Structural changes of TasA in biofilm formation of Bacillus subtilis.

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, ß-sheet-rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level.

"I don't believe it." Acceptance and skepticism of genetic health information among African-American and White smokers.

RATIONALE: Effective translation of genomics research into practice depends on public acceptance of genomics-related health information. OBJECTIVE: To explore how smokers come to accept or reject information about the relationship between genetics and nicotine addiction. METHODS: Thirteen focus groups (N = 84) were stratified by education (seven < Bachelor's degree, six ≥ Bachelor's degree) and race (eight black, five white). Participants viewed a 1-min video describing the discovery of a genetic variant associated with increased risk of nicotine addiction and lung cancer. Next, they provided their opinions about the information. Two coders analyzed the data using grounded theory. RESULTS: Pre-video knowledge about why people smoke cigarettes and what genetic risk means informed beliefs about the relationship between genes and addiction. These beliefs were not always consistent with biomedical explanations, but formed the context through which participants processed the video's information. This, in turn, led to information acceptance or skepticism. Participants explained their reactions in terms of the scientific merits of the research and used their existing knowledge and beliefs to explain their acceptance of or skepticism about the information. CONCLUSION: Laypeople hold complex understandings of genetics and addiction. However, when lay and biomedical explanations diverge, genetics-related health information may be rejected.

Using an Internet-Based Breast Cancer Risk Assessment Tool to Improve Social-Cognitive Precursors of Physical Activity.

BACKGROUND: Internet-based cancer risk assessment tools might serve as a strategy for translating epidemiological risk prediction research into public health practice. Understanding how such tools affect key social-cognitive precursors of behavior change is crucial for leveraging their potential into effective interventions. PURPOSE: To test the effects of a publicly available, Internet-based, breast cancer risk assessment tool on social-cognitive precursors of physical activity. METHODS: Women (N = 132) aged 40-78 with no personal cancer history indicated their perceived risk of breast cancer and were randomly assigned to receive personalized ( www.yourdiseaserisk.wustl.edu ) or nonpersonalized breast cancer risk information. Immediately thereafter, breast cancer risk perceptions and physical activity-related behavioral intentions, self-efficacy, and response efficacy were assessed. RESULTS: Personalized information elicited higher intentions, self-efficacy, and response efficacy than nonpersonalized information, P values < 0.05. Self-efficacy and response efficacy mediated the effect of personalizing information on intentions. Women who received personalized information corrected their inaccurate risk perceptions to some extent, P values < 0.05, but few fully accepted the information. CONCLUSION: Internet-based risk assessment tools can produce beneficial effects on important social-cognitive precursors of behavior change, but lingering skepticism, possibly due to defensive processing, needs to be addressed before the effects can be maximized.

Smokers' beliefs about the tobacco control potential of "a gene for smoking": a focus group study.

BACKGROUND: Several genetic variations associated with nicotine dependence and lung cancer exist. Translating this knowledge into tobacco control policy relies on smokers' perceptions of the implications of the research. This study explored smokers' beliefs about the tobacco control uses for research examining genomics, smoking, and addiction. METHOD: Smokers (N = 85) participated in one of thirteen focus groups and one interview, stratified by race (eight black, six white) and education (seven < Bachelor's degree, seven ≥ Bachelor's degree). Data were analyzed by two independent coders using standard analysis and validation techniques. RESULTS: Nearly all groups suggested using genetic information for youth-oriented tobacco prevention education. Beliefs about the effectiveness of such actions varied. Many participants believed that providing smokers personalized genetic testing results or informing them about the existence of a gene would not motivate people to quit. All smokers emphasized the need for improved smoking cessation treatment options. Using genomics research to develop gene therapies and personalized drug treatments were also mentioned, yet perceptions of such treatments were mixed. Whereas some participants viewed the possibility positively, others expressed concern about cost and access. Participants who were skeptical of the effectiveness of using genetic information for tobacco control noted that the harms of tobacco use are widely known and genetic information does not add much of a deterrent. CONCLUSION: Participants generated several possible tobacco control uses for genomics research findings. Our findings suggest that tobacco control experts should consult with smokers prior to implementing tobacco control measures. The potential public health benefits of genetics and genomics research related to tobacco use cannot be realized until communication strategies that are most likely to encourage and support tobacco avoidance decisions, and minimize mistrust and backlash, are identified.

