Freeze Fracture Approach to Directly Visualize Wetting Transitions on Nanopatterned Superhydrophobic Silicon Surfaces: More than a Proof of Principle

Freeze fracturing is applied to make the wetting behavior of artificially nanopatterned Si surfaces directly visible. For this purpose, almost hexagonally arranged nanopillars of fixed areal density (127 μm^–2) and diameters (35 nm) but varying heights (40–150 nm) were fabricated on silicon. Measurement of contact angles (CAs) including hysteresis allowed to distinguish between the Wenzel (W) and the Cassie–Baxter (CB) states with droplets completely wetting the pillars or residing on top of them, respectively. Providing additional depth contrast by evaporating the ice replica with thin carbon and (typically 3 nm) platinum layers under 45° allowed resolving 3D features of 5 nm within the ice replica. In this way, laterally sharp transitions from CB- to W-states could be revealed, indicating the formation of zero-curvature water surfaces even on the nanoscale

Enhanced Selectivity by Passivation: Molecular Imprints for Viruses with Exceptional Binding Properties

Inspired by the recognition processes found in biology such as enzyme–substrate and antibody–antigen interactions, synthetic systems with comparable molecular recognition properties have been investigated during recent years based on molecular imprinting strategies. While materials with recognition capabilities for small molecules (i.e., with low molecular weight) have achieved substantial advancements, the synthesis of molecularly imprinted materials with virus recognition properties remains challenging to date. Likewise, protein–surface and protein–protein interactions are essential for a wide variety of biological applications in biotechnology. In biological sensor technology the coating of surfaces to prevent nonspecific adsorption interactions plays an important role. Particularly, polyethylene glycol (PEG) stands out for its high performance in preventing proteins from nonspecifically interactions. However, blocking agents such as the protein bovine serum albumin (BSA) can also be useful as unspecific binding prevention agents for passivation, without modification of the surface. Herein the influence of blocking agents as unspecific reaction components is investigated on the enhancements of selectivity from adenovirus-imprinted particles, whereas adenovirus was used as target species in molecular imprinting. Furthermore, quantitative polymerase chain reaction (qPCR) was used for the first time as virus quantification approach in this context

APP processing in HEK293 cells overexpressing APP or BACE1.

<p><b>a)</b> APP and BACE1 expression in HEK293 cells transduced with APP pUltra-hot, BACE pUltra-hot or the empty control vector pUltra-hot detected by western blotting using 6E10 α-Aβ antibody (Covance) and D10E5 α-BACE1 antibody. β-actin as a loading control was detected using AC-15 α-β-actin antibody (Sigma). <b>b)</b> Fold change of protein expression in HEK APP pUltra and HEK BACE pUltra versus control cells (HEK pUltra) by quantification of western blots. Western blots of three independent protein samples were analyzed with a total of 9 images. APP and BACE1 levels were corrected for β-actin loading control and normalized to the levels of control cells, which were set to 1. Error bars indicate standard error. <b>c)</b> Quantification of sAPP and Aβ secretion rate using ELISA and corrected for the collection time and the mean cell number during collection time. Results were normalized to the mean values of control cells, which were set to 1. The mean values of 3 independent experiments measured in duplicates are shown. Error bars indicate standard error.</p

APP overexpression reduces mitochondrial respiration in Neuro-2a cells.

<p><b>a)</b> APP and BACE1 overexpression in N2a cells stably transfected with HA-phCMV3, APP695-CMV(myc) or BACE-phCMV3(HA) detected by western blotting using α-HA antibody (Sigma) and 9E10 α-myc antibody (Sigma). β-actin as a loading control was detected using AC-15 α-β-actin antibody (Sigma). <b>b)</b> Calculated doubling time in the 48h period between seeding of the cells and high-resolution respirometry. The mean of 5 independent experiments is shown. Error bars indicate confidence intervals (95%). <b>c)</b> High-resolution respirometry of stably transfected N2a cells in an Oxygraph-2k. Titration protocol: addition of 2mio cells in their conditioned medium (Routine respiration), 1.25μM oligomycin (Leak respiration), titration of FCCP to a final concentration of ~2.5μM (ETS capacity), 0.5μM rotenone and 5μM antimycin A (ROX). Oxygen consumption of the cells was corrected for ROX and normalized to the Routine respiration of N2a HA, which was set to 1. The means of 10 independent experiments, each measured in duplicate, are shown. Error bars indicate standard error. Significance was calculated using Kruskal-Wallis test and Dunn´s multiple comparison test. <b>d)</b> Mitochondrial mass measured as Mitotracker Green FM fluorescence using FACS analysis. The mean fluorescence in the FL1 channel was normalized to control cells, which was set to 1. The mean values of 5 independent experiments measured in triplicate are shown. Error bars indicate confidence intervals (95%).</p

APP as against BACE1 overexpression results in higher intracellular Aβ and a reallocation towards mitochondria.

