He was a Veteran of the United States Army and then later served in the National Guard Reverend Caldwell was a licensed minister with the South Carolina International Pentecostal Holiness Conference member of Lake City Pentecostal Holiness Church along with serving in various positions with the SCIPHC and serving as a supply Pastor for several churches in the area of Lake City and Joshua Mark (Morgan) Caldwell of Odessa Ester Bella Caldwell and Julia Caldwell; brother Joy Moore of Fountain Inn; numerous nieces Reverend Caldwell was preceded in death by his son Reverend Johnathan Edward Caldwell; daughters Elizabeth Caldwell and Grace Caldwell; brothers Curtis Woodrow Caldwell and Nealy Caldwell; sister to follow at Camp Branch Pentecostal Holiness Church Cemetery The family will receive friends from 2:00 – 3:00 PM A total of 84 new fast-charging points for EVs have been rolled out in Lower Saxony EnBW has put 84 new fast-charging points for electric vehicles (EV) into operation across five fast-charging parks in Germany The five new fast-charging parks are located in Mitterteich (Bavaria) Dorfmark (Lower Saxony) and Bensheim (Hesse) the company’s nationwide charging network now comprises more than 6,000 charging points across the country Each new location is equipped with high power charging points capable of delivering up to 400 kilowatts of power “With now 1,500 locations and over 6,500 charging points we will continue to consistently drive forward our expansion strategy and make electromobility even more suitable for everyday use with our charging infrastructure,” said Volker Rimpler Chief Technology Officer for e-mobility at EnBW 24 vehicles can charge at the same time while the site  in Münchberg is equipped with 16 fast charging points Drivers will find 12 new charging points in Ingolstadt and eight in Mitterteich The locations can be retrofitted with additional charging points as capacity increases and operates with 100% green electricity Each of the fast-charging parks also has a built-in photovoltaic system which will be installed in the coming weeks Christ Wash Systems Let there be light! VEGA, the next generation. Book your stand NOW! The leading service station and car wash trade fair in Europe. Driving future of mobility – Meet us at P2D to experience EV charging in a whole new dimension Get Ahead of Flow Rate Issues Detect and Fix Anomalies Fast with Fuel Analytics. Get the eBook! Copyright © 2025. All rights reserved. Use of this website signifies your agreement to the Privacy policy Your request has been blocked by our security system due to potential security concerns Please contact us for assistance. Introduction: Bioproduction of plant-derived triterpenoids in recombinant microbes is receiving great attention to make these biologically active compounds industrially accessible as nutraceuticals, pharmaceutics, and cosmetic ingredients. So far, there is no direct method for detecting triterpenoids under physiological conditions on a cellular level, information yet highly relevant to rationalizing microbial engineering. Methods: Here, we show in a proof-of-concept study, that triterpenoids can be detected and monitored in living yeast cells by combining coherent anti-Stokes Raman scattering (CARS) and second-harmonic-generation (SHG) microscopy techniques. We applied CARS and SHG microscopy measurements, and for comparison classical Nile Red staining, on immobilized and growing triterpenoid-producing, and non-producing reference Saccharomyces cerevisiae strains. Results and Discussion: We found that the SHG signal in triterpenoid-producing strains is significantly higher than in a non-producing reference strain, correlating with lipophile content as determined by Nile red staining. In growing cultures, both CARS and SHG signals showed changes over time, enabling new insights into the dynamics of triterpenoid production and storage inside cells. Volume 11 - 2023 | https://doi.org/10.3389/fbioe.2023.1106566 Introduction: Bioproduction of plant-derived triterpenoids in recombinant microbes is receiving great attention to make these biologically active compounds industrially accessible as nutraceuticals there is no direct method for detecting triterpenoids under physiological conditions on a cellular level information yet highly relevant to rationalizing microbial engineering that triterpenoids can be detected and monitored in living yeast cells by combining coherent anti-Stokes Raman scattering (CARS) and second-harmonic-generation (SHG) microscopy techniques We applied CARS and SHG microscopy measurements and for comparison classical Nile Red staining on immobilized and growing triterpenoid-producing and non-producing reference Saccharomyces cerevisiae strains Results and Discussion: We found that the SHG signal in triterpenoid-producing strains is significantly higher than in a non-producing reference strain correlating with lipophile content as determined by Nile red staining both CARS and SHG signals showed changes over time enabling new insights into the dynamics of triterpenoid production and storage inside cells the post-synthesis localization of triterpenoids is unclear Schematic overview of the heterologous biosynthesis of lupane-type triterpenoids in S The native yeast metabolism converts farnesyl pyrophosphate (FPP) to squalene (SQ) by squalene synthase (Erg9) Squalene epoxidase (Erg1) catalyses the conversion of squalene to 2,3-oxidosqualene (OSQ) The heterologous pathway constitutes an oxidosqualene cyclase (OSC) catalyzing the cyclization of 2,3-oxidosqualene to lupeol as well as a cytochrome P450 monooxygenase (CYP) and corresponding cytochrome P450 reductase (CPR) Dashed arrows correspond to a hypothetical transfer of triterpenoids from the endoplasmic reticulum (ER) membrane into lipid droplets (LDs) and further to the plasma membrane In addition to the unclear intracellular triterpenoid distribution between membranes and