VitroJet: new features and case studies.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

Reducing risk for chronic disease: evaluation of a collective community approach to sustainable evidence-based health programming.

BACKGROUND: Community initiatives can shape health behaviors, such as physical activity and dietary habits, across a population and help reduce the risk of developing chronic disease. To achieve this goal and impact health outcomes, Pasadena Vibrant Community aimed to engage communities in an ongoing dialogue about the importance of healthy behaviors, implement and advance community-based strategies to promote health, and improve diet and physical activity behaviors. The initiative was centered around a collaboration between a backbone organization, steering committee, and 7 collaborating organizations funded to implement multicomponent, evidence-based programs.. The common agenda was detailed in a community action plan, which included 19 interventions targeting healthy eating and active living among adults and youth in Pasadena, Texas. METHODS: A mixed methods evaluation of the initiative was conducted over 4 years. Data sources included document reviews of quarterly progress reports (n = 86) and supplemental data reports (n = 16) provided by collaborating organizations, annual Steering Committee surveys (n = 4), and interviews conducted with staff from a subset of Collaborating Organizations (n = 4). RESULTS: The initiative reached over 50,000 community members per year through 19 evidence-based interventions and impacted health outcomes, including knowledge and adoption of healthy eating practices and increased physical activity. Thirty-one systems-level changes were implemented during the initiative, including 16 environmental changes. Steering Committee meetings and shared goals enabled connections, communication, and cooperation, which allowed Collaborating Organizations to address challenges and combine resources to deliver their programs. CONCLUSIONS: Community initiatives can effectively permeate the community by reaching individuals, improving physical activity and dietary habits, and ensuring sustainability. Based on the experience reported here, the success of a community initiative can be facilitated if collaborating organizations come together to implement evidence-based interventions and tailor them to the community, and if they are empowered by significant leadership and supportive collaboration and aligned by a common agenda.

Implementation of Session Zero as a Recruitment Strategy in the National Diabetes Prevention Program's Lifestyle Change Program.

PURPOSE: The purpose of the study was to understand the extent to which organizations offering the Centers for Disease Control and Prevention's (CDC) National Diabetes Prevention Program (National DPP) lifestyle change program implement session zero (a pre-enrollment session designed to recruit, engage, and enroll participants in programs), the stated purpose(s) for offering session zero, the content of session zero, and best practices for using session zero for recruitment. METHODS: Researchers conducted a survey of all organizations offering the National DPP lifestyle change program that were registered with the CDC's Diabetes Prevention Recognition Program and their affiliated delivery locations. RESULTS: Most (79.5%) delivery locations reported implementing session zero; of these, most used session zero as a recruitment strategy (81.1%) and orientation session (72.8%), whereas few (17.7%) used session zero solely to complete participant enrollment paperwork. Most (60.7%) delivery locations that implement session zero offer all their sessions at the same location, offer one session per upcoming participant cohort (66.7%), and use a consistent agenda (83.0%). Out of a list of activities informed by behavior change theory, the most common was offering an opportunity to enroll in the year-long lifestyle change program at the end of session zero (71.1%). CONCLUSIONS: Most National DPP delivery locations implement session zero as a recruitment and orientation session. Most delivery locations reported including some activities informed by behavior change theory, but delivery locations could offer more theoretically informed activities during their session zero. The findings provide practice-based considerations for implementing session zero for recruitment into lifestyle change programs.

Message Framing Strategies to Promote the Uptake of PrEP: Results from Formative Research with Diverse Adult Populations in the United States.

There are no evidence-based recommendations for communicating about pre-exposure prophylaxis (PrEP) as part of a broader HIV-prevention messaging approach. To inform future message development related to PrEP uptake, we interviewed 235 individuals across ten locations in the U.S. to explore their understanding and perceptions of draft HIV prevention messages and assess their overall preferences for a broad or PrEP-focused messaging approach. Participants responded favorably to and related to both draft messages. Participants who were not aware of PrEP were more likely to say the broad HIV-prevention message was personally relevant than those aware of PrEP. There were no significant differences in perceived personal relevance for the PrEP-focused message. Qualitative findings suggest that HIV prevention messages should use specific well-defined terms, include links to additional information, and use choice-enhancing language that emphasizes personal agency and frames the call to action as an informed decision among an array of effective prevention options.

