Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2–4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.     

Identification and design of a novel series of MGAT2 inhibitors

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to na?ve, p <0.01) in plasma triacylglycerol (TAG) concentration.     

Measures of Quality of Care for People with HIV: A Scoping Review of Performance Indicators for Primary Care

The healthcare of people with HIV is transitioning from specialty care to the primary healthcare (PHC) system. However, many of the performance indicators used to measure the quality of HIV care pre-date this transition. The goal of this work was to examine how existing HIV care performance indicators measure the comprehensive and longitudinal care offered in a PHC setting. A scoping review consisting of peer-reviewed and grey literature searches was performed. Two reviewers evaluated study eligibility and indicators in documents meeting inclusion criteria were extracted into a database. Indicators were matched to a PHC performance measurement framework to determine their applicability for evaluating quality of care in the PHC setting. The literature search identified 221 publications, of which 47 met inclusion criteria. 1184 indicators were extracted and removal of duplicates left 558 unique indicators. A majority of the 558 indicators fell under the ‘secondary prevention’ (12%) and ‘care of chronic conditions’ (33%) domains when indicators were matched to the PHC performance framework. Despite the imbalance, nearly all performance domains in the PHC framework were populated by at least one indicator with significant concentrations in domains such as patient-provider relationship, patient satisfaction, population and community characteristics, and access to care. Existing performance frameworks for the care of people with HIV provide a comprehensive set of indicators that align well with a PHC performance framework. Nonetheless, some important elements of care, such as patient-reported outcomes, are poorly covered by existing indicators. Advancing our understanding of how the experience of care for people with HIV is impacted by changes in health services delivery, specifically more care within the PHC system, will require performance indicators to capture this aspect of HIV care.     

Evidence for ligand- and pH-dependent conformational changes in liposome-associated mannose 6-phosphate receptor

Digestion of mannose 6-phosphate receptor preparations with trypsin and chymotrypsin was found to produce characteristic polypeptide "fingerprints" of the receptor. Lengthy digestions with both proteases produced fragments of the receptor which appeared to be resistant to further proteolysis. This suggests the occurrence of distinct structural domains within the receptor protein. Liposome-associated mannose 6-phosphate receptor preparations were made using phosphatidylcholine and purified receptor. Receptor molecules were oriented in the liposomes with greater than 90% of ligand-binding sites on the outside surfaces of the liposomes. Liposome-associated mannose 6-phosphate receptor was labeled with 125I at pH 7.5 and 5.4 in the presence or absence of sugar phosphate ligands. Limited trypsin digestion was used to analyze 125I-labeled receptor preparations. Peptide fragments having molecular weights of approximately 60,000 and 23,000 were found to be most prominently labeled. At pH 7.5 the labeling of the 60-kDa fragment was enhanced strongly by the presence of mannose 6-phosphate. This ligand-induced enhancement of 125I-labeling was saturable, had a K1/2 value of 0.4 mM, required the presence of phosphatidylcholine, and did not occur at pH 5.4. Incorporation of 125I into both polypeptide fragments was significantly reduced at pH 5.4. These results suggest the occurrence of ligand- and pH-dependent conformational changes in domains of the mannose 6-phosphate receptor which may be necessary for proper function of this membrane receptor in receptor-mediated endocytosis.     

The beta 1----2-D-xylose and alpha 1----3-L-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Isolation, characterisation and comparison with other legume lectins

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 1H-NMR showed that the major component was the fucose-containing heptasaccharide Man alpha 3(Man alpha 6)(Xyl beta 2)Man beta 4GlcNAc beta 4(Fuc alpha 3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components. The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Man alpha 3(Man alpha 6)Man beta 4GlcNAc beta 4GlcNAc] by a D-xylose residue linked beta 1----2 to the beta-mannosyl residue and an L-fucose residue linked alpha 1----3 to the reducing terminal N-acetylglucosamine residue. The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.