A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

Concerns about aging and caregiving among middle-aged and older lesbian and gay adults.

OBJECTIVES: Despite the increasing number of lesbian and gay older adults, research geared towards health and well-being of this population is limited. Many lesbian and gay seniors experience health disparities and are at risk for poor health outcomes. The aims of this study were to gather in-depth information on the concerns of lesbian and gay elders with respect to aging and care needs. METHOD: The sample included 124 gay men and lesbian women aged 50+ years. Data were gathered via focus groups and questionnaires. The focus groups addressed: (1) concerns about aging in the LGBT community, (2) barriers to needed support and services, (3) concerns about caregiving and (4) needed programs for lesbian and gay seniors. RESULTS: Concerns expressed about aging included: lack of financial security, lack of family or social support, fears about the lack of someone to provide needed care, and discrimination in healthcare or service communities. Participants also indicated concerns about being alone and vulnerable and a need for resources and support programs, specifically for lesbian and gay older adults and for lesbian and gay caregivers. CONCLUSIONS: These findings suggest needed areas of support and programs for older gay men and lesbian women. They also suggest that healthcare professionals might need more training regarding the particular needs and concerns of this community.

H2A.Z and H2B.Z double-variant nucleosomes define intergenic regions and dynamically occupy var gene promoters in the malaria parasite Plasmodium falciparum.

Histone variants are important components of eukaryotic chromatin and can alter chromatin structure to confer specialized functions. H2B variant histones are rare in nature but have evolved independently in the phyla Apicomplexa and Trypanasomatida. Here, we investigate the apicomplexan-specific Plasmodium falciparum histone variant Pf H2B.Z and show that within nucleosomes Pf H2B.Z dimerizes with the H2A variant Pf H2A.Z and that Pf H2B.Z and Pf H2A.Z occupancy correlates in the subset of genes examined. These double-variant nucleosomes also carry common markers of euchromatin like H3K4me3 and histone acetylation. Pf H2B.Z levels are elevated in intergenic regions across the genome, except in the var multigene family, where Pf H2A.Z/Pf H2B.Z double-variant nucleosomes are only enriched in the promoter of the single active var copy and this enrichment is developmentally regulated. Importantly, this pattern seems to be specific for var genes and does not apply to other heterochromatic gene families involved in red blood cell invasion which are also subject to clonal expression. Thus, Pf H2A.Z/Pf H2B.Z double-variant nucleosomes appear to have a highly specific function in the regulation of P. falciparum virulence.

Expression of P. falciparum var genes involves exchange of the histone variant H2A.Z at the promoter.

Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

Implementation of a Psychosocial Intervention Program for Working Caregivers.

The overall aim of this pilot study was to examine the feasibility and acceptability of a multicomponent, psychosocial intervention specifically designed to meet the unique needs of caregivers who are balancing caregiving duties with work responsibilities. Seventy-one family caregivers employed at a private, nonprofit institution in South Florida were randomized to either the Caregiver Workstation condition (n = 35) or a control condition (n = 36). Sixty-two caregivers completed the 5-month follow-up. Our results indicate that an intervention tailored to the time demands of a working caregiver is feasible, acceptable to caregivers, and has the potential to have positive long-term effects. Currently, there are limited data available regarding the benefits of employer programs for caregivers or the type of programs caregivers find most useful. This pilot study is the first step in developing a working caregiver intervention program that can be implemented on a broad-scale basis.

Attitudes Toward Computers Across Adulthood From 1994 to 2013.

Background and Objectives: Regardless of the increased deployment of technologies in everyday living domains, barriers remain that hamper technology adoption by older adults. Understanding barriers to adoption such as individual differences in attitudes toward computers is important to the design of strategies to reduce age-related digital disparities. Research Design and Methods: This article reports a time-sequential analysis of data from the Edward R. Roybal Center on Human Factors and Aging Research and the Center for Research and Education on Aging and Technology Enhancement (CREATE) on computer attitudes among a large (N = 3,917), diverse sample of community-dwelling adults aged from 18 to 98 years. The data were gathered from 1994 to 2013. Results: The findings indicated that there are still age disparities in attitudes; older adults report less comfort with and less efficacy about using computers than younger people. We also found a cohort (birth year) effect; attitudes are generally more positive among more recent birth cohorts. Those who have more education and experience with computers also have more positive attitudes. Males generally have more positive attitudes than females; however, the gender difference decreases with increased age. Discussion and Implications: Technology affords potential benefits for older people, but lack of uptake in technology clearly puts older adults at a disadvantage in terms of negotiating today's digital world. This article provides insight into attitudinal barriers that may affect on technology uptake among older adults. The findings have implications for the design of technology training programs, design of technology systems, and policy.

