Dietrich, Daniel R.

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Daniel R.
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PODO/TERT256 : A promising human immortalized podocyte cell line and its potential use for in vitro research at different oxygen levels

2024, Schlichenmaier, Nadja, Zielinski, Alexander, Beneke, Sascha, Dietrich, Daniel R.

Podocytes are of key interest for the prediction of nephrotoxicity as they are especially sensitive to toxic insults due to their central role in the glomerular filtration apparatus. However, currently, prediction of nephrotoxicity in humans remains insufficiently reliable, thus highlighting the need for advanced in vitro model systems using human cells with improved prediction capacity. Recent approaches for refining in vitro model systems focus on closely replicating physiological conditions as observed under the in vivo situation typical of the respective nephron section of interest. PODO/TERT256, a human immortalized podocyte cell line, were employed in a semi-static transwell system to evaluate its potential use as a human podocyte in vitro system for modelling potential human glomerular toxicity. Furthermore, the impact of routinely employed excessive oxygen tension (21 % - AtmOx), when compared to the physiological oxygen tensions (10 % - PhysOx) observed in vivo, was analyzed. Generally, cultured PODO/TERT256 formed a stable, contact-inhibited monolayer with typical podocyte morphology (large cell body, apical microvilli, finger-like cytoplasmic projections (reminiscent of foot processes), and interdigitating cell-cell junctions) and developed a size-selective filtration barrier. PhysOx, however, induced a more pronounced in vivo like phenotype, comprised of significantly larger cell bodies, significantly enhanced filtration barrier size-selectivity, and a remarkable re-localization of nephrin to the cell membrane, thus suggesting an improved in vitro replication of in vivo characteristics. Preliminary toxicity characterization with the known glomerulotoxin doxorubicin (DOX) suggested an increasing change in filtration permeability, already at the lowest DOX concentrations tested (0.01 μM) under PhysOx, whereas obvious changes under AtmOx were observed as of 0.16 μM and higher with a near all or nothing effect. The latter findings suggested that PODO/TERT256 could serve as an in vitro human podocyte model for studying glomerulotoxicity, whereby culturing at PhyOx tension appeared critical for an improved in vivo-like phenotype and functionality. Moreover, PODO/TERT256 could be incorporated into advanced human glomerulus systems in vitro, recapitulating microfluidic conditions and multiple cell types (endothelial and mesenchymal cells) that can even better predict human glomerular toxicity.

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Investigation of microcystin conformation and binding towards PPP1 by molecular dynamics simulation

2022-01-05, Jaeger, Sabrina, Nitschke, Jahn, Altaner, Stefan, Klein, Karsten, Dietrich, Daniel R., Schreiber, Falk

Microcystins (MC) are a group of structurally similar cyanotoxins with currently 279 described structural variants. Human exposure is frequent by consumption of contaminated water, food or food supplements. MC can result in serious intoxications, commensurate with ensuing pathology in various organs or in rare cases even mortality. The current WHO risk assessment primarily considers MC-LR, while all other structural variants are treated as equivalent to MC-LR, despite that current data strongly suggest that MC-LR is not the most toxic MC, and toxicity can be very different for MC congeners. To investigate and analyse binding and conformation of different MC congeners, we applied for the first time Molecular Dynamics (MD) simulation to four MC congeners (MC-LR, MC-LF, [Enantio-Adda5]MC-LF, [β-D-Asp3,Dhb7]MC-RR). We could show that ser/thr protein phosphatase 1 is stable in all MD simulations and that MC-LR backbone adopts to a second conformation in solvent MD simulation, which was previously unknown. We could also show that MC congeners can adopt to different backbone conformation when simulated in solvent or in complex with ser/thr protein phosphatase 1 and differ in their binding behaviour. Our findings suggest that MD Simulation of different MC congeners aid in understanding structural differences and binding of this group of structurally similar cyanotoxins.

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Interdisciplinary Reservoir Management : a Tool for Sustainable Water Resources Management

2021-04-18, Daus, Milan, Koberger, Katharina, Koca, Kaan, Beckers, Felix, Encinas Fernández, Jorge, Weisbrod, Barbara, Dietrich, Daniel R., Hofmann, Hilmar, Martin-Creuzburg, Dominik, Peeters, Frank