lipid droplets there is little knowledge about triterpenoid production among individual cells of a population Such insights would be valuable for understanding hydrophobic molecule trafficking and guiding strain engineering strategies Due to the hydrophobic structure of triterpenoids we assumed that Nile red staining is also a suitable method for detecting these compounds produced intracellularly by engineered yeast cells We first applied Nile red staining to show that triterpenoid-producing strains differ from the reference strain regarding the accumulation of intracellular hydrophobic compounds we tested the applicability of CARS and SHG microscopy to monitor triterpenoid accumulation in recombinant yeast We performed CARS and SHG analyses for both fixed and live (growing) cells to evaluate the applicability of both methods for determining intracellular triterpenoid accumulation with temporal and spatial resolution cerevisiae BA6∆ (unpublished) is a pah1 deletion mutant of BA6 generated through replacement of PAH1 with a hygromycin resistance cassette (YMR165C∆:hphMX) Batch cultivations in 500 ml shake flasks started with a filling volume of 20 ml A pre-culture was used to inoculate the main cultures to a start optical density measured at 600 nm (OD600) of 0.1 The optical density of the cultures was determined using a spectrophotometer (Ultrospec 10 Germany) with 0.5% phosphate-buffered saline (PBS) used as blank A predetermined correlation factor of 0.29 g cell dry weight (CDW) per unit OD600 for strain BA6∆ and 0.2 for BA6 and the reference was used to derive the CDW concentration The cultivations took place in a rotary shaker (New Brunswick Innova 44R) at 30°C samples were taken during exponential growth on glucose and after the diauxic shift during growth on ethanol For static, non-shaken cultivations, we designed the experimental setup shown in Figure 2 Experimental setup of static culture experiments Static culture experiments took place in a glass-bottom microscopy chamber with a coverslip (ibidiplates μ-Slide 4 Well Glass Bottom Three of the four chambers were filled with 1 ml of the yeast culture at a starting OD600 of 0.4 The chamber was fitted onto the microscope directly below the objective lens to take microscopy images of each chamber at regular intervals The cultures were aerated with ambient air to prevent oxygen limitation sterile air was moisturized with water and ethanol by passing it through an ethanol:water solution (50:50 (v/v)) Two holes on both ends of the lid served as gas inlet and outlet; a third opening in the dividing wall ensured gas exchange between the two chambers Arrows indicate airflow through the chambers Samples of 800 μL of culture broth were transferred into 2 ml sample tubes for the analysis of intracellular triterpenoid concentrations and stored at −20°C until further processing or immediately analyzed Cells were disrupted by adding 250 μL of glass beads (smooth 500 μm and 800 μl chloroform:methanol (ratio 80:20 v/v) and vigorously agitating in the Mini-Beadbeater 16 (BioSpec Products Samples were centrifuged for 10 min at 13,000 rpm (16,089 x g) and 5°C 200 μl of the lower organic phase were transferred into a glass vial (VVial Phenomenex) containing an insert (15 mm tip KG Germany) for High-Performance Liquid Chromatography (HPLC) analysis Product quantification was conducted with a reverse HPLC system (Thermo Fisher UltiMate 3,000 Dionex) Samples were kept a 4°C in an autosampler (Thermo Fisher UltiMate 3,000) Five µL of the sample were injected into a C18 column (Merck Spherisorb ODS-2 (5 μm) Germany) with a guard column (EC 4/3 NUCLEODUR C18 Gravity while the column temperature was kept at 40°C in a thermostatic column compartment (Thermo Fisher UltiMate 3,000) Analytes were eluted using gradient chromatography at 1.2 mL/min flow rate using a binary pump (Ultimate 3,000 Pump Solvent B was 0.2% (v/v) formic acid of high purity (≥98%) The linear gradient method was as follows: 20% B from 0 to 1 min then back to 20% B from 23 to 26 min Solvents were degassed using an online degasser Analytes of interest were monitored using a charged aerosol detector (CAD esa ERC GmbH) driven by a nitrogen generation system (Parker two times 800 μl were taken from the same cultivation serial dilutions (df = 2) of standards for lupeol (Sigma Aldrich and betulin aldehyde (Cfm Oskar Tropitzsch GmbH Yeast cells were immobilized on agarose beds for microscopic analyses One ml of warm 1% agarose gel was applied onto a microscope slide and covered with a second microscopy slide to force the formation of a planar surface Ten µL of a culture diluted to an OD600 of 1.0 was applied on the agarose layer and protected with a cover glass Desiccation of samples was prevented by covering the edges of the coverslip with clear lacquer and analyzing samples immediately Nile Red staining was performed based on a protocol described in Zuriani et al. (2013) was centrifuged at 12,000 g for 5 min and the pellet was resuspended in 1 mL 0.5% PBS 60 μl of Nile red (80 μg/mL dissolved in acetone) was added to the cell suspension The samples were incubated for 30 min at RT in the dark samples were centrifuged again at 12,000 g for 5 min Pellets were carefully resuspended in 1 mL 0.5% PBS and 100 μl of the sample were transferred to a 96-well microplate Fluorescence was measured at different excitation (Ex) and emission (Em) wavelengths using the Multi-Mode Microplate Reader Synergy; Ex/Em 550/638 nm for phospholipids 23°C) with the following parameters: read height 8 mm ≥95%) were analyzed in crystalline conditions and squalene in liquid form Saturated solutions of ergosterol and the main triterpenoid products betulin and betulinic acid in ethanol were prepared to analyze the CARS and SHG spectra in their dissolved state Assuming additivity of the signal intensities the mean intensities of dissolved substances were corrected by subtracting the mean CARS signal