Quality of colonoscopy in an emerging country: A prospective, multicentre study in Russia.

BACKGROUND: The quality of colonoscopy has been related to a higher risk of interval cancer, and this issue has been addressed extensively in developed countries. The aim of our study was to explore the main quality indicators of colonoscopy in a large emerging country. METHODS: Consecutive patients referred for colonoscopy in 14 centres were prospectively included between July and October 2014. Before colonoscopy, several clinical and demographic variables were collected. Main quality indicators (i.e. caecal intubation rate, (advanced) adenoma detection rate, rate of adequate cleansing and sedation) were collected. Data were analysed at per patient and per centre level (only for those with at least 100 cases). Factors associated with caecal intubation rate and adenoma detection rate were explored at multivariate analysis. RESULTS: A total of 8829 (males: 35%; mean age: 57 + 14 years) patients were included, with 11 centres enrolling at least 100 patients. Screening (including non-alarm symptoms) accounted for 59% (5188/8829) of the indications. Sedation and split preparation were used in 26% (2294/8829) and 25% (2187/8829) of the patients. Caecal intubation was achieved in 7616 patients (86%), and it was ≥85% in 8/11 (73%) centres. Adenoma detection rate was 18% (1550/8829), and it was higher than 20% in five (45%) centres, whilst it was lower than 10% in four (33%) centres. At multivariate analysis, age (OR: 1.020, 95% CI: 1.015-1.024), male sex (OR: 1.2, 95% CI: 1.1-1.3), alarm symptoms (OR: 1.8, 95% CI: 1.7-2), split preparation (OR: 1.4, 95% CI: 1.2-1.6), caecal intubation rate (OR: 1.6, 95% CI: 1.3-1.9) and withdrawal time measurement (OR: 1.2, 95% CI: 1.6-2.1) were predictors of a higher adenoma detection rate, while adequate preparation (OR: 3.4: 95% CI: 2.9-3.9) and sedation (OR: 1.3; 95% CI: 1.1-1.6) were the strongest predictors of caecal intubation rate. CONCLUSIONS: According to our study, there is a substantial intercentre variability in the main quality indicators. Overall, the caecal intubation rate appears to be acceptable in most centres, whilst the overall level of adenoma detection appears low, with less than half of the centres being higher than 20%. Educational and quality assurance programs, including higher rates of sedation and split regimen of preparation, may be necessary to increase the key quality indicators.

Measuring the energetics of membrane protein dimerization in mammalian membranes.

Thus far, quantitative studies of lateral protein interactions in membranes have been restricted peptides or simplified protein constructs in lipid vesicles or bacterial membranes. Here we show how free energies of membrane protein dimerization can be measured in mammalian plasma membrane-derived vesicles. The measurements, performed in single vesicles, utilize the quantitative imaging FRET (QI-FRET) method. The experiments are described in a step-by-step protocol. The protein characterized is the transmembrane domain of glycophorin A, the most extensively studied membrane protein, known to form homodimers in hydrophobic environments. The results suggest that molecular crowding in cellular membranes has a dramatic effect on the strength of membrane protein interactions.

Hill coefficient analysis of transmembrane helix dimerization.

Here, we employed the Hill equation, used broadly to characterize cooperativity in protein-ligand binding, to describe the dimerization of transmembrane (TM) helices in hydrophobic environments. The Hill analysis of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domain dimerization gives a Hill coefficient of approximately 1 for lipid bilayers but only approximately 0.2 for sodium dodecyl sulfate (SDS) micelles. We propose that this finding is indicative of heterogeneity in FGFR3 TM dimer structure and stability in SDS micelles. We further speculate that (1) the Hill equation can be used as a tool to assess the existence of multiple structural states of TM dimers in different hydrophobic environments and (2) the structural heterogeneity, detectable by Hill analysis, may be the underlying reason for the broad peaks and the low resolution NMR studies of peptides in detergents.

Energetics of ErbB1 transmembrane domain dimerization in lipid bilayers.

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as -2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.