Community REACH: An Implementation of an Evidence-Based Caregiver Program.

Background and Objectives: Family caregivers (CGs) are critical to the provision of long-term services and support for older adults. Numerous intervention programs to alleviate CG distress have been developed and evaluated yet few have been implemented in community settings. This paper describes and presents outcomes from Community REACH, a community implementation of the evidence-based Resources for Enhancing Alzheimer's Caregiver Health (REACH) II program. Research Design and Methods: Community REACH involved a partnership between REACH II investigators and United HomeCare Services (UHCS), a nonprofit home health organization that provides home health, personal care, companion, and respite services. The intervention program, an adapted version of an evidence-based program, was a 6-month multicomponent psychosocial intervention, which involved six individual face-to-face and six individual telephone sessions, and telephone support groups. One hundred and forty-six CGs who were primarily female (76%) and Latino, and providing care for an individual with Alzheimer's disease (AD) were enrolled. Program effectiveness was assessed by examining changes in perceived social support, burden, and depression, and CG self-efficacy. Results: At 6 months, CGs reported significant decreases in depression, burden, being and bothered by the care recipient's memory problems. There was also a significant decline in the number of CGs at risk for clinical depression. These improvements were maintained at 12 months and there was an increase in feelings of social support. Discussion and Implications: The findings indicate that evidence-based CG programs can be successfully implemented in community settings and benefit CGs of AD patients. A continued partnership between the program developers and community partners is key to implementation success.

Exploring Older Adults' Video Game Use in the PRISM Computer System.

BACKGROUND AND OBJECTIVE: As part of the PRISM (Personal Reminder Information & Social Management) randomized field trial, a large group of older adults (N = 150) received a computer system in their home that presented them with the opportunity to play eleven different video games. While researchers have often assessed older adults' gaming preferences and habits through survey data and focus groups, this trial represented a unique opportunity to study gaming behavior "in the wild" over an entire year. RESEARCH DESIGN AND METHODS: We present an exploration of game usage data, individual differences in game preferences and gaming habits, and individual difference predictors of game use. RESULTS: Although few individual difference variables consistently predicted game use and preferences, there were clear favorites among the different games, and results demonstrate that given the opportunity and training many older adults may become active and long-term gamers. DISCUSSION AND IMPLICATIONS: Findings have implications for designing video games that older adults enjoy, supporting enjoyable and meaningful interactions with video games across the life span, and for designing cognitive, social, and health interventions involving games.

Enhancing the quality of life of dementia caregivers from different ethnic or racial groups: a randomized, controlled trial.

BACKGROUND: Caring for a family member with dementia is extremely stressful, contributes to psychiatric and physical illness among caregivers, and increases the risk for caregiver death. Finding better ways to support family caregivers is a major public health challenge. OBJECTIVE: To test the effects of a structured multicomponent intervention on quality of life and clinical depression in caregivers and on rates of institutional placement of care recipients in 3 diverse racial or ethnic groups. DESIGN: Randomized, controlled trial. SETTING: In-home caregivers in 5 U.S. cities. PARTICIPANTS: 212 Hispanic or Latino, 219 white or Caucasian, and 211 black or African-American caregivers and their care recipients with Alzheimer disease or related disorders. INTERVENTION: Caregivers within each racial or ethnic group were randomly assigned to an intervention or to a control group. The intervention addressed caregiver depression, burden, self-care, and social support and care recipient problem behaviors through 12 in-home and telephone sessions over 6 months. Caregivers in the control group received 2 brief "check-in" telephone calls during the 6-month intervention. MEASUREMENTS: The primary outcome was a quality-of-life indicator comprising measures of 6-month caregiver depression, burden, self-care, and social support and care recipient problem behaviors. Secondary outcomes were caregiver clinical depression and institutional placement of the care recipient at 6 months. RESULTS: Hispanic or Latino and white or Caucasian caregivers in the intervention group experienced significantly greater improvement in quality of life than those in the control group (P < 0.001 and P = 0.037, respectively). Black or African-American spouse caregivers also improved significantly more (P = 0.003). Prevalence of clinical depression was lower among caregivers in the intervention group (12.6% vs. 22.7%; P = 0.001). There were no statistically significant differences in institutionalization at 6 months. LIMITATIONS: The study used only a single 6-month follow-up assessment, combined heterogeneous cultures and ethnicities into a single group, and excluded some ethnic groups. CONCLUSIONS: A structured multicomponent intervention adapted to individual risk profiles can increase the quality of life of ethnically diverse dementia caregivers. ClinicalTrials.gov identifier: NCT00177489.

