Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.

Exploitation of Other Social Amoebae by Dictyostelium caveatum

Dictyostelium amoebae faced with starvation trigger a developmental program during which many cells aggregate and form fruiting bodies that consist of a ball of spores held aloft by a thin stalk. This developmental strategy is open to several forms of exploitation, including the remarkable case of Dictyostelium caveatum, which, even when it constitutes 1/10(3) of the cells in an aggregate, can inhibit the development of the host and eventually devour it. We show that it accomplishes this feat by inhibiting a region of cells, called the tip, which organizes the development of the aggregate into a fruiting body. We use live-cell microscopy to define the D. caveatum developmental cycle and to show that D. caveatum amoebae have the capacity to ingest amoebae of other Dictyostelid species, but do not attack each other. The block in development induced by D. caveatum does not affect the expression of specific markers of prespore cell or prestalk cell differentiation, but does stop the coordinated cell movement leading to tip formation. The inhibition mechanism involves the constitutive secretion of a small molecule by D. caveatum and is reversible. Four Dictyostelid species were inhibited in their development, while D. caveatum is not inhibited by its own compound(s). D. caveatum has evolved a predation strategy to exploit other members of its genus, including mechanisms of developmental inhibition and specific phagocytosis

'Correction:'Peer chart audits: A tool to meet Accreditation Council on Graduate Medical Education (ACGME) competency in practice-based learning and improvement

<p>Abstract</p> <p>Background</p> <p>The Accreditation Council on Graduate Medical Education (ACGME) supports chart audit as a method to track competency in Practice-Based Learning and Improvement. We examined whether peer chart audits performed by internal medicine residents were associated with improved documentation of foot care in patients with diabetes mellitus.</p> <p>Methods</p> <p>A retrospective electronic chart review was performed on 347 patients with diabetes mellitus cared for by internal medicine residents in a university-based continuity clinic from May 2003 to September 2004. Residents abstracted information pertaining to documentation of foot examinations (neurological, vascular, and skin) from the charts of patients followed by their physician peers. No formal feedback or education was provided.</p> <p>Results</p> <p>Significant improvement in the documentation of foot exams was observed over the course of the study. The percentage of patients receiving neurological, vascular, and skin exams increased by 20% (from 13% to 33%) (p = 0.001), 26% (from 45% to 71%) (p < 0.001), and 18% (51%–72%) (p = 0.005), respectively. Similarly, the proportion of patients receiving a well-documented exam which includes all three components – neurological, vascular and skin foot exam – increased over time (6% to 24%, p < 0.001).</p> <p>Conclusion</p> <p>Peer chart audits performed by residents in the absence of formal feedback were associated with improved documentation of the foot exam in patients with diabetes mellitus. Although this study suggests that peer chart audits may be an effective tool to improve practice-based learning and documentation of foot care in diabetic patients, evaluating the actual performance of clinical care was beyond the scope of this study and would be better addressed by a randomized controlled trial.</p

Representation and misrepresentation of scientific evidence in contemporary tobacco regulation:a review of tobacco industry submissions to the UK Government consultation on standardised packaging

BACKGROUND: Standardised packaging (SP) of tobacco products is an innovative tobacco control measure opposed by transnational tobacco companies (TTCs) whose responses to the UK government's public consultation on SP argued that evidence was inadequate to support implementing the measure. The government's initial decision, announced 11 months after the consultation closed, was to wait for 'more evidence', but four months later a second 'independent review' was launched. In view of the centrality of evidence to debates over SP and TTCs' history of denying harms and manufacturing uncertainty about scientific evidence, we analysed their submissions to examine how they used evidence to oppose SP. METHODS AND FINDINGS: We purposively selected and analysed two TTC submissions using a verification-oriented cross-documentary method to ascertain how published studies were used and interpretive analysis with a constructivist grounded theory approach to examine the conceptual significance of TTC critiques. The companies' overall argument was that the SP evidence base was seriously flawed and did not warrant the introduction of SP. However, this argument was underpinned by three complementary techniques that misrepresented the evidence base. First, published studies were repeatedly misquoted, distorting the main messages. Second, 'mimicked scientific critique' was used to undermine evidence; this form of critique insisted on methodological perfection, rejected methodological pluralism, adopted a litigation (not scientific) model, and was not rigorous. Third, TTCs engaged in 'evidential landscaping', promoting a parallel evidence base to deflect attention from SP and excluding company-held evidence relevant to SP. The study's sample was limited to sub-sections of two out of four submissions, but leaked industry documents suggest at least one other company used a similar approach. CONCLUSIONS: The TTCs' claim that SP will not lead to public health benefits is largely without foundation. The tools of Better Regulation, particularly stakeholder consultation, provide an opportunity for highly resourced corporations to slow, weaken, or prevent public health policies

Fetal Myocardium in the Kidney Capsule: An In Vivo Model of Repopulation of Myocytes by Bone Marrow Cells

