The Delivery Science Rapid Analysis Program: A Research and Operational Partnership at Kaiser Permanente Northern California.

INTRODUCTION: Health care researchers and delivery system leaders share a common mission to improve health care quality and outcomes. However, differing timelines, incentives, and priorities are often a barrier to research and operational partnerships. In addition, few funding mechanisms exist to generate and solicit analytic questions that are of interest to both research and to operations within health care settings, and provide rapid results that can be used to improve practice and outcomes. METHODS: The Delivery Science Rapid Analysis Program (RAP) was formed in 2013 within the Kaiser Permanente Northern California Division of Research, sponsored by The Permanente Medical Group. A Steering Committee consisting of both researchers and clinical leaders solicits and reviews proposals for rapid analytic projects that will use existing data and are feasible within 6 months and with up to $30,000 (approximately 25-50% full-time equivalent) of programmer/analyst effort. Review criteria include the importance of the analytic question for both research and operations, and the potential for the project to have a significant impact on care delivery within 12 months of completion. RESULTS: The RAP funded 5 research and operational analytic projects between 2013 and 2017. These projects spanned a wide range of clinical areas, including lupus, pediatric obesity, diabetes, e-cigarette use, and hypertension. The hypertension RAP project, which focused on optimizing thiazide prescribing in Black/African-American patients with hypertension, led to new insights that inform an equitable care quality metric designed to reduce blood pressure control disparities throughout the KPNC region. CONCLUSIONS: Programs that actively encourage research and operational analytic partnerships have significant potential to improve care, enhance research collaborations, and contribute to the building and sustaining of learning health care systems.

Resin supported acyl carrier protein labeling strategies.

The post-translational modifying enzymes phophopantetheinyl transferase and acyl carrier protein hydrolase have shown utility in the functional modification of acyl carrier proteins. Here we develop these tools as immobilized biocatalysts on agarose supports. New utility is imparted through these methods, enabling rapid and label-independent protein purification. Immobilization of acyl carrier protein is also demonstrated for rapid activity assays of these 4'-phosophopantetheine modifying enzymes, displaying a particular advantage in the case of phosphopantetheine removal, where few orthogonal techniques have been demonstrated. These tools further enrich the suite of functional utility of 4'-phosophopantetheine chemistry, with applications to protein functionalization, materials, and natural product biosynthetic studies.

Metabolic perturbation of an essential pathway: evaluation of a glycine precursor of coenzyme A.

Pantetheine and its corresponding disulfide pantethine play a key role in metabolism as building blocks of coenzyme A (CoA), an essential cofactor utilized in ~4% of primary metabolism and central to fatty acid, polyketide, and nonribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we show that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one fewer carbon than pantetheine, can rescue a mutant E. coli strain MG1655ΔpanC lacking a functional pantothenate synthetase. Using mass spectrometry, we show that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogues.

Resin-based investigation of acyl carrier protein interaction networks in Escherichia coli.

Protein-protein interactions play an integral role in metabolic regulation. Elucidation of these networks is complicated by the changing identity of the proteins themselves. Here we demonstrate a resin-based technique that leverages the unique tools for acyl carrier protein (ACP) modification with non-hydrolyzable linkages. ACPs from Escherichia coli and Shewanella oneidensis MR-1 are bound to Affigel-15 with varying acyl groups attached and introduced to proteomic samples. Isolation of these binding partners is followed by MudPIT analysis to identify each interactome with the variable of ACP-tethered substrates. These techniques allow for investigation of protein interaction networks with the changing identity of a given protein target.

Genomic insights into the physiology and ecology of the marine filamentous cyanobacterium Lyngbya majuscula.

Filamentous cyanobacteria of the genus Lyngbya are important contributors to coral reef ecosystems, occasionally forming dominant cover and impacting the health of many other co-occurring organisms. Moreover, they are extraordinarily rich sources of bioactive secondary metabolites, with 35% of all reported cyanobacterial natural products deriving from this single pantropical genus. However, the true natural product potential and life strategies of Lyngbya strains are poorly understood because of phylogenetic ambiguity, lack of genomic information, and their close associations with heterotrophic bacteria and other cyanobacteria. To gauge the natural product potential of Lyngbya and gain insights into potential microbial interactions, we sequenced the genome of Lyngbya majuscula 3L, a Caribbean strain that produces the tubulin polymerization inhibitor curacin A and the molluscicide barbamide, using a combination of Sanger and 454 sequencing approaches. Whereas ∼ 293,000 nucleotides of the draft genome are putatively dedicated to secondary metabolism, this is far too few to encode a large suite of Lyngbya metabolites, suggesting Lyngbya metabolites are strain specific and may be useful in species delineation. Our analysis revealed a complex gene regulatory network, including a large number of sigma factors and other regulatory proteins, indicating an enhanced ability for environmental adaptation or microbial associations. Although Lyngbya species are reported to fix nitrogen, nitrogenase genes were not found in the genome or by PCR of genomic DNA. Subsequent growth experiments confirmed that L. majuscula 3L is unable to fix atmospheric nitrogen. These unanticipated life history characteristics challenge current views of the genus Lyngbya.

Practical 4'-phosphopantetheine active site discovery from proteomic samples.

Polyketide and nonribosomal peptides constitute important classes of small molecule natural products. Due to the proven biological activities of these compounds, novel methods for discovery and study of the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymes responsible for their production remains an area of intense interest, and proteomic approaches represent a relatively unexplored avenue. While these enzymes may be distinguished from the proteomic milieu by their use of the 4'-phosphopantetheine (PPant) post-translational modification, proteomic detection of PPant peptides is hindered by their low abundance and labile nature which leaves them unassigned using traditional database searching. Here we address key experimental and computational challenges to facilitate practical discovery of this important post-translational modification during shotgun proteomics analysis using low-resolution ion-trap mass spectrometers. Activity-based enrichment maximizes MS input of PKS/NRPS peptides, while targeted fragmentation detects putative PPant active sites. An improved data analysis pipeline allows experimental identification and validation of these PPant peptides directly from MS² data. Finally, a machine learning approach is developed to directly detect PPant peptides from only MS² fragmentation data. By providing new methods for analysis of an often cryptic post-translational modification, these methods represent a first step toward the study of natural product biosynthesis in proteomic settings.