Toward Good In Vitro Reporting Standards

A good experiment reported badly is worthless. Meaningful contributions to the body of science are made by sharing the full methodology and results so that they can be evaluated and reproduced by peers. Erroneous and incomplete reporting does not do justice to the resources spent on conducting the experiment and the time peers spend reading the article. In theory peer-review should ensure adequate reporting – in practice it does not. Many areas have developed reporting standards and checklists to support the adequate reporting of scientific efforts, but in vitro research still has no generally accepted criteria. It is characterized by a “Wild West” or “anything goes” attitude. Such a culture may undermine trust in the reproducibility of animal-free methods, and thus parallel the “reproducibility crisis” discussed for other life science fields. The increasing data retrieval needs of computational approaches (in extreme as “big data” and artificial intelligence) makes reporting quality even more important so that the scientific community can take full advantage of the results. The first priority of reporting standards is to ensure the completeness and transparency of information provided (data focus). The second tier is a quality of data display that makes information digestible and easy to grasp, compare and further analyze (information focus). This article summarizes a series of initiatives geared towards improving the quality of in vitro work and its reporting. This shall ultimately lead to Good In Vitro Reporting Standards (GIVReSt)

Women in Alternatives

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t4 Workshop Report: Integrated Testing Strategies (ITS) for Safety Assessment

Integrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and “Good ITS Practices”.JRC.I.5-Systems Toxicolog

The Baltimore declaration toward the exploration of organoid intelligence

We, the participants of the First Organoid Intelligence Workshop - "Forming an OI Community" (22-24 February 2022), call on the international scientific community to explore the potential of human brain-based organoid cell cultures to advance our understanding of the brain and unleash new forms of biocomputing while recognizing and addressing the associated ethical implications. The term "organoid intelligence" (OI) has been coined to describe this research and development approach (1) in a manner consistent with the term "artificial intelligence" (AI) - used to describe the enablement of computers to perform tasks normally requiring human intelligence. OI has the potential for diverse and far-reaching applications that could benefit humankind and our planet, and which urge the strategic development of OI as a collaborative scientific discipline. OI holds promise to elucidate the physiology of human cognitive functions such as memory and learning. It presents game-changing opportunities in biological and hybrid computing that could overcome significant limitations in silicon-based computing. It offers the prospect of unparalleled advances in interfaces between brains and machines. Finally, OI could allow breakthroughs in modeling and treating dementias and other neurogenerative disorders that cause an immense and growing disease burden globally. Realizing the world-changing potential of OI will require scientific breakthroughs. We need advances in human stem cell technology and bioengineering to recreate brain architectures and to model their potential for pseudo-cognitive capabilities. We need interface breakthroughs to allow us to deliver input signals to organoids, measure output signals, and employ feedback mechanisms to model learning processes. We also need novel machine learning, big data, and AI technologies to allow us to understand brain organoids

Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Regulation und Funktion der microRNA während der neuronalen Entwicklung und Spezifizierung von Stammzellen

Title, Acknowledgements and Contents Abbreviations and Summary Introduction Materials and Methods Results Discussion Reference List AppendixIn the present study the role of microRNAs (miRNAs) in the control of developmental timing in the mammalian nervous system and in the specification of neural cell fate was investigated. miRNAs are a recently discovered class of small, 21-22 nt, regulatory RNA molecules. They inhibit translation of target mRNAs by binding to sites of imperfect anti-sense complementarity in 3 untranslated regions (UTRs). Many miRNAs are evolutionarily conserved, which has allowed their identification in various species. In the model organisms C. elegans and D. melanogaster, miRNAs regulate genes involved in fundamental developmental processes including cell proliferation, apoptosis, and the timing of cell fate decisions in the CNS (e.g. let-7 and lin-4 for C. elegans and Bantam and mir-14 for D. melanogaster). Hundreds of miRNA genes are expressed in humans and mice, and a substantial fraction of these genes has been identified in neural cells. Although the biological functions of most miRNAs are unknown, miRNAs are predicted to regulate about 30% of the human genes. Disruption of miRNA biogenesis is definitely associated with severe disturbances in neural development in model organisms and most likely with human clinical syndromes (Fragile X Mental Retardation Syndrome, Spinal Motor Atrophy, DiGeorge Syndrome). This fact, together with the well established role of miRNA genes in C. elegans and D. melanogaster development, points to the relevance of this newly emerging field for the understanding of developmental disorders. In this work the regulation of a set of highly expressed neural miRNAs, and in particular the let-7 family during mouse brain development and neural differentiation of embryonic stem (ES) cells has been studied. Significant differences were observed in the onset and magnitude of induction for individual miRNAs. miRNAs were strongly induced during neural differentiation of ES cells, suggesting the validity of the stem cell model for studying miRNA regulation in neural development. In undifferentiated ES and embryonal carcinoma (EC) cells, both the let-7 primary transcript and precursor were detected in the absence of mature miRNA accumulation, suggesting an important post-transcriptional component in the regulation of let-7 expression. An in vitro assay for precursor processing revealed developmental regulation of let-7 as well as mir-128 and mir-30 maturation. Precursor processing activity increased during neural differentiation of ES and EC cells and was greater in primary neurons compared to astrocytes. Neuron-specific binding activity of pre-miRNAs was shown by antibody challenge to contain the Fragile X Mental Retardation Protein (FMRP). As further evidence for developmental regulation of the miRNA processing pathway, it was shown that Argonaute proteins and FMRP failed to localize to cytoplasmic foci identified as processing bodies (P-bodies) in self-renewing ES or EC cells. Comparing expression in cultures of embryonic neurons and astrocytes, marked lineage specificity was found for many of the miRNAs studied. Two of the most highly expressed miRNAs in adult brain (mir-124, mir-128) were preferentially expressed in neurons. In contrast, mir-23, a miRNA previously implicated in neural specification, was restricted to astrocytes. Lineage specificity was further explored using reporter constructs for three miRNAs of particular interest (let-7, mir-125 and mir-128). miRNA-mediated suppression of these reporters was observed after their transfection into neurons but not astrocytes. Furthermore, reporter constructs containing let-7 or mir-125 target sites were downregulated in EC-derived neurons, reflecting the upregulation of miRNAs during neuronal development. In addition, mRNA target degradation was observed in response to let-7 and mir-125, opening new questions regarding the mechanism of miRNA-mediated mRNA silencing. Disrupting the interaction of let-7 and mir-125 with their target genes during neural differentiation led to an increase in astrocyte marker expression (GFAP and A2B5), implicating let 7 and mir-125 in neuronal lineage commitment. Finally, a functional let-7/mir-125 response element in the 3 UTR of a mouse lin-41 homolog was identified, revealing a conserved let-7/target gene interaction that is active during early neural differentiation.In dieser vorgelegten Studie sollte der Einfluss von microRNAs (miRNAs) auf die zeitspezifische Entwicklung des Nervensystems sowie die neuronale Spezifizierung von Stammzellen untersucht werden. miRNAs gehören zu einer kürzlich entdeckten Klasse regulatorischer RNA-Moleküle mit einer Länge von ca. 22 NT. Diese inhibieren die Translation durch unvollständig komplementäre Bindung an 3 -gelegenen untranslatierten Bereich (3 UTR) ihrer Ziel-mRNAs. Viele miRNAs und deren Zielregionen sind hochkonserviert und konnten in einer Vielzahl von Arten nachgewiesen werden. Anhand von entwicklungsbiologischen Studien an C. elegans und D. melanogaster, wurde gezeigt, dass miRNAs an fundamentalen, entwicklungsspezifischen Prozessen wie Proliferation, Apoptose sowie an der zeit- und gewebespezifischen Differenzierung des zentralen Nervensystems (ZNS) beteiligt sind (z.B. let-7 und lin-4 bei C. elegans sowie Bantam and mir-14 bei D. melanogaster). Beim Menschen und der Maus konnten über hundert miRNAs identifiziert werden, von denen eine beträchtliche Anzahl im Nervensystem vorkommt. Obwohl die genaue biologische Funktion der meisten miRNAs noch unbekannt ist, wird angenommen, dass ca. 30% der proteincodierenden Gene von ihnen reguliert werden. Viele klinische Krankheiten wie z.B. Fragiles-X-Syndrom, Spinale Muskelatrophie, DiGeorge Syndrom und neurospezifische Entwicklungsstörungen sind u.a. auf eine defekte miRNA-Biogenese zurückzuführen. miRNA-Entwicklungsstudien an C. elegans und D. Melanogaster und die oben genannten Tatsachen weisen auf die Relevanz dieses neuen Forschungsgebiets für das Verständnis von Entwicklungsstörungen. In dieser Arbeit wurden einige miRNAs untersucht, die eine starke neuralspezifische Expression während der Gehirnentwicklung und der neuralen Differenzierung von embryonalen Stammzellen (ES-Zellen) und embryonalen Karzinomzellen (EC-Zellen) der Maus zeigten. Es konnten signifikante Unterschiede hinsichtlich des Expressionsstarts und der Expressionsstärke für einzelne miRNAs nachgewiesen werden. Da diese miRNAs während der neuralen Differenzierung von ES-Zellen stark exprimiert werden, empfiehlt sich das Stammzellenmodel für die Untersuchung der miRNA-Regulation in der neuralen Entwicklung. In undifferenzierten ES- und EC-Zellen konnten zwar das primäre let-7 Transkript (pri-let-7) und das 70 NT lange Precursor-Transkript (pre- let-7), jedoch kaum reife let-7 miRNA nachgewiesen werden. Dies weist auf wichtige, noch unbekannte posttranskriptionale regulatorische Komponenten für die Prozessierung der reifen let-7 miRNA hin. Weitere in vitro Studien über die posttranskriptionale Prozessierung der Precursor-miRNA zeigten, dass die Reifung von let-7 sowie von mir-128 und mir-30 entwicklungsspezifisch reguliert wird. Die Aktivität dieser Prozessierung steigt während der Neuraldifferenzierung von ES und EC stark an. Diese Aktivität war in entstehenden primären Neuronen höher als in Astrozyten. Durch Inkubation der in vitro Reaktion mit einem Antikörper gegen das Fragile X Mental Retardation Protein (FMRP) konnte eine neuronenspezifische Bindungsaktivität von pre- miRNAs an das FMRP nachgewiesen werden. Des Weiteren konnte durch Antikörperfärbungen gezeigt werden, dass in undifferenzierten ES- und EC- Zellen Argonauteproteine und FMRP nicht in processing-Bodies (P-Bodies) vorhanden sind. Die oben genannten Ergebnisse weisen darauf hin, dass die miRNA-Prozessierung während der Entwicklung reguliert wird. Durch vergleichende Expressionsstudien zwischen embryonalen Neuronen und Astrozyten konnte gezeigt werden, dass viele miRNAs zelllinienspezifisch sind. Zum Beispiel werden die im adulten Gehirn am stärksten exprimierten miRNAs, mir-124 und mir-128, bevorzugt in Neuronen exprimiert, dagegen mir-23, welches zunächst als neuralspezifische miRNA impliziert wurde, rein astrozytenspezifisch ist. Die Zelllinienspezifität und Funktionalität von drei miRNAs (let-7, mir-125 and mir-128) wurde mittels GFP-Reporterkonstrukten untersucht. Ein schwaches GFP-Signal bzw. eine miRNA induzierte Hemmung der Translation konnte nach der Transfektion in Neuronen jedoch nicht in Astrozyten beobachtet werden. Des Weiteren konnte eine Inhibierung der Translation für let-7- und mir-125-Sensorkonstrukte in EC-induzierten Neuronen gezeigt werden. Dies ist auf eine Aktivierung dieser miRNAs während der neuralen Differenzierung zurückzuführen. Zusätzlich konnte hier eine Degradierung der mRNA von let-7- und mir-125-Zielgenen festgestellt werden, was neue Fragen hinsichtlich der miRNA induzierten Stilllegung von Zielgenen aufwirft. In einem anderen Versuch wurde die natürliche Interaktion von let-7 und mir-125 mit deren Zielgenen durch Überexpression von exogenen Sensorkonstrukten verhindert. Das führte zu einer verstärkten Expression von Astrozyten-spezifischen Proteinen (GFAP und A2B5), was die Bedeutung von let-7 und mir-125 für die Differenzierung von neuralen Vorläuferzellen unterstreicht. Schließlich konnten hochkonservierte funktionelle let-7- und mir-125 Bindestellen in der 3' UTR des Maus lin-41 Homologs identifiziert werden. Diese Untersuchungen zeigen, dass konservierte let-7-Zielgen- Interaktionen während der frühe neuralen Differenzierung stattfinden