<p><b>a)</b> Reduction of transduction strength quantified by measuring mCherry fluorescence of living cells by FACS analysis. Fluorescence was normalized to the first day of measurement (day 0) which was set to 1. Means of 3 independent experiments each with at least 2 time points of analysis per time interval are displayed. Error bars indicate standard error. Significance versus control cells was calculated using One-way ANOVA with Bonferroni´s Multiple comparison. <b>b)</b> Intracellular Aβ40 levels measured by ELISA and displayed as absolute raw counts normalized to control cells, which were set to 1. Error bars indicate standard error. Significance was calculated using unpaired two-tailed t-test. <b>c)</b> High-resolution respirometry of naïve HEK293 cells treated with conditioned medium from HEK pUltra, HEK APP pUltra or HEK BACE pUltra for 18h, Oxygen consumption of the cells was corrected for ROX and normalized to the Routine respiration of HEK sn pUltra, which was set to 1. Means of 6 independent experiments measured in duplicates are shown. Error bars indicate standard error. Significance was evaluated versus control using Kruskal-Wallis test with Dunn´s multiple comparison for routine respiration and One-way ANOVA with Bonferroni´s multiple comparison for Leak respiration and ETS capacity. <b>d)</b> Quantification of mitochondrially localized portion of APP/Aβ determined by immunoelectron microscopy. Per experiment, at least 20 images of mitochondria-rich regions were taken and all immunogold particles were counted manually. Means of 3 independent experiments are displayed. Error bars indicate standard error. Significance was calculated using unpaired two-tailed t-test. <b>e)</b> Exemplary image for the immunogold-staining of Aβ/APP in HEK293 cells.</p

Intracellular but not extracellular Aß correlates with the rescue of mitochondrial respiration following GSI treatment.

<p><b>a)</b> Quantification of Aβ38, Aβ40 and Aβ42 secretion rate using ELISA. Secretion rates were corrected for the collection time and the mean cell number during collection time. Results were normalized to the mean values of untreated control cells, which were set to 1. The mean values of at least 3 independent experiments measured in technical duplicates are shown. Error bars indicate standard error. <b>b)</b> Intracellular Aβ40 levels measured by ELISA and displayed as absolute raw counts normalized to untreated control cells, which were set to 1. Error bars indicate standard error. <b>c)</b> Accumulation of APP-CTF detected by western blotting using α APP C-terminus-antibody (Sigma). <b>d)</b> Fold change of APP-CTF accumulation in HEK pUltra and HEK APP pUltra normalized to untreated cells (set to 1) by quantification of western blots. Western blots of three independent protein samples per condition were analyzed with 3 images each. Equal loading was ensured by BCA protein assay. Error bars indicate standard error.</p

Gamma secretase inhibitor treatment rescues mitochondrial respiration in HEK APP pUltra.

<p><b>a)</b> High-resolution respirometry of HEK pUltra and HEK APP treated with 0/0.1/1/5nM GSI, performed in an Oroboros Oxygraph-2k. Titration protocol: addition of 2mio cells in their conditioned medium (Routine respiration), 1.25μM oligomycin (Leak respiration), titration of FCCP to a final concentration of ~3μM (ETS capacity), 0.5μM rotenone and 5μM antimycin A (ROX). Oxygen consumption of the cells was corrected for ROX and normalized to the Routine respiration of HEK pUltra, which was set to 1. Means of at least 8 independent experiments measured in duplicates are shown. Error bars indicate standard error. Significance was evaluated for different GSI concentrations versus untreated control using Kruskal-Wallis test and Dunn´s multiple comparison test. <b>b)</b> Differences in respiration (normalized to Routine of control, which was set to 1) of HEK APP pUltra to the respective control cells treated with the same concentration of GSI. Lower asterisks indicate significances of HEK APP pUltra versus respective control, upper asterisks indicate significances between the differences (HEK APP pUltra to HEK pUltra) of GSI treated to untreated cells. Significance was calculated using Kruskal-Wallis test and Dunn´s multiple comparison test.</p

High-resolution respirometry reveals a reduced mitochondrial respiration following APP but not BACE overexpression.