Polar residues in transmembrane helices can decrease electrophoretic mobility in polyacrylamide gels without causing helix dimerization.

There are only a few available methods to study lateral interactions and self assembly of transmembrane helices. One of the most frequently used methods is sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) which can report on strong interactions between peptides in SDS solution. Here we offer a cautionary tale about studying the folding and assembly of membrane proteins using peptides and SDS-PAGE experiments as a membrane mimetic system. At least for the specific peptide and detergent systems studied here, we show that a polar asparagine residue in the 12th position of an otherwise hydrophobic helical segment of 20 amino acids causes a peptide to migrate on SDS-PAGE gels with an apparent molecular weight that is twice its true molecular weight, suggesting dimerization. However when examined carefully in SDS solutions and in situ in the polyacrylamide gel itself using Forster resonance energy transfer no interaction can be detected. Instead we show evidence suggesting that differential interactions between peptide and detergent drive the differences in electrophoretic mobility without any interaction between peptides. These results emphasize the need to apply multiple independent techniques to the study of membrane protein folding, and they highlight the usefulness of studying folding and structure of membrane proteins in lipid membranes rather than in detergents.

Utility of surface-supported bilayers in studies of transmembrane helix dimerization.

This review focuses on the methods that are available to study transmembrane (TM) helix dimerization in membrane-like environments (either bacterial membranes or lipid bilayers, as mimics of the eukaryotic cellular membrane), with an emphasis on the utility of surface-supported bilayers in such studies.

Quantitative measurements of protein interactions in a crowded cellular environment.

Quantitative measurements of protein interaction strengths, crucial for describing signaling networks and predicting cellular responses to environmental stimuli, are typically performed in dilute buffer solutions. However, protein-protein interactions in cells occur within the context of a crowded system, which is characterized by a high macromolecular concentration. In this paper, we explore the utility of cell-derived vesicles as a model crowded environment for quantitative FRET measurements of protein-protein interactions. We show that the FRET efficiency, and the donor and acceptor concentrations, can be calculated in each vesicle. We also introduce the "quantitative imaging Foster resonance energy transfer" method as a tool that can yield protein interaction strengths within these vesicles.

Characterization of antimicrobial peptide activity by electrochemical impedance spectroscopy.

Electrochemical impedance spectroscopy performed on surface-supported bilayer membranes allows for the monitoring of changes in membrane properties, such as thickness, ion permeability, and homogeneity, after exposure to antimicrobial peptides (AMPs). We show that two model cationic peptides, very similar in sequence but different in activity, induce dramatically different changes in membrane properties as probed by impedance spectroscopy. Moreover, the impedance results excluded the "barrel-stave" and the "toroidal pore" models of AMP mode of action, and are more consistent with the "carpet" and the "detergent" models. The impedance data provide important new insights about the kinetics and the scale of the peptide action which currently are not addressed by the "carpet" and the "detergent" models. The method presented not only provides additional information about the mode of action of a particular AMP, but offers a means of characterizing AMP activity in reproducible, well-defined quantitative terms.

A simple "proximity" correction for Förster resonance energy transfer efficiency determination in membranes using lifetime measurements.

Accurate measurements of oligomerization in membranes by Förster resonance energy transfer (FRET) are always compromised by a substantial contribution from random chance colocalization of donors and acceptors. Recently, Li and coworkers demonstrated the use of computer simulation in estimating the contribution of this "proximity" component to correct the FRET efficiency and estimate the free energy of dimer formation of the G380R mutants of fibroblast growth factor receptor 3 (FGFR3) transmembrane domain immersed into lipid bilayer. Because tight dimerization will result in complete energy transfer from donor to acceptor, we have used the same experimental system of fluorescein- and rhodamine-labeled G380R mutants of FGFR3 for the experimental assessment of the proximity FRET corrections using fluorescence lifetime measurements. The experimental proximity FRET correction, based on time-resolved fluorescence measurements, is expected to have general advantages over theoretical correction, especially in the case of nonrandomly distributed monomers.

