Physical and Functional Coupling of CFTR and PDE3A

Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrated that phosphodiesterase type 3A (PDE3A) is physically and functionally coupled to cystic fibrosis transmembrane conductance regulator (CFTR). PDE3A inhibition increases cyclic adenosine 3′, 5′-monophosphate (cAMP) levels in a compartmentalized manner at the plasma membrane, which potentiates CFTR channel function and further clusters PDE3A and CFTR into microdomains. Actin skeleton disruption reduces PDE3A-CFTR interaction; segregates PDE3A from its interacting partners thus compromise the integrity of the macromolecular complex. Consequently, PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. Physiologically, formation of the CFTR-PDE3A-containing macromolecular complexes was investigated using pig trachea submucosal gland secretion model. PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion. These findings are important in understanding the regulation of CFTR function by phosphodiesterases

Modeling and Simulation of Enzymatic Biofuel Cells with Three-Dimensional Microelectrodes

The enzymatic biofuel cells (EBFCs) are considered as an attractive candidate for powering future implantable medical devices. In this study, a computational model of EBFCs based on three-dimensional (3-D) interdigitated microelectrode arrays was conducted. The main focus of this research is to investigate the effect of different designs and spatial distributions of the microelectrode arrays on mass transport of fuels, enzymatic reaction rate, open circuit output potential and current density. To optimize the performance of the EBFCs, numerical simulations have been performed for cylindrical electrodes with various electrode heights and well widths. Optimized cell performance was obtained when the well width is half of the height of the 3-D electrode. In addition, semi-elliptical shaped electrode is preferred based on the results from current density and resistive heating simulation

Asserting The Security Restrictions Applicable To Images Posted By Users To Information Platforms

It's becoming more difficult to maintain privacy in the age of social media, as seen by the recent rash of high-profile examples in which people have inadvertently released private information online. All of these incidents show why it's crucial to have user access management tools for freely available information. To address this requirement, we propose an Adaptive Privacy Policy Prediction (A3P) system that may provide users with guidance on how to organise their picture privacy settings. Here, we investigate if and how a user's privacy preferences may be revealed via their social network settings, image content, and metadata. Our two-tiered method takes into account the user's prior activity on the site to determine the most fitting privacy options for their future picture uploads. Our method employs a policy prediction algorithm to automatically build a policy for each newly submitted image, taking into consideration users' social qualities, and an image classification framework to find groups of photos that may be associated by similar rules. Rulemaking will evolve over time to accommodate shifting public attitudes towards personal data privacy. We provide the results of a large-scale analysis of more than 6,000 policies, demonstrating that our method achieves prediction accuracy of 93% or better

FAST DISSOLVING TABLETS OF PIMOZIDE: DESIGN, OPTIMIZATION AND INVITRO CHARACTERIZATION

As precision of dosing and patient compliance become an important prerequisite for a long-term treatment of Tourette’s syndrome there is a need to develop formulation for this drug, which overcomes problems such as difficulty in swallowing, inconvenience in administration while travelling and patient’s acceptability. The present work was undertaken with a view to develop a fast dissolving tablets of Pimozide  using Kyron T-314 as super-disintegrant along with Avicel PH 102 as diluent by response surface method using direct compression. Drug-excipient compatibility studies were confirmed by FTIR Spectroscopy. The tablets were evaluated for hardness, friability, weight variation, wetting time, disintegration time and uniformity of content and invitro dissolution. Based on evaluating parameters, formulation prepared by using 4.5% Kyron T-314 with 11.5% Avicel PH-102 was selected as optimized formulation and Formulation (F3) had disintegration time of 7.63±0.25s. and percentage cumulative drug release of   81.60 after 10min. The formulations were further studied and confirmed for their stability. Hence it was concluded that direct compression using Kyron T-314 superdisintegrant and Avicel PH 102 was simple and economic technique which can be used for formulation of fast dissolving tablets of Pimozide.Key word: Pimozide, Tourette’s syndrome, fast dissolving tablets, Kyron T 314, direct compression

Glucose transport by epithelia prepared from harvested enterocytes

Transformed and cultured cell lines have significant shortcomings for investigating the characteristics and responses of native villus enterocytes in situ. Interpretations of results from intact tissues are complicated by the presence of underlying tissues and the crypt compartment. We describe a simple, novel, and reproducible method for preparing functional epithelia using differentiated enterocytes harvested from the small intestine upper villus of adult mice and preterm pigs with and without necrotizing enterocolitis. Concentrative, rheogenic glucose uptake was used as an indicator of epithelial function and was demonstrated by cellular accumulation of tracer 14C d-glucose and Ussing chamber based short-circuit currents. Assessment of the epithelia by light and immunofluorescent microscopy revealed the harvested enterocytes remain differentiated and establish cell–cell connections to form polarized epithelia with distinct apical and basolateral domains. As with intact tissues, the epithelia exhibit glucose induced short-circuit currents that are increased by exposure to adenosine and adenosine 5′-monophosphate (AMP) and decreased by phloridzin to inhibit the apical glucose transporter SGLT-1. Similarly, accumulation of 14C d-glucose by the epithelia was inhibited by phloridzin, but not phloretin, and was stimulated by pre-exposure to AMP and adenosine, apparently by a microtubule-based mechanism that is disrupted by nocodazole, with the magnitudes of responses to adenosine, forskolin, and health status exceeding those we have measured using intact tissues. Our findings indicate that epithelia prepared from harvested enterocytes provide an alternative approach for comparative studies of the characteristics of nutrient transport by the upper villus epithelium and the responses to different conditions and stimuli

