure prediction is often used to develop hypotheses about structure-function relationships for newly discovered RNA sequences, to identify unknown functional RNAs, and to design sequences. Secondary structure prediction methods typically use a thermodynamic model that estimates the free energy change of possible structures based on a set of nearest neighbor parameters. These parameters were derived from optical melting experiments of small model oligonucleotides. This work aims to better understand the precision of structure prediction. Here, the experimental errors in optical melting experiments were propagated to errors in the derived nearest neighbor parameter values and then to errors in RNA secondary structure prediction. To perform this analysis, the optical melting experimental values were systematically perturbed within the estimates of experimental error and alternative sets of nearest neighbor parameters were then derived from these error-bounded values. Secondary structure predictions using either the perturbed or reference parameter sets were then compared. This work demonstrated that the precision of RNA secondary structure prediction is more robust than suggested by previous work based on perturbation of the nearest neighbor parameters. This robustness is due to correlations between parameters. Additionally, this work identified weaknesses in the parameter derivation that makes accurate assessment of parameter uncertainty difficult. Considerations for experimental design are provided to mitigate these weaknesses are provided.

Comparison of clinical pharmacy specialists and primary care physicians in treatment of type 2 diabetes mellitus in rural Veterans Affairs facilities.

PURPOSE: Results of a study to compare the impact of clinical pharmacy specialist (CPS) interventions with primary care physician (PCP) interventions in veterans with type 2 diabetes mellitus (T2D) in a rural setting are presented. METHODS: A retrospective analysis was performed examining veterans diagnosed with T2D with a glycosylated hemoglobin (HbA1c) of ≥8% receiving treatment at a rural community-based outpatient clinic associated with the Memphis Veterans Affairs Medical Center. Propensity score matching was used to create a 1:1 cohort of patients managed by physicians or clinical pharmacy specialists. Patients were evaluated as their own control and as compared cohorts. The primary outcome was the difference in HbA1c. Secondary outcomes included changes in total cholesterol, low-density lipoprotein cholesterol, triglycerides, and body mass index. RESULTS: Data were collected from 124 patients (n = 62 CPS patients, n = 62 PCP patients). Baseline HbA1c in the CPS and PCP groups were 10.2% ± 1.9% and 9.6% ± 1.6%, respectively. Postintervention HbA1c in the CPS cohort was 7.5% ± 1.1% (range, 6-11.7%), indicating an absolute reduction of 2.7% (p < 0.001). Postintervention HbA1c in the PCP cohort was 8.5% ± 1.5% (range, 5.4-12.6%), resulting in an absolute reduction of 1.1% (p < 0.001). The CPS intervention resulted in a greater mean HbA1c absolute reduction of 1.6 percentage points compared to physician intervention (p < 0.001). CONCLUSION: Compared with physician intervention, clinical pharmacy intervention in the treatment of T2D led to a greater mean HbA1c reduction in patients receiving care through VA facilities in rural settings.

Metformin Has Positive Therapeutic Effects in Colon Cancer and Lung Cancer.

BACKGROUND: Metformin (MF), a diabetic drug, has antineoplastic activity as adjuvant therapy for breast cancer and prostate cancer. MF is thought to work via inhibition of mammalian target of rapamycin and activation of p53 and liver kinase B1 via adenosine 5'-monophosphate-activated protein kinase. We investigated survival, recurrences and metastasis in patients with type 2 diabetes mellitus (DM2) along with colorectal cancer (CC) or lung cancer (LC) taking MF using the electronic medical record in Memphis Veterans Affairs Medical Center (colon, n = 202; lung, n = 180). MATERIALS AND METHODS: Patients with CC or LC and DM2 on MF were compared to controls taking any medication except MF. Recurrences, metastases, secondary cancers, survival and carcinoembryonic antigen levels were compared using t test and chi-squared test. Inclusion criteria were based on MF use, CC or LC diagnosis and DM2. RESULTS: For CC, the MF group noted fewer deaths (48% versus 76%, P < 0.001), recurrences (4% versus 19%, P = 0.002), metastases (23% versus 46%, P = 0.001), better 5-year survival rates (57% versus 37%, P = 0.004), overall survival years (5.7 versus 4.1, P = 0.007) and greater carcinoembryonic antigen decrease (72% versus 47%, P = 0.015). MF was associated with improved 5-year survival rates (29% versus 15%, P = 0.023) and overall survival years (3.4 versus 1.8, P < 0.001) in LC. CONCLUSIONS: Our study shows that MF therapy is associated with significantly better prognosis in patients with CC and improved survival in LC. Patients with CC on MF had fewer recurrences and metastases. Differences in metabolic pathways between CC and LC likely account for the differences in the effect of MF.