Reservoirs are a common way to store and retain water serving for a multitude of purposes like storage of drinking and irrigation water, recreation, flood protection, navigation, and hydropower production, and have been built since centuries. Today, few reservoirs serve only one purpose, which requires management of present demands and interests. Since each reservoir project will cause negative impacts alongside desired advantages both on a local, regional and global scale, it is even more urgent to develop a common management framework in an attempt to mitigate negative impacts, incorporate different demands and make them visible within the discourse in order to avoid conflicts from early on. The scientific publications on reservoirs are manifold, yet a comprehensive and integrative holistic tool about management of this infrastructure is not available. Therefore, a comprehensive and integrated conceptual tool was developed and proposed by the authors of this paper that can contribute to the sustainable management of existing reservoirs. The tool presented herein is based on the results from the interdisciplinary CHARM (CHAllenges of Reservoir Management) project as well as the condensed outcome of relevant literature to aid and enhance knowledge of reservoir management. The incorporated results are based on field, laboratory and empirical social research. The project CHARM focused on five different aspects related to existing reservoirs in southern Germany (Schwarzenbachtalsperre, Franconian Lake District), namely: sedimentation of reservoirs, biostabilisation of fine sediments, toxic cyanobacteria(l) (blooms), greenhouse gas emissions from reservoirs and social contestation, respectively consent. These five research foci contributed to the topics and setup of a conceptual tool, put together by the research consortium via delphi questioning, which can be found alongside this publication to provide insights for experts and laymen. Conceptualising and analysing the management in combination with quantitative and qualitative data in one descriptive tool presents a novelty for the case studies and area of research. The distribution within the scientific community and interested public will possibly make a positive contribution to the goal of sustainable water resources management in the future.

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Variability in microcystin quotas during a Microcystis bloom in a eutrophic lake

2021, Wood, Susanna A., Puddick, Jonathan, Hawes, Ian, Steiner, Konstanze, Dietrich, Daniel R., Hamilton, David P.

Microcystis is a bloom-forming genus of cyanobacteria with some genotypes that produce highly toxic microcystin hepatotoxins. In waterbodies where biological and physical factors are relatively homogenous, toxin quotas (the average amount of toxin per cell), at a single point in time, are expected to be relatively constant. In this study we challenged this assumption by investigating the spatial distribution of microcystin quotas at a single point in time on two separate occasions in a lake with a major Microcystis bloom. Microcystis cell concentrations varied widely across the lake on both sampling occasions (730- and 137-fold) together with microcystin quotas (148- and 362-fold). Cell concentrations and microcystin quotas were strongly positively correlated (R2 = 0.89, P < 0.001, n = 28; R2 = 0.67, P < 0.001, n = 25). Analysis of Microcystis strains using high-throughput sequencing of the 16S-23S rRNA intergenic spacer region showed no relationship between microcystin quota and the relative abundance of specific sequences. Collectively, the results of this study indicate an association between microcystin production and cell density that magnifies the potential for bloom toxicity at elevated cell concentrations.

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US regulations to curb alleged cancer causes are ineffectual and compromised by scientific, constitutional and ethical violations

2023-04-08, Gori, Gio B., Aschner, Michael, Borgert, Christopher J., Cohen, Samuel M., Dietrich, Daniel R., Galli, Corrado L., Greim, Helmut, Heslop-Harrison, John S., Kacew, Sam, Kaminski, Norbert E.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

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Is Toxin-Producing Planktothrix sp. an Emerging Species in Lake Constance?

2021-09-17, Fournier, Corentin, Riehle, Eva, Dietrich, Daniel R., Schleheck, David

Recurring blooms of filamentous, red-pigmented and toxin-producing cyanobacteria Planktothrix rubescens have been reported in numerous deep and stratified prealpine lakes, with the exception of Lake Constance. In a 2019 and 2020 Lake Constance field campaign, we collected samples from a distinct red-pigmented biomass maximum below the chlorophyll-a maximum, which was determined using fluorescence probe measurements at depths between 18 and 20 m. Here, we report the characterization of these deep water red pigment maxima (DRM) as cyanobacterial blooms. Using 16S rRNA gene-amplicon sequencing, we found evidence that the blooms were, indeed, contributed by Planktothrix spp., although phycoerythrin-rich Synechococcus taxa constituted most of the biomass (>96% relative read abundance) of the cyanobacterial DRM community. Through UPLC–MS/MS, we also detected toxic microcystins (MCs) in the DRM in the individual sampling days at concentrations of ≤1.5 ng/L. Subsequently, we reevaluated the fluorescence probe measurements collected over the past decade and found that, in the summer, DRM have been present in Lake Constance, at least since 2009. Our study highlights the need for a continuous monitoring program also targeting the cyanobacterial DRM in Lake Constance, and for future studies on the competition of the different cyanobacterial taxa. Future studies will address the potential community composition changes in response to the climate change driven physiochemical and biological parameters of the lake.

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Label-Free Identification and Differentiation of Different Microplastics Using Phasor Analysis of Fluorescence Lifetime Imaging Microscopy (FLIM)-Generated Data

2021-04-15, Monteleone, Adrian, Schary, Weronika, Wenzel, Folker, Langhals, Heinz, Dietrich, Daniel R.

