Membrane-Lipid Therapy in Operation: The HSP Co-Inducer BGP-15 Activates Stress Signal Transduction Pathways by Remodeling Plasma Membrane Rafts

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in ‘membrane-lipid therapy’ to combat many various protein-misfolding diseases associated with aging

Microbial Community Shifts during Biogas Production from Biowaste and/or Propionate

Propionate is the most delicate intermediate during anaerobic digestion as its degradation is thermodynamically unfavorable. To determine its maximum possible degradation rates during anaerobic digestion, a reactor was fed Monday to Friday with an organic loading rate (OLR) of 12/14 kg CODbiowaste·m−3·d−1 plus propionate up to a final OLR of 18 kg COD·m−3·d−1. No feed was supplied on weekends as it was the case in full-scale. To maintain permanently high propionate oxidizing activity (POA), a basic OLR of 3 kg CODpropionate·m−3·d−1 all week + 11 kg CODbiowaste·m−3·d−1 from Monday to Friday was supplied. Finally a reactor was operated with an OLR of 12 kg CODbiowaste·m−3·d−1 from Monday to Friday and 5 kg CODpropionate·m−3·d−1 from Friday night to Monday morning to maintain a constant gas production for permanent operation of a gas engine. The propionate degradation rates (PDRs) were determined for biowaste + propionate feeding. Decreasing PDRs during starvation were analyzed. The POA was higher after propionate supply than after biowaste feeding and decreased faster during starvation of a propionate-fed rather than a biowaste-fed inoculum. Shifts of the propionate-oxidizing and methanogenic community were determined

Fast method for calibrated self-discharge measurement of lithium-ion batteries including temperature effects and comparison to modelling

The self-discharge rate is an important parameter to assess the quality of lithium-ion batteries (LIBs). This paper presents an accurate, efficient, and comprehensive method for measuring and understanding the self-discharge behaviour of LiB cells, considering factors such as temperature and cell to cell variability, as well as underlying electrochemical mechanisms. A method for precise potentiostatic self-discharge measurement (SDM) is demonstrated that is validated by measuring 21 commercial cylindrical 4.7 Ah cells at a state of charge (SoC) of 30%. The self-discharge current ranges between 3 and 6 μA at 23 °C, with an experimental noise level of 0.25 μA. At higher temperatures of 40 °C the self-discharge current increases to 97 μA. The temperature coefficient of voltage (TCV) is experimentally obtained by exposing the cells to a temperature profile with positive and negative step polarities and following the open circuit voltage (OCV) response. Observed TCVs range from +180 µV/K at 40% SoC to −320 µV/K at 0% SoC. For SDM temperature experiments, the cells were set to an SoC with a minimum TCV. From the SDM currents at different temperatures the Arrhenius kinetics and the electrochemical activation energy barrier is determined as 0.94 ± 0.14 eV, indicating chemical side reactions as source of self-discharge. For SDM modelling the electrochemical processes are coupled with a 3D temperature finite element model (FEM) and an electric circuit model resulting in a good overlap with the dynamics and time-constants of the experimental self-discharge curves. The primary challenges addressed are accurately measuring microampere (µA) discharge currents of high-quality cells, reducing measurement time, understanding the temperature dependence of self-discharge, determining activation energy, and demonstrating the applicability and generalization of SDM

Resting State Orai1 Diffuses as Homotetramer in the Plasma Membrane of Live Mammalian Cells*

Store-operated calcium entry is essential for many signaling processes in nonexcitable cells. The best studied store-operated calcium current is the calcium release-activated calcium (CRAC) current in T-cells and mast cells, with Orai1 representing the essential pore forming subunit. Although it is known that functional CRAC channels in store-depleted cells are composed of four Orai1 subunits, the stoichiometric composition in quiescent cells is still discussed controversially: both a tetrameric and a dimeric stoichiometry of resting state Orai1 have been reported. We obtained here robust and similar FRET values on labeled tandem repeat constructs of Orai1 before and after store depletion, suggesting an unchanged tetrameric stoichiometry. Moreover, we directly visualized the stoichiometry of mobile Orai1 channels in live cells using a new single molecule recording modality that combines single molecule tracking and brightness analysis. By alternating imaging and photobleaching pulses, we recorded trajectories of single, fluorescently labeled Orai1 channels, with each trajectory consisting of bright and dim segments, corresponding to higher and lower numbers of colocalized active GFP label. The according brightness values were used for global fitting and statistical analysis, yielding a tetrameric subunit composition of mobile Orai1 channels in resting cells

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule