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In the embryonic stem cell test (EST), differentiation of mouse embryonic stem cells (mESCs) is used as a model to assess embryotoxicity in vitro . The test was successfully validated by the European Center for the Validation of Alternative Methods (ECVAM) and models fundamental mechanisms in embryotoxicity, such as cytotoxicity and differentiation. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. To predict the embryotoxic potential of a test substance, three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts. A special feature of the EST is that it is solely based on permanent cell lines so that primary embryonic cells and tissues from pregnant animals are not needed. In this protocol, we describe the ECVAM-validated method, in which the morphological assessment of contracting cardiomyocytes is used as an endpoint for differentiation, and the molecular-based FACS-EST method, in which highly predictive protein markers specific for developing heart tissue were selected. With these methods, the embryotoxic potency of a compound can be assessed in vitro within 10 or 7 d, respectively.

Studying chemical disturbances during neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative in vitro testing approach for the identification of developmental neurotoxicants. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development and ESC differentiation and specification. Thus, neural differentiation of mESCs in vitro allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed changes in miRNome and transcriptome during neural differentiation of mESCs exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neurally differentiating mESCs upon VPA treatment. Based on miRNA profiling we observed that VPA shifts the lineage specification from neural to myogenic differentiation (upregulation of muscle-abundant miRNAs, mir-206, mir-133a and mir-10a, and downregulation of neural-specific mir-124a, mir-128 and mir-137). These findings were confirmed on the mRNA level and via immunochemistry. Particularly, the expression of myogenic regulatory factors (MRFs) as well as muscle-specific genes (Actc1, calponin, myosin light chain, asporin, decorin) were found elevated, while genes involved in neurogenesis (e.g. Otx1, 2, and Zic3, 4, 5) were repressed. These results were specific for valproate treatment and--based on the following two observations--most likely due to the inhibition of histone deacetylase (HDAC) activity: (i) we did not observe any induction of muscle-specific miRNAs in neurally differentiating mESCs exposed to the unrelated developmental neurotoxicant sodium arsenite; and (ii) the expression of muscle-abundant mir-206 and mir-10a was similarly increased in cells exposed to the structurally different HDAC inhibitor trichostatin A (TSA). Based on our results we conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. The observed lineage shift into myogenesis, where miRNAs may play an important role, could be one of the developmental neurotoxic mechanisms of VPA.

Apart from the first family member, uncoupling protein 1 (UCP1), the functions of other UCPs (UCP2-UCP5) are still unknown. In analyzing our own results and those previously published by others, we have assumed that UCP's cellular expression pattern coincides with a specific cell metabolism and changes if the latter is altered. To verify this hypothesis, we analyzed the expression of UCP1-5 in mouse embryonic stem cells before and after their differentiation to neurons. We have shown that only UCP2 is present in undifferentiated stem cells and it disappears simultaneously with the initiation of neuronal differentiation. In contrast, UCP4 is simultaneously up-regulated together with typical neuronal marker proteins TUJ-1 and NeuN during mESC differentiation in vitro as well as during murine brain development in vivo. Notably, several tested cell lines express UCP2, but not UCP4. In line with this finding, neuroblastoma cells that display metabolic features of tumor cells express UCP2, but not UCP4. UCP2's occurrence in cancer, immunological and stem cells indicates that UCP2 is present in cells with highly proliferative potential, which have a glycolytic type of metabolism as a common feature, whereas UCP4 is strongly associated with non-proliferative highly differentiated neuronal cells.

Epilepsy is characterized by the occurrence of epileptic events, ranging from brief bursts of interictal epileptiform brain activity to their most dramatic manifestation as clinically overt bilateral tonic–clonic seizures. Epileptic events are often modulated in a patient-specific way, for example by sleep. But they also reveal temporal patterns not only on ultra- and circadian, but also on multidien scales. Thus, to accurately track the dynamics of epilepsy and to thereby enable and improve personalized diagnostics and therapies, user-friendly systems for long-term out-of-hospital recordings of electrical brain signals are needed. Here, we present two wearable devices, namely ULTEEM and ULTEEMNite, to address this unmet need. We demonstrate how the usability concerns of the patients and the signal quality requirements of the clinicians have been incorporated in the design. Upon testbench verification of the devices, ULTEEM was successfully benchmarked against a reference EEG device in a pilot clinical study. ULTEEMNite was shown to record typical macro- and micro-sleep EEG characteristics in a proof-of-concept study. We conclude by discussing how these devices can be further improved and become particularly useful for a better understanding of the relationships between sleep, epilepsy, and neurodegeneration.

Objective Clinical trials on drug safety and efficacy are understood to be valid for all humans, but are not equally distributed worldwide. We aimed to analyze the global distribution of clinical drug trials (phase I–IV) in epilepsy and its correlation with key demographical and epidemiological factors. Methods We combined data on industry‐funded interventional drug trials in epilepsy from clinicaltrials.gov with epidemiological, geographical, socioeconomic, and human development data. This allowed us to analyze the distribution of trials and trial sites in relation to population, epilepsy incidence, and prevalence. Results Among 459 trials conducted in 69 countries, a vast majority of sites were situated in high‐income regions and countries with very high Human Development Index (HDI). There was a striking underrepresentation of low‐income countries and those with low HDI. Specifically, we found a 40‐ to 145‐fold underrepresentation of trial sites in the WHO regions of Africa and the Eastern Mediterranean when comparing the actual number of sites to the expected number, assuming an even distribution by population, incidence, or prevalence. Significance Our findings highlight inequalities in the distribution of epilepsy trial sites based on population size and disease burden. They underscore the need for more equitable trial placement strategies that consider both disease epidemiology and demographic diversity to ensure adequate representation of all affected populations. Plain Language Summary This study looked at where clinical trials for epilepsy medications are being done around the world. We found that most trials take place in high‐income countries, while many areas with a high number of people living with epilepsy—especially in Africa and the Eastern Mediterranean—are left out. This shows a big gap between where the disease is most common and where research happens. Highlighting this mismatch can help guide future efforts to make clinical research more inclusive and globally relevant.

Background: In biomedical research, the past two decades have seen the advent of in vitro model systems based on stem cells, humanized cell lines, and engineered organotypic tissues, as well as numerous cellular assays based on primarily established tumor-derived cell lines and their genetically modified derivatives. Objective: There are high hopes that these systems might replace the need for animal testing in regulatory toxicology. However, despite increasing pressure in recent years to reduce animal testing, regulators are still reluctant to adopt in vitro approaches on a large scale. It thus seems appropriate to consider how we could realistically perform regulatory toxicity testing using in vitro assays only. Discussion and Conclusion: Here, we suggest an in vitro—only approach for regulatory testing that will benefit consumers, industry, and regulators alike.

Sleep disturbances are common in acute stroke patients and are linked with a negative stroke outcome. However, it is also unclear which and how such changes may be related to stroke outcome. To explore this link, we performed a sleep electroencephalogram (EEG) study in animals and humans after ischemic stroke. (1) Animal study: 12 male rats were assigned to two groups: ischemia (IS) and sham surgery (Sham). In both groups, sleep architecture was investigated 24 h before surgery and for the following 3 days. (2) Human study: 153 patients with ischemic stroke participating in the SAS-CARE prospective, multicenter cohort study had a polysomnography within 9 days after stroke onset. Functional stroke outcome was assessed by the modified Rankin Scale (mRS) at hospital discharge (short-term outcome) and at a 3-month follow-up (long-term outcome). (1) Animal study: rapid eye movement (REM) sleep was significantly reduced in the IS group compared to the Sham group. (2) Human study: patients with poor short-term functional outcome had a reduction of REM sleep and prolonged REM latency during the acute phase of stroke. REM latency was the only sleep EEG variable found to be significantly related to short- and long-term functional impairment in a multiple linear regression analysis. Acute ischemic stroke is followed by a significant reduction of REM sleep in animals and humans. In humans, this reduction was linked with a bad stroke outcome; in addition, REM latency was found to be an independent predictor of stroke evolution. Potential explanations for this role of REM sleep in stroke are discussed. http://clinicaltrials.gov. Unique identifier: NCT01097967.

On the occasion of the 20th anniversary of the Center for Alternative Methods to Animal Experiments (ZEBET), an international symposium was held at the German Federal Institute for Risk Assessment (BfR) in Berlin. At the same time, this symposium was meant to celebrate the 50th anniversary of the publication of the book “The Principles of Humane Experimental Technique” by Russell and Burch in 1959 in which the 3Rs principle (that is, Replacement, Reduction, and Refinement ) has been coined and introduced to foster the development of alternative methods to animal testing. Another topic addressed by the symposium was the new vision on “Toxicology in the twenty-first Century”, as proposed by the US-National Research Council, which aims at using human cells and tissues for toxicity testing in vitro rather than live animals. An overview of the achievements and current tasks, as well as a vision of the future to be addressed by ZEBET@BfR in the years to come is outlined in the present paper.

Volumetric and morphometric studies have demonstrated structural abnormalities related to chronic epilepsies on a cohort- and population-based level. On a single-patient level, specific patterns of atrophy or cortical reorganization may be widespread and heterogeneous but represent potential targets for further personalized image analysis and surgical therapy. The goal of this study was to compare morphometric data analysis in 37 patients with temporal lobe epilepsies with expert-based image analysis, pre-informed by seizure semiology and ictal scalp EEG. Automated image analysis identified abnormalities exceeding expert-determined structural epileptogenic lesions in 86% of datasets. If EEG lateralization and expert MRI readings were congruent, automated analysis detected abnormalities consistent on a lobar and hemispheric level in 82% of datasets. However, in 25% of patients EEG lateralization and expert readings were inconsistent. Automated analysis localized to the site of resection in 60% of datasets in patients who underwent successful epilepsy surgery. Morphometric abnormalities beyond the mesiotemporal structures contributed to subtype characterisation. We conclude that subject-specific morphometric information is in agreement with expert image analysis and scalp EEG in the majority of cases. However, automated image analysis may provide non-invasive additional information in cases with equivocal radiological and neurophysiological findings.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.