Pre-enrichment of modified low density lipoproteins with alpha-tocopherol mitigates adverse effects on cultured retinal capillary cells

We determined whether pre-enrichment of low density lipoproteins (LDL) with alpha-tocopherol mitigates their adverse effects, following in vitro glycation, oxidation or glycoxidation, towards cultured bovine retinal capillary endothelial cells (RCEC) and pericytes.

Toxicity of mildly modified low-density lipoproteins to cultured retinal capillary endothelial cells and pericytes

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P <0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal <glycated <or = minimally oxidized <glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.

Impact of Testing Procedure on Permeability of Compacted Soils

The commonly used British Standard constant head triaxial permeability (BS) test, for permeability testing of fine grained soils, is known to have a relatively long test duration. Consequently, a reduction in the required time for permeability test provides potential cost savings, to the construction industry (specifically, for use during Construction Quality Control (CQA) of landfill mineral liners). The purpose of this article is to investigate and evaluate alternative short duration testing methods for the measurement of the permeability of fine grained soils.

As part of the investigation the feasibility of an existing method of short duration permeability test, known as the Accelerated Permeability (AP) test was assessed and compared with permeability measured using British Standard method (BS) and Ramp Accelerated Permeability (RAP). Four different fine grained materials, of a variety of physical properties were compacted at various moisture contents to produced analogous samples for testing using three the three different methodologies. Fabric analysis was carried out on specimens derived from post-test samples using Mercury Intrusion Porosimetry (MIP) and Scanning Electron Microscope (SEM) to assess the effects of testing methodology on soil structure. Results showed that AP testing in general under predicts permeability values derived from the BS test due to large changes in structure of the soil caused by AP test methodology, which is also validated using MIP and SEM observations. RAP testing, in general provides an improvement to the AP test but still under-predicts permeability values. The potential savings in test duration are shown to be relatively minimal for both the AP and RAP tests.

Measurement of permeability using a bench-top centrifuge

The commonly used British Standard constant head triaxial permeability test for testing of fine-grained soils is relatively time consuming. A reduction in the required time for soil permeability testing would provide potential cost savings to the construction industry, particularly in the construction quality assurance of landfill clay liners. The purpose of this paper is to evaluate an alternative approach of measuring permeability of fine-grained soils benefiting from accelerated time scaling for seepage flow when testing specimens in elevated gravity conditions provided by a centrifuge. As part of the investigation, an apparatus was designed and produced to measure water flow through soil samples under conditions of elevated gravitational acceleration using a small desktop laboratory centrifuge. A membrane was used to hydrostatically confine the test sample. A miniature data acquisition system was designed and incorporated in the apparatus to monitor and record changes in head and flow throughout the tests. Under enhanced gravity in the centrifuge, the flow through the sample was under ‘variable head' conditions as opposed to ‘constant head' conditions as in the classic constant head permeability tests conducted at 1 g . A mathematical model was developed for analysis of Darcy's coefficient of permeability under conditions of elevated gravitational acceleration and verified using the results obtained. The test data compare well with the results on analogous samples obtained using the classical British Standard constant head permeability tests.

Estimation of the surface area and volume of the retinal capillary basement membrane using the stereologic method of vertical sections

The modern stereologic method of vertical sections was applied to the retina as a means of generating unbiased estimates of three-dimensional structure. The method is illustrated with real data on the volume and surface area of the capillary basement membrane from the central retina of the rat. Novel methods of estimating the volume of retina sampled and of creating accurate vertical sections are described. The advantages of using stereologic methods to generate quantitative information on the three-dimensional structure of the retina are discussed and compared to those of previous quantitative methods that provide data on two-dimensional structure only.

Assessment of the effectiveness of the guard ring in obtaining a uni-directional flow in an in situ water permeability test

The non-destructive evaluation of the water permeability of concrete structures is a long standing challenge, principally due to the difficulty of achieving a uni-direction flow for computing the water permeability coefficient. The use of a guard ring (GR) was originally proposed for the in situ sorptivity test, but little information can be found for the water permeability test. In this study, the effect of a GR was carefully examined through the flow simulation, which was verified by carrying out experiments. It was observed that the GR can confine the flow near the surface, but cannot achieve a uni-directional flow across the whole depth of flow. To achieve a better performance, it is essential to consider the effects of the size of the inner seal and the GR and the significant interaction between these two. The analysis of the experimental data has indicated that the GR influences the flow for porous concretes, but there is no significant effect for dense concretes. Further investigation, validated using the flow-net theory, has shown a strong correlation between the water permeability coefficients obtained with the GR (K w-GR) and without it (K w-No GR), suggesting that one dimensional flow is not essential for interpreting data for site tests. Another practical issue was that more than 30 % of the tests with GR failed due to the difficulty of achieving a good seal between the inner and the outer chambers. Based on the work reported in this paper, a new water permeability test is proposed.

