Accelerating Colorectal Cancer Screening and Follow-up through Implementation Science (ACCSIS) in Appalachia: protocol for a group randomized, delayed intervention trial.

Appalachian regions of Kentucky and Ohio are hotspots for colorectal cancer (CRC) mortality in the USA. Screening reduces CRC incidence and mortality; however, screening uptake is needed, especially in these underserved geographic areas. Implementation science offers strategies to address this challenge. The aim of the current study was to conduct multi-site, transdisciplinary research to evaluate and improve CRC screening processes using implementation science strategies. The study consists of two phases (Planning and Implementation). In the Planning Phase, a multilevel assessment of 12 health centers (HC) (one HC from each of the 12 Appalachian counties) was conducted by interviewing key informants, creating community profiles, identifying HC and community champions, and performing HC data inventories. Two designated pilot HCs chose CRC evidence-based interventions to adapt and implement at each level (i.e., patient, provider, HC, and community) with evaluation relative to two matched control HCs. During the Implementation Phase, study staff will repeat the rollout process in HC and community settings in a randomized, staggered fashion in the remaining eight counties/HCs. Evaluation will include analyses of electronic health record data and provider and county surveys. Rural HCs have been reluctant to participate in research because of concerns about capacity; however, this project should demonstrate that research does not need to be burdensome and can adapt to local needs and HC abilities. If effective, this approach could be disseminated to HC and community partners throughout Appalachia to encourage the uptake of effective interventions to reduce the burden of CRC.

We conducted a multi-site study to evaluate and improve CRC screening processes using implementation science strategies at multiple levels including the patient, provider, health center, and community. Our goals were to increase rates of guideline-recommended CRC screening, follow-up, and referral-to-care in an Appalachian, medically underserved population.

Deficiency of lncRNA SNHG12 impairs ischemic limb neovascularization by altering an endothelial cell cycle pathway.

SNHG12, a long noncoding RNA (lncRNA) dysregulated in atherosclerosis, is known to be a key regulator of vascular senescence in endothelial cells (ECs). However, its role in angiogenesis and peripheral artery disease has not been elucidated. Hind-limb ischemia studies using femoral artery ligation (FAL) in mice showed that SNHG12 expression falls readily in the acute phase of the response to limb ischemia in gastrocnemius muscle and recovers to normal when blood flow recovery is restored to ischemic muscle, indicating that it likely plays a role in the angiogenic response to ischemia. Gain- and loss-of-function studies demonstrated that SNHG12 regulated angiogenesis - SNHG12 deficiency reduced cell proliferation, migration, and endothelial sprouting, whereas overexpression promoted these angiogenic functions. We identified SNHG12 binding partners by proteomics that may contribute to its role in angiogenesis, including IGF-2 mRNA-binding protein 3 (IGF2BP3, also known as IMP3). RNA-Seq profiling of SNHG12-deficient ECs showed effects on angiogenesis pathways and identified a strong effect on cell cycle regulation, which may be modulated by IMP3. Knockdown of SNHG12 in mice undergoing FAL using injected gapmeRs) decreased angiogenesis, an effect that was more pronounced in a model of insulin-resistant db/db mice. RNA-Seq profiling of the EC and non-EC compartments in these mice revealed a likely role of SNHG12 knockdown on Wnt, Notch, and angiopoietin signaling pathways. Together, these findings indicate that SNHG12 plays an important role in the angiogenic EC response to ischemia.

Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates.

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.

Novel Lesional Transcriptional Signature Separates Atherosclerosis With and Without Diabetes in Yorkshire Swine and Humans.

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IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses.

T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation.

Provider Practice and Perceptions of Pediatric Obesity in Appalachian Kentucky.

OBJECTIVES: This study assessed providers' present practices and perceived needs in Appalachian Kentucky to identify the standard of care and implementation of expert recommendations for managing pediatric obesity. METHODS: Questionnaire data were gathered from 28 providers at a pediatric obesity continuing medical education workshop in eastern Kentucky. We assessed current practices, perceived barriers to treatment, and needed resources for managing pediatric obesity. RESULTS: Respondents reported mixed adherence to expert recommendations, with providers less frequently addressing family-reported barriers to change and assessing a family's readiness to change behaviors related to pediatric obesity. Respondents also reported service barriers related to patient motivation, lack of time with patients, and a lack of referral options. Finally, providers reported needing multiple community resources to better address pediatric obesity, including improved physical education programs, access to community recreation centers, additional referral resources for multidisciplinary care, and additional training in motivational techniques. CONCLUSIONS: There remains a significant need for education and guidance regarding the implementation of expert recommendations for addressing pediatric obesity in Appalachian Kentucky. Providers reported needing multiple community resources, including improved physical education programs, access to community recreation centers, additional referral resources for multidisciplinary care, and additional training in motivational techniques. We discuss the implications for disseminating and implementing expert recommendations in rural eastern Kentucky, with an emphasis on the roles of behavioral health experts.

Dual muscle-liver transduction imposes immune tolerance for muscle transgene engraftment despite preexisting immunity.

Immune responses to therapeutic transgenes are a potential hurdle to treat monogenic muscle disorders. These responses result from the neutralizing activity of transgene-specific B cells and cytotoxic T cells recruited upon gene transfer. We explored here how dual muscle-liver expression of a foreign transgene allows muscle transgene engraftment after adenoassociated viral vector delivery. We found in particular that induction of transgene-specific tolerance is imposed by concurrent muscle and liver targeting, resulting in the absence of CD8+ T cell responses to the transgene. This tolerance can be temporally decoupled, because transgene engraftment can be achieved in muscle weeks after liver transduction. Importantly, transgene-specific CD8+ T cell tolerance can be established despite preexisting immunity to the transgene. Whenever preexisting, transgene-specific CD4+ and CD8+ memory T cell responses are present, dual muscle-liver transduction turns polyclonal, transgene-specific CD8+ T cells into typically exhausted T cells with high programmed cell death 1 (PD-1) expression and lack of IFN-γ production. Our results demonstrate that successful transduction of muscle tissue can be achieved through liver-mediated control of humoral and cytotoxic T cell responses, even in the presence of preexisting immunity to the muscle-associated transgene.

Priming the Physician Pipeline: A Regional AHEC's Use of in-state Medical School Data to Guide Its Health Careers Programming.

Amid evidence of its rural, Appalachian students' low application and matriculation rates to in-state medical schools, the Northeast Kentucky Area Health Education Center developed two physician pipeline programs. This report describes the programs' early successes and provides a basis for heightened regional responsibility in the recruitment of medical school applicants.

DFT: A Theory Full of Holes?

This article is a rough, quirky overview of both the history and present state of the art of density functional theory. The field is so huge that no attempt to be comprehensive is made. We focus on the underlying exact theory, the origin of approximations, and the tension between empirical and nonempirical approaches. Many ideas are illustrated on the exchange energy and hole. Features unique to this article include how approximations can be systematically derived in a nonempirical fashion and a survey of warm dense matter.

Cytosolic lipid droplets: from mechanisms of fat storage to disease.

The lipid droplet (LD) is a phylogenetically conserved organelle. In eukaryotes, it is born from the endoplasmic reticulum, but unlike its parent organelle, LDs are the only known cytosolic organelles that are micellar in structure. LDs are implicated in numerous physiological and pathophysiological functions. Many aspects of the LD has captured the attention of diverse scientists alike and has recently led to an explosion in information on the LD biogenesis, expansion and fusion, identification of LD proteomes and diseases associated with LD biology. This review will provide a brief history of this fascinating organelle and provide some contemporary views of unanswered questions in LD biogenesis.

Purification of integral membrane proteins and lipid-binding assays.

The lipid droplet (LD) is an evolutionarily conserved organelle composed primarily of triglycerides (TAG) and cholesteryl esters. Recently, Fat storage-Inducing Transmembrane proteins 1 & 2 (FITM1/FIT1 and FITM2/FIT2) were discovered as a conserved family of proteins involved in fat storage. FIT1 and FIT2 are both localized to the endoplasmic reticulum, but have distinct tissue distributions. FIT proteins mediate TAG LD accumulation when overexpressed, but do not synthesize TAG. FIT proteins function by partitioning newly synthesized TAG into LDs. In order to understand the mechanism by which this occurs, a method was developed to purify FIT proteins from insect cells in detergent micelles. The ability of purified FIT proteins to bind TAG and other neutral lipids was tested in detergent micelles, demonstrating lipid specificity and saturation binding. These techniques can be applied to a variety of proteins in lipid biology in an effort to try to reconstitute a mechanism of action or protein activity. The methods that will be discussed here can also be scaled to either screen a library of mutant proteins for binding to a particular compound or utilized to delineate structural requirements of ligands that are important for protein-ligand interactions. Here, we present a detailed description of the purification protocol and micellar protein-ligand binding experiments and their possible applications.

SCS3 and YFT2 link transcription of phospholipid biosynthetic genes to ER stress and the UPR.

The ability to store nutrients in lipid droplets (LDs) is an ancient function that provides the primary source of metabolic energy during periods of nutrient insufficiency and between meals. The Fat storage-Inducing Transmembrane (FIT) proteins are conserved ER-resident proteins that facilitate fat storage by partitioning energy-rich triglycerides into LDs. FIT2, the ancient ortholog of the FIT gene family first identified in mammals has two homologs in Saccharomyces cerevisiae (SCS3 and YFT2) and other fungi of the Saccharomycotina lineage. Despite the coevolution of these genes for more than 170 million years and their divergence from higher eukaryotes, SCS3, YFT2, and the human FIT2 gene retain some common functions: expression of the yeast genes in a human embryonic kidney cell line promotes LD formation, and expression of human FIT2 in yeast rescues the inositol auxotrophy and chemical and genetic phenotypes of strains lacking SCS3. To better understand the function of SCS3 and YFT2, we investigated the chemical sensitivities of strains deleted for either or both genes and identified synthetic genetic interactions against the viable yeast gene-deletion collection. We show that SCS3 and YFT2 have shared and unique functions that connect major biosynthetic processes critical for cell growth. These include lipid metabolism, vesicular trafficking, transcription of phospholipid biosynthetic genes, and protein synthesis. The genetic data indicate that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and that deletion of SCS3 and YFT2 impacts a regulatory mechanism that coordinates these processes. Part of this mechanism involves a role for SCS3 in communicating changes in the ER (e.g. due to low inositol) to Opi1-regulated transcription of phospholipid biosynthetic genes. We conclude that SCS3 and YFT2 are required for normal ER membrane biosynthesis in response to perturbations in lipid metabolism and ER stress.

Mining for future health care providers in Appalachian Kentucky.

Recent studies project a significant shortage of U.S. health care providers within the next decade. To address southeastern Kentucky's persistent undersupply of medical professionals, a multi-county Health Career Enrichment Camp for rising 9th and 10th grade students was implemented in 2008. This report describes the annual camp's development and preliminary outcomes.

Direct binding of triglyceride to fat storage-inducing transmembrane proteins 1 and 2 is important for lipid droplet formation.

The process of lipid droplet (LD) formation is an evolutionarily conserved process among all eukaryotes and plays an important role in both cellular physiology and disease. Recently, fat storage-inducing transmembrane proteins 1 and 2 (FIT1/FITM1 and FIT2/FITM2) were discovered as an evolutionarily conserved family of proteins involved in fat storage. In mammals, FIT1 is expressed primarily in skeletal muscle and FIT2 is expressed primarily in adipose, raising the possibility that FIT1 and FIT2 have unique functions. These proteins are exclusively localized to the endoplasmic reticulum (ER) and mediate triglyceride-rich LD accumulation when overexpressed in cells, mouse liver, or muscle. Unlike the ER-resident diacylglycerol O-acyltransferase family of triglyceride-synthesizing enzymes, FITs do not synthesize triglyceride, but rather partition triglyceride into LDs. The mechanism by which FIT proteins mediate this process has not been determined. A simple hypothesis was tested that FIT proteins bind to triglyceride to mediate LD formation. Here, it is shown that FIT proteins purified in detergent micelles directly bind triolein with specificity and saturation-binding kinetics. A FIT2 gain-of-function mutant that formed larger LDs, FLL(157-9)AAA, showed increased binding to triolein relative to wild-type FIT2, whereas FIT1 and a FIT2 partial loss-of-function mutant, N80A, had significantly lower triolein binding and produced smaller LDs. In summary, FIT proteins are transmembrane domain-containing proteins shown to bind triglyceride. These findings indicate that FITs have a unique biochemical mechanism in mediating LD formation and implicates triglyceride binding as important for FIT-mediated LD formation.