Debate surrounds the question of whether the heart is a post-mitotic organ in part due to the lack of an in vivo model in which myocytes are able to actively regenerate. The current study describes the first such mouse model — a fetal myocardial environment grafted into the adult kidney capsule. Here it is used to test whether cells descended from bone marrow can regenerate cardiac myocytes. One week after receiving the fetal heart grafts, recipients were lethally irradiated and transplanted with marrow from green fluorescent protein (GFP)-expressing C57Bl/6J (B6) donors using normal B6 recipients and fetal donors. Levels of myocyte regeneration from GFP marrow within both fetal myocardium and adult hearts of recipients were evaluated histologically. Fetal myocardium transplants had rich neovascularization and beat regularly after 2 weeks, continuing at checkpoints of 1, 2, 4, 6, 8 and12 months after transplantation. At each time point, GFP-expressing rod-shaped myocytes were found in the fetal myocardium, but only a few were found in the adult hearts. The average count of repopulated myocardium with green rod-shaped myocytes was 996.8 cells per gram of fetal myocardial tissue, and 28.7 cells per adult heart tissue, representing a thirty-five fold increase in fetal myocardium compared to the adult heart at 12 months (when numbers of green rod-shaped myocytes were normalized to per gram of myocardial tissue). Thus, bone marrow cells can differentiate to myocytes in the fetal myocardial environment. The novel in vivo model of fetal myocardium in the kidney capsule appears to be valuable for testing repopulating abilities of potential cardiac progenitors

A new model for health care delivery

<p>Abstract</p> <p>Background</p> <p>The health care delivery system in the United States is facing cost and quality pressures that will require fundamental changes to remain viable. The optimal structures of the relationships between the hospital, medical school, and physicians have not been determined but are likely to have a large impact on the future of healthcare delivery. Because it is generally agreed that academic medical centers will play a role in the sustainability of this future system, a fundamental understanding of the relative contributions of the stakeholders is important as well as creativity in developing novel strategies to achieve a shared vision.</p> <p>Discussion</p> <p>Core competencies of each of the stakeholders (the hospital, the medical school and the physicians) must complement the others and should act synergistically. At the same time, the stakeholders should determine the common core values and should be able to make a meaningful contribution to the delivery of health care.</p> <p>Summary</p> <p>Health care needs to achieve higher quality and lower cost. Therefore, in order for physicians, medical schools, and hospitals to serve the needs of society in a gratifying way, there will need to be change. There needs to be more scientific and social advances. It is obvious that there is a real and urgent need for relationship building among the professionals whose duty it is to provide these services.</p

The IGNITE (investigation to guide new insight into translational effectiveness) trial: Protocol for a translational study of an evidenced-based wellness program in fire departments

<p>Abstract</p> <p>Background</p> <p>Worksites are important locations for interventions to promote health. However, occupational programs with documented efficacy often are not used, and those being implemented have not been studied. The research in this report was funded through the American Reinvestment and Recovery Act Challenge Topic 'Pathways for Translational Research,' to define and prioritize determinants that enable and hinder translation of evidenced-based health interventions in well-defined settings.</p> <p>Methods</p> <p>The IGNITE (investigation to guide new insights for translational effectiveness) trial is a prospective cohort study of a worksite wellness and injury reduction program from adoption to final outcomes among 12 fire departments. It will employ a mixed methods strategy to define a translational model. We will assess decision to adopt, installation, use, and outcomes (reach, individual outcomes, and economic effects) using onsite measurements, surveys, focus groups, and key informant interviews. Quantitative data will be used to define the model and conduct mediation analysis of each translational phase. Qualitative data will expand on, challenge, and confirm survey findings and allow a more thorough understanding and convergent validity by overcoming biases in qualitative and quantitative methods used alone.</p> <p>Discussion</p> <p>Findings will inform worksite wellness in fire departments. The resultant prioritized influences and model of effective translation can be validated and manipulated in these and other settings to more efficiently move science to service.</p

Better Few than Hungry: Flexible Feeding Ecology of Collared Lemurs Eulemur collaris in Littoral Forest Fragments

Frugivorous primates are known to encounter many problems to cope with habitat degradation, due to the fluctuating spatial and temporal distribution of their food resources. Since lemur communities evolved strategies to deal with periods of food scarcity, these primates are expected to be naturally adapted to fluctuating ecological conditions and to tolerate a certain degree of habitat changes. However, behavioral and ecological strategies adopted by frugivorous lemurs to survive in secondary habitats have been little investigated. Here, we compared the behavioral ecology of collared lemurs (Eulemur collaris) in a degraded fragment of littoral forest of south-east Madagascar, Mandena, with that of their conspecifics in a more intact habitat, Sainte Luce.Lemur groups in Mandena and in Sainte Luce were censused in 2004/2007 and in 2000, respectively. Data were collected via instantaneous sampling on five lemur groups totaling 1,698 observation hours. The Shannon index was used to determine dietary diversity and nutritional analyses were conducted to assess food quality. All feeding trees were identified and measured, and ranging areas determined via the minimum convex polygon. In the degraded area lemurs were able to modify several aspects of their feeding strategies by decreasing group size and by increasing feeding time, ranging areas, and number of feeding trees. The above strategies were apparently able to counteract a clear reduction in both food quality and size of feeding trees.Our findings indicate that collared lemurs in littoral forest fragments modified their behavior to cope with the pressures of fluctuating resource availability. The observed flexibility is likely to be an adaptation to Malagasy rainforests, which are known to undergo periods of fruit scarcity and low productivity. These results should be carefully considered when relocating lemurs or when selecting suitable areas for their conservation

Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL

Introduction It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal. Methods Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation. Results Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10%-30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment. Conclusions We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes. [PROVISIONAL