Interaction of HIV protease inhibitors with OATP1B1, 1B3, and 2B1

The effects of human immunodeficiency virus (HIV) protease inhibitors (PI) on the accumulation of the fluorescent bile salt analogue cholyl-glycylamido-fluorescein (CGamF) were determined in organic anion transporting polypeptide (OATP)-1B1 and -1B3-expressing Chinese hamster ovary (CHO) cells. In addition, interaction studies in Caco-2 monolayers, known only to express the OATP2B1 isoform, were conducted using the established OATP substrate estrone 3-sulfate (E3S), since no CGamF accumulation was observed in Caco-2 monolayers. CGamF appeared an excellent substrate for the OATP1B subfamily, with net accumulation clearance values of 7.8 and 142 microl min(-1) mg(-1) protein in OATP1B1 and OATP1B3-transfected cells, respectively. K(i)-values reflecting inhibition of CGamF accumulation by HIV PI correlated well between OATP1B1 and OATP1B3-expressing cells. Lopinavir was the most potent inhibitor (K(i) = 0.5-1.4 microM) of OATP1B-mediated CGamF accumulation compared with atazanavir, darunavir, ritonavir, and saquinavir (K(i) between 1.4 and 3.3 microM). Inhibitory profiles towards OATP2B1-mediated E3S accumulation were different with only indinavir, saquinavir, and ritonavir showing substantial effects. In conclusion, OATP1B3 appears to be a major transport mechanism mediating sodium-independent CGamF accumulation in human liver, and CGamF could be used as a probe substrate for in vitro drug interaction studies. The remarkably potent inhibition of OATP1B1 by lopinavir may explain some clinically relevant drug interactions between lopinavir and OATP1B substrates such as fexofenadine

Computer-Assisted Precision Surgery in the Ear

Chirurgische Eingriffe am Ohr stellen aufgrund der komplexen Anatomie und der Grössenverhältnisse der beteiligten anatomischen Strukturen eine Herausforderung für den HNO-Chirurgen dar. In diesem Beitrag wird ein Ansatz für die roboterbasierte Navigation zur Hörgeräteimplantation vorgestellt. Insbesondere wird auf die Möglichkeit des Fräsens von Implantatlagern im Felsenbein eingegangen. Je präziser ein Implantat im Schädel verankert werden kann, desto einfacher ist der chirurgischen Ablauf. Weiterhin, profitieren Patienten von verkürzten Operationszeiten und weniger schmerzhaften Eingriffen.Traditional surgical procedures involving the implantation of artificial hearing devices in the inner ear are challenging due to the size and complexity of anatomical structures within the temporal bone. To date, no stereotactic instrument guidance technology providing the necessary levels of accuracy is available. This work presents an approach to robot assisted implantation of hearing devices. Specifically, the robot system was used to milla cavity to for a direct acoustical stimulation implant. As the precision of such cavities increases, so also can future implant generations improve in terms of size, complexity and cost effectiveness. Additionally, patients themselves would profit from shorter procedure times and less painful interventions

Pharmacological manipulation of L-carnitine transport into L6 cells with stable overexpression of human OCTN2

Abstract.: The high-affinity Na+-dependent carnitine transporter OCTN2 (SLC22A5) has a high renal expression and reabsorbs most filtered carnitine. To gain more insight into substrate specificity of OCTN2, we overexpressed hOCTN2 in L6 cells and characterized the structural requirements of substances acting as human OCTN2 (hOCTN2) inhibitors. A 1905-bp fragment containing the hOCTN2 complete coding sequence was introduced into the pWpiresGFP vector, and L6 cells were stably transduced using a lentiviral system. The transduced L6 cells revealed increased expression of hOCTN2 on the mRNA, protein and functional levels. Structural requirements for hOCTN2 inhibition were predicted in silico and investigated in vitro. Essential structural requirements for OCTN2 inhibition include a constantly positively charged nitrogen atom and a carboxyl, nitrile or ester group connected by a 2-4-atom linker. Our cell system is suitable for studying in vitro interactions with OCTN2, which can subsequently be investigated in viv