Impedance spectroscopy of bilayer membranes on single crystal silicon.

Bilayer membranes on solid supports are being developed as electrically addressable, robust, surface-supported membrane mimetics. These platforms are being explored for basic ion channel research as well as for detection and analyte sensing. The formation of bilayer membranes on semiconductor surfaces is an important step in device integration for transistor and sensor arrays. Here, the authors review the contributions to the impedance response of bilayer membranes on semiconductors, and highlight the important issues for experimental measurements. The authors also present experimental results for diphytanoyl phosphocholine bilayers formed on moderately doped and highly doped n-type silicon using Langmuir-Blodgett-based deposition techniques. The authors demonstrate that a detailed understanding of the contributions to the impedance response is important in developing silicon-based membrane platforms. The authors further report on the bias dependence of the impedance, and show that on highly doped n-type silicon, the membrane impedance can be measured over a 2 V range.

Surface supported bilayer platform for studies of lateral association of proteins in membranes (Mini Review).

Here, the authors review how surface supported bilayers can be engineered and how Forster resonance energy transfer (FRET) can be used to quantify interactions between transmembrane peptides in these bilayers. The requirements for the surface supported platform are (1) lateral mobility of the peptides, (2) transmembrane orientation of the peptides, and (3) capabilities for FRET measurements. To satisfy these requirements, a new assembly method, termed "directed assembly" was developed. This assembly method could have broad utility in basic studies of proteins in membranes and in biotechnological applications.

Forster resonance energy transfer measurements of transmembrane helix dimerization energetics.

Lateral interactions between hydrophobic transmembrane (TM) helices in membranes underlie the folding of multispan membrane proteins and signal transduction by receptor tyrosine kinases (RTKs). Quantitative measurements of dimerization energetics in membranes are required to uncover the physical principles behind these processes. Here, we overview how FRET measurements can be used to determine the thermodynamics of TM helix homo- and heterodimerization in vesicles and in supported bilayers. Such measurements can shed light on the molecular mechanism behind pathologies arising due to single-amino acid mutations in membrane proteins.

Electrical measurements of bilayer membranes formed by Langmuir-Blodgett deposition on single-crystal silicon.

Bilayer membranes on solid supports are used for fundamental studies of biophysical properties and for the development of biosensors and other devices. Here we report on electrically addressable bilayer membranes formed by Langmuir-Blodgett (LB)-based deposition on single-crystal silicon. The incorporation of a polymer cushion ensures high lipid mobility in both the lower and upper leaflet, allowing the potential for combined investigations of electrical, structural, and dynamic characteristics of membrane-associated proteins. Impedance spectroscopy is used to demonstrate that the lipid bilayers are robust and reproducible with an impedance of about 10(4) Omega cm2 and a capacitance of about 0.8 microF cm(-2). The ability to characterize ion channels is demonstrated using the model system gramicidin. These results demonstrate that artificial bilayers formed by LB deposition have many unique advantages for electrical measurements of membranes and their components.

Studies of receptor tyrosine kinase transmembrane domain interactions: the EmEx-FRET method.

The energetics of transmembrane (TM) helix dimerization in membranes and the thermodynamic principles behind receptor tyrosine kinase (RTK) TM domain interactions during signal transduction can be studied using Förster resonance energy transfer (FRET). For instance, FRET studies have yielded the stabilities of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domains and two FGFR3 pathogenic mutants, Ala391Glu and Gly380Arg, in the native bilayer environment. To further our understanding of the molecular mechanisms of deregulated FGFR3 signaling underlying different pathologies, we determined the effect of the Gly382Asp FGFR3 mutation, identified in a multiple myeloma cell line, on the energetics of FGFR3 TM domain dimerization. We measured dimerization energetics using a novel FRET acquisition and processing method, termed "emission-excitation FRET (EmEx-FRET)," which improves the precision of thermodynamic measurements of TM helix association. The EmEx-FRET method, verified here by analyzing previously published data for wild-type FGFR3 TM domain, should have broad utility in studies of protein interactions, particularly in cases when the concentrations of fluorophore-tagged molecules cannot be controlled.

