Evaluation of Multi-Level Barriers and Facilitators in a Large Diabetic Retinopathy Screening Program in Federally Qualified Health Centers: A Qualitative Study

BACKGROUND: Recommended annual diabetic retinopathy (DR) screening for people with diabetes has low rates in the USA, especially in underserved populations. Telemedicine DR screening (TDRS) in primary care clinics could expand access and increase adherence. Despite this potential, studies have observed high variability in TDRS rates among clinics and over time, highlighting the need for implementation supports. Previous studies of determinants of TDRS focus on patients\u27 perspectives, with few studies targeting upstream multi-level barriers and facilitators. Addressing this gap, this qualitative study aimed to identify and evaluate multi-level perceived determinants of TDRS in Federally Qualified Health Centers (FQHCs), to inform the development of targeted implementation strategies. METHODS: We developed a theory-based semi-structured interview tool based on the Consolidated Framework for Implementation Research (CFIR). We conducted 22 key informant interviews with professionals involved in TDRS (administrators, clinicians, staff). The interviews were audio-recorded and transcribed verbatim. Reported barriers and facilitators were organized into emergent themes and classified according to CFIR constructs. Constructs influencing TDRS implementation were rated for each study site and compared across sites by the investigators. RESULTS: Professionals identified 21 main barriers and facilitators under twelve constructs of the five CFIR domains. Several identified themes were novel, whereas others corroborated previous findings in the literature (e.g., lack of time and human resources, presence of a champion). Of the 21 identified themes, 13 were classified under the CFIR’s Inner Setting domain, specifically under the constructs Compatibility and Available Resources. Themes under the Outer Setting domain (constructs External Incentives and Cost) were primarily perceived by administrators, whereas themes in other domains were perceived across all professional categories. Two Inner Setting (Leadership Engagement, Goals and Feedback) and two Process (Champion, Engaging) constructs were found to strongly distinguish sites with high versus low TDRS performance. CONCLUSIONS: This study classified barriers and facilitators to TDRS as perceived by administrators, clinicians, and staff in FQHCs, then identified CFIR constructs that distinguished high- and low-performance clinics. Implementation strategies such as academic detailing and collection and communication of program data and successes to leadership; engaging of stakeholders through involvement in implementation planning; and appointment of intervention champions may therefore improve TDRS implementation and sustainment in resource-constrained settings

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other α/β-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.

The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

The Repertoire of Heterotrimeric G Proteins and RGS Proteins in Ciona intestinalis

BACKGROUND:Heterotrimeric G proteins and regulators of G protein signaling (RGS) proteins are key downstream interacting partners in the G protein coupled receptor (GPCR) signaling pathway. The highly versatile GPCR transmembrane signaling system is a consequence of the coupling of a diverse set of receptors to downstream partners that include multiple subforms of G proteins and regulatory proteins including RGS proteins, among others. While the GPCR repertoire of Ciona intestinalis, representing the basal chordate is known, the repertoire of the heterotrimeric G proteins and RGS proteins is unknown. METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we performed an in-silico genome-wide search of C. intestinalis for its complement of G proteins and RGS proteins. The identification of several one-to-one orthologs of human G proteins at the levels of families, subfamilies and types and of homologs of the human RGS proteins suggests an evolutionarily conserved structure function relationship of the GPCR signaling mechanism in the chordates. CONCLUSIONS:The C. intestinalis genome encodes a highly conserved, albeit, limited repertoire of the heterotrimeric G protein complexes with the size of subunit types comparable with that in lower eukaryotes

Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy

The authors of this research article demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle, and show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein. These movements are of crucial importance for understanding the mechanistic principles of this GAD

Free Energy Simulations of a GTPase: GTP and GDP Binding to Archaeal Initiation Factor 2

