BASHY Dye Platform Enables the Fluorescence Bioimaging of Myelin Debris Phagocytosis by Microglia during Demyelination

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the presence of demyelinated regions with accumulated myelin lipid debris. Importantly, to allow effective remyelination, such debris must be cleared by microglia. Therefore, the study of microglial activity with sensitive tools is of great interest to better monitor the MS clinical course. Using a boronic acid-based (BASHY) fluorophore, specific for nonpolar lipid aggregates, we aimed to address BASHY’s ability to label nonpolar myelin debris and image myelin clearance in the context of demyelination. Demyelinated ex vivo organotypic cultures (OCSCs) and primary microglia cells were immunostained to evaluate BASHY’s co-localization with myelin debris and also to evaluate BASHY’s specificity for phagocytosing cells. Additionally, mice induced with experimental autoimmune encephalomyelitis (EAE) were injected with BASHY and posteriorly analyzed to evaluate BASHY+ microglia within demyelinated lesions. Indeed, in our in vitro and ex vivo studies, we showed a significant increase in BASHY labeling in demyelinated OCSCs, mostly co-localized with Iba1-expressing amoeboid/phagocytic microglia. Most importantly, BASHY’s presence was also found within demyelinated areas of EAE mice, essentially co-localizing with lesion-associated Iba1+ cells, evidencing BASHY’s potential for the in vivo bioimaging of myelin clearance and myelin-carrying microglia in regions of active demyelination.

Accurate Lipid Quantification of Tissue Homogenates Requires Suitable Sample Concentration, Solvent Composition, and Homogenization Procedure—A Case Study in Murine Liver

Lipidomics aim to quantify lipid species in all kinds of samples, including tissues. To subject a fixed amount of sample to various workflows, tissue homogenates were frequently prepared at defined concentrations in water or by addition of organic solvents. Here, we investigated this first step of tissue lipidomics by quantitative flow injection analysis coupled to Fourier-Transform mass spectrometry (FTMS). The influence of sample concentration, solvent composition, and homogenization procedure on the recovery of lipids was studied in murine liver. Liver homogenates were prepared either by grinding tissue in liquid nitrogen or by bead-based homogenization. Ground samples were dissolved at different concentrations in water, methanol, and water/methanol = 1/1 (v/v). Here, lipid recovery depends on solvent composition and sample concentration. The recovery of nonpolar lipid classes, including triglycerides and cholesteryl ester, was decreased in methanolic homogenates. In contrast, due to superior dispersion of precipitates, bead-based homogenization resulted in efficient lipid recovery independent of the solvent composition. However, lipid distribution within samples, i.e., lipid content of supernatant and pellet following centrifugation, was altered substantially by solvent composition. In conclusion, accurate lipid quantification of tissue homogenates requires evaluation of solvent composition, sample concentration, as well as the homogenization method to guarantee efficient lipid recovery. Due to a potential loss of lipids, removal of precipitates by centrifugation prior to lipid extraction should be avoided.

Direct evidence of acid-base interactions in gecko adhesion

While it is generally accepted that van der Waals (vdW) forces govern gecko adhesion, several studies indicate contributions from non-vdW forces and highlight the importance of understanding the adhesive contact interface. Previous work hypothesized that the surface of gecko setae is hydrophobic, with nonpolar lipid tails exposed on the surface. However, direct experimental evidence supporting this hypothesis and its implications on the adhesion mechanism is lacking. Here, we investigate the sapphire-setae contact interface using interface-sensitive spectroscopy and provide direct evidence of the involvement of acid-base interactions between polar lipid headgroups exposed on the setal surface and sapphire. During detachment, a layer of unbound lipids is left as a footprint due to cohesive failure within the lipid layer, which, in turn, reduces wear to setae during high stress sliding. The absence of this lipid layer enhances adhesion, despite a small setal-substrate contact area. Our results show that gecko adhesion is not exclusively a vdW-based, residue-free system.

Investigation of DPPC liposomes reveals their capability to entrap Aroclor 1260, an emerging environmental pollutant

Persistent organic pollutants (POPs) are a class of organic compounds that can accumulate in biological and ecological environments due to their resistive nature to chemical, thermal and photo degradation. Polychlorinated biphenyls (PCBs) are a class of man-made POPs that saw wide-spread use in commercial and industrial infrastructure as both an insulator and coolant in electrical transformers and capacitors. 2,2’,3,3’,4,4’-hexachlorobiphenyl (HCBP) was one of the most widely produced PCBs. As these mechanical structures fail or are decommissioned, PCBs are released into the soil, migrate to the water table, and eventually spread to nearby ecosystems by rain and wind. The stability of POPs and specifically PCBs leave few options for environmental waste removal. Conventionally, liposomes have been used for their drug delivery capabilities, but here we have chosen to investigate their capability in removing this class of emerging environmental pollutants. Liposomes are small, nonpolar lipid bi-layered aggregates capable of capturing a wide variety of both polar and nonpolar compounds. Dipalmitoylphosphatidylcholine (DPPC) is a well-characterized lipid that can be derived from natural sources. It is a phospholipid typically found as a major component of pulmonary surfactant mixtures. Liposomes were prepared using probe-tip sonication for both direct and passive incorporation of the HCBP compound. Assimilation was assessed using both differential scanning calorimetry and UV-Vis spectroscopy. After direct incorporation of HCBP the phase transition temperature, Tm, decreased from 40.8 °C to 37.4 °C. A subsequent UV-Vis analysis of HCBP by both direct and passive incorporation showed an increase in HCBP incorporation proportionate to the length of exposure time up to 24 hours and relative to the initial quantity present during the direct incorporation. Together the decrease in Tm and increase in absorbance are indicative of HCBP incorporation and further demonstrate the potential for their use as a method of sustainable environmental cleanup.

Abstract P200: Elevated Triacylglycerols and Diacylglycerols Had Association With Increased Carotid Arterial Stiffness, Independent of Cvd Risk Factors, in Women With or at High Risk of HIV Infection

Background: Metabolomics has provided new insights into mechanistic knowledge of CVD. However, this approach has limited use for studying arterial disease in high-risk women with and without HIV infection. Methods: Using liquid chromatography-tandem mass spectrometry, we profiled plasma levels of 114 cationic polar and 211 nonpolar lipid metabolites among 411 women (72% HIV+; 60% Black and 31% Hispanic) aged 35-50 from the Women’s Interagency HIV Study. Carotid arterial distensibility, a direct measure of carotid stiffness, was calculated from ultrasound measurements of the right common carotid artery diameter at systole and diastole and brachial artery pulse pressure measured. We performed partial least squares discriminant analysis (PLS-DA) to identify metabolite clusters associated with carotid stiffness (lowest vs. the other 3 quartiles of distensibility index). We used multivariate linear regression models to examine associations of individual metabolites with the distensibility index. Results: PLS-DA identified two major metabolite clusters associated with carotid stiffness. In the lipid metabolite cluster, triacylglycerols (TAGs 52:3, 52:4, 54:4), diacylglycerols (DAGs 36:2, 36:3) and sphingomyelins (16:1, 18:1, 18:2) were associated with decreased distensibility, while lysophosphatidylcholines (18:2, 20:5) were associated with increased distensibility. In the cationic polar metabolite cluster, urate, C4-OH carnitine, C5-DC carnitine, pseudouridine and 1-methyladenosine were associated with decreased distensibility. The associations of TAGs 52:3, 52:4, 54:4 and DAG 36:3 with carotid stiffness remained significant after further adjustment for conventional CVD risk factors ( Table ). No interaction by HIV infection was found. Conclusions: Among women with or at risk of HIV infection from predominantly race-ethnic minority groups, plasma TAGs and DAG of higher carbon number and double bond content are associated with carotid stiffness independent of conventional CVD risk factors.