intensity of pure ethanol The difference in mean intensities is the estimated CARS signal caused by these substances While betulin and betulinic acid generated only a weak CARS signal ergosterol produced a stronger CARS signal in solution We also determined the number of SHG pixels not colocalizing with CARS. The term was normalized for each object using Eq. 3 by dividing with the total object pixel size The total object pixel size refers to the total pixels independent of the threshold of 25% of the maximum intensity The altered lipophilic molecule profiles (lipids triterpenoids) make these strains excellent test objects for evaluating techniques for analyzing the intracellular compositions in yeast We used this stain to confirm differences in the hydrophobic compound composition of the chosen yeast strains a prerequisite for CARS and SHG measurements We measured the fluorescence at two different excitation and emission wavelengths: Ex/Em 515/583 nm for neutral lipids and 550/638 nm to detect polar phospholipids Since this method falls short of distinguishing the different heterologous and native lipophilic compounds more definitive explanations cannot be given Qualitative determination of the hydrophobic molecule content of engineered yeast B): Fluorescence intensity [-] at Ex/Em 515/583 nm (A) 550/638 nm (B) of Nile red-stained cell suspensions of the reference strain (Ref) and the two betulinic acid-producing strains BA6 and BA6∆ sampled from a batch cultivation during growth on glucose (I) and after the diauxic shift during growth on ethanol (II) The OD600 of all samples was adjusted to 1 (C) Triterpenoid yield [mg/gCDW] of the reference strain (Ref) and the two betulinic acid-producing strains BA6 and BA6∆ sampled from a batch cultivation during growth on glucose (I) and after the diauxic shift during growth on ethanol (II) Nile red staining indicated compositional differences between the reference and triterpenoid-producing yeast strains Single-cell microscopy imaging techniques are required for subcellular localization of triterpenoids or time-resolved tracking of triterpenoid distribution in individual cells and molecule partitioning during cell proliferation we tested the applicability of CARS and SHG microscopy techniques as non-invasive methods for analyzing living yeast cells we analyzed pure standards of triterpenoids and ergosterol to observe their detectability by CARS and SHG techniques Ergosterol was included to estimate interference with triterpenoid signals because of its structural and biophysical similarity and the differential accumulation in the tested yeast strains Evaluating CARS and SHG measurements for triterpenoid detection and an overlay of both signals of solid ergosterol Solid compounds were measured in xy and liquid squalene in xz dimension Yellow areas in the overlay images emerge through colocalization of CARS and SHG signals green or red are areas with solely or predominantly SHG or CARS signal respectively; edge length = 75 µm Likewise, ethanolic solutions of ergosterol, betulin, and betulinic acid showed a CARS signal but no SH activity (Figure 5) Transmission microscopy showing laser reflection (T) (white) and SHG (green) signals of pure ethanol and solutions of ergosterol and betulinic acid in ethanol at 817 nm edge length = 75 µm total triterpenoids (sum of betulinic acid and triterpenoid content of the investigated strains during growth in batch culture on glucose (I) and after the diauxic shift on ethanol (II) CARS and SHG measurements of triterpenoids in yeast and the overlay of CARS and SHG signals for the reference (Ref) and triterpenoid producing strains BA6 and BA6∆ at early (I) and late fermentation stage (II) Measurements were performed at 816 nm; edge length is 25 µm the SHG signal in the reference strain was noticeably lower than in the triterpenoid-producing strains Together with the SHG signal of solid triterpenoid standards the SH activity of BA6 and BA6∆ cells suggests that it is caused by triterpenoid agglomerates or (pseudo) crystals BA6∆ cells showed cell-to-cell heterogeneity in SHG, especially during stage I. Similarly, previous studies reported variability in endoplasmic reticulum morphology and lipid droplet abundance between single BA6∆ cells (Walter, 2018) which might therefore differ in lipid composition if the here observed variability in SHG signal relates to this phenotype or differences in triterpenoid accumulation or lipid content cannot be stated Immobilized cells of the reference and engineered strains sampled during stage II showed comparable CARS signals. Spherical structures were detected by CARS in all three strains (Figure 6) the SHG signal in triterpenoid-producing strains was much higher than in the non-producing strain The overlay of the CARS and SHG signals allows speculation of analyte distribution While most regions show a colocalization (indicated by the yellow color) there are also regions with only or predominantly SHG or CARS activity areas of presumed lipid droplet localizations showed no SHG signal suggesting that triterpenoids are either not present in these organelles or are not in a crystal-like form required for exerting SH activity regions with a predominant SH signal suggest the agglomeration of solid triterpenoid particles in a low-lipid environment The overlay of CARS and SHG of solid sterols/terpenoids shows a predominance of SHG at least for some compounds Hence pixels with SHG non-colocalized with CARS point to the enrichment of these molecules in solid/pseudocrystal form and in a low-lipid environment when located in lipid membranes and lipid droplets always result in both CARS and SHG signals our results hypothesize that triterpenoids might also accumulate in other cellular spaces or be enriched in microdomains (lipid raft-like structures) within membranes Box plot representation of colocalization analysis for the reference (Ref) and triterpenoid