Dyspepsia: A stepwise approach to evaluation and management.

Let a patient's age and specific symptoms steer your initial investigation. Consider treatment options beyond antibiotics for H pylori gastritis and PPIs.

Purification of recombinant apolipoproteins A-I and A-IV and efficient affinity tag cleavage by tobacco etch virus protease.

The expression of recombinant apolipoproteins provides experimental avenues that are not possible with plasma purified protein. The ability to specifically mutate residues or delete entire regions has proven to be a valuable tool for understanding the structure and function of apolipoproteins. A common feature of many recombinant systems is an affinity tag that allows for straightforward and high-yield purification of the target protein. A specific protease can then cleave the tag and yield the native recombinant protein. However, the application of this strategy to apolipoproteins has proven somewhat problematic because of the tendency for these highly flexible proteins to be nonspecifically cleaved at undesired sites within the native protein. Although systems have been developed using a variety of proteases, many suffer from low yield and, especially, the high cost of the enzyme.We developed a method that utilizes the tobacco etch virus protease to cleave a histidine-tag from apolipoproteins A-I and A-IV expressed in Escherichia coli. This protease can be easily and inexpensively expressed within most laboratories. We found that the protease efficiently cleaved the affinity tags from both apolipoproteins without nonspecific cleavage. All structural and functional measurements showed that the proteins were equivalent to native or previously characterized protein preparations. In addition to cost-effectiveness, advantages of the tobacco etch virus protease include a short cleavage time, low reaction temperature, and easy removal using the protease's own histidine-tag.

Clinical evaluation of e-Quit worRx: a mobile app to enhance smoking cessation shared decision making in primary care.

BACKGROUND: Smoking is the leading preventable cause of morbidity and mortality in the United States. Primary care providers (PCPs) have a unique opportunity to engage patients to quit smoking, but to be effective, clinicians must be able to personalize evidence-based interventions that are useful and appealing to patients in a time efficient manner. We pilot tested a novel iPad application (app), called e-Quit worRx™, designed to enhance patient-centered shared-decision making (SDM) about smoking cessation, with the primary goal of determining feasibility in primary care offices. METHODS: A total of 73 patients from three offices within a local diverse primary care network were enrolled in a pragmatic single crossover-controlled trial. The decision aid app was incorporated into current smokers' waiting time for their PCP in the exam room, and their PCP reviewed their personal responses and selections to finalize treatment choices. Mixed methods were used in the evaluation and the primary outcomes were app feasibility in primary care and enhanced SDM. RESULTS: Our app was determined to be feasible for use in primary care for both patients and PCPs. It significantly increased time spent discussing smoking cessation with their PCP and the likelihood that a decision was made at the time of the visit. While not significant, mean differences were observed in other study measures including SDM, decisional conflict, quality of patient-provider communication, and stage of change progression at 12 weeks post-trial. CONCLUSIONS: We created a usable and acceptable iPad app-based decision aid for use in primary care offices. The design process presented several challenges including integration into a clinical setting. Despite these challenges, we successfully ran a pragmatic pilot trial in three primary care offices using a technology novel to many of the users.

Challenges in the Development of e-Quit worRx: An iPad App for Smoking Cessation Counseling and Shared Decision Making in Primary Care.

BACKGROUND: Smoking is the leading preventable cause of morbidity and mortality in the United States, killing more than 450,000 Americans. Primary care physicians (PCPs) have a unique opportunity to discuss smoking cessation evidence in a way that enhances patient-initiated change and quit attempts. Patients today are better equipped with technology such as mobile devices than ever before. OBJECTIVE: The aim of this study was to evaluate the challenges in developing a tablet-based, evidence-based smoking cessation app to optimize interaction for shared decision making between PCPs and their patients who smoke. METHODS: A group of interprofessional experts developed content and a graphical user interface for the decision aid and reviewed these with several focus groups to determine acceptability and usability in a small population. RESULTS: Using a storyboard methodology and subject matter experts, a mobile app, e-Quit worRx, was developed through an iterative process. This iterative process helped finalize the content and ergonomics of the app and provided valuable feedback from both patients and provider teams. Once the app was made available, other technical and programmatic challenges arose. CONCLUSIONS: Subject matter experts, although generally amenable to one another's disciplines, are often challenged with effective interactions, including language, scope, clinical understanding, technology awareness, and expectations. The successful development of this app and its evaluation in a clinical setting highlighted those challenges and reinforced the need for effective communications and team building.

Enhanced placental cholesterol efflux by fetal HDL in Smith-Lemli-Opitz syndrome.

Previous studies from this laboratory have shown that maternal-derived cholesterol can be effluxed from trophoblasts to fetal HDL and plasma. We had the opportunity to study for the first time the ability of HDL and plasma from a fetus with the Smith-Lemli-Opitz syndrome (SLOS) to efflux cholesterol from trophoblasts. It was unclear whether cholesterol could be effluxed to fetuses with SLOS since lipoprotein levels are often very low. To answer this question, cord blood was collected from the placentas of an SLOS fetus and unaffected fetuses just after delivery. Plasma cholesterol concentrations were very low in the affected fetus; cholesterol, 7-dehydrocholesterol, and 8-dehydocholesterol concentrations were 14.1, 4.5, and 5.2 mg/dl, respectively. The HDL from the fetal SLOS effluxed approximately 50% more cholesterol from a trophoblast cell line, were smaller in size, and had a lower cholesterol to phospholipid ratio as compared to HDL from unaffected fetuses or adults. Plasma from the SLOS fetus effluxed cholesterol to a similar percentage as unaffected fetal plasma or adult plasma, possibly due to fewer HDL particles as demonstrated in previous SLOS patients. These novel data demonstrate that the cholesterol-deficient SLOS fetus is able to obtain cholesterol from trophoblasts at a time when cholesterol is playing a critical role in development, and has implications for design of treatments for cholesterol deficiency syndromes as well as understanding of prenatal cholesterol transport in humans.

Diabetes in homeless persons: barriers and enablers to health as perceived by patients, medical, and social service providers.

The ways homelessness and diabetes affect each other is not well known. The authors sought to understand barriers and enablers to health for homeless people with diabetes as perceived by homeless persons and providers. The authors performed semistructured interviews with a sample of participants (seven homeless persons, six social service providers, and five medical providers) in an urban Midwest community. Data analysis was performed with the qualitative editing method. Participants described external factors (chaotic lifestyle, diet/food availability, access to care, and medications) and internal factors (competing demands, substance abuse, stress) that directly affect health. Social service providers were seen as peripheral to diabetes care, although all saw their primary functions as valuable. These factors and relationships are appropriately modeled in a complex adaptive chronic care model, where the framework is bottom up and stresses adaptability, self-organization, and empowerment. Adapting the care of homeless persons with diabetes to include involvement of patients and medical and social service providers must be emergent and responsive to changing needs.

The structure of dimeric apolipoprotein A-IV and its mechanism of self-association.

Apolipoproteins are key structural elements of lipoproteins and critical mediators of lipid metabolism. Their detergent-like properties allow them to emulsify lipid or exist in a soluble lipid-free form in various states of self-association. Unfortunately, these traits have hampered high-resolution structural studies needed to understand the biogenesis of cardioprotective high-density lipoproteins (HDLs). We derived a crystal structure of the core domain of human apolipoprotein (apo)A-IV, an HDL component and important mediator of lipid absorption. The structure at 2.4 Å depicts two linearly connected 4-helix bundles participating in a helix swapping arrangement that offers a clear explanation for how the protein self-associates as well as clues to the structure of its monomeric form. This also provides a logical basis for antiparallel arrangements recently described for lipid-containing particles. Furthermore, we propose a "swinging door" model for apoA-IV lipid association.

Mass spectrometric determination of apolipoprotein molecular stoichiometry in reconstituted high density lipoprotein particles.

Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL particles.

Effect of intraperitoneal and intravenous administration of cholecystokinin-8 and apolipoprotein AIV on intestinal lymphatic CCK-8 and apo AIV concentration.