Cellular resilience

Cellular resilience describes the ability of a cell to cope with environmental changes such as toxicant exposure. If cellular metabolism does not collapse directly after the hit or end in programmed cell death, the ensuing stress responses promote a new homeostasis under stress. The processes of reverting "back to normal" and reversal of apoptosis ("anastasis") have been studied little at the cellular level. Cell types show astonishingly similar vulnerability to most toxicants, except for those that require a very specific target, metabolism or mechanism present only in specific cell types. The majority of chemicals triggers "general cytotoxicity" in any cell at similar concentrations. We hypothesize that cells differ less in their vulnerability to a given toxicant than in their resilience (coping with the "hit"). In many cases, cells do not return to the naive state after a toxic insult. The phenomena of "pre-conditioning", "tolerance" and "hormesis" describe this for low-dose exposures to toxicants that render the cell more resistant to subsequent hits. The defense and resilience programs include epigenetic changes that leave a "memory/scar" - an alteration as a consequence of the stress the cell has experienced. These memories might have long-term consequences, both positive (resistance) and negative, that contribute to chronic and delayed manifestations of hazard and, ultimately, disease. This article calls for more systematic analyses of how cells cope with toxic perturbations in the long-term after stressor withdrawal. A technical prerequisite for these are stable (organotypic) cultures and a characterization of stress response molecular networks.publishe

In Situ Measurement Methods for the CO2-Induced Gelation of Biopolymer Systems

This work presents two novel methods to investigate in situ the carbon dioxide (CO2)-induced gelation of biopolymer-based solutions. The CO2-induced gelation is performed in a viewing cell at room temperature under CO2 pressure (20 to 60 bar), whereby calcium precursors are used as cross-linkers. The novel methods allow the in situ optical observation and evaluation of the gelation process via the change in turbidity due to dissolution of dispersed calcium carbonate (CaCO3) particles and in situ pH measurements. The combination of both methods enables the determination of the gelation direction, gelation rate, and the pH value in spatial and temporal resolution. The optical gelation front and pH front both propagate equally from top to bottom through the sample solutions, indicating a direct link between a decrease in the pH value and the dissolution of the CaCO3 particles. Close-to-vertical movement of both gelation front and pH front suggests almost one dimensional diffusion of CO2 from the contact surface (gel–CO2) to the bottom of the sample. The gelation rate increases with the increase in CO2 pressure. However, the increase in solution viscosity and the formation of a gel layer result in a strong decrease in the gelation rate due to a hindrance of CO2 diffusion. Released carbonate ions from CaCO3 dissolution directly influence the reaction equilibrium between CO2 and water and therefore the change in pH value of the solution. Increasing the CaCO3 concentrations up to the solubility results in lower gelation rates.This work presents two novel methods to investigate in situ the carbon dioxide (CO2)-induced gelation of biopolymer-based solutions. The CO2-induced gelation is performed in a viewing cell at room temperature under CO2 pressure (20 to 60 bar), whereby calcium precursors are used as cross-linkers. The novel methods allow the in situ optical observation and evaluation of the gelation process via the change in turbidity due to dissolution of dispersed calcium carbonate (CaCO3) particles and in situ pH measurements. The combination of both methods enables the determination of the gelation direction, gelation rate, and the pH value in spatial and temporal resolution. The optical gelation front and pH front both propagate equally from top to bottom through the sample solutions, indicating a direct link between a decrease in the pH value and the dissolution of the CaCO3 particles. Close-to-vertical movement of both gelation front and pH front suggests almost one dimensional diffusion of CO2 from the contact surface (gel–CO2) to the bottom of the sample. The gelation rate increases with the increase in CO2 pressure. However, the increase in solution viscosity and the formation of a gel layer result in a strong decrease in the gelation rate due to a hindrance of CO2 diffusion. Released carbonate ions from CaCO3 dissolution directly influence the reaction equilibrium between CO2 and water and therefore the change in pH value of the solution. Increasing the CaCO3 concentrations up to the solubility results in lower gelation rates

Developmental neurotoxicity : challenges in the 21st century and in vitro opportunities

In recent years neurodevelopmental problems in children have increased at a rate that suggests lifestyle factors and chemical exposures as likely contributors. When environmental chemicals contribute to neurodevelopmental disorders developmental neurotoxicity (DNT) becomes an enormous concern. But how can it be tackled? Current animal test-based guidelines are prohibitively expensive, at $1.4 million per substance, while their predictivity for human health effects may be limited, and mechanistic data that would help species extrapolation are not available. A broader screening for substances of concern requires a reliable testing strategy, applicable to larger numbers of substances, and sufficiently predictive to warrant further testing. This review discusses the evidence for possible contributions of environmental chemicals to DNT, limitations of the current test paradigm, emerging concepts and technologies pertinent to in vitro DNT testing and assay evaluation, as well as the prospect of a paradigm shift based on 21st century technologies