RESUMEN: No existen recommendaciones basadas en evidencia para comunicar sobre la profilaxis prexposición (PrEP) como parte de un efoque más amplio de mensajes de prevención del VIH. Para informar el desarrollo de mansajes relacionados con el consumo de la PrEP, entrevistamos a 235 personas en 10 ubicaciones en los EE.UU. para explorar su comprensión y percepciones de los borradores de mensajes de prevención del VIH y evaluar sus preferencias generales por un enfoque de mensajeria amplio o centrado en la PrEP. Los participantes respondieron favorablemente y relacionadoscon ambos barradores de mensajes. Los participantes que no conocían la PrEP tenían más probabilidades de decir que el mansaje general de prevención amplia de VIH era personalmente relevent que aquellos que conocían la PrEP. No existe differencias significativas en la relevancia personal percibida para el mensaje centrado en la PrEP. Los hallazgos cualitativos sugieren que los mensajes de prevención del VIH deben utilizar términos especificos bien definidos, incluir enlaces a información adicional y utilizar un lenguaje que mejore las opciones, que enfatice la agencia personal y enmarque el llamado a la acción como una decisión informada entre una variedad de opciones de prevención efectivas.

Be Well Communities™: mobilizing communities to promote wellness and stop cancer before it starts.

PURPOSE: Increasingly, cancer centers are delivering population-based approaches to narrow the gap between known cancer prevention strategies and their effective implementation. Leveraging successful healthy community initiatives, MD Anderson developed Be Well Communities™, a model that implements evidence-based actions to directly impact people's lives. METHODS: In partnership with local organizations, MD Anderson's Be Well Communities team executed and evaluated 16 evidence-based interventions to address community priorities in healthy diets, physical activity, and sun safety. Evaluation included assessing the effectiveness of evidence-based interventions, stakeholders' perceptions of collaboration, and the population-level impact on dietary and physical activity behaviors among students using the School Physical Activity and Nutrition Survey and the System for Observing Fitness Instruction Time. Two-tailed t-tests were used to compare tested parameters at baseline and follow-up. p values less than .05 were considered significant. RESULTS: This model achieved its early outcomes, including effectively implementing evidence-based interventions, building strong partnerships, increasing access to healthy foods, improving the built environment, and increasing healthy food and water consumption and moderate to vigorous physical activity among students (p < .001). CONCLUSIONS: Be Well Communities is an effective model for positively impacting community health which could be leveraged by others to deliver evidence-based actions to improve population health.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Strategies to address anaemia among pregnant and lactating women in India: a formative research study.

OBJECTIVE: Over half of pregnant women in India are affected by anaemia, which can lead to premature birth, low birth weight and maternal and child mortality. Using formative research, we aimed to understand social and cultural factors around iron and folic acid (IFA) supplement provision and adherence to identify potential strategies for improving adherence and behaviours to prevent and treat anaemia among pregnant and lactating Indian women. DESIGN: In-depth interviews and focus group discussions with women and key informant interviews with health officials and workers. SETTING: Four districts in two Indian states: Allahabad and Bara Banki districts in Uttar Pradesh and Chikkballapura and Mandya districts in Karnataka. PARTICIPANTS: Pregnant and lactating women (n 65) and district officials and community health workers (n 14). RESULTS: Most women were aware of anaemia but did not understand its seriousness and consequences. All women received IFA supplements (predominantly for free), but many were not adherent because of side effects; lack of information from healthcare providers on the causes of anaemia, its seriousness and solutions and low social support. To address anaemia, women were most confident in their ability to prepare and eat healthier foods but lacked control over resources such as appropriate food availability. CONCLUSIONS: Based on the findings, we recommend multicomponent interventions to train healthcare providers, address systemic barriers and involve family members to support IFA supplement adherence and dietary changes. Future research will determine which strategies are most effective to reduce the burden of anaemia in India among pregnant and lactating women.

Fragment-based drug discovery using cryo-EM.

Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. Here, we review the use of cryo-EM in fragment-based drug discovery (FBDD) based on in-house method development. We demonstrate not only that cryo-EM can reveal details of the molecular interactions between fragments and a protein, but also that the current reproducibility, quality, and throughput are compatible with FBDD. We exemplify this using the test system ß-galactosidase (Bgal) and the oncology target pyruvate kinase 2 (PKM2).

Using Trials of Improved Practices to identify practices to address the double burden of malnutrition among Rwandan children.

OBJECTIVE: Low- and middle-income countries (LMIC) are increasingly experiencing the double burden of malnutrition. Studies to identify 'double-duty' actions that address both undernutrition and overweight in sub-Saharan Africa are needed. We aimed to identify acceptable behaviours to achieve more optimal feeding and physical activity practices among both under- and overweight children in Rwanda, a sub-Saharan LMIC with one of the largest recent increases in child overweight. DESIGN: We used the Trials of Improved Practices (TIPs) method. During three household visits over 1·5 weeks, we used structured interviews and unstructured observations to collect data on infant and young child feeding practices and caregivers' experiences with testing recommended practices. SETTING: An urban district and a rural district in Rwanda. PARTICIPANTS: Caregivers with an under- or overweight child from 6 to 59 months of age (n 136). RESULTS: We identified twenty-five specific recommended practices that caregivers of both under- and overweight children agreed to try. The most frequently recommended practices were related to dietary diversity, food quantity, and hygiene and food handling. The most commonly cited reason for trying a new practice was its benefits to the child's health and growth. Financial constraints and limited food availability were common barriers. Nearly all caregivers said they were willing to continue the practices and recommend them to others. CONCLUSIONS: These practices show potential for addressing the double burden as part of a broader intervention. Still, further research is needed to determine whether caregivers can maintain the behaviours and their direct impact on both under- and overweight.

Cryo-EM in drug discovery.

The impact of structural biology on drug discovery is well documented, and the workhorse technique for the past 30 years or so has been X-ray crystallography. With the advent of several technological improvements, including direct electron detectors, automation, better microscope vacuums and lenses, phase plates and improvements in computing power enabled by GPUs, it is now possible to record and analyse images of protein structures containing high-resolution information. This review, from a pharmaceutical perspective, highlights some of the most relevant and interesting protein structures for the pharmaceutical industry and shows examples of how ligand-binding sites, membrane proteins, both big and small, pseudo symmetry and complexes are being addressed by this technique.

A Fragment-Derived Clinical Candidate for Antagonism of X-Linked and Cellular Inhibitor of Apoptosis Proteins: 1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1 H,2 H,3 H-pyrrolo[3,2- b]pyridin-1-yl)-2-[(2 R,5 R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660).

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP-XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

Implementing a STEMI system of care in urban Bangalore: Rationale and Study Design for heart rescue India.

BACKGROUND: A system of care designed to measure and improve process measures such as symptom recognition, emergency response, and hospital care has the potential to reduce mortality and improve quality of life for patients with ST-elevation myocardial infarction (STEMI). OBJECTIVE: To document the methodology and rationale for the implementation and impact measurement of the Heart Rescue India project on STEMI morbidity and mortality in Bangalore, India. STUDY DESIGN: A hub and spoke STEMI system of care comprised of two interventional, hub hospitals and five spoke hospitals will build and deploy a dedicated emergency response and transport system covering a 10 Km. radius area of Bangalore, India. High risk patients will receive a dedicated emergency response number to call for symptoms of heart attack. A dedicated operations center will use geo-tracking strategies to optimize response times including first responder motor scooter transport, equipped with ECG machines to transmit ECG's for immediate interpretation and optimal triage. At the same time, a dedicated ambulance will be deployed for transport of appropriate STEMI patients to a hub hospital while non-STEMI patients will be transported to spoke hospitals. To enhance patient recognition and initiation of therapy, school children will be trained in basic CPR and signs and symptom of chest pain. Hub hospitals will refine their emergency department and cardiac catheterization laboratory protocols using continuous quality improvement techniques to minimize treatment delays. Prior to hospital discharge, secondary prevention measures will be initiated to enhance long-term patient outcomes.

Survey of Afterschool Programs Suggests Most Offer Fruit and Vegetables Daily.

INTRODUCTION: Most children underconsume fruit and vegetables. This study estimated the frequency and quality of fruit and vegetables offered during snack in US afterschool programs and examined program-level factors associated with offering them, including awareness and use of the National AfterSchool Association Healthy Eating and Physical Activity standards. METHODS: We conducted descriptive analyses and regression modeling by using data collected from 684 National AfterSchool Association members and their colleagues via a 2015 online survey. RESULTS: At the previous snack, 63% of respondents offered fruit, a vegetable, or both, with 42% offering only fruit, 18% offering fruit and vegetables, and 3% offering only vegetables. The quality of the items offered showed that most respondents selected the healthiest options, such as fresh fruit and vegetables. Controlling for other factors, we found that factors independently associated with offering fruit, vegetables, or both were membership in the National AfterSchool Association, using the standards for menu planning, and training staff members in healthy eating more than once a year. Programs run by school districts were less likely to offer fruit than programs run by other organizations. CONCLUSION: Membership in the National AfterSchool Association and use of its Healthy Eating and Physical Activity standards are associated with offering fruit and vegetables during snack at afterschool programs staffed by National AfterSchool Association members and their colleagues across the United States. With over a third of sites surveyed offering neither a fruit nor a vegetable at the previous snack, additional implementation of the standards is still needed.