producing strains BA6 and BA6∆ during growth stage I and II (A): Ratio of SHG/CARS signal colocalization (B): Ratio of CARS signal colocalizing with SHG signal (C): Ratio of SHG signal not colocalizing with CARS normalized to the total object pixel size Dots outside the box: Outliers; Vertical lines: Whiskers; Dots inside the box: Means; Horizontal line inside the box: median The colocalization analysis was performed for single objects (representing single cells and in some cases cell agglomerates) in the microscopy image of each strain The number of cells per image for the different strains was as follows: BA6 (I) The large variance in some data sets indicated by the whiskers and outliers may have been caused by biological heterogeneity between individual cells and analytical challenges when cell agglomerates are formed these results indicate a correlation of SHG signal with triterpenoid accumulation and the subcellular distribution Overall and important for the scope of this study The potentially higher ergosterol content could also have contributed to the higher SHG signal excreted by cells under these growth conditions compared to cells from glucose batch cultivations Time resolved triterpenoid production in yeast Measurement of CARS (red) and SHG (green) signals and the overlay for the reference strain (Ref) and triterpenoid producing strains BA6 and BA6∆ during growth on ethanol in a static-culture Overlay images show yellow in areas of strong colocalization of CARS and SHG Cultivation took place at room temperature The culture was aerated with a water and ethanol solution (50:50 v/v) Measurements were performed at 816 nm; edge length = 25 µm Also, considerable differences in the CARS and SHG signals were observed throughout the cultivation of the triterpenoid-producing strains BA6 and BA6∆. The SHG signal increased in both cultures over time, indicating accumulation of triterpenoids. While the SHG signal in BA6 was nearly equal in all cells, for BA6∆, some differences were observed in the SHG signal of individual cells. Figure 8 shows 3 cells of BA6∆ that form a cluster The larger size of the upper cell suggests it to be an old cell explaining its high SHG signal from the beginning the two smaller cells had a weak SHG signal at the beginning likely correlating with intracellular triterpenoid accumulation The overlay of SHG and CARS pictures revealed no or very few colocalizations indicating that the triterpenoids were mostly not present in soluble form in a lipid rich environment Whether they are locally enriched in lipid membranes or even present in an aqueous surrounding the vacuole cannot be distinguished with the current measurements One limitation of the here presented methodology is that CARS also detects triterpenoids (Yu et al., 2008) the signals of both methods should be considered for a deeper evaluation of the results These data show clear differences in producing and non-producing strains and importantly allow time-resolved observation on single cell level—the greatest advantages of the applied non-invasive microscopy technique compared to analytical methods which provide an averaged triterpenoid concentration of an entire population we used three techniques (Nile red staining CARS and SHG microscopy) to analyze lipid compounds and triterpenoids in yeast cells We could show that the combination of the non-invasive techniques CARS and SHG allow detection of lipids and triterpenoids in living yeast cells This highlights the great potential of these methods for future analysis of natural product synthesis with special and temporal resolution Other than conventional methods like Nile red the application of CARS and SHG allows the detection of both triterpenoids and lipids in individual cells The application of CARS and SHG signal measurement shows potential for further development and an advanced quantification detection of the individual triterpenoid components at different wavelengths would be an option to further improve the existing method towards more specific and quantitative analysis Using the current state-of-the art techniques we were able to monitor triterpenoid accumulation in cells of a population over the entire cultivation time this is the first time that the combination of both CARS and SHG techniques are applied for the analysis of intracellular product accumulation in living yeast cells This proof-of-principle study shows the potential of application of CARS and SHG microscopy techniques in applied microbiology allowing new insights into the dynamics of triterpenoid production and storage by recombinant yeasts The raw data supporting the conclusion of this article will be made available by the authors and MD conceptualized and designed of the study All authors contributed to manuscript revision This research was performed within the project PRODIGY (process-directed drug generation in yeast) funded by the German Federal Ministry of Education and Research BMBF (grant No We would like to share our gratitude to our project partners Prof Markus Nett and Anna Tippelt (Department of Biochemical and Chemical Engineering TU Dortmund University) for their support and enabling us to perform our yeast cultivations in their laboratory The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1106566/full#supplementary-material The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets PubMed Abstract | CrossRef Full Text | Google Scholar Visualization of β-carotene and starch granules in plant 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BE (2023) Non-invasive monitoring of microbial triterpenoid production using nonlinear microscopy techniques Received: 23 November 2022; Accepted: 23 January 2023;Published: 28 February 2023 Copyright © 2023 Dianat, Münchberg, Blank, Freier and Ebert. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Birgitta E. Ebert, YmlyZ2l0dGEuZWJlcnRAdXEuZWR1LmF1; Erik Freier, ZXJpay5mcmVpZXJAdW5pLXd1cHBlcnRhbC5kZQ==; Lars M. Blank, bGFycy5ibGFua0Byd3RoLWFhY2hlbi5kZQ== †ORCID: Mariam Dianat, orcid.org/0000-0001-6430-6146; Lars M. Blank, orcid.org/0000-0003-0961-4976; Erik Freier, orcid.org/0000-0002-0559-4210; Birgitta E. Ebert, orcid.org/0000-0001-9425-7509 Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details Redox-active films were proposed as protective matrices for preventing oxidative deactivation of oxygen-sensitive catalysts such as hydrogenases for their use in fuel cells the theoretical models predict quasi-infinite protection from oxygen and the aerobic half-life for hydrogenase-catalyzed hydrogen oxidation within redox films lasts only about a day we employ operando confocal microscopy to elucidate the deactivation processes The hydrogen peroxide generated from incomplete reduction of oxygen induces the decomposition of the redox matrix rather than deactivation of the biocatalyst We show that efficient dismutation of hydrogen peroxide by iodide extends the aerobic half-life of the catalytic film containing an oxygen-sensitive [NiFe] hydrogenase to over one week approaching the experimental anaerobic half-life our data support the theory that redox films make the hydrogenases immune against the direct deactivation by oxygen and highlight the importance of suppressing hydrogen peroxide production in order to reach complete protection from oxidative stress a Structure of the [NiFe]-hydrogenase and the viologen-modified polymer (polyethylenimine backbone) b Proposed mechanism for protection from O2 The hydrogenase catalyzes the reversible oxidation of H2 in the inner parts of the film and generates electrons that are transferred to the electrode via the viologen (Vred/Vox) moieties (pathway 1) which produces the catalytic current when an oxidative potential is applied to the electrode the presence of O2 in the electrolyte creates an oxidative driving force that diverts some of the electrons towards the electrolyte/film interface (pathway 2) where the viologen moieties act as catalysts for O2 reduction thus protecting the catalyst in the inner film domain but also producing H2O2 Iodide catalyzes the subsequent H2O2 dismutation to H2O and half a molecule of O2 and demonstrate that suppression of reactive oxygen species is also necessary to avoid oxidative degradation of the catalytic films While dismutation of two molecules of H2O2 generates one molecule of O2 successive cycles of the viologen-catalyzed O2 reduction reaction and of the iodide-catalyzed H2O2 decomposition results in H2O as the sole end product 3 mm in diameter) were coated with viologen-modified polymer with a surface coverage of 2.2 mg cm−2 pH 7) with 10 µL stock solution of Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit The fluorescence of the oxidized viologen-modified films was collected around its maximum emission of 590 nm upon excitation at 488 nm The emission of the H2O2 fluorescent probe was determined around its maximum emission of 660 nm upon excitation at 633 nm All measurements were performed under ambient air and at 300 K This demonstrates that O2 reduction catalyzed by the viologen-modified film generates H2O2 and that H2O2 can be efficiently dismutated when iodide is present in the electrolyte Error bars are defined as standard deviation Average values and standard deviations were obtained from measurements at three individually prepared electrodes and then normalized by average ip values before O2 reduction All measurements were performed with GCE (3 mm in diameter) coated with viologen-modified polymer with a surface coverage of 0.3 mg cm−2 Source data are provided as a Source Data file the conclusion from the CV results is that loss of viologen peak current is associated with H2O2 generation These observations highlight the need for efficient dismutation of H2O2 generated from the viologen-catalyzed O2 reduction reaction in order to avoid decomposition of the redox hydrogel film The polymer surface coverage for all hydrogenase modified electrodes is 2.26 mg cm−2 complete reduction of O2 to water is necessary to bypass additional deactivation pathways involving reactive oxygen species that degrade the redox film the use of iodide is particularly advantageous since the dismutation of H2O2 leads to water and half a molecule of O2 that is further reduced by the viologen and thus eventually leads to water as the sole final product The iodide being used as a catalyst in this reaction is not used up and therefore represents a practical solution for long-term operation of energy converting systems in which H2O2 production is often an undesired side reaction of the system This approach for H2O2 removal being exclusively based on tuning the composition of the electrolyte rather than tuning the catalytic system itself is directly applicable for enhancing the stability of other low potential electrochemical or photochemical systems suffering from degradation induced by H2O2 generation an aqueous suspension of the viologen-modified polymer (4 µL 40 mg ml−1) and [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (3 µL pH 6.8) were mixed and applied to the GCE (3 mm diameter) the polymer was drop-cast onto the GCE with surface coverages indicated in the captions of the figures Tris buffer was added to the solution droplet (1 µL pH 9) immediately after drop-casting to accelerate the polymer cross-linking via disulfide bond formation Electrodes were stored at 4 °C in a closed vessel overnight After 12 h the solution became turbid and was then left to dry in air for another hour before the electrochemical measurements Fluorescence measurements were performed on a Leica Microsystems TCS SP8 CARS laser scanning microscope in a fully confocal optical setup An argon gas laser excited the samples at 488 nm and an He–Ne laser was used for excitations at 633 nm through an HC PL Fluotar ×10/0.3 dry or Fluotar VISIR ×25/0.95 water objective The emission was detected in EPI