A strategy to advance the evidence base in palliative medicine: formation of a palliative care research cooperative group.

BACKGROUND: Palliative medicine has made rapid progress in establishing its scientific and clinical legitimacy, yet the evidence base to support clinical practice remains deficient in both the quantity and quality of published studies. Historically, the conduct of research in palliative care populations has been impeded by multiple barriers including health care system fragmentation, small number and size of potential sites for recruitment, vulnerability of the population, perceptions of inappropriateness, ethical concerns, and gate-keeping. METHODS: A group of experienced investigators with backgrounds in palliative care research convened to consider developing a research cooperative group as a mechanism for generating high-quality evidence on prioritized, clinically relevant topics in palliative care. RESULTS: The resulting Palliative Care Research Cooperative (PCRC) agreed on a set of core principles: active, interdisciplinary membership; commitment to shared research purposes; heterogeneity of participating sites; development of research capacity in participating sites; standardization of methodologies, such as consenting and data collection/management; agile response to research requests from government, industry, and investigators; focus on translation; education and training of future palliative care researchers; actionable results that can inform clinical practice and policy. Consensus was achieved on a first collaborative study, a randomized clinical trial of statin discontinuation versus continuation in patients with a prognosis of less than 6 months who are taking statins for primary or secondary prevention. This article describes the formation of the PCRC, highlighting processes and decisions taken to optimize the cooperative group's success.

Development of Stimulus-Responsive Ligands for the Modulation of Copper and Iron Coordination

The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.

Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N′-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.

Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.

Development and usability testing of a Web-based decision aid for families of patients receiving prolonged mechanical ventilation.

BACKGROUND: Web-based decision aids are increasingly important in medical research and clinical care. However, few have been studied in an intensive care unit setting. The objectives of this study were to develop a Web-based decision aid for family members of patients receiving prolonged mechanical ventilation and to evaluate its usability and acceptability. METHODS: Using an iterative process involving 48 critical illness survivors, family surrogate decision makers, and intensivists, we developed a Web-based decision aid addressing goals of care preferences for surrogate decision makers of patients with prolonged mechanical ventilation that could be either administered by study staff or completed independently by family members (Development Phase). After piloting the decision aid among 13 surrogate decision makers and seven intensivists, we assessed the decision aid's usability in the Evaluation Phase among a cohort of 30 surrogate decision makers using the Systems Usability Scale (SUS). Acceptability was assessed using measures of satisfaction and preference for electronic Collaborative Decision Support (eCODES) versus the original printed decision aid. RESULTS: The final decision aid, termed 'electronic Collaborative Decision Support', provides a framework for shared decision making, elicits relevant values and preferences, incorporates clinical data to personalize prognostic estimates generated from the ProVent prediction model, generates a printable document summarizing the user's interaction with the decision aid, and can digitally archive each user session. Usability was excellent (mean SUS, 80 ± 10) overall, but lower among those 56 years and older (73 ± 7) versus those who were younger (84 ± 9); p = 0.03. A total of 93% of users reported a preference for electronic versus printed versions. CONCLUSIONS: The Web-based decision aid for ICU surrogate decision makers can facilitate highly individualized information sharing with excellent usability and acceptability. Decision aids that employ an electronic format such as eCODES represent a strategy that could enhance patient-clinician collaboration and decision making quality in intensive care.

Adaptive intervention design in mobile health: Intervention design and development in the Cell Phone Intervention for You trial.

BACKGROUND/AIMS: The obesity epidemic has spread to young adults, and obesity is a significant risk factor for cardiovascular disease. The prominence and increasing functionality of mobile phones may provide an opportunity to deliver longitudinal and scalable weight management interventions in young adults. The aim of this article is to describe the design and development of the intervention tested in the Cell Phone Intervention for You study and to highlight the importance of adaptive intervention design that made it possible. The Cell Phone Intervention for You study was a National Heart, Lung, and Blood Institute-sponsored, controlled, 24-month randomized clinical trial comparing two active interventions to a usual-care control group. Participants were 365 overweight or obese (body mass index≥25 kg/m2) young adults. METHODS: Both active interventions were designed based on social cognitive theory and incorporated techniques for behavioral self-management and motivational enhancement. Initial intervention development occurred during a 1-year formative phase utilizing focus groups and iterative, participatory design. During the intervention testing, adaptive intervention design, where an intervention is updated or extended throughout a trial while assuring the delivery of exactly the same intervention to each cohort, was employed. The adaptive intervention design strategy distributed technical work and allowed introduction of novel components in phases intended to help promote and sustain participant engagement. Adaptive intervention design was made possible by exploiting the mobile phone's remote data capabilities so that adoption of particular application components could be continuously monitored and components subsequently added or updated remotely. RESULTS: The cell phone intervention was delivered almost entirely via cell phone and was always-present, proactive, and interactive-providing passive and active reminders, frequent opportunities for knowledge dissemination, and multiple tools for self-tracking and receiving tailored feedback. The intervention changed over 2 years to promote and sustain engagement. The personal coaching intervention, alternatively, was primarily personal coaching with trained coaches based on a proven intervention, enhanced with a mobile application, but where all interactions with the technology were participant-initiated. CONCLUSION: The complexity and length of the technology-based randomized clinical trial created challenges in engagement and technology adaptation, which were generally discovered using novel remote monitoring technology and addressed using the adaptive intervention design. Investigators should plan to develop tools and procedures that explicitly support continuous remote monitoring of interventions to support adaptive intervention design in long-term, technology-based studies, as well as developing the interventions themselves.

Sea Urchin Body Plan Development and Evolution: An Integrative Transcriptomic Approach

My dissertation work integrates comparative transcriptomics and functional analyses to investigate gene expression changes underlying two significant aspects of sea urchin evolution and development: the dramatic developmental changes associated with an ecologically significant shift in life history strategy and the development of the unusual radial body plan of adult sea urchins.

In Chapter 2, I investigate evolutionary changes in gene expression underlying the switch from feeding (planktotrophic) to nonfeeding (lecithotrophic) development in sea urchins. In order to identify these changes, I used Illumina RNA-seq to measure expression dynamics across 7 developmental stages in three sea urchin species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris tuberculata, and an outgroup planktotroph Lytechinus variegatus. My analyses draw on a well-characterized developmental gene regulatory network (GRN) in sea urchins to understand how the ancestral planktotrophic developmental program was altered during the evolution of lecithotrophic development. My results suggest that changes in gene expression profiles occurred more frequently across the transcriptome during the evolution of lecithotrophy than during the persistence of planktotrophy. These changes were even more pronounced within the GRN than across the transcriptome as a whole, and occurred in each network territory (skeletogenic, endomesoderm and ectoderm). I found evidence for both conservation and divergence of regulatory interactions in the network, as well as significant changes in the expression of genes with known roles in larval skeletogenesis, which is dramatically altered in lecithotrophs. I further explored network dynamics between species using coexpression analyses, which allowed me to identify novel players likely involved in sea urchin neurogenesis and endoderm patterning.

In Chapter 3, I investigate developmental changes in gene expression underlying radial body plan development and metamorphosis in H. erythrogramma. Using Illumina RNA-seq, I measured gene expression profiles across larval, metamorphic, and post-metamorphic life cycle phases. My results present a high-resolution view of gene expression dynamics during the complex transition from pre- to post-metamorphic development and suggest that distinct sets of regulatory and effector proteins are used during different life history phases.

Collectively, my investigations provide an important foundation for future, empirical studies to investigate the functional role of gene expression change in the evolution of developmental differences between species and also for the generation of the unusual radial body plan of sea urchins.

Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Regulates Dendritic Cells and Myeloid Derived Suppressor Cells Development in the Lymphoma Microenvironment

Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.

We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.

In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.

Biological and Physical Factors Affecting the Natural History and Evolution of Encapsulated Development

The evolution of reproductive strategies involves a complex calculus of costs and benefits to both parents and offspring. Many marine animals produce embryos packaged in tough egg capsules or gelatinous egg masses attached to benthic surfaces. While these egg structures can protect against environmental stresses, the packaging is energetically costly for parents to produce. In this series of studies, I examined a variety of ecological factors affecting the evolution of benthic development as a life history strategy. I used marine gastropods as my model system because they are incredibly diverse and abundant worldwide, and they exhibit a variety of reproductive and developmental strategies.

The first study examines predation on benthic egg masses. I investigated: 1) behavioral mechanisms of predation when embryos are targeted (rather than the whole egg mass); 2) the specific role of gelatinous matrix in predation. I hypothesized that gelatinous matrix does not facilitate predation. One study system was the sea slug Olea hansineensis, an obligate egg mass predator, feeding on the sea slug Haminoea vesicula. Olea fed intensely and efficiently on individual Haminoea embryos inside egg masses but showed no response to live embryos removed from gel, suggesting that gelatinous matrix enables predation. This may be due to mechanical support of the feeding predator by the matrix. However, Haminoea egg masses outnumber Olea by two orders of magnitude in the field, and each egg mass can contain many tens of thousands of embryos, so predation pressure on individuals is likely not strong. The second system involved the snail Nassarius vibex, a non-obligate egg mass predator, feeding on the polychaete worm Clymenella mucosa. Gel neither inhibits nor promotes embryo predation for Nassarius, but because it cannot target individual embryos inside an egg mass, its feeding is slow and inefficient, and feeding rates in the field are quite low. However, snails that compete with Nassarius for scavenged food have not been seen to eat egg masses in the field, leaving Nassarius free to exploit the resource. Overall, egg mass predation in these two systems likely benefits the predators much more than it negatively affects the prey. Thus, selection for environmentally protective aspects of egg mass production may be much stronger than selection for defense against predation.

In the second study, I examined desiccation resistance in intertidal egg masses made by Haminoea vesicula, which preferentially attaches its flat, ribbon-shaped egg masses to submerged substrata. Egg masses occasionally detach and become stranded on exposed sand at low tide. Unlike adults, the encased embryos cannot avoid desiccation by selectively moving about the habitat, and the egg mass shape has high surface-area-to-volume ratio that should make it prone to drying out. Thus, I hypothesized that the embryos would not survive stranding. I tested this by deploying individual egg masses of two age classes on exposed sand bars for the duration of low tide. After rehydration, embryos midway through development showed higher rates of survival than newly-laid embryos, though for both stages survival rates over 25% were frequently observed. Laboratory desiccation trials showed that >75% survival is possible in an egg mass that has lost 65% of its water weight, and some survival (<25%) was observed even after 83% water weight lost. Although many surviving embryos in both experiments showed damage, these data demonstrate that egg mass stranding is not necessarily fatal to embryos. They may be able to survive a far greater range of conditions than they normally encounter, compensating for their lack of ability to move. Also, desiccation tolerance of embryos may reduce pressure on parents to find optimal laying substrata.

The third study takes a big-picture approach to investigating the evolution of different developmental strategies in cone snails, the largest genus of marine invertebrates. Cone snail species hatch out of their capsules as either swimming larvae or non-dispersing forms, and their developmental mode has direct consequences for biogeographic patterns. Variability in life history strategies among taxa may be influenced by biological, environmental, or phylogenetic factors, or a combination of these. While most prior research has examined these factors singularly, my aim was to investigate the effects of a host of intrinsic, extrinsic, and historical factors on two fundamental aspects of life history: egg size and egg number. I used phylogenetic generalized least-squares regression models to examine relationships between these two egg traits and a variety of hypothesized intrinsic and extrinsic variables. Adult shell morphology and spatial variability in productivity and salinity across a species geographic range had the strongest effects on egg diameter and number of eggs per capsule. Phylogeny had no significant influence. Developmental mode in Conus appears to be influenced mostly by species-level adaptations and niche specificity rather than phylogenetic conservatism. Patterns of egg size and egg number appear to reflect energetic tradeoffs with body size and specific morphologies as well as adaptations to variable environments. Overall, this series of studies highlights the importance of organism-scale biotic and abiotic interactions in evolutionary patterns.