Development of an online-coupled MARGA upgrade for the 2 h interval quantification of low-molecular-weight organic acids in the gas and particle phases

A method is presented to quantify the lowmolecular- weight organic acids such as formic, acetic, propionic, butyric, pyruvic, glycolic, oxalic, malonic, succinic, malic, glutaric, and methanesulfonic acid in the atmospheric gas and particle phases, based on a combination of the Monitor for AeRosols and Gases in ambient Air (MARGA) and an additional ion chromatography (Compact IC) instrument. Therefore, every second hourly integrated MARGA gas and particle samples were collected and analyzed by the Compact IC, resulting in 12 values per day for each phase. A proper separation of the organic target acids was initially tackled by a laboratory IC optimization study, testing different separation columns, eluent compositions and eluent flow rates for both isocratic and gradient elution. Satisfactory resolution of all compounds was achieved using a gradient system with two coupled anion-exchange separation columns. Online pre-concentration with an enrichment factor of approximately 400 was achieved by solid-phase extraction consisting of a methacrylate-polymer-based sorbent with quaternary ammonium groups. The limits of detection of the method range between 0.5 ngm3 for malonate and 17.4 ngm3 for glutarate. Precisions are below 1.0 %, except for glycolate (2.9 %) and succinate (1.0 %). Comparisons of inorganic anions measured at the TROPOS research site in Melpitz, Germany, by the original MARGA and the additional Compact IC are in agreement with each other (R2 D0.95-0.99). Organic acid concentrations from May 2017 as an example period are presented. Monocarboxylic acids were dominant in the gas phase with mean concentrations of 306 ngm3 for acetic acid, followed by formic (199 ngm3), propionic (83 ngm3), pyruvic (76 ngm3), butyric (34 ngm3) and glycolic acid (32 ngm3). Particulate glycolate, oxalate and methanesulfonate were quantified with mean concentrations of 26, 31 and 30 ngm3, respectively. Elevated concentrations of gas-phase formic acid and particulate oxalate in the late afternoon indicate photochemical formation as a source

Measurements of PM10 ions and trace gases with the online system MARGA at the research station Melpitz in Germany – A five-year study

An hourly quantification of inorganic water-soluble PM10 ions and corresponding trace gases was performed using the Monitor for AeRosols and Gases in ambient Air (MARGA) at the TROPOS research site in Melpitz, Germany. The data availability amounts to over 80% for the five-year measurement period from 2010 to 2014. Comparisons were performed for the evaluation of the MARGA, resulting in coefficients of determinations (slopes) of 0.91 (0.90) for the measurements against the SO2 gas monitor, 0.84 (0.88), 0.79 (1.39), 0.85 (1.20) for the ACSM NO3 −, SO4 2− and NH4 + measurements, respectively, and 0.85 (0.65), 0.88 (0.68), 0.91 (0.83), 0.86 (0.82) for the filter measurements of Cl−, NO3 −, SO4 2− and NH4 +, respectively. A HONO comparison with a batch denuder shows large scatter (R2 = 0.41). The MARGA HNO3 is underestimated compared to a batch and coated denuder with shorter inlets (slopes of 0.16 and 0.08, respectively). Less NH3 was observed in coated denuders for high ambient concentrations. Long-time measurements show clear daily and seasonal variabilities. Potential Source Contribution Function (PSCF) analysis indicates the emission area of particulate ions Cl−, NO3 −, SO4 2−, NH4 +, K+ and gaseous SO2 to lie in eastern European countries, predominantly in wintertime. Coarse mode sea salt particles are transported from the North Sea to Melpitz. The particles at Melpitz are nearly neutralised with a mean molar ratio of 0.90 for the five-year study. A slight increase of the neutralization ratio over the last three years indicates a stronger decrease of the anthropogenically emitted NO3 − and SO4 2− compared to NH4 +