Structural insights into the activation of the RhoA GTPase by the lymphoid blast crisis (Lbc) oncoprotein.

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and (1)H,(13)C,(15)N-based NMR spectroscopy. The two domains assume a "chair" shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.

The Nursing Home Falls Self-Efficacy Scale: development and testing.

We examined a brief measure of falls self-efficacy in nursing home residents participating in a pilot randomized controlled trial to study the effects of hip protectors on the prevention of fractures (N = 116, mean age 82 ± 8, 72% female). Internal consistency reliability was acceptable with Cronbach's alpha of .79. Factor analysis supported two factors representing self-efficacy expectations and outcome expectancy. Contrasted groups comparisons and construct validity were examined. We found lower falls self-efficacy in participants who needed help with mobility, in people with lower executive function, and in participants who reported fear of falling. Scores were not associated with prospective falls or adherence with hip protector use. The findings of this study provide preliminary support for the reliability and validity of the scale for future research.

A qualitative exploration of a health trainer programme in two UK primary care trusts.

AIMS: World Health Organization data illustrate a worldwide re-emergence of interest in the scope of lay health workers for extending services to 'hard-to-reach' community groups. In the UK, the health trainer model of service delivery represents one such innovative way of working, first described in the White Paper Choosing Health: Making Healthier Choices Easier and more recently in the Kings Fund report. The scheme was introduced into selected primary care settings in England from 2005 and rolled out nationally from 2007. The aim of this study was to examine the perceived value of the health trainer scheme. METHODS: This paper describes qualitative data from two studies undertaken in 2007-2009, comprising in-depth consultations with key primary care stakeholders, health trainers and their clients in two primary care trusts in northern and central England. Data was collected via 12 semi-structured interviews with key stakeholders and service users and from 8 focus groups with a total of 33 trainee and qualified health trainers. RESULTS: The UK health trainer approach was regarded as effective in contributing to the support of a broad spectrum of health and welfare issues across widely diverse communities in the two primary care trusts evaluated. Study data also indicated a wide-ranging impact of the health trainer service, extending not only to the lay health workers themselves, but also to their families, friends and colleagues. CONCLUSIONS: The health trainer service appears to be not only 'fit for purpose', but also to bring with it certain 'added value', which was not predicted by the two primary care service providers at the outset. A critical factor in this success appears to be the unique combination of time, the 'person next door' and a 'one-to-one' approach, which facilitated an innovative and highly productive connection between the health trainer and client. However, participants in this evaluation perceived that the current format and constituents of service performance data were significantly failing to credit the health trainer scheme with the full extent of this impact.

Resonance assignments of the human AKAP13-PH domain and stabilizing DH helix.

The human AKAP13 protein contains DH and PH domains, which are responsible for its cell transforming activity. Despite its biomedical importance, the contribution of the PH domain to AKAP13 activity remains unclear and no three dimensional structure is available to date. Here we report the backbone and side chain (1)H, (13)C and (15)N resonance assignments of a 20 kDa construct comprising the uniformly (13)C and( 15)N labeled AKAP13-PH domain and an associated helix from the DH domain which is required for its stable expression. Resonance assignment has been achieved using conventional triple resonance experiments; 95% of all back bone resonances and more than 90% of side chain resonances have been successfully assigned. The (1)H, (13)C and (15)N chemical shifts have been deposited in BMRB with accession number of 16195.

The Hip Impact Protection Project: design and methods.