<p><b>a)</b> High-resolution respirometry performed in an Oroboros Oxygraph-2k. The blue curve shows the oxygen concentration in the sealed chamber and the red curve shows the oxygen consumption of the cells. Titration protocol: addition of 2mio cells in their conditioned medium (Routine respiration), 1.25μM oligomycin (Leak respiration), titration of FCCP to a final concentration of ~3μM (ETS capacity), 0.5μM rotenone and 5μM antimycin A (residual oxygen consumption; ROX). <b>b)</b> Measurement of complex IV capacity after blockage of complex III. Addition of 2mM ascorbate and 0.5mM TMPD (Asc+TMPD) results in maximum complex IV respiration plus auto-oxidation (intervals 1–3). Addition of 50μM sodium sulfide (Sulf) results in oxygen consumption just by auto-oxidation, which is dependent on oxygen concentration (intervals 4–9). <b>c)</b> Calculation of complex IV capacity. Pairs of values of oxygen flux and the corresponding oxygen concentration are displayed. The black rhombs show the dependence of the auto-oxidation on the oxygen concentration, whereas the blue squares show the sum of auto-oxidation and complex IV respiration in relation to the oxygen concentration. Thus, complex IV capacity can be seen as the distance between the blue and black regression curves. <b>d)</b> Oxygen consumption of the cells was corrected for ROX and normalized to the Routine respiration of HEK pUltra, which was set to 1. The mean of 7 (HEK pUltra), 8 (HEK APP pUltra) and 8 (HEK BACE) independent experiments, each measured in duplicate, are shown. Error bars indicate standard error. Significance versus control was calculated using Kruskal-Wallis test with Dunn´s method for correction of multiple comparison. <b>e)</b> Mitochondrial mass measured as Mitotracker Green FM fluorescence (MT Green) using FACS analysis or citrate synthase activity (CS activity). The mean values of 5 independent experiments measured in triplicate (MT Green) and 6 independent experiments measured in technical duplicates (CS activity) are shown, all normalized to control cells. Error bars indicate confidence intervals (95%). Significance versus control was calculated using Kruskal-Wallis test with Dunn´s method for correction of multiple comparison.</p

APP overexpression results in an increased mitochondrial membrane potential.

<p><b>a)</b> Mitochondrial membrane potential was measured by staining the cells with the fluorescent dyes DiOC6, TMRM or JC-1 and subsequent analysis using FACS. Dead cells were excluded by gating in the FSC/SSC plot. Mean fluorescent intensities or the ratio of FL1/FL2 for JC-1 were corrected for the uncoupled control cells and normalized to control cells, which were set to 1. Means of 7 (DiOC6), 6 (TMRM) and 3 (JC-1) independent experiments are shown, each measured in triplicates. Error bars indicate standard error. Significance versus control was calculated using Kruskal-Wallis test with Dunn´s multiple comparison for DiOC6 and TMRM and One-way ANOVA with Bonferroni´s multiple comparison for JC-1. <b>b)</b> Calculated doubling time in the 48h period between seeding of the cells and high-resolution respirometry. The mean of 10 independent experiments is shown. Error bars indicate confidence intervals (95%). <b>c)</b> Measurement of lactic acid concentration in the conditioned medium in which also respirometry is performed in. Results of 5 independent experiments are shown, each measured in technical duplicates. Outcomes were corrected for collection time and mean cell number during collection time, and were finally normalized to the release rate of control cells, which was set to 1. Error bars indicate confidence intervals (95%).</p

MV9G cells allow focal growth of HCMV and are suitable for drug resistance assays.

<p>(A) MV9G or MeWo cells were co-cultured with infected cells, either HFF or homotypic cells as indicated, for 7 days. Infected cells were detected by immunostaining of viral immediate early antigens and the focus expansion (FE) value (left) was determined by counting the number of cells per focus as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169580#pone.0169580.ref042" target="_blank">42</a>]. The graph integrates data from three independent experiments. Statistical significance was assessed by student’s t-test. Example images of immunostained foci are shown (right). (B-C) MV9G cells were incubated with HFF cultures infected with clinical HCMV isolates for two days. The cultures were exposed to serial dilutions of either GCV (left) or FOS (right) and the luciferase activity was measured. The drug resistance assays were performed in quadruplicates. A sigmoidal dose response curve was fitted onto the normalized inhibition values to determine the effective drug concentration yielding 50% inhibition (EC50). Error bars: SEM.</p