Surface-supported bilayers with transmembrane proteins: role of the polymer cushion revisited.

Protein lateral mobility in surface-supported bilayers is often much lower than the mobility of the lipids. In the present study we explore whether the incorporation of a PEG cushion between the bilayer and the substrate increases the lateral mobility of transmembrane proteins in bilayers produced via directed assembly, a method based on Langmuir-Blodgett deposition techniques. In our experiments, the PEG cushions were incorporated by adding PEG lipids to the protein/lipid monolayer at the air/water interface, at the first step of bilayer assembly. The protein and lipid mobilities in 160 different bilayers, with various PEG molecular weights and PEG lipid concentrations, were measured and compared. We found that the measured diffusion coefficients do not depend on the PEG molecular weight or the PEG lipid concentration and are very similar to the values measured in the absence of PEG. Therefore, contrary to our expectations, we found that a PEG cushion does not necessarily increase protein mobility, suggesting that the low protein mobility is not a consequence of protein-substrate interactions. Furthermore, we showed that the low protein mobility is not due to protein aggregation. The major determinant of protein mobility in surface-supported bilayer systems appears to be the method of bilayer assembly. While proteins were always mobile if the bilayers were prepared using the directed assembly method, in the presence and absence of a PEG cushion, other bilayer assembly protocols resulted in complete lack of protein mobility.

Spectral Förster resonance energy transfer detection of protein interactions in surface-supported bilayers.

Förster resonance energy transfer (FRET), a fluorescence detection technique, is often used for sensing molecular interactions in solution and in membranes. Here we show that (1) FRET spectra can be recorded in single bilayers, supported on a surface, and (2) the fluorescein/rhodamine dye pair is an adequate reporter of FRET when spectral detection is used. Thus, measurements pertaining to molecular interactions in membranes can be carried out in supported bilayers. Spectral FRET has advantages over imaging FRET, which monitors only signal amplitudes at certain wavelength. There are also advantages to performing spectral FRET measurements in supported bilayers as compared to free liposomes in suspension. However, the spectral properties of dyes can be altered in an unexpected manner in an ordered bilayer structure on a surface, such that fluorescence detection in surface-supported bilayers is not always trivial.

Transmembrane helix heterodimerization in lipid bilayers: probing the energetics behind autosomal dominant growth disorders.

Here, we show that the energetics of transmembrane helix heterodimer formation can be characterized in liposomes using Förster resonance energy transfer (FRET). We present the theory and the protocol for measuring the free energy of heterodimerization, and the total (hetero and homo-dimeric) dimer fraction. We use the presented methodology to determine the propensity for heterodimer formation between wild-type fibroblast growth factor receptor 3 (FGFR3) transmembrane domain and the Ala391Glu mutant, linked to Crouzon syndrome with acanthosis nigricans.

Directed assembly of surface-supported bilayers with transmembrane helices.

The lateral assembly of transmembrane (TM) helices gives rise to membrane proteins with complex folds, which play important roles in biochemical processes. Therefore, the assembly of surface-supported bilayers containing TM helices is the first step toward the development of functional biomembrane mimetics. Here we report novel directed assembly of surface-supported lipid bilayers with laterally mobile TM helices. The TM helices were incorporated into lipid monolayers at the air/water interface, and the monolayers were then transferred onto glass substrates using Langmuir-Blodgett (LB) deposition. Finally, bilayers were assembled using lipid vesicle fusion on top of the LB monolayers. The novelty is the incorporation of the peptides into the monolayer at the first step of bilayer assembly, which allows control over the peptide concentration and orientation. The transmembrane orientation of the peptides was confirmed using oriented circular dichroism (OCD), lateral mobility was assessed using fluorescence recovery after photobleaching (FRAP), and diffusion coefficients were determined using a novel boundary profile evolution (BPE) method. The described directed-assembly approach can be used to develop versatile bilayer platforms for studying membrane proteins interactions in native bilayer environments.