Population relevance of neurotoxic effects in refined and alternative behavior tests with zebrafish (Danio rerio)

Water supplies are widely, but unobtrusively contaminated with numerous substances of largely unknown biological properties. A particularly worrisome group are neurotoxic substances, which may, in the long term, not only affect human health, but also wildlife. Neurotoxic effects have become an issue of emerging concern in ecotoxicology, since they may have multiple underlying mechanisms, are often hard to detect, but have the potential to give rise to a severe adverse outcome. As neurotoxicity is even more difficult to detect without extensive animal testing, it presents a major challenge to modern ecotoxicology which is striving to reduce and replace animal studies. My model species, the zebrafish (Danio rerio), is widely used in aquatic ecotoxicology but room for refinement remains especially where tests are carried out with adult individuals instead of potentially less perceptive early-life stages. Since zebrafish, like many other small fish, naturally form shoals and likely behave differently in isolation, I developed a shoal-based approach. In brief, early-life stage tests according to OECD TG 210 were augmented by two behavior tests that are typically carried out with single adult fish, but could be adapted to groups of juveniles with acceptable limitations: a novel tank test and a predator response assay. The selective serotonin reuptake inhibitor fluoxetine ((RS)-N-Methyl-3-phenyl-3-(4-trifluoromethylphenoxy) propylamine) served as model substance during a proof-of-concept study. In a follow-up study, I verified the suitability of this approach using a selection of other substances with different modes-of-action: carbamazepine (sodium channel inhibition), paraoxon-methyl (acetylcholine esterase inhibition), and tris(1,3-dichlorisopropyl) phosphate (TDCPP; endocrine disruption). Finally, in order to assess whether existing alternative methods correlate to immediately population relevant endpoints, I carried out several other experiments across the life-stages of zebrafish with the same model substances. Fluoxetine produced adverse effects down to concentrations three orders of magnitude below the EC10 from acute fish embryo toxicity tests (OECD TG 236). The known neurotoxicants carbamazepine and paraoxon-methyl caused significant effects on zebrafish behavior both upon release into a novel tank and after presentation of a predator dummy. TDCPP, which is thought to disrupt neural development at much earlier stages than those exposed in my experiments, only caused minor behavioral changes. Histopathology of the test fish confirmed the absence of acute organ damage at the concentrations used (always ≤ EC10 from fish embryo tests). The suitability of shoal-based behavioral changes in juvenile zebrafish as sensitive endpoints of neurotoxicity could thus be confirmed. The deviations in behavior compared to the control groups permit conclusions about the “anxiety state”, which arguably influences the fish’s survival chances in the wild. An early and more abstract behavior endpoint, larval motility (6 dpf), also proved to be very efficient and held up well in a comparison with adult and juvenile behavior tests. Finally, a reproduction assay with adult fish exposed to fluoxetine revealed decreased fecundity as another directly population relevant effect of this chemical. 2 Correlation with embryonic and further adult data from literature revealed the good predictive power of 24-h spontaneous coiling tests for later behavior defects, leading me to propose a set of embryonic tests (FET + coiling) for neurotoxicity range-finding and screening in the future. If the results from these “alternative methods” are negative or inconclusive, in vivo testing is indispensable to assess neurotoxicity; as such, larval motility and juvenile behavior assays might follow

Tensile Stiffness of Laminated Segment Timber

Laminated Segment Timber (LST) is an engineered wood product. It is panelshaped and glued of trapezoidal spruce lamellas. As intermediate product, it is provided for the processing to layered LST, a glulam-like building material. The source material for LST are mechanically graded logs. This study aims at developing modelling and simulation techniques to assess the mechanical properties of LST. It is reported about 294 nondestructive tensile tests on specimens from LST and about the corresponding results. Digital image correlation was used to examine the tensile stiffness. Both global and local stiffness of the LST specimens were analysed in terms of tensile MOE. It was found that the local MOE of modelling units for LST can be predicted by the dynamic MOE of the source material and a newly developed knot ratio referring to the cross-section of LST specimens. Using this correlation, two stiffness models were derived for the local MOE. Their response shows good agreement with that of reference models from the literature. The models are, therefore, suitable for incorporation into a layered LST finite element model which is intended for predicting the mechanical properties

Stabilization and Incipient Carbonization of Electrospun Polyacrylonitrile Nanofibers Fixated on Aluminum Substrates

Storck JL, Grothe T, Tuvshinbayar K, et al. Stabilization and Incipient Carbonization of Electrospun Polyacrylonitrile Nanofibers Fixated on Aluminum Substrates. Fibers. 2020;8(9): 55.Polyacrylonitrile (PAN) nanofibers, prepared by electrospinning, are often used as a precursor for carbon nanofibers. The thermal carbonization process necessitates a preceding oxidative stabilization, which is usually performed thermally, i.e., by carefully heating the electrospun nanofibers in an oven. One of the typical problems occurring during this process is a strong deformation of the fiber morphologies—the fibers become thicker and shorter, and show partly undesired conglutinations. This problem can be solved by stretching the nanofiber mat during thermal treatment, which, on the other hand, can lead to breakage of the nanofiber mat. In a previous study, we have shown that the electrospinning of PAN on aluminum foils and the subsequent stabilization of this substrate is a simple method for retaining the fiber morphology without breaking the nanofiber mat. Here, we report on the impact of different aluminum foils on the physical and chemical properties of stabilized PAN nanofibers mats, and on the following incipient carbonization process at a temperature of max. 600 °C, i.e., below the melting temperature of aluminum

Comparative Study of Metal Substrates for Improved Carbonization of Electrospun PAN Nanofibers