CCK and apolipoprotein AIV (apo AIV) are gastrointestinal satiety signals whose synthesis and secretion by the gut are stimulated by fat absorption. Intraperitoneally administered CCK-8 is more potent in suppressing food intake than a similar dose administered intravenously, but the reason for this disparity is unclear. In contrast, both intravenous and intraperitoneally administered apo AIV are equally as potent in inhibiting food intake. When we compared the lymphatic concentration of CCK-8 and apo AIV, we found that neither intraperitoneally nor intravenously administered CCK-8 or apo AIV altered lymphatic flow rate. Interestingly, intraperitoneal administration of CCK-8 produced a significantly higher lymphatic concentration at 15 min than did intravenous administration. Intraperitoneal injection of apo AIV also yielded a higher lymphatic concentration at 30 min than did intravenous administration. Intraperitoneal administration of CCK-8 and apo AIV also resulted in a much longer period of elevated CCK-8 and apo AIV peptide concentration in lymph than intravenous administration. Furthermore, enzymatic activity of dipeptidyl peptidase IV (DPPIV) and aminopeptidase was higher in plasma than in lymph during fasting, and so, satiation peptides, such as CCK-8 and apo AIV in the lymph, are protected from degradation by the significantly lower DPPIV and aminopeptidase activity levels in lymph than in plasma. Therefore, the higher potency of intraperitoneally administered CCK-8 compared with intravenously administered CCK-8 in inhibiting food intake may be explained by both its higher concentration in lymph and the prolonged duration of its presence in the lamina propria.

A three-dimensional homology model of lipid-free apolipoprotein A-IV using cross-linking and mass spectrometry.

Human apolipoprotein A-IV (apoA-IV) is a 46-kDa exchangeable plasma protein with many proposed functions. It is involved in chylomicron assembly and secretion, protection from atherosclerosis through a variety of mechanisms, and inhibition of food intake. There is little structural basis for these proposed functions due to the lack of a solved three-dimensional structure of the protein by x-ray crystallography or NMR. Based on previous studies, we hypothesized that lipid-free apoA-IV exists in a helical bundle, like other apolipoprotein family members and that regions near the N and C termini may interact. Utilizing a homobifunctional lysine cross-linking agent, we identified 21 intramolecular cross-links by mass spectrometry. These cross-links were used to constrain the building of a sequence threaded homology model using the I-TASSER server. Our results indicate that lipid-free apoA-IV does indeed exist as a complex helical bundle with the N and C termini in close proximity. This first structural model of lipid-free apoA-IV should prove useful for designing studies aimed at understanding how apoA-IV interacts with lipids and possibly with unknown protein partners.

Modulation of apolipoprotein A-IV lipid binding by an interaction between the N and C termini.

Apolipoprotein A-IV (apoA-IV) is a 376-amino acid exchangeable apolipoprotein made in the small intestine of humans. Although it has many proposed roles in vascular disease, satiety, and chylomicron metabolism, there is no known structural basis for these functions. The ability to associate with lipids may be a key step in apoA-IV functionality. We recently identified a single amino acid, Phe(334), which seems to inhibit the lipid binding capability of apoA-IV. We also found that an intact N terminus was necessary for increased lipid binding of Phe(334) mutants. Here, we identify Trp(12) and Phe(15) as the N-terminal amino acids required for the fast lipid binding seen with the F334A mutant. Furthermore, we found that individual disruption of putative amphipathic alpha-helices 3-11 had little effect on lipid binding, suggesting that the N terminus of apoA-IV may be the operational site for initial lipid binding. We also provide three independent pieces of experimental evidence supporting a direct intramolecular interaction between sequences near amino acids 12/15 and 334. This interaction could represent a unique "switch" mechanism by which apoA-IV changes lipid avidity in vivo.

The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins.

The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism. Mutagenesis techniques have been widely used to modulate apolipoprotein lipid affinity for studying biological function, but the approach requires rapid and reliable lipid affinity assays to compare the mutants. Here, we describe a novel method that measures apolipoprotein binding to a standardized preparation of small unilamellar vesicles (SUVs) containing trace biotinylated and fluorescent phospholipids. After a 30 min incubation at various apolipoprotein concentrations, vesicle-bound protein is rapidly separated from free protein on columns of immobilized streptavidin in a 96-well microplate format. Vesicle-bound protein and lipid are eluted and measured in a fluorescence microplate reader for calculation of a dissociation constant and the maximum number of potential binding sites on the SUVs. Using human apolipoprotein A-I (apoA-I), apoA-IV, and mutants of each, we show that the assay generates binding constants that are comparable to other methods and is reproducible across time and apolipoprotein preparations. The assay is easy to perform and can measure triplicate binding parameters for up to 10 separate apolipoproteins in 3.5 h, consuming only 120 microg of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.

Specific sequences in the N and C termini of apolipoprotein A-IV modulate its conformation and lipid association.

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.