Enhancers and long noncoding RNAs (lncRNAs) are key determinants of lineage specification during development. Here, we evaluate remodeling of the enhancer landscape and modulation of the lncRNA transcriptome during mesendoderm specification. We sort mesendodermal progenitors from differentiating embryonic stem cells (ESCs) according to Eomes expression, and find that enhancer usage is coordinated with mesendoderm-specific expression of key lineage-determining transcription factors. Many of these enhancers are associated with the expression of lncRNAs. Examination of ESC-specific enhancers interacting in three-dimensional space with mesendoderm-specifying transcription factor loci identifies MesEndoderm Transcriptional Enhancer Organizing Region (Meteor). Genetic and epigenetic manipulation of the Meteor enhancer reveal its indispensable role during mesendoderm specification and subsequent cardiogenic differentiation via transcription-independent and -dependent mechanisms. Interestingly, Meteor-deleted ESCs are epigenetically redirected towards neuroectodermal lineages. Loci, topologically associating a transcribed enhancer and its cognate protein coding gene, appear to represent therefore a class of genomic elements controlling developmental competence in pluripotency.

The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A1, B1 and E1, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A1, B1, E1 and F1 were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.

Clostridium botulinum neurotoxins (BoNTs) are the most poisonous naturally occurring protein toxins known to mankind and are the causative agents of the severe and potentially life-threatening disease botulism. They are also known for their application as cosmetics and as unique bio-pharmaceuticals to treat an increasing number of neurological and non-neurological disorders. Currently, the potency of biologically active BoNT for therapeutic use is mainly monitored by the murine LD50-assay, an ethically disputable test causing suffering and death of a considerable number of mice. The aim of this study was to establish an in vitro assay as an alternative to the widely used in vivo mouse bioassay. We report a novel BoNT detection assay using mouse embryonic stem cell-derived neurons (mESN) cultured on multi-electrode arrays (MEAs). After 21 days in culture, the mESN formed a neuronal network showing spontaneous bursting activity based on functional synapses and express the necessary target proteins for BoNTs. Treating cultures for 6 h with 16.6 pM of BoNT serotype A and incubation with 1.66 pM BoNT/A or 33 Units/ml of Botox® for 24 h lead to a significant reduction of both spontaneous network bursts and average spike rate. This data suggests that mESN cultured on MEAs pose a novel, biologically relevant model that can be used to detect and quantify functional BoNT effects, thus accelerating BoNT research while decreasing animal use.

Background. The incidence of diabetes mellitus (both pregestational and gestational) is increasing worldwide, and hyperglycemia during pregnancy is associated with adverse pregnancy outcomes. Evidence on the safety and efficacy of metformin during pregnancy has accumulated resulting in an increase in its prescription in many reports. Aims. We aimed to determine the prevalence of antidiabetic drug use (insulins and blood glucose-lowering drugs) before and during pregnancy in Switzerland and the changes therein during pregnancy and over time. Methods. We conducted a descriptive study using Swiss health insurance claims (2012-2019). We established the MAMA cohort by identifying deliveries and estimating the last menstrual period. We identified claims for any antidiabetic medication (ADM), insulins, blood glucose-lowering drugs, and individual substances within each class. We defined three groups of pattern use based on timing of dispensation: (1) dispensation of at least one ADM in the prepregnancy period and in or after trimester 2 (T2) (pregestational diabetes); (2) dispensation for the first time in or after T2 (GDM); and (3) dispensation in the prepregnancy period and no dispensation in or after T2 (discontinuers). Within the pregestational diabetes group, we further defined continuers (dispensation for the same group of ADM) and switchers (different ADM group dispensed in the prepregnancy period and in or after T2). Results. MAMA included 104,098 deliveries with a mean maternal age at delivery of 31.7. Antidiabetic dispensations among pregnancies with pregestational and gestational diabetes increased over time. Insulin was the most dispensed medication for both diseases. Between 2017 and 2019, less than 10% of pregnancies treated for pregestational diabetes continued metformin rather than switching to insulin. Metformin was offered to less than 2% of pregnancies to treat gestational diabetes (2017-2019). Conclusion. Despite its position in the guidelines and the attractive alternative that metformin represents to patients who may encounter barriers with insulin therapy, there was reluctance to prescribe it.