Functional identification of Arabidopsis ATSIP2 (At3g57520) as an alkaline α-galactosidase with a substrate specificity for raffinose and an apparent sink-specific expression patter

Arabidopsis ATSIP2 has recently been suggested to be a raffinose synthase gene. However, it has high amino acid identity to functionally characterized alkaline α-galactosidases from Cucumis melo and Zea mays. Using the Sf9 insect cell expression system, we demonstrate that recombinant ATSIP2 is a genuine alkaline α-galactosidase with a distinct substrate specificity for raffinose, and not a raffinose synthase. A β-glucuronidase reporter construct using the ATSIP2 promoter shows that ATSIP2 is strongly expressed in sink tissues of Arabidopsis, i.e. sink leaves and non-xylem parts of the root stele, suggesting a physiological function in raffinose phloem unloading

Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features

Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases

Review on fat replacement using protein-based microparticulated powders or microgels: a textural perspective

Background Due to the growing rise in obesity and food-linked diseases, the replacement of calorie-dense fat has been a key focus of food industries in the last few decades with proteins being identified as promising fat replacers (FRs). Scope and approach This review aims to provide an overview of animal and plant protein-based FR studies that have been performed in the last 5 years. Protein isolates/concentrates, their microparticulated forms and protein microgels in model and real foods have been examined. Special emphasis has been given on the characterisation techniques that have been used to compare the full fat (FF) and low fat (LF) versions of the foods using FRs. Key findings and conclusions Microparticulated whey protein (MWP) has been the preferred choice FR with some success in replacing fat in model foods and dairy applications. Plant proteins on the other hand have attracted limited research attention as FRs, but show success similar to that of animal proteins. Key characterisation techniques used to compare full fat with low fat products containing FRs have been apparent viscosity, texture profile analysis, microscopy, particle size and sensory properties with oral tribology being a relatively recent undertaking. Coupling tribology with adsorption techniques (muco-adhesion) can be effective to bridge the instrumental-sensory property gap and might accelerate the development cycle of designing low/no fat products. From a formulation viewpoint, sub-micron sized microgels that show shear-thinning behaviour and have boundary lubrication properties offer promises with respect to exploiting their fat replacement potential in the future

Evaluation of tolerance to lentiviral LV-RPE65 gene therapy vector after subretinal delivery in non-human primates.

Several approaches have been developed for gene therapy in RPE65-related Leber congenital amaurosis. To date, strategies that have reached the clinical stages rely on adeno-associated viral vectors and two of them documented limited long-term effect. We have developed a lentiviral-based strategy of RPE65 gene transfer that efficiently restored protein expression and cone function in RPE65-deficient mice. In this study, we evaluated the ocular and systemic tolerances of this lentiviral-based therapy (LV-RPE65) on healthy nonhuman primates (NHPs), without adjuvant systemic anti-inflammatory prophylaxis. For the first time, we describe the early kinetics of retinal detachment at 2, 4, and 7 days after subretinal injection using multimodal imaging in 5 NHPs. We revealed prolonged reattachment times in LV-RPE65-injected eyes compared to vehicle-injected eyes. Low- (n = 2) and high-dose (n = 2) LV-RPE65-injected eyes presented a reduction of the outer nuclear and photoreceptor outer segment layer thickness in the macula, that was more pronounced than in vehicle-injected eyes (n = 4). All LV-RPE65-injected eyes showed an initial perivascular reaction that resolved spontaneously within 14 days. Despite foveal structural changes, full-field electroretinography indicated that the overall retinal function was preserved over time and immunohistochemistry identified no difference in glial, microglial, or leucocyte ocular activation between low-dose, high-dose, and vehicle-injected eyes. Moreover, LV-RPE65-injected animals did not show signs of vector shedding or extraocular targeting, confirming the safe ocular restriction of the vector. Our results evidence a limited ocular tolerance to LV-RPE65 after subretinal injection without adjuvant anti-inflammatory prophylaxis, with complications linked to this route of administration necessitating to block this transient inflammatory event