Implementing Evidence-Based Programs: Lessons Learned From the Field

Evidence-based programs (EBPs) are used in many health promotion efforts to ensure that the intended positive behavioral and health outcomes will be achieved. However, because EBPs are developed and tested in research settings, the contextual elements of real world implementation play an important role in their successful delivery in communities. As an intermediary charged with supporting the translation of research to practice for a statewide teen pregnancy prevention initiative, the ACT for Youth Center of Excellence conducted a mid-course evaluation to identify factors that contribute to the successful implementation of EBPs. The findings indicate that successful implementation involves systematic planning that addresses three critical contextual factors: community readiness, organizational capacity, and setting characteristics. By attending both to factors identified from local practice, and to theory and evidence identified through implementation science, practitioners can strengthen programming and enhance the quality of EBP delivery.     

Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.     

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

Background

The combinatorial library strategy of using multiple candidate ligands in mixtures as library members is ideal in terms of cost and efficiency, but needs special screening methods to estimate the affinities of candidate ligands in such mixtures. Herein, a new method to screen candidate ligands present in unknown molar quantities in mixtures was investigated.

Results

The proposed method involves preparing a processed-mixture-for-screening (PMFS) with each mixture sample and an exogenous reference ligand, initiating competitive binding among ligands from the PMFS to a target immobilized on magnetic particles, recovering target-ligand complexes in equilibrium by magnetic force, extracting and concentrating bound ligands, and analyzing ligands in the PMFS and the concentrated extract by chromatography. The relative affinity of each candidate ligand to its reference ligand is estimated via an approximation equation assuming (a) the candidate ligand and its reference ligand bind to the same site(s) on the target, (b) their chromatographic peak areas are over five times their intercepts of linear response but within their linear ranges, (c) their binding ratios are below 10%. These prerequisites are met by optimizing primarily the quantity of the target used and the PMFS composition ratio. The new method was tested using the competitive binding of biotin derivatives from mixtures to streptavidin immobilized on magnetic particles as a model. Each mixture sample containing a limited number of candidate biotin derivatives with moderate differences in their molar quantities were prepared via parallel-combinatorial-synthesis (PCS) without purification, or via the pooling of individual compounds. Some purified biotin derivatives were used as reference ligands. This method showed resistance to variations in chromatographic quantification sensitivity and concentration ratios; optimized conditions to validate the approximation equation could be applied to different mixture samples. Relative affinities of candidate biotin derivatives with unknown molar quantities in each mixture sample were consistent with those estimated by a homogenous method using their purified counterparts as samples.

Conclusions

This new method is robust and effective for each mixture possessing a limited number of candidate ligands whose molar quantities have moderate differences, and its integration with PCS has promise to routinely practice the mixture-based library strategy.     

Developmental basis of mammalian digit reduction: a case study in pigs

Digit reduction has occurred in parallel in many mammalian lineages. However, despite this pattern's prevalence, the developmental mechanisms underlying mammalian digit reduction remain controversial. We therefore undertook a study of digit development in the pig (Sus scrofa), a mammal with reduced first, second, and fifth digits. Our results indicate that from its earliest formation, the pig limb bud is significantly narrower than that of the model pentadactyl mammal, mouse. Furthermore, the cartilage condensations of the pig's reduced digits are noticeably smaller than those of their nonreduced counterparts from the time of their formation. In addition, growth rates of pig digits are comparable, as are the patterns of cell death in developing pig and mouse limbs. Taken together, results suggest that pig's first, second, and fifth digits are primarily reduced through evolutionary modifications in the early developmental patterning of their limbs. Results of this study, coupled with those from study of limb development in other mammals, suggest that although major developmental reorganizations (e.g., complete digit or limb loss) during early limb development may be selected against, it may be common for more subtle evolutionary modifications in limb development (e.g., changes in relative digit size) to occur at this time.     

A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.     

Inactivation of plant infecting fungal and viral pathogens to achieve biological containment in drainage water using UV treatment

Aim: To explore whether ultraviolet (UV) light treatment within a closed circulating and filtered water drainage system can kill plant pathogenic species. Methods and Results: Ultraviolet experiments at 254 nm were conducted to determine the inactivation coefficients for seven plant pathogenic species. At 200 mJ cm^?2, the individual species log reductions obtained for six Ascomycete fungi and a cereal virus were as follows: Leptosphaeria maculans (9·9‐log), Leptosphaeria biglobosa (7·1‐log), Barley stripe mosaic virus (BSMV) (4·1‐log), Mycosphaerella graminicola (2·9‐log), Fusarium culmorum (1·2‐log), Fusarium graminearum (0·6‐log) and Magnaporthe oryzae (0·3‐log). Dilution experiments showed that BSMV was rendered noninfectious when diluted to >1/512. Follow‐up large‐scale experiments using up to 400 l of microbiologically contaminated waste water revealed that the filtration of drainage water followed by UV treatment could successfully be used to inactivate several plant pathogens. Conclusions: By combining sedimentation, filtration and UV irradiation within a closed system, plant pathogens can be successfully removed from collected drainage water. Significance and Impact of the Study: Ultraviolet irradiation is a relatively low cost, energy efficient and labour nonintensive method to decontaminate water arising from a suite of higher biological containment level laboratories and plant growth rooms where genetically modified and/or quarantine fungal and viral plant pathogenic organisms are being used for research purposes.     

Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets alpha and delta cells resided at the periphery of the beta-cell core. However, human islets were markedly different in that alpha, beta, and delta cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of alpha, beta, and delta cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.     

Four base recognition by triplex-forming oligonucleotides at physiological pH

We have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU [2′-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine] recognizes AT base pairs with high affinity, ^MeP (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while ^APP (6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one) and S [N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide)] bind to CG and TA base pairs, respectively. Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, ^MeP and ^APP retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA.     

Design and synthesis of potent pyridazine inhibitors of p38 MAP kinase

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.