Carbon nanofibers are used for a broad range of applications, from nano-composites to energy storage devices. They are typically produced from electrospun poly(acrylonitrile) nanofibers by thermal stabilization and carbonization. The nanofiber mats are usually placed freely movable in an oven, which leads to relaxation of internal stress within the nanofibers, making them thicker and shorter. To preserve their pristine morphology they can be mechanically fixated, which may cause the nanofibers to break. In a previous study, we demonstrated that sandwiching the nanofiber mats between metal sheets retained their morphology during stabilization and incipient carbonization at 500 °C. Here, we present a comparative study of stainless steel, titanium, copper and silicon substrate sandwiches at carbonization temperatures of 500 °C, 800 °C and 1200 °C. Helium ion microscopy revealed that all metals mostly eliminated nanofiber deformation, whereas silicone achieved the best results in this regard. The highest temperatures for which the metals were shown to be applicable were 500 °C for silicon, 800 °C for stainless steel and copper, and 1200 °C for titanium. Fourier transform infrared and Raman spectroscopy revealed a higher degree of carbonization and increased crystallinity for higher temperatures, which was shown to depend on the substrate material

Metallic Supports Accelerate Carbonization and Improve Morphological Stability of Polyacrylonitrile Nanofibers during Heat Treatment

Storck JL, Hellert C, Brockhagen B, et al. Metallic Supports Accelerate Carbonization and Improve Morphological Stability of Polyacrylonitrile Nanofibers during Heat Treatment. Materials. 2021;14(16): 4686.Electrospun poly(acrylonitrile) (PAN) nanofibers are typical precursors of carbon nanofibers. During stabilization and carbonization, however, the morphology of pristine PAN nanofibers is not retained if the as-spun nanofiber mats are treated without an external mechanical force, since internal stress tends to relax, causing the whole mats to shrink significantly, while the individual fibers thicken and curl. Stretching the nanofiber mats during thermal treatment, in contrast, can result in fractures due to inhomogeneous stress. Previous studies have shown that stabilization and carbonization of PAN nanofibers electrospun on an aluminum substrate are efficient methods to retain the fiber mat dimensions without macroscopic cracks during heat treatment. In this work, we studied different procedures of mechanical fixation via metallic substrates during thermal treatment. The influence of the metallic substrate material as well as different methods of double-sided covering of the fibers, i.e., sandwiching, were investigated. The results revealed that sandwich configurations with double-sided metallic supports not only facilitate optimal preservation of the original fiber morphology but also significantly accelerate the carbonization process. It was found that unlike regularly carbonized nanofibers, the metal supports allow complete deoxygenation at low treatment temperature and that the obtained carbon nanofibers exhibit increased crystallinity

On the reliability of highly magnified micrographs for structural analysis in materials science.

Wortmann M, Layland AS, Frese N, et al. On the reliability of highly magnified micrographs for structural analysis in materials science. Scientific reports. 2020;10(1): 14708.Highly magnified micrographs are part of the majority of publications in materials science and related fields. They are often the basis for discussions and far-reaching conclusions on the nature of the specimen. In many cases, reviewers demand and researchers deliver only the bare minimum of micrographs to substantiate the research hypothesis at hand. In this work, we use heterogeneous poly(acrylonitrile) nanofiber nonwovens with embedded nanoparticles to demonstrate how an insufficient or biased micrograph selection may lead to erroneous conclusions. Different micrographs taken by transmission electron microscopy and helium ion microscopy with sometimes contradictory implications were analyzed and used as a basis for micromagnetic simulations. With this, we try to raise awareness for the possible consequences of cherry-picking for the reliability of scientific literature

QSEA-modelling of genome-wide DNA methylation from sequencing enrichment experiments

Genome-wide enrichment of methylated DNA followed by sequencing (MeDIP-seq) offers a reasonable compromise between experimental costs and genomic coverage. However, the computational analysis of these experiments is complex, and quantification of the enrichment signals in terms of absolute levels of methylation requires specific transformation. In this work, we present QSEA, Quantitative Sequence Enrichment Analysis, a comprehensive workflow for the modelling and subsequent quantification of MeDIP-seq data. As the central part of the workflow we have developed a Bayesian statistical model that transforms the enrichment read counts to absolute levels of methylation and, thus, enhances interpretability and facilitates comparison with other methylation assays. We suggest several calibration strategies for the critical parameters of the model, either using additional data or fairly general assumptions. By comparing the results with bisulfite sequencing (BS) validation data, we show the improvement of QSEA over existing methods. Additionally, we generated a clinically relevant benchmark data set consisting of methylation enrichment experiments (MeDIP-seq), BS-based validation experiments (Methyl-seq) aswell as gene expression experiments (RNA-seq) derived from non-small cell lung cancer patients, and show that the workflow retrieves well-known lung tumour methylation markers that are causative for gene expression changes, demonstrating the applicability of QSEA for clinical studies. QSEA is implemented in R and available from the Bioconductor repository 3.4 (www.bioconductor.org/packages/qsea)

Functional Therapeutic Target Validation Using Pediatric Zebrafish Xenograft Models

The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing. In such an approach, patient-derived tumor cells are exposed to a library of approved oncological drugs in a physiological setting, e.g., in the form of animal avatars injected with patient tumor cells. We used molecularly fully characterized tumor samples from the INFORM study to compare drug screen results of individual patient-derived cell models in functional assays: (i) patient-derived spheroid cultures within a few days after tumor dissociation; (ii) tumor cells reisolated from the corresponding mouse PDX; (iii) corresponding long-term organoid-like cultures and (iv) drug evaluation with the corresponding zebrafish PDX (zPDX) model. Each model had its advantage and complemented the others for drug hit and drug combination selection. Our results provide evidence that in vivo zPDX drug screening is a promising add-on to current functional drug screening in precision medicine platforms