direction with photomultiplier tubes (PMTs) from 500 to 630 nm During fluorescence measurements the temperature in the sample compartment was stabilized at 300 K The stock solutions of Amplite™ fluorimetric hydrogen peroxide assay kit were prepared according to the manufacturer’s protocol CARS measurements were performed on a Leica Microsystems TCS SP8 CARS laser scanning microscope A picoEmerald laser system from APE Berlin generated two synchronized pulsed laser beams at 817 and 1064 nm which were temporally and spatially overlapped at the measurement position on the sample The resulting CARS signal at 663 nm was detected via a non-descanned PMT This corresponds to a Raman intensity at 2841 cm−1 The detection window of the PMT ranges from 560 to 750 nm During CARS measurements the temperature in the sample compartment was stabilized at 300 K The CARS lasers and signal detection were focused and collected via the same objectives as for the fluorescence measurements The measurements followed each other in close succession (very few seconds) All electrochemical measurements were carried out with BA Metrohm Autolab as well as a rotating-disc electrode setup and O2 were controlled with mass-flow controllers The reference electrode was Ag/AgCl/3 M KCl Potentials are converted to the SHE using the correction ESHE = EAg/AgCl + 210 mV All electrochemical measurements were performed at 298 K the O2 concentration was monitored with FireStingGO2 Power curves were calculated from the steady-state currents obtained from stepped potential chronoamperometric experiments A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage A redox hydrogel protects the O2-sensitive FeFe-hydrogenase from Chlamydomonas reinhardtii from oxidative damage Mechanism of protection of catalysts supported in redox hydrogel films Preventing the coffee-ring effect and aggregate sedimentation by in situ gelation of monodisperse materials Heterogeneous ammonia and hydrogen production by MoFe protein High-power formate/dioxygen biofuel cell based on mediated electron transfer type bioelectrocatalysis Engineered electron-transfer chain in photosystem 1 based photocathodes outperforms electron-transfer rates in natural photosynthesis Rational wiring of photosystem II to hierarchical indium tin oxide electrodes using redox polymers Wiring of photosystem I and hydrogenase on an electrode for photoelectrochemical H2 production by using redox polymers for relatively positive onset potential Cytochrome c-coupled photosystem I and photosystem II (PSI/PSII) photo-bioelectrochemical cells Photosystem I (PSI)/Photosystem II (PSII)-based photo-bioelectrochemical cells revealing directional generation of photocurrents A poly(cobaloxime)/carbon nanotube electrode: freestanding buckypaper with polymer-enhanced H2-evolution performance Dual properties of a hydrogen oxidation Ni-catalyst entrapped within a polymer promote self-defense against oxygen Polymer-bound DuBois-type molecular H2 oxidation Ni catalysts are protected by redox polymer matrices The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster Oxygen-tolerant proton reduction catalysis Dynamics of electron hopping in assemblies of redox centers Rational design of quinones for high power density biofuel cells A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus Impact of oxygen on glucose oxidation kinetics in a redox polymer mediated glucose oxidase electrode On the parameters affecting the characteristics of the “wired” glucose oxidase anode Oxygen reduction on redox mediators may affect glucose biosensors based on “wired” enzymes How the reduction of O2 on enzymes and/or redox mediators affects the calibration curve of “wired” glucose oxidase and glucose dehydrogenase biosensors Complete protection of O2-sensitive catalysts in thin films Use of ‘split waves’ for the measurement of electrocatalytic kinetics Methyl viologen mediated oxygen reduction on a boron-doped diamond electrode The catalytic decomposition of hydrogen peroxide by the iodine-iodide couple at 25° The iodide-catalyzed decomposition of hydrogen peroxide Mechanistic details of an old reaction as revealed by electrospray ionization mass spectrometry monitoring Cyclic voltammetry of electrocatalytic films: fast catalysis regimes Cyclic voltammetry analysis of electrocatalytic films Enhancing the biocompatibility and biodegradability of linear poly(ethylene imine) through controlled oxidation Theoretical treatment of diffusion and kinetics in amperometric immobilized enzyme electrodes Part I Spectroelectrochemical Characterization of the [NiFe] Hydrogenase of Miyazaki F Properties of Purified Hydrogenase from the Particulate Fraction of Desulfovibrio vulgaris Diazonium functionalisation of carbon nanotubes for specific orientation of multicopper oxidases: controlling electron entry points and oxygen diffusion to the enzyme Download references Tobias Vöpel for discussions about the H2O2 reporter Steffen Hardt for help with the fuel cell experiments and Nina Breuer for the preparation of the DvMF [NiFe] hydrogenase funded by the Deutsche Forschungsgemeinschaft (DFG German Research Foundation) under Germany ́s Excellence Strategy—EXC-2033—Projektnummer 390677874 by the ERC starting grant 715900 and by the ANR-DFG project SHIELDS (PL 746/2-1) is grateful for the support by the China Scholarship Council (CSC) acknowledge funding by the “Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen” the “Regierender Bürgermeister von Berlin-inkl and the “Bundesministerium für Bildung und Forschung” also in the form of the Leibniz-Research-Cluster (grant number: 031A360E) Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V Max-Planck-Institut für Chemische Energiekonversion H.L performed all electrochemical experiments involving mediated electron transfer and contributed to the confocal fluorescence experiments A.A.O contributed with direct electron transfer electrodes performed the electrochemical modeling and simulations conceived and performed the spectroscopic experiments All authors contributed to writing the manuscript The authors declare no competing interests Peer review information Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-020-14673-7 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Receive our weekly Newsletterand set tailored daily news alerts Medical/Hygiene a leading manufacturer of wood-based cellulosic specialty fibres and Hof University opened their new Nonwoven Development Center (VEZ) Lenzing has access to a state-of-the-art development line at the campus in Münchberg with immediate effect This offers new opportunities for sustainable fibre and nonwoven innovations for a wide range of applications including hygiene In line with its sCore TEN corporate strategy the Lenzing Group is focusing on sustainable innovations which are agreed in an optimum manner to the needs of the value chain “We offer our customers and partners a decisive competitive advantage - agility The pilot plant in the VEZ allows the resource-efficient development of fibre and nonwoven innovations on a small scale” Vice President Business Unit Nonwovens at Lenzing The VEZ was completed according to schedule in September 2020 after Lenzing and the University of Hof signed a cooperation agreement for its use in 2019 such as the directive (EU) 2019/904 of the European Parliament and of the Council of 5 June 2019 on the reduction of the impact of certain plastic products on the environment increase demand for responsibly manufactured nonwovens The so-called Single-Use Plastics Directive aims at building awareness and greater transparency with regard to wet wipes and feminine hygiene products With its Veocel branded wood-based cellulose fibres Lenzing has been laying the foundation for many years for sustainable nonwoven applications and will test and develop innovative ideas using the new possibilities offered by the VEZ “We are noticing increasing interest in sustainable concepts from biodegradable cellulose fibres” Jürgen Eizinger sums up the market development of the last months and adds: “We are aware that the fibres used have an enormous influence on the final product our commitment extends beyond fibre production.” Lenzing will support customers and partners more intensively in the development of new nonwoven applications and at the same time promote cooperation in the field of marketing the company already established new certification standards for the Veocel brand Since then certified manufacturers can only use the Veocel logo with blends of biodegradable cellulosic fibres the Veocel brand allows consumers to make a more conscious product selection With its #ItsInOurHands environmental initiative the Veocel brand also actively contributes to creating awareness More detailed information can be obtained on itsinourhands.com www.lenzing.com Lenzing opens Center of Excellence in Indonesia Lenzing applies for 25 patents for Lenzing Web Technology Please enable JS and disable any ad blocker son of Thomas Keith Caldwell and Joy Munchberg Caldwell.  He was a graduate of Williston-Elko High School Reverend Caldwell was a member of Lake City Pentecostal Holiness Church recently received his Ministerial Credentials and was an Honorary Discipleship Ministries Council Member with the South Carolina Conference IPHC Inc.  He was an avid Clemson fan and Carolina Panthers fan.  Tom Caldwell and Joy Caldwell of Turbeville; brothers of Turbeville and Joshua (Morgan) Caldwell of Conway; nephews Reverend Caldwell was preceded in death by his sisters Elizabeth Caldwell and Grace Caldwell; paternal grandmother The family will receive friends from 3:00 – 4:00 PM The two bestseller “Why Doesn’t Anyone Blush Anymore?” by Rabbi Manis Friedman and “The Book of Purpose” by Rabbi Tzvi Freeman have now been published in German by Books&Bagels in Switzerland in German “Wird denn niemand mehr rot?” and “Das Buch vom Sinn” were designed by the renowned book designer professor Ralf de Jong easily understood and entertaining language how modesty can become a powerful tool for meaningful and intimate relationships The publication of this book was made possible by the help of Bettina and Lutz Münchberg “Das Buch vom Sinn” by Rabbi Freeman contains his condensed and sometimes directly quoted thoughts of the Rebbe about how to find purpose in life. It deals with questions about who we are, if our existence is an end to itself or if we can fill our lives with purpose and meaning. This book was published with the support of chabad.org More information on the books can be found here: www.booksnbagels.com/liebe and here: www.booksnbagels.com/sinn Supporting a well-founded textile-specific education in the new institute Sandler AG has donated a professorship worth a total of EUR 600,000 Transport/​Aerospace a new textile research centre was inaugurated at Hof University’s Münchberg campus together with other entrepreneurs and industry associations has been decisively advocating the establishment of a textile research centre at Hof University’s Münchberg facility the Bavarian state government approved the establishment of the institute thus laying the foundation for the expansion of textile-specific education which is deemed essential for Sandler and other companies in the region it was a cause and at the same time a responsibility to further promote this field of education Sandler decided to provide EUR 600,000 for an endowed professorship The company thereby further contributes to the educational infrastructure in the region,” Sandler said The new professorship has been filled since 2019 - Prof Claus-Ekkehard Koukal holds a lectureship at the University of Applied Sciences Hof in the field of Innovative Textiles teaching in the areas of nonwoven production including the textile industry and research and development he continues to be active in consultancy and research The research