Assessing functional performance using computer-based simulations of everyday activities.

Current functional capacity (FC) measures for patients with schizophrenia typically involve informant assessments or are in paper and pencil format, requiring in-person administration by a skilled assessor. This approach presents logistic problems and limits the possibilities for remote assessment, an important issue for these patients. This study evaluated the feasibility of using a computer-based assessment battery, including simulations of everyday activities. The battery was compared to in-person standard assessments of cognition and FC with respect to baseline convergence and sensitivity to group differences. The battery, administered on a touch screen computer, included measures of critical everyday activities, including: ATM Banking/Financial Management, Prescriptions Refill via Telephone/Voice Menu System, and Forms Completion (simulating a clinic and patient history form). The sample included 77 older adult patients with schizophrenia and 24 older adult healthy controls that were administered the battery at two time points. The results indicated that the battery was sensitive to group differences in FC. Performance on the battery was also moderately correlated with standard measures of cognitive abilities and showed convergence with standard measures of FC, while demonstrating good test-retest reliability. Our results show that it is feasible to use technology-based assessment protocols with older adults and patients with schizophrenia. The battery overcomes logistic constraints associated with current FC assessment protocols as the battery is computer-based, can be delivered remotely and does not require a healthcare professional for administration.

The usability of electronic personal health record systems for an underserved adult population.

OBJECTIVE: The goals of this study were to identify the demands associated with using electronic personal health records (PHRs) and to evaluate the ability of adults of lower socioeconomic status and low health literacy to use PHRs to perform health management activities. BACKGROUND: PHRs are proliferating in clinical practices and health care organizations. These systems offer the potential of increasing the active involvement of patients in health self-management. However, little is known about the actual usability of these tools for health consumers. METHOD: We used task analysis and health literacy load analysis to identify the cognitive and literacy demands inherent in the use of PHRs and evaluated the usability of three currently available PHR systems with a sample of 54 adults. Participants used the systems to perform tasks related to medication management, interpretation of lab/test results, and health maintenance. Data were also gathered on the participants' perception of the potential value of using a PHR. RESULTS: The results indicated that a majority of the participants had difficulty completing the tasks and needed assistance. There was some variability according to task and PHR system. However, most participants perceived the use of PHRs as valuable. CONCLUSIONS: Although considered a valuable tool by consumers, the use of PHR systems may be challenging for many people. Strategies are needed to enhance the usability of these systems, especially for people with low literacy, low health literacy, or limited technology skills. APPLICATION: The data from this study have implications for the design of PHRs.

Computer proficiency questionnaire: assessing low and high computer proficient seniors.