Temporal Changes in Collagen Cross-Links in Spontaneous Articular Cartilage Repair

Objective: Little is known about how the biochemical properties of collagen change during tissue regeneration following cartilage damage. In the current study, temporal changes in cartilage repair tissue biochemistry were assessed in a rabbit osteochondral defect. Design: Bilateral full-thickness 3-mm osteochondral trochlear groove defects were created in 54 adult male skeletally mature New Zealand white rabbits, and tissue repair was monitored over 16 weeks. Collagen content, cross-links, lysyl hydroxylation, gene expression, histological grading, and Fourier transform infrared analyses were performed at 2, 4, 6, 8, 12, and 16 weeks. Results: Defect fill occurred at ~4 weeks postinjury; however, histological grading showed that the repair tissue never became normal, primarily due to the presence of fibrocartilage. Gene expression levels of Col1a1 and Col IIaI were higher in the defect compared with adjacent regions. Collagen content in the repair tissue reached the level of normal cartilage at 6 weeks, but it took 12 weeks for the extent of lysine hydroxylation to return to normal. Divalent immature cross-links markedly increased in the early stages of repair. Though the levels gradually diminished thereafter, they never returned to the normal levels. The mature cross-link, pyridinoline, gradually increased with time and nearly reached normal levels by week 16. Infrared imaging data of protein content paralleled the biochemical data. However, collagen maturity, a parameter previously shown to reflect collagen cross-link ratios in bone, did not correlate with the biochemical determination of cross-links in the repair tissue. Conclusion: Collagen biochemical data could provide markers for clinical monitoring in a healing defect

Monitoring the Progression of Spontaneous Articular Cartilage Healing with Infrared Spectroscopy

Objective. Evaluation of early compositional changes in healing articular cartilage is critical for understanding tissue repair and for therapeutic decision-making. Fourier transform infrared imaging spectroscopy (FT-IRIS) can be used to assess the molecular composition of harvested repair tissue. Furthermore, use of an infrared fiber-optic probe (IFOP) has the potential for translation to a clinical setting to provide molecular information in situ. In the current study, we determined the feasibility of IFOP assessment of cartilage repair tissue in a rabbit model, and assessed correlations with gold-standard histology. Design. Bilateral osteochondral defects were generated in mature white New Zealand rabbits, and IFOP data obtained from defect and adjacent regions at 2, 4, 6, 8, 12, and 16 weeks postsurgery. Tissues were assessed histologically using the modified O’Driscoll score, by FT-IRIS, and by partial least squares (PLS) modeling of IFOP spectra. Results. The FT-IRIS parameters of collagen content, proteoglycan content, and collagen index correlated significantly with modified O’Driscoll score (P = 0.05, 0.002, and 0.02, respectively), indicative of their sensitivity to tissue healing. Repair tissue IFOP spectra were distinguished from normal tissue IFOP spectra in all samples by PLS analysis. However, the PLS model for prediction of histological score had a high prediction error, which was attributed to the spectral information being acquired from the tissue surface only. Conclusion. The strong correlations between FT-IRIS data and histological score support further development of the IFOP technique for clinical applications, although further studies to optimize data collection from the full sample depths are required

Combined in vitro and in silico approaches to the assessment of stimulant properties of novel psychoactive substances – The case of the benzofuran 5-MAPB

Novel psychoactive substances (NPS) are increasingly prevalent world-wide although their pharmacological characteristics are largely unknown; those with stimulant properties, due to interactions with the dopamine transporter (DAT), have addictive potential which their users may not realise. We evaluated the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, and the effects of 5-MAPB on electrically-evoked dopamine efflux by fast-cyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a concentration-dependent manner, with significant effects at 10 and 30 μM. The voltammetry data suggest that 5-MAPB reduces the rate of dopamine reuptake; while the peak dopamine efflux was not increased, the area under the curve was augmented. 5-MAPB can also cause reverse dopamine transport consistent with stimulant properties, more similar to amphetamine than cocaine. Molecular modelling and docking studies compared the binding site of DAT in complex with 5-MAPB to dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI-121. This structural comparison reveals a binding mode for 5-MAPB found in the primary binding (S1) site, central to transmembrane domains 1, 3, 6 and 8, which overlaps with the binding modes of dopamine, cocaine and its analogues. Atomistic molecular dynamics simulations further show that, when in complex with 5-MAPB, DAT can exhibit conformational transitions that spontaneously isomerize the transporter into inward-facing state, similarly to that observed in dopamine-bound DAT. These novel insights, offered by the combination of computational methods of biophysics with neurobiological procedures, provide structural context for NPS at DAT and relate them with their functional properties at DAT as the molecular target of stimulants

Stabilization of a prokaryotic LAT transporter by random mutagenesis

Altres ajuts: Fundació La Marató TV3 (20132330)A fluorescence-based screen was used to analyze 70 LAT transporter mutants and identify variants with improved stability and monodispersity. The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis -serine/-threonine exchanger is the best-known prokaryotic paradigm of the mammalian -amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest