Structural Diversity of the Active N-Terminal Kinase Domain of p90 Ribosomal S6 Kinase 2

The p90 ribosomal protein kinase 2 (RSK2) is a highly expressed Ser/Thr kinase activated by growth factors and is involved in cancer cell proliferation and tumor promoter-induced cell transformation. RSK2 possesses two non-identical kinase domains, and the structure of its N-terminal domain (NTD), which is responsible for phosphorylation of a variety of substrates, is unknown. The crystal structure of the NTD RSK2 was determined at 1.8 Å resolution in complex with AMP-PNP. The N-terminal kinase domain adopted a unique active conformation showing a significant structural diversity of the kinase domain compared to other kinases. The NTD RSK2 possesses a three-stranded βB-sheet inserted in the N-terminal lobe, resulting in displacement of the αC-helix and disruption of the Lys-Glu interaction, classifying the kinase conformation as inactive. The purified protein was phosphorylated at Ser227 in the T-activation loop and exhibited in vitro kinase activity. A key characteristic is the appearance of a new contact between Lys216 (βB-sheet) and the β-phosphate of AMP-PNP. Mutation of this lysine to alanine impaired both NTDs in vitro and full length RSK2 ex vivo activity, emphasizing the importance of this interaction. Even though the N-terminal lobe undergoes structural re-arrangement, it possesses an intact hydrophobic groove formed between the αC-helix, the β4-strand, and the βB-sheet junction, which is occupied by the N-terminal tail. The presence of a unique βB-sheet insert in the N-lobe suggests a different type of activation mechanism for RSK2

Proactive and integrated primary care for frail older people: design and methodological challenges of the Utrecht primary care PROactive frailty intervention trial (U-PROFIT)

<p>Abstract</p> <p>Background</p> <p>Currently, primary care for frail older people is reactive, time consuming and does not meet patients' needs. A transition is needed towards proactive and integrated care, so that daily functioning and a good quality of life can be preserved. To work towards these goals, two interventions were developed to enhance the care of frail older patients in general practice: a screening and monitoring intervention using routine healthcare data (U-PRIM) and a nurse-led multidisciplinary intervention program (U-CARE). The U-PROFIT trial was designed to evaluate the effectiveness of these interventions. The aim of this paper is to describe the U-PROFIT trial design and to discuss methodological issues and challenges.</p> <p>Methods/Design</p> <p>The effectiveness of U-PRIM and U-CARE is being tested in a three-armed, cluster randomized trial in 58 general practices in the Netherlands, with approximately 5000 elderly individuals expected to participate. The primary outcome is the effect on activities of daily living as measured with the Katz ADL index. Secondary outcomes are quality of life, mortality, nursing home admission, emergency department and out-of-hours General Practice (GP), surgery visits, and caregiver burden.</p> <p>Discussion</p> <p>In a large, pragmatic trial conducted in daily clinical practice with frail older patients, several challenges and methodological issues will occur. Recruitment and retention of patients and feasibility of the interventions are important issues. To enable broad generalizability of results, careful choices of the design and outcome measures are required. Taking this into account, the U-PROFIT trial aims to provide robust evidence for a structured and integrated approach to provide care for frail older people in primary care.</p> <p>Trial registration</p> <p><a href="http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2288">NTR2288</a></p

Internet Usage by Low-Literacy Adults Seeking Health Information: An Observational Analysis

BACKGROUND: Adults with low literacy may encounter informational obstacles on the Internet when searching for health information, in part because most health Web sites require at least a high-school reading proficiency for optimal access. OBJECTIVE: The purpose of this study was to 1) determine how low-literacy adults independently access and evaluate health information on the Internet, 2) identify challenges and areas of proficiency in the Internet-searching skills of low-literacy adults. METHODS: Subjects (n=8) were enrolled in a reading assistance program at Bidwell Training Center in Pittsburgh, PA, and read at a 3rd to 8th grade level. Subjects conducted self-directed Internet searches for designated health topics while utilizing a think-aloud protocol. Subjects' keystrokes and comments were recorded using Camtasia Studio screen-capture software. The search terms used to find health information, the amount of time spent on each Web site, the number of Web sites accessed, the reading level of Web sites accessed, and the responses of subjects to questionnaires were assessed. RESULTS: Subjects collectively answered 8 out of 24 questions correctly. Seven out of 8 subjects selected "sponsored sites"-paid Web advertisements-over search engine-generated links when answering health questions. On average, subjects accessed health Web sites written at or above a 10th grade reading level. Standard methodologies used for measuring health literacy and for promoting subjects to verbalize responses to Web-site form and content had limited utility in this population. CONCLUSION: This study demonstrates that Web health information requires a reading level that prohibits optimal access by some low-literacy adults. These results highlight the low-literacy adult population as a potential audience for Web health information, and indicate some areas of difficulty that these individuals face when using the Internet and health Web sites to find information on specific health topics

Natural killer cell and hepatic cell interaction via NKG2A leads to dendritic cell-mediated induction of CD4+ CD25+ T cells with PD-1-dependent regulatory activities

Natural killer (NK) cells have the ability to control dendritic cell (DC)-mediated T cell responses. However, the precise mechanisms by which NK receptor-mediated regulation of NK cells determines the magnitude and direction of DC-mediated T cell responses remain unclear. In the present study, we applied an in vitro co-culture system to examine the impact of NK cells cultured with hepatic cells on DC induction of regulatory T cells. We found that interaction of NK cells and non-transformed hepatocytes (which express HLA-E) via the NKG2A inhibitory receptor resulted in priming of DCs to induce CD4+ CD25+ T cells with regulatory properties. NKG2A triggering led to characteristic changes of the cytokine milieu of co-cultured cells; an increase in the transforming growth factor (TGF)-β involved in the generation of this specific type of DC, and a decrease in the tumour necrosis factor-α capable of antagonizing the effect of TGF-β. The regulatory cells induced by NK cell-primed DCs exert their suppressive actions through a negative costimulator programmed death-1 (PD-1) mediated pathway, which differs from freshly isolated CD4+ CD25+ T cells. These findings provide new insight into the role of NK receptor signals in the DC-mediated induction of regulatory T cells