Relationships Between Theoretically Derived Short-Term Outcomes and Support for Policy Among the Public and Decision-Makers.

PURPOSE AND OBJECTIVES: Policy change is a lengthy and complex process. Thus, it is important to articulate hypothesized causal pathways between advocacy activities and policy change outcomes and to identify and monitor early indicators of progress toward policy change. INTERVENTION APPROACH: The Kansas Health Foundation supports grantee efforts to address the public health effects of obesity through evidence-based policy, systems, and environmental change interventions. To build support for policy, systems, and environmental changes in schools, workplaces, and health care and retail settings, grantees mobilize communities, educate government policy makers, and advocate with organizational decision makers. EVALUATION METHODS: To understand whether early outcomes from obesity-prevention advocacy efforts predict interim outcomes related to eventual policy change, we conducted surveys of the general public and of opinion leaders in Kansas, which were designed to measure components of Kansas Health Foundation's theory of change. We then used structural equation modeling to test the theory of change's underlying relationships by using support for obesity prevention policies as the outcome. RESULTS: Our findings supported the hypothesized model: perceptions of obesity as a serious community problem influence beliefs about causes of the problem. Beliefs about causes predict beliefs about who is responsible for the solution to the problem, which in turn predicts support for obesity prevention policies. IMPLICATIONS FOR PUBLIC HEALTH: Evaluators of advocacy for policy change interventions can use this approach to monitor proximal changes in public and opinion leader beliefs related to eventual policy change and to determine whether efforts are likely to be successful or need to be adapted or abandoned.

ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNFα-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth.

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I-II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381-91. ©2018 AACR.

Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-Linked Inhibitor of Apoptosis Protein (XIAP).

XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.

Identifying Synergies in Multilevel Interventions.

Social ecological models of health often describe multiple levels of influence that interact to influence health. However, it is still common for interventions to target only one or two of these levels, perhaps owing in part to a lack of guidance on how to design multilevel interventions to achieve optimal impact. The convergence strategy emphasizes that interventions at different levels mutually reinforce each other by changing patterns of interaction among two or more intervention audiences; this strategy is one approach for combining interventions at different levels to produce synergistic effects. We used semistructured interviews with 65 representatives in a cross-site national initiative that enhanced health and outcomes for patients with diabetes to examine whether the convergence strategy was a useful conceptual model for multilevel interventions. Using a framework analysis approach to analyze qualitative interview data, we found three synergistic themes that match the convergence strategy and support how multilevel interventions can be successful. These three themes were (1) enhancing engagement between patient and provider and access to quality care; (2) supporting communication, information sharing, and coordination among providers, community stakeholders, and systems; and (3) building relationships and fostering alignment among providers, community stakeholders, and systems. These results support the convergence strategy as a testable conceptual model and provide examples of successful intervention strategies for combining multilevel interventions to produce synergies across levels and promote diabetes self-management and that may extend to management of other chronic illnesses as well.

Public and Opinion Leader Willingness to Fund Obesity-Focused Policies in Kansas.

Obesity increases the risk for leading causes of death, including cardiovascular disease and some cancers. Midwestern and southern states have the highest obesity rates-in Kansas, one in every three adults is obese. We compared the willingness of Kansas adults and opinion leaders to pay more in taxes to fund obesity prevention policies. In 2014, we asked a representative sample of 2,203 Kansas adults (response rate 15.7%) and 912 opinion leaders (response rate 55%) drawn from elected office and other sectors, including business and health, whether they would pay an additional $50 in annual taxes to support five policies that improve access to healthy foods and opportunities for physical activity. We used adjusted Wald tests to compare public and opinion leaders' responses, and regression analysis to assess whether differences in respondents' gender, age, location (urban/rural), race/ethnicity, and political stance affected results. Adjusting for demographic differences, Kansas adults were more willing than opinion leaders to pay $50 in taxes for each of the five policy interventions. This study demonstrates a willingness among residents of a fiscally conservative state to pay increased taxes for policies that could reduce population obesity rates. Health professionals, including nurses, can use these findings to educate policy makers in Kansas and geopolitically similar states about widespread public support for obesity prevention policies. Public health and other nurses could also apply our methods to assess support for obesity prevention policies in their jurisdictions.