Re-patterning of skeletal muscle energy metabolism by fat storage-inducing transmembrane protein 2.

Triacylglyceride stored in cytosolic lipid droplets (LDs) constitutes a major energy reservoir in most eukaryotes. The regulated turnover of triacylglyceride in LDs provides fatty acids for mitochondrial ß-oxidation and ATP generation in physiological states of high demand for energy. The mechanisms for the formation of LDs in conditions of energy excess are not entirely understood. Fat storage-inducing transmembrane protein 2 (FIT2/FITM2) is the anciently conserved member of the fat storage-inducing transmembrane family of proteins implicated to be important in the formation of LDs, but its role in energy metabolism has not been tested. Here, we report that expression of FIT2 in mouse skeletal muscle had profound effects on muscle energy metabolism. Mice with skeletal muscle-specific overexpression of FIT2 (CKF2) had significantly increased intramyocellular triacylglyceride and complete protection from high fat diet-induced weight gain due to increased energy expenditure. Mass spectrometry-based metabolite profiling suggested that CKF2 skeletal muscle had increased oxidation of branched chain amino acids but decreased oxidation of fatty acids. Glucose was primarily utilized in CKF2 muscle for synthesis of the glycerol backbone of triacylglyceride and not for glycogen production. CKF2 muscle was ATP-deficient and had activated AMP kinase. Together, these studies indicate that FIT2 expression in skeletal muscle plays an unexpected function in regulating muscle energy metabolism and indicates an important role for lipid droplet formation in this process.

Structural insights into triglyceride storage mediated by fat storage-inducing transmembrane (FIT) protein 2.

Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2) belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9)AAA) in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9)AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation.

Effects of N-glycosylation on the activity and localization of GlcNAc-6-sulfotransferase 1.

N-Acetylglucosamine-6-sulfotransferase-1 (GlcNAc6ST-1) is a Golgi-resident glycoprotein that is responsible for sulfation of the l-selectin ligand on endothelial cells. Here, we report the sites at which GlcNAc6ST-1 is modified with N-linked glycans and the effects that each glycan has on enzyme activity, specificity, and localization. We determined that glycans are added at three of four potential N-linked glycosylation sites: N196, N410, and N428. The N428 glycan is required for the production of sulfated cell surface glycans: cells expressing a mutant enzyme lacking this glycan were unable to sulfate the sialyl Lewis X tetrasaccharide or a putative extended core 1 O-linked glycan. The N196 and N410 glycans differentially affect sulfation of two different substrates: cells that express an enzyme lacking the N410 glycan are able to sulfate the sialyl Lewis X substrate, but produce reduced levels of a sulfated peripheral lymph node addressin epitope and cells that express an enzyme lacking the N196 glycan are able to produce a sulfated peripheral lymph node addressin epitope, but are impaired in their ability to sulfate sialyl Lewis X. The glycans' effects on enzyme activity may be mediated, in part, by changes in enzyme localization. While most mutants that lacked glycans localized normally within the Golgi, the N428A mutant and a mutant lacking all glycans were also found to localize ectopically. Altered trafficking of mutants may be associated with the mechanisms by which misglycosylated enzyme is degraded.

CD8 T cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized HLA-A*0201-transgenic mice.

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8(+) T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8(+) T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG(181)) generated a cytotoxic CD8(+) T cell response able to aggravate CD4(+)-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8(+) T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG(181) epitope as a primary candidate.