Effects of D- and L-glucose and mannitol on retinal capillary cells:Inhibition by nanomolar aminoguanidine

Hyperglycemia may contribute directly to pericyte loss and capillary leakage in early diabetic retinopathy. To elucidate relative contributions of glycation, glycoxidation, sugar autoxidation, osmotic stress and metabolic effects in glucose-mediated capillary damage, we tested the effects of D-glucose, L-glucose, mannitol and the potentially protective effects of aminoguanidine on cultured bovine retinal capillary pericytes and endothelial cells. Media (containing 5 mM D-glucose) were supplemented to increase the concentration of each sugar by 5, 10, or 20 mM. Subconfluent pericytes and endothelial cells were exposed to the supplemented media in the presence or absence of aminoguanidine (1 nM-100 µM) for three days. Cell counts, viability and protein were determined. For both cell types, all three sugars produced concentration-dependent decreases in cell counts and protein content (p

Investigation of moisture condition and Autoclam sensitivity on air permeability measurements for both normal concrete and high performance concrete

While on site measurement of air permeability provides a useful approach for assessing the likely long term durability of concrete structures, no existing test method is capable of effectively determining the relative permeability of high performance concrete (HPC). Lack of instrument sensitivity and the influence of concrete moisture are proposed as two key reasons for this phenomenon. With limited systematic research carried out in this area to date, the aim if this study was to investigate the influence of instrument sensitivity and moisture condition on air permeability measurements for both normal concrete and HPC. To achieve a range of moisture conditions, samples were dried initially for between one and 5 weeks and then sealed in polythene sheeting and stored in an oven at 50 C to internally distribute moisture evenly. Moisture distribution was determined throughout using relative humidity probe and electrical resistance measurements. Concrete air permeability was subsequently measured using standardised air permeability (Autoclam) and water penetration (BS EN: 12390-8) tests to assess differences between the HPCs tested in this study. It was found that for both normal and high performance concrete, the influence of moisture on Autoclam air permeability results could be eliminated by pre-drying (50 ± 1 C, RH 35%) specimens for 3 weeks. While drying for 5 weeks alone was found not to result in uniform internal moisture distributions, this state was achieved by exposing specimens to a further 3 weeks of sealed pre-conditioning at 50 ± 1 C. While the Autoclam test was not able to accurately identify relative HPC quality due to low sensitivity at associated performance levels, an effective preconditioning procedure to obtain reliable air permeability of HPC concretes was identified. © 2013 The Authors

Immune complex formation in human diabetic retina enhances toxicity of oxidized LDL towards retinal capillary pericytes

Recently it has been shown that levels of circulating oxidized LDL immune complexes (ox-LDL-IC) predict the development of diabetic retinopathy (DR). This study aimed to investigate whether ox-LDL-IC are actually present in the diabetic retina, and to define their effects on human retinal pericytes vs. ox-LDL. In retinal sections from people with type 2 diabetes, co-staining for ox-LDL and IgG was present, proportionate to DR severity, and detectable even in the absence of clinical DR. In contrast, no such staining was observed in retinas from non-diabetic subjects. In vitro, human retinal pericytes were treated with native (N-) LDL, ox-LDL, and ox-LDL-IC (0-200 mg protein/l), and measures of viability, receptor expression, apoptosis, ER and oxidative stresses, and cytokine secretion were evaluated. Ox-LDL-IC exhibited greater cytotoxicity than ox-LDL towards retinal pericytes. Acting through the scavenger (CD36) and IgG (CD64) receptors, low concentrations of ox-LDL-IC triggered apoptosis mediated by oxidative and ER stresses, and enhanced inflammatory cytokine secretion. The data suggest that IC formation in the diabetic retina enhances the injurious effects of ox-LDL. These findings offer new insights into pathogenic mechanisms of DR, and may lead to new preventive measures and treatments.

An assay for angiotensin-converting enzyme using capillary zone electrophoresis

A sensitive and rapid method was developed for angiotensin-converting enzyme (ACE) activity determination by capillary zone electrophoresis. Hippuryl-View the MathML source-histidyl-View the MathML source-leucine, a synthetic tripeptide, was used as the ACE-specific substrate. Capillary zone electrophoresis was employed to separate the products of the enzymatic reaction and the ACE activity was determined by quantification of hippuric acid, a result of the enzymatic reaction on the tripeptide. The capillary electrophoresis was performed in a 27 cm × 75 μm i.d. fused-silica capillary using 200 mM boric acid–borate buffer (pH 9.0) as a run buffer with an applied voltage of 8.1 kV at a capillary temperature of 23°C. The electrophoresis was monitored at 228 nm. Each electrophoretic run requires only a nanoliter of the enzymatic reactant solution, at only 6 min, rendering a powerful tool for the ACE assay.

Hard coatings on elastomers for reduced permeability and increased wear resistance

In this study, three different elastomers, namely hydrogenated nitrile butadiene rubber, fluoroelastomer and silicone, have been subjected to two different hard metallised coatings by ion implantation process. The three different elastomers are commonly used in various seal applications, where reduced wear and gas permeability are essential in maintaining seal performance and functionality. Samples of these rubbers have been coated with chromium coating in one set of tests. In the second set of tests, samples of elastomers have been coated with tungsten carbide coating being deposited on all the three different elastomers. Wear, gas permeability and mechanical behaviour of the coated samples were compared with each other and with the control uncoated elastomers. All the coated samples showed good reduction in gas permeability. With the use of metallised coatings, there has been improved resistance to wear in all the coated samples. Adhesion strength and effect of coating on the elastomer have been investigated by mechanical testing. Mechanical tests revealed good adhesion of metal coatings on all the rubber samples, and there was no detrimental effect on the mechanical properties after coating. © 2012 Institute of Materials, Minerals and Mining.