A sensitivity analysis of RNA folding nearest neighbor parameters identifies a subset of free energy parameters with the greatest impact on RNA secondary structure prediction.

Nearest neighbor parameters for estimating the folding energy changes of RNA secondary structures are used in structure prediction and analysis. Despite their widespread application, a comprehensive analysis of the impact of each parameter on the precision of calculations had not been conducted. To identify the parameters with greatest impact, a sensitivity analysis was performed on the 291 parameters that compose the 2004 version of the free energy nearest neighbor rules. Perturbed parameter sets were generated by perturbing each parameter independently. Then the effect of each individual parameter change on predicted base-pair probabilities and secondary structures as compared to the standard parameter set was observed for a set of sequences including structured ncRNA, mRNA and randomized sequences. The results identify for the first time the parameters with the greatest impact on secondary structure prediction, and the subset which should be prioritized for further study in order to improve the precision of structure prediction. In particular, bulge loop initiation, multibranch loop initiation, AU/GU internal loop closure and AU/GU helix end parameters were particularly important. An analysis of parameter usage during folding free energy calculations of stochastic samples of secondary structures revealed a correlation between parameter usage and impact on structure prediction precision.

Impact of the REACH II and REACH VA Dementia Caregiver Interventions on Healthcare Costs.

OBJECTIVE: Examine caregiver and care recipient healthcare costs associated with caregivers' participation in Resources for Enhancing Alzheimer's Caregivers Health (REACH II or REACH VA) behavioral interventions to improve coping skills and care recipient management. DESIGN: RCT (REACH II); propensity-score matched, retrospective cohort study (REACH VA). SETTING: Five community sites (REACH II); 24 VA facilities (REACH VA). PARTICIPANTS: Care recipients with Alzheimer's disease and related dementias (ADRD) and their caregivers who participated in REACH II study (analysis sample of 110 caregivers and 197 care recipients); care recipients whose caregivers participated in REACH VA and a propensity matched control group (analysis sample of 491). MEASUREMENTS: Previously collected data plus Medicare expenditures (REACH II) and VA costs plus Medicare expenditures (REACH VA). RESULTS: There was no increase in VA or Medicare expenditures for care recipients or their caregivers who participated in either REACH intervention. For VA care recipients, REACH was associated with significantly lower total VA costs of care (33.6%). VA caregiver cost data was not available. CONCLUSION: In previous research, both REACH II and REACH VA have been shown to provide benefit for dementia caregivers at a cost of less than $5/day; however, concerns about additional healthcare costs may have hindered REACH's widespread adoption. Neither REACH intervention was associated with additional healthcare costs for caregivers or patients; in fact, for VA patients, there were significantly lower healthcare costs. The VA costs savings may be related to the addition of a structured format for addressing the caregiver's role in managing complex ADRD care to an existing, integrated care system. These findings suggest that behavioral interventions are a viable mechanism to support burdened dementia caregivers without additional healthcare costs.

Effect of Clinical Pharmacist Intervention on Hemoglobin A1C Reduction in Veteran Patients With Type 2 Diabetes in a Rural Setting.

BACKGROUND: Type 2 diabetes mellitus (T2DM), if left uncontrolled, is associated with significant morbidity and mortality. Patients in rural areas may not have access to adequate resources to successfully treat diabetes. Clinical pharmacists may be utilized to bridge this gap. OBJECTIVE: To evaluate the impact of a clinical pharmacist on glycemic control in veterans with T2DM enrolled in a rural, outpatient clinic. METHODS: Retrospective chart review was performed on veterans with T2DM referred to the pharmacist-managed therapeutic monitoring clinic at a community-based outpatient clinic located in rural Jackson, TN. Patients served as their own controls. Patients with hemoglobin A1C (A1C) ≥8% were included. The primary outcome was A1C change from baseline in patients managed by the clinical pharmacist. Secondary end points included blood pressure, cholesterol, and weight. RESULTS: Of 111 veterans identified as having a A1C ≥8% in the pharmacist-managed clinic, 86 met inclusion criteria. At baseline, mean ± SD A1C was 10.5% ± 2.0% (range = 8.7%-16.2%). By the end of the intervention period, mean A1C had decreased by 2.8 percentage points to 7.7% ± 1.4% (P < 0.001). At the end of the intervention, 34% (n = 29) had a A1C of <7%, 40% (n = 34) between 7% and 7.9%, and only 6% (n = 5) >10% (P < 0.001). Improvements in diastolic blood pressure (P = 0.001), total cholesterol (P = 0.001), and triglyceride levels (P = 0.036) were also statistically significant when baseline and intervention period values were compared. CONCLUSION: Pharmacist interventions at a rural, outpatient clinic had a statistically significant impact on A1C reduction in veterans with T2DM.

Metformin Has a Positive Therapeutic Effect on Prostate Cancer in Patients With Type 2 Diabetes Mellitus.

OBJECTIVE: Prostate cancer and type 2 diabetes mellitus (DM2) are both common diseases found in the elderly male population. The diabetic drug, metformin, has been shown to have antineoplastic properties and demonstrated better treatment outcomes when used as adjuvant therapy in patients with breast cancer. The hormonally-sensitive cancer analogous to breast cancer in men is prostate cancer. We investigated improved survival, lower risks of recurrences, and lower, more stable levels of prostate-specific antigen (PSA) in patients with DM2 along with prostate cancer on metformin. METHODS: Patients with prostate cancer along with DM2 who remained on metformin were compared with controls who were not on metformin matched by age, weight, race and Gleason score cancer staging. The endpoints of our study included final PSA values, number of recurrences, metastases and number living for each group. RESULTS: There were significantly fewer deaths (23% versus 10%), fewer recurrences (15% versus 8%), fewer metastases (5% versus 0%) and fewer secondary cancers (17% versus 6%) in the metformin group (P < 0.004). The final PSA value was lower in the metformin-treated group with a result approaching significance (P = 0.067). The primary treatments for prostate cancer (ie, surgery, radiation and androgen depletion) were found to be comparable in both the groups. CONCLUSIONS: Our retrospective study shows that adjuvant metformin therapy leads to a better prognosis in prostate cancer. Not only are PSA levels controlled for several years but also there are significantly fewer cancer recurrences in metformin-treated patients. Overall, these results are promising and should be followed up with a prospective study to assess long-term survival.

Dementia Caregivers' Use of Services for Themselves.

PURPOSE OF THE STUDY: Health care and social services such as physician visits and support groups used by dementia caregivers for themselves were examined. Caregivers (N = 642) were from the Resources for Enhancing Alzheimer's Caregivers Health (REACH II) study. DESIGN AND METHODS: Caregiver predisposing, enabling, and need variables were examined using chi-squared and t-tests to characterize service users. Stagewise linear regression was used to explain numbers of services used. RESULTS: Predisposing, enabling, and need variables were significantly related to dementia caregivers' service use. In regression, caregivers who were older, more educated, married, not employed, depressed, with functional disability needs, more illness-related reduced activity days, more medications, more symptoms, and fewer hours on duty per day used significantly more services for themselves. The statistically significant model explained 22.2% variance in numbers of services used. Service users, compared with nonusers, evidenced greater burden, bother with behaviors, and more desire to institutionalize. IMPLICATIONS: This study shows that caregiver service use is related to caregiver characteristics. Future work should examine the impact of caregiving on health care and social service use and costs. The societal costs of caregiving may be better understood when we account for additional service use by caregivers themselves. A significant clinical and policy issue is who should assess and support the caregiver. Possibilities include the care recipient's health care practitioner, the caregiver's health care practitioner, or a formal caregiver-focused program based in the health care system or the social service network.

REACH VA: Moving from Translation to System Implementation.

PURPOSE OF THE STUDY: Resources for Enhancing All Caregivers Health in the Department of Veterans Affairs (REACH VA) has been implemented in the VA system as a national program for caregivers. DESIGN AND METHODS: We describe the trajectory of REACH VA from national randomized clinical trial through translation to national implementation. The implementation is examined through the six stages of the Fixsen and Blasé implementation process model: exploration and adoption, program installation, initial implementation, full operation, innovation, and sustainability. Different drivers that move the implementation process forward are important at each stage, including staff selection, staff training, consultation and coaching, staff evaluation, administrative support, program evaluation/fidelity, and systems interventions. RESULTS: Caregivers in the REACH VA 4 session intervention currently implemented in the VA had similar outcomes to longer REACH interventions, including Resources for Enhancing Alzheimer's Caregivers Health (REACH II). Caregivers experienced significant decreases in burden, depression, anxiety, number of troubling patient behaviors reported, caregiving frustrations, stress symptoms (feeling overwhelmed, feeling like crying, being frustrated as a result of caregiving, being lonely), and general stress. Effect sizes (Cohen's d) for these significant variables were between small and medium ranging from .24 to .46. IMPLICATIONS: The implementation of REACH VA provides a road map for implementation of other behavioral interventions in health care delivery settings. Lessons learned include the importance of implementing a proven, needed intervention, support from both leadership and clinical staff, willingness to respond to staff and organization needs and modify the intervention while preserving its integrity, and fitting the intervention into ongoing routines and practices.

Identification of destabilizing and stabilizing mutations of Ste2p, a G protein-coupled receptor in Saccharomyces cerevisiae.

The isolation of mutations affecting the stabilities of transmembrane proteins is useful for enhancing the suitability of proteins for structural characterization and identification of determinants of membrane protein stability. We have pursued a strategy for the identification of stabilized variants of the yeast α-factor receptor Ste2p. Because it was not possible to screen directly for mutations providing thermal stabilization, we first isolated a battery of destabilized temperature-sensitive variants, based on loss of signaling function and decreased levels of binding of the fluorescent ligand, and then screened for intragenic second-site suppressors of these phenotypes. The initial screens recovered singly and multiply substituted mutations conferring temperature sensitivity throughout the predicted transmembrane helices of the receptor. All of the singly substituted variants exhibit decreases in cell-surface expression. We then screened randomly mutagenized libraries of clones expressing temperature-sensitive variants for second-site suppressors that restore elevated levels of binding sites for fluorescent ligand. To determine whether any of these were global suppressors, and thus likely stabilizing mutations, they were combined with different temperature-sensitive mutations. Eight of the suppressors exhibited the ability to reverse the defect in ligand binding of multiple temperature-sensitive mutations. Combining certain suppressors into a single allele resulted in levels of suppression greater than that seen with either suppressor alone. Solubilized receptors containing suppressor mutations in the absence of temperature-sensitive mutations exhibit a reduced tendency to aggregate during immobilization on an affinity matrix. Several of the suppressors also exhibit allele-specific behavior indicative of specific intramolecular interactions in the receptor.