As plastic pollution is becoming an increasing worldwide problem, a variety of different techniques for the detection and in-depth characterization of plastics, including spectroscopy and chromatography methods, were introduced to the public. Recently we presented fluorescence lifetime imaging microscopy (FLIM) a new approach for the identification and characterization of microplastics using their fluorescence lifetime (τ) for differentiation. A very powerful extension of the recently established FLIM could be phasor analysis, which allows data representation in an interactive 2D graphical phasor plot thereby enabling a global view of the fluorescence decay in each pixel of the measured image. Microplastic particles generated from six different types of plastics were subjected to excitation wavelengths of 440 nm, upon which specific fluorescence lifetimes as well as the photon yield were determined using FLIM and phasor analysis. We could show that phasor analysis for FLIM with a laser pulse repetition frequency of 40 MHz was able to generate specific locations in the phasor plot for the plastics for fast differentiation, e.g. resulting in well-defined phasor plot positions for ABS at 3.019 ns, PPE at 6.239 ns, PET bottle from Germany at 2.703 ns and PET bottle from USA at 2.711 ns. Phasor analysis for FLIM proves to be a fast, label-free, and sensitive method for the identification and differentiation of plastics also with the aid of visualization variation enabling techniques such as heat treatment of plastics.

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Physiological oxygen and co-culture with human fibroblasts facilitate in vivo-like properties in human renal proximal tubular epithelial cells

2022-05-06, Piossek, Felicitas, Beneke, Sascha, Schlichenmaier, Nadja, Mucic, Goran, Drewitz, Sabine, Dietrich, Daniel R.

Reliable prediction of compound mediated nephrotoxicity in humans is still unsatisfactory irrespective of the recent advancements in in silico, in vitro and in vivo models. Therefore, current in vitro approaches need refinement to better match the human in vivo situation, specifically with regard to the potential influence of other cell types (e.g. fibroblasts) and to the potential biases introduced by the excessive 21% O2 (AtmOx) as employed in routine cell culturing. We used a transwell co-culture model combining human renal proximal tubule epithelial cells (RPTEC/TERT1) and human fibroblasts (fHDF/TERT166) to compare the functional properties and expression of selected marker proteins at 21% O2 and at the physiologically normal 10% O2 tension (PhysOx) commensurate with in vivo conditions. Culturing at PhysOx and co-culturing with fibroblasts significantly improved epithelial barrier integrity, expression of transporters (e.g. aquaporin 2; OCT-MATE; MRP-OAT) and metabolism. Moreover, beyond culturing these human cells in co-culture for up to 41 days, we were able to demonstrate increased functionality of cation transport, as shown via ASP+ (OCT-MATE axis), and anion transport, as shown via LY (MRP-OAT axis). Thus, adjusting the in vitro system to near physiological conditions had a major impact on functionality and provides the basis for the future development of true flow-through microfluidic renal testing systems with better predictability of human renal proximal toxicity.

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The EU chemicals strategy for sustainability : in support of the BfR position

2021-08-07, Barile, Frank A., Berry, Sir Colin, Blaauboer, Bas, Boobis, Alan, Bolt, Herrmann M., Borgert, Christopher, Dekant, Wolfgang, Dietrich, Daniel R., Domingo, Jose L., Greim, Helmut

The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.

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Examination of microcystin neurotoxicity using central and peripheral human neurons

2021, Klima, Stefanie, Suciu, Ilinca, Hoelting, Lisa, Gutbier, Simon, Waldmann, Tanja, Dietrich, Daniel R., Leist, Marcel

Microcystins (MC) are a group of cyanobacterial toxins that comprises MC-LF and other cyclic heptapeptides, best known as potent hepatotoxicants. Cell culture and epidemiological studies suggest that MC might also affect the nervous system, when there is systemic exposure e.g. via drinking water or food. We asked whether in vitro studies with human neurons could provide estimates on the neurotoxicity hazard of MC-LF. First, we used LUHMES neurons, a well-established test system for neurotoxicants and neuropathological processes. These central nervous system cells expressed OATP1A2, a presumed carrier of MC-LF, and we observed selective neurite toxicity in the µM range (EC20 = 3.3 µM ≈ 3.3 µg/ml). Toxicity paralleled transcriptome changes pointed towards attenuated cell maintenance and biosynthetic processes. Prolonged exposure for up to four days did not increase toxicity. As a second model, we used human dorsal root ganglia-like neurons. These peripheral nervous system cells represent parts of the nervous system not protected by the blood brain barrier in humans. Toxicity was observed in a similar concentration range (EC20 = 7.4 µM). We conclude that MC-LF poses a potential neurotoxic hazard in humans. The adverse effect concentrations observed here were orders of magnitude higher than those presumed to be encountered after normal nutritional or environmental exposure. However, the low µM concentrations found to be toxic are close to levels that may be reached after very excessive algae supplement intake.