BACKGROUND: Nearly 340,000 hip fractures occur each year in the U.S. With current demographic trends, the number of hip fractures is expected to double at least in the next 40 years. PURPOSE: The Hip Impact Protection Project (HIP PRO) was designed to investigate the efficacy and safety of hip protectors in an elderly nursing home population. This paper describes the innovative clustered matched-pair research design used in HIP PRO to overcome the inherent limitations of clustered randomization. METHODS: Three clinical centers recruited 37 nursing homes to participate in HIP PRO. They were randomized so that the participating residents in that home received hip protectors for either the right or left hip. Informed consent was obtained from either the resident or the resident's responsible party. The target sample size was 580 residents with replacement if they dropped out, had a hip fracture, or died. One of the advantages of the HIP PRO study design was that each resident was his/her own case and control, eliminating imbalances, and there was no confusion over which residents wore pads (or on which hip). LIMITATIONS: Generalizability of the findings may be limited. Adherence was higher in this study than in other studies because of: (1) the use of a run-in period, (2) staff incentives, and (3) the frequency of adherence assessments. The use of a single pad is not analogous to pad use in the real world and may have caused unanticipated changes in behavior. Fall assessment was not feasible, limiting the ability to analyze fractures as a function of falls. Finally, hip protector designs continue to evolve so that the results generated using this pad may not be applicable to other pad designs. However, information about factors related to adherence will be useful for future studies. CONCLUSIONS: The clustered matched-pair study design avoided the major problem with previous cluster-randomized investigations of this question - unbalanced risk factors between the experimental group and the control group. Because each resident served as his/her own control, the effects of unbalanced risk factors on treatment effect were virtually eliminated. In addition, the use of frequent adherence assessments allowed us to study the effect of various demographic and environmental factors on adherence, which was vital for the assessment of efficacy.

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.

Backbone and sidechain 1H, 13C and 15N resonance assignments of the Bright/ARID domain from the human JARID1C (SMCX) protein.

We have assigned 1H, 13C and 15N resonances of the Bright/ARID DNA-binding domain from the human JARID1C protein, a newly discovered histone demethylase belonging to the JmjC domain-containing protein family.

The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins.

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.

Efficacy of a hip protector to prevent hip fracture in nursing home residents: the HIP PRO randomized controlled trial.

CONTEXT: Past studies of the efficacy of hip protectors to prevent hip fracture in nursing home residents have had conflicting results, possibly due to potential biases from clustered randomization designs and modest adherence to intervention. OBJECTIVE: To determine whether an energy-absorbing and energy-dispersing hip protector would reduce the risk of hip fracture when worn by nursing home residents. DESIGN, SETTING, AND PARTICIPANTS: Multicenter, randomized controlled clinical trial in which 37 nursing homes were randomly assigned to having residents wear a 1-sided hip protector on the left or right hip. Participants were 1042 nursing home residents (mean [SD] aged 85 [7] years; 79% women) who consented and adhered to the hip protector use during a 2-week run-in period and were enrolled. Participating facilities were in greater Boston, Massachusetts, St Louis, Missouri, and Baltimore, Maryland from October 2002 to October 2004. Mean duration of participation for nursing home residents was 7.8 months. None were withdrawn because of adverse effects. INTERVENTION(S): Undergarments with a 1-sided hip protector made of a 0.32-cm outer layer of polyethylene (2.7 kg/m3) backed by a hard high-density polyethylene shield (0.95 cm) that was backed by 0.9 kg/m3 of 1.27-kg ethylene vinyl acetate foam. Each facility was visited 3 times per week to assess adherence and provide staff support. MAIN OUTCOME MEASURE: Adjudicated hip fracture occurrences on padded vs unpadded hips. RESULTS: After a 20-month follow-up (676 person-years of observation), the study was terminated due to a lack of efficacy. The incidence rate of hip fracture on protected vs unprotected hips did not differ (3.1%; 95% confidence interval [CI], 1.8%-4.4% vs 2.5%; 95% CI, 1.3%-3.7%; P = .70). For the 334 nursing home residents with greater than 80% adherence to hip protector use, the incidence rate of hip fracture on protected vs unprotected hips did not differ (5.3%; 95% CI, 2.6%-8.8% vs 3.5%; 95% CI, 1.3%-5.7%; P = .42). Overall adherence was 73.8%. CONCLUSIONS: In this clinical trial of an energy-absorbing/shunting hip protector conducted in US nursing homes, we were unable to detect a protective effect on the risk of hip fracture, despite good adherence to protocol. These results add to the increasing body of evidence that hip protectors, as currently designed, are not effective for preventing hip fracture among nursing home residents. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00058864.