International audienceArchaeal initiation factor 2 (aIF2) is a protein involved in the initiation of protein biosynthesis. In its GTP-bound, "ON" conformation, aIF2 binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and its dependence on the ON or OFF conformational state of aIF2, molecular dynamics free energy simulations (MDFE) are a tool of choice. However, the validity of the computed free energies depends on the simulation model, including the force field and the boundary conditions, and on the extent of conformational sampling in the simulations. aIF2 and other GTPases present specific difficulties; in particular, the nucleotide ligand coordinates a divalent Mg(2+) ion, which can polarize the electronic distribution of its environment. Thus, a force field with an explicit treatment of electronic polarizability could be necessary, rather than a simpler, fixed charge force field. Here, we begin by comparing a fixed charge force field to quantum chemical calculations and experiment for Mg(2+):phosphate binding in solution, with the force field giving large errors. Next, we consider GTP and GDP bound to aIF2 and we compare two fixed charge force fields to the recent, polarizable, AMOEBA force field, extended here in a simple, approximate manner to include GTP. We focus on a quantity that approximates the free energy to change GTP into GDP. Despite the errors seen for Mg(2+):phosphate binding in solution, we observe a substantial cancellation of errors when we compare the free energy change in the protein to that in solution, or when we compare the protein ON and OFF states. Finally, we have used the fixed charge force field to perform MDFE simulations and alchemically transform GTP into GDP in the protein and in solution. With a total of about 200 ns of molecular dynamics, we obtain good convergence and a reasonable statistical uncertainty, comparable to the force field uncertainty, and somewhat lower than the predicted GTP/GDP binding free energy differences. The sign and magnitudes of the differences can thus be interpreted at a semiquantitative level, and are found to be consistent with the experimental binding preferences of ON- and OFF-aIF2

G-protein signaling: back to the future

Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Gα·GDP/Gβγ heterotrimers to promote GDP release and GTP binding, resulting in liberation of Gα from Gβγ. Gα·GTP and Gβγ target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Gα and heterotrimer reformation — a cycle accelerated by ‘regulators of G-protein signaling’ (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) β is activated by Gαq and Gβγ, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Gα nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways

A Computational Study of Elongation Factor G (EFG) Duplicated Genes: Diverged Nature Underlying the Innovation on the Same Structural Template

BACKGROUND: Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG's evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function. METHODOLOGY/PRINCIPAL FINDINGS: We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif "RGITI"; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV. CONCLUSIONS/SIGNIFICANCE: Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes

Modeling of Human Prokineticin Receptors: Interactions with Novel Small-Molecule Binders and Potential Off-Target Drugs

The Prokineticin receptor (PKR) 1 and 2 subtypes are novel members of family A GPCRs, which exhibit an unusually high degree of sequence similarity. Prokineticins (PKs), their cognate ligands, are small secreted proteins of ∼80 amino acids; however, non-peptidic low-molecular weight antagonists have also been identified. PKs and their receptors play important roles under various physiological conditions such as maintaining circadian rhythm and pain perception, as well as regulating angiogenesis and modulating immunity. Identifying binding sites for known antagonists and for additional potential binders will facilitate studying and regulating these novel receptors. Blocking PKRs may serve as a therapeutic tool for various diseases, including acute pain, inflammation and cancer.Ligand-based pharmacophore models were derived from known antagonists, and virtual screening performed on the DrugBank dataset identified potential human PKR (hPKR) ligands with novel scaffolds. Interestingly, these included several HIV protease inhibitors for which endothelial cell dysfunction is a documented side effect. Our results suggest that the side effects might be due to inhibition of the PKR signaling pathway. Docking of known binders to a 3D homology model of hPKR1 is in agreement with the well-established canonical TM-bundle binding site of family A GPCRs. Furthermore, the docking results highlight residues that may form specific contacts with the ligands. These contacts provide structural explanation for the importance of several chemical features that were obtained from the structure-activity analysis of known binders. With the exception of a single loop residue that might be perused in the future for obtaining subtype-specific regulation, the results suggest an identical TM-bundle binding site for hPKR1 and hPKR2. In addition, analysis of the intracellular regions highlights variable regions that may provide subtype specificity