project EFRE: The Future – Textile Industry 4.0 which focusses on the transfer of know-how from research to small and medium-sized companies; or the Traveltex project which involves the development of functional knitted fabrics for travel clothing “The close cooperation with universities and research centres is very important to us we have been cultivating these partnerships for many years," explains Dr "We are happy to be able to continue supporting textile-specific education in the region." www.sandler.de This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Joy Buck of Northville is a fan of garage sales the one-time shop owner often headed to the Upper Peninsula sightseeing with visitors “Many friends and family came to visit,” she wrote in an email to the column “My sister and her husband came to see us one time and our husbands went to the Sault to gamble at the casino.” While they did that she and her sister would sometimes head out on the garage sale trail she told appraiser Brian Thomczek at a recent Trash or Treasure day held at Judy Frankel Antiques Buck came across a painting she had taken with her on two moves since Titled “Pheasants,” it has a registration number of 19868 and is signed by “Majewicz,” she told the appraiser “I have researched his work and feel that this may have value,” she continued or if it really has any,” she told the appraiser “I could never really find out more information about what it’s worth The back of the oil on canvas is marked “G Majewicz” and it is signed in the lower right It measures 211/2 by 25 and features three pheasants in a landscape Buck said that she found out a little more about the artist and that while her information showed he was born in Germany he was Polish and his forte was birds and wildlife Internet research revealed a variety of wildlife and bird images by artist George Majewicz The website askart.com list the artist’s birth date as 1897 and place as Polkowice/Munchberg and that he died in 1965 moreover especially in hunting and animal painting He derives his motives mostly from the African animal world and devoted himself preferably to the representation of animals in the wilderness in connection with an impressive landscape description.” Thomczek said the work could use a bit of restoration and cleaning and recommended former DIA employee Ken Katz who runs the downtown’s Conservation and Museum Services (conservationandmuseumservices.com) He told Buck that the frame alone is worth $150-$200; with the painting he’d appraise it at $300-$500 adding “that could go up if we had time to do more research on the artist and his sales records.” he told her that she has a good eye and she should continue her garage sale hobby It’s nicely done and definitely by a well-trained hand Landscape scenes are popular paintings and it would be something that collectors Do you have an object you would like to know more about Send a photo and description that includes how you acquired the object to: The Detroit News Include your name and daytime telephone number You may also send your photo and description to trashortreas@aol.com If chosen you’ll need to bring the items to an appraisal session Polish death metal act Hate are in mourning after the death of their bassist Slawek 'Mortifer' Archangielskij The musician passed away near the German town of Munchberg on the night of April 5 while the band was en route to their next gig on their European tour Hate, who had been on the road in support of their latest album, 'Solarflesh,' posted about the loss of their comrade and stated that they'll immediately stop their current tour as a result of his death. Archangielskij was also the bass player for Naumachia and the guitarist for Saltus. Both of those bands reposted Hate's original Facebook message on his passing on their sites as well The posting on the bassist's death reads as follows: SŁAWEK „MORTIFER” ARCHANGIELSKIJ IS DEAD At night 5th/6th April near a German town of Munchberg best comrade and longtime bass player unexpectedly passed away After the show in Stuttgart last night he went to sleep and never woke up We found him lifeless early in the morning and immediately called an ambulance Results of Sławek’s autopsy should be known soon we decided to cancel the remaining shows and return home We gave detailed testimonies to the German police We are shocked and shattered by his sudden We mourn together with Slawek’s family and friends The death metal act has been around since 1990 but has gone through a number of bassists since their start Archangielskij had been a touring member since 2007 and an official band member since 2009 Frontman Adam "ATF Sinner" Buszko remains the band's only original member 'Solarflesh' is Hate's eighth studio album Watch the video for 'Alchemy of Blood' from Hate's 'Solarflesh' album below: Polish death metal act Hate are in mourning after the death of their bassist Slawek 'Mortifer' Archangielskij. The musician passed away near the German town of Munchberg on the night of April 5 while the band was en route to their next gig on their European tour.\nRead More Besiktas eye Hajduk’s Marko Livaja, but talks wait for title race to finish Shock in Split: Gennaro Gattuso set to leave Hajduk after Dinamo defeat has set off on the adventure of a lifetime by deciding to walk to the Euro in Germany with Detić planning to cover 40-50 km each day with the use of public transport considered only in the case of serious injury Detić will walk during the day and rest at night in a tent and he hopes to receive support from fellow Croatians in the cities along the way The thousand-kilometer journey began from Črešnjevo Detić's ambitious trek aims to show his passion for football and his determination to support his team at the European Championship Nogomania.com is a premier destination for football enthusiasts delivering fresh and in-depth content from the heart of the Ex-Yu region's football scene.