Backbone and sidechain 1H, 13C and 15N resonance assignments of the RGS domain from human RGS14.

We have assigned 1H, 13C and 15N resonances of the RGS domain from the human RGS14 protein, a multi-domain member of the RGS (Regulators of G-protein signalling) family of proteins, important in the down-regulation of specific G-protein signalling pathways.

Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination.

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.

Geriatric syndromes in elderly patients admitted to an oncology-acute care for elders unit.

PURPOSE: The goal of this study was to characterize an elderly population admitted to a novel Oncology-Acute Care for Elders (OACE) unit, determine the prevalence of functional dependencies and geriatric syndromes, and examine their suitability for an interdisciplinary model of care. PATIENTS AND METHODS: We conducted a retrospective review of 119 patients age 65 years or older who had a primary oncologic or hematologic diagnosis and were admitted to the OACE Unit. Standard geriatric screens were administered to assess mood, functional, and cognitive status. Demographic and medical data were compiled by review of patients' medical records. RESULTS: The mean age of the patients was 74.1 years (standard deviation, 5.9 years). The sample was predominantly white, of equal sex, had limitations in instrumental and basic activities of daily living, and a mean length of stay of 6 days. Geriatric syndromes detected by the OACE interdisciplinary team included cognitive impairment (dementia and/or delirium), depression, weight loss, and use of high-risk medications. Adverse events such as falls, restraint use, and pressure sores were rare. CONCLUSION: In this descriptive study, many older cancer patients were found to have geriatric syndromes by the OACE team and these patients were considered appropriate for an interdisciplinary model of care. Additional studies are needed to compare the outcomes of hospitalized older oncology patients receiving an OACE intervention with those patients receiving usual care.

Structural and biochemical study of effector molecule recognition by the E.coli glyoxylate and allantoin utilization regulatory protein AllR.

The interaction of Escherichia coli AllR regulator with operator DNA is disrupted by the effector molecule glyoxylate. This is a general, yet uncharacterized regulatory mechanism for the large IclR family of transcriptional regulators to which AllR belongs. The crystal structures of the C-terminal effector-binding domain of AllR regulator and its complex with glyoxylate were determined at 1.7 and 1.8 A, respectively. Residues involved in glyoxylate binding were explored in vitro and in vivo. Altering the residues Cys217, Ser234 and Ser236 resulted in glyoxylate-independent repression by AllR. Sequence analysis revealed low conservation of amino acid residues participating in effector binding among IclR regulators, which reflects potential chemical diversity of effector molecules, recognized by members of this family. Comparing the AllR structure to that of Thermotoga maritima TM0065, the other representative of the IclR family that has been structurally characterized, indicates that both proteins assume similar quaternary structures as a dimer of dimers. Mutations in the tetramerization region, which in AllR involve the Cys135-Cys142 region, resulted in dissociation of AllR tetramer to dimers in vitro and were functionally inactive in vivo. Glyoxylate does not appear to function through the inhibition of tetramerization. Using sedimentation velocity, glyoxylate was shown to conformationally change the AllR tetramer as well as monomer and dimer resulting in altered outline of AllR molecules.