PURPOSE OF THE STUDY: Computers and the Internet have the potential to enrich the lives of seniors and aid in the performance of important tasks required for independent living. A prerequisite for reaping these benefits is having the skills needed to use these systems, which is highly dependent on proper training. One prerequisite for efficient and effective training is being able to gauge current levels of proficiency. We developed a new measure (the Computer Proficiency Questionnaire, or CPQ) to measure computer proficiency in the domains of computer basics, printing, communication, Internet, calendaring software, and multimedia use. Our aim was to develop a measure appropriate for individuals with a wide range of proficiencies from noncomputer users to extremely skilled users. DESIGN AND METHODS: To assess the reliability and validity of the CPQ, a diverse sample of older adults, including 276 older adults with no or minimal computer experience, was recruited and asked to complete the CPQ. RESULTS: The CPQ demonstrated excellent reliability (Cronbach's α = .98), with subscale reliabilities ranging from .86 to .97. Age, computer use, and general technology use all predicted CPQ scores. Factor analysis revealed three main factors of proficiency related to Internet and e-mail use; communication and calendaring; and computer basics. Based on our findings, we also developed a short-form CPQ (CPQ-12) with similar properties but 21 fewer questions. IMPLICATIONS: The CPQ and CPQ-12 are useful tools to gauge computer proficiency for training and research purposes, even among low computer proficient older adults.

The personalized reminder information and social management system (PRISM) trial: rationale, methods and baseline characteristics.

Technology holds promise in terms of providing support to older adults. To date, there have been limited robust systematic efforts to evaluate the psychosocial benefits of technology for older people and identify factors that influence both the usability and uptake of technology systems. In response to these issues, we developed the Personal Reminder Information and Social Management System (PRISM), a software application designed for older adults to support social connectivity, memory, knowledge about topics, leisure activities and access to resources. This trail is evaluating the impact of access to the PRISM system on outcomes such as social isolation, social support and connectivity. This paper reports on the approach used to design the PRISM system, study design, methodology and baseline data for the trial. The trial is multi-site randomized field trial. PRISM is being compared to a Binder condition where participants received a binder that contained content similar to that found on PRISM. The sample includes 300 older adults, aged 65-98 years, who lived alone and at risk for being isolated. The primary outcome measures for the trial include indices of social isolation and support and well-being. Secondary outcomes measures include indices of computer proficiency, technology uptake and attitudes towards technology. Follow-up assessments occurred at 6 and 12 months post-randomization. The results of this study will yield important information about the potential value of technology for older adults. The study also demonstrates how a user-centered iterative design approach can be incorporated into the design and evaluation of an intervention protocol.

A methodology for describing and decomposing complex psychosocial and behavioral interventions.

This article presents a comprehensive conceptual framework and methodology for characterizing multidimensional psychosocial and behavioral interventions for family caregivers of people with Alzheimer's disease. The methodology involved conducting a detailed task analysis of the interventions, and the application of the analytical hierarchy process. The authors were able to achieve high levels of consistency and reliability using this methodology to decompose and describe the interventions according to a complex conceptual framework comprising 3 dimensions: target domain, functional domain, and delivery system characteristics. Application of this methodology will enhance the ability to describe and measure interventions consistently, make comparisons across studies, isolate causal factors that achieve desired outcomes, and more generally elevate the science of psychosocial intervention research.

An evaluation of performance by older persons on a simulated telecommuting task.

Telecommuting work represents a strategy for managing the growing number of older people in the workforce. This study involved a simulated customer service telecommuting task that used e-mail to answer customer queries about media-related products and company policies. Participants included 27 "younger" older adults (50-65 years) and 25 "older" older adults (66-80 years). The participants performed the task for two 2-hr sessions a day over 4 consecutive days. Although both age groups showed significant improvement across sessions on many of the performance criteria, in general the improvements were more marked for the older age-group participants. However, the participants from both age groups had difficulty meeting some of the task performance requirements. These results are discussed in terms of training strategies for older workers.

A cost-benefit analysis methodology for assessing product adoption by older user populations.

A methodology for assessing perceptions of a product through cost-benefit analysis is demonstrated in an experimental study involving use of one of two products for supporting interaction with telephone voice menu systems. The method emphasizes ratings of constructs related to costs and benefits considered relevant to decisions regarding product adoption, and the use of the analytic hierarchy process technique to derive subjective importance weights associated with those constructs. Twenty-five younger subjects (18-39 years of age) and 27 older subjects (60-82 years of age) interacted with simulated telephone voice menu systems supported by either a screen phone or a graphical device. Although analysis of the benefit-cost ratios revealed that benefits were perceived as more important than costs, the analysis of the constituent item ratings and importance weights provided potentially more useful information for predicting product adoption.

Use of a telehealth system by older adults with hypertension.

We conducted a feasibility study of a home telehealth system that monitored blood pressure and bodyweight. A total of 34 participants with hypertension (10 males and 24 females) had the telemedicine system installed in their homes. Their mean age was 72 years and 94% of them self-identified as Hispanic. The telehealth system transmitted blood pressure and bodyweight data to a server. There was also a messaging function that was used to send a daily reminder to the participants. Participants used the telehealth system for six months. Ten participants were lost to follow up and 24 participants completed the entire study. Participants had strong positive perceptions regarding the usability and usefulness of the telemedicine system. Most of them (92%) found the device easy to use and 96% felt that the training they received prepared them to use the device. The providers indicated that use of the system improved their ability to manage their patients. The results of the study suggest that use of the telehealth system for home monitoring in Hispanic patients with hypertension is feasible.

Identification of dengue-specific human antibody fragments using phage display.

High-affinity antibodies are valuable tools for dengue research. A method for the selection of dengue-specific, human antibody fragments using naïve repertoires displayed on M13 filamentous bacteriophage is described. Naïve repertoires are unbiased, thus enabling the identification of antibodies to dengue structural and nonstructural proteins from the same library. Dengue-specific clones are enriched by binding to an immobilized dengue antigen, followed by washing, elution, and amplification of phage for subsequent rounds of selection. Dengue virus has four antigenically related serotypes, and the serotype of the antigen can be kept constant or alternated during the selection process depending on whether serotype-specific or cross-reactive antibodies are required. After the selection process, clones are screened, and specific clones are identified by phage ELISA and Western blot.

Identification and molecular characterization of human antibody fragments specific for dengue NS5 protein.

The multifunctional dengue nonstructural (NS) protein 5 from the four serotypes of dengue virus (DENV1-4) is essential for viral replication and harbors a methyl transferase (MTase) and a RNA-dependent RNA-polymerase domain (RdRp). There are limited comparative studies of NS5 from the four DENV serotypes and this is further hampered by a lack of cross-reactive NS5 antibodies. In this study, recombinant NS5 proteins were expressed, purified, enzymatically characterized, and used strategically as bait in biopanning experiments with a naïve human Fab phage-display library to identify serotype specific or cross-reactive Fab fragments. Using a combination of peptide competition ELISA and peptide phage display the epitopes of the cross-reactive Fabs were mapped to the first alpha helix of the MTase domain (5M1) and the priming loop of the RdRp domain (5R3). The epitope of a third, serotype-specific Fab (5M3) was mapped to aa19-30 of the DENV3 MTase domain. Together the recombinant proteins and specific antibodies will facilitate further mechanistic studies of the DENV replication complex.

Factors influencing use of an e-health website in a community sample of older adults.

OBJECTIVE: The use of the internet as a source of health information and link to healthcare services has raised concerns about the ability of consumers, especially vulnerable populations such as older adults, to access these applications. This study examined the influence of training on the ability of adults (aged 45+ years) to use the Medicare.gov website to solve problems related to health management. The influence of computer experience and cognitive abilities on performance was also examined. DESIGN: Seventy-one participants, aged 47-92, were randomized into a Multimedia training, Unimodal training, or Cold Start condition and completed three healthcare management problems. MEASUREMENT AND ANALYSES: Computer/internet experience was measured via questionnaire, and cognitive abilities were assessed using standard neuropsychological tests. Performance metrics included measures of navigation, accuracy and efficiency. Data were analyzed using analysis of variance, χ(2) and regression techniques. RESULTS: The data indicate that there was no difference among the three conditions on measures of accuracy, efficiency, or navigation. However, results of the regression analyses showed that, overall, people who received training performed better on the tasks, as evidenced by greater accuracy and efficiency. Performance was also significantly influenced by prior computer experience and cognitive abilities. Participants with more computer experience and higher cognitive abilities performed better. CONCLUSIONS: The findings indicate that training, experience, and abilities are important when using complex health websites. However, training alone is not sufficient. The complexity of web content needs to be considered to ensure successful use of these websites by those with lower abilities.