Strategic aims for improving the regulatory assessment of Developmental Neurotoxicity (DNT) using non-animal methods

Currently, the identification of chemicals that have the potential to induce developmental neurotoxicity (DNT) is based on animal testing, since there are no regulatory accepted alternative methods for this purpose. Since at the regulatory level, systematic testing of DNT is not a standard requirement within the EU legislation of chemical safety assessment, DNT testing is only performed in higher tiered tests triggered based on structure activity relationships or evidence of neurotoxicity in systemic adult studies. However, these triggers are rarely used and in addition do not always serve as reliable indicators of DNT as they are observed in an adult rodent animal. Consequently, to date only a limited amount of chemicals (Grandjean and Landrigan, 2006; Smirnova et al., 2014), mainly pesticides (Bjørling-Poulsen et al., 2008) have been tested under US EPA (OPPTS 870.630) or OECD DNT TG 426. Therefore, there is the pressing need for developing alternative methodologies that can more rapidly and cost-effectively screen large numbers of chemicals for their potential to cause DNT. In this report we propose that in vitro studies could contribute to the identification of potential triggers for DNT evaluation since existing cellular models permit the evaluation of a chemical impact on key neurodevelopmental processes, mimicking different windows of human brain development, especially if human models derived from induced pluripotent stem cells are applied. Furthermore, the battery of currently available DNT alternative test methods anchored to critical neurodevelopmental processes and key events identified in DNT Adverse Outcome Pathways (AOPs) could be applied to generate in vitro data useful for various regulatory purposes. Incorporation of in vitro mechanistic information would increase scientific confidence in decision making, by decreasing uncertainty and leading to refinement of chemical grouping according to biological activity. In this report development of IATA (Integrated Approaches to Testing and Assessment) based on key neurodevelopmental processes and AOP-informed is proposed as a tool for not only speeding up chemical screening, but also providing mechanistic data in support of hazard assessment and in the evaluation of chemical mixtures. Such mechanistically informed IATA for DNT evaluation could be developed integrating various sources of information (e.g., non-testing methods, in vitro approaches, as well as in vivo animal and human data), contributing to screening for prioritization, hazard identification and characterization, and possibly safety assessment of chemicals, speeding up the evaluation of thousands of compounds present in industrial, agricultural and consumer products that lack safety data on DNT potential. It is planned that the data and knowledge generated from such testing will be fed into the development of an OECD guidance document on alternative approaches to DNT testing.JRC.F.3-Chemicals Safety and Alternative Method

10 Years After

September 11, 2011, marks 10 years since the terrorist attacks that changed the nation and the world. In recognition of this noteworthy anniversary, five alumni offer reflections and reminiscences about the event and its impact on their lives

EURL ECVAM strategy to replace, reduce and refine the use of animals in the assessment of acute mammalian systemic toxicity

Information on acute systemic toxicity represents a standard requirement within several pieces of chemicals legislation in the EU. One of the main drivers of conducting the test is classification and labelling. Currently, only in vivo tests are accepted by regulatory bodies and most of the standard tests use lethality as endpoint. Based on an assessment of the regulatory needs and the scientific state-of-the art in the area, EURL ECVAM considers that efforts should be directed towards a) the reduction and replacement of animal tests for the identification and classification of acute systemic toxicity, and b) the refinement of in vivo studies. Consideration should be given to collecting, organising and applying mechanistic knowledge related to this endpoint, to provide a strong mechanistic basis for the design and validation of integrated prediction models. EURL ECVAM proposes to evaluate promising components of integrated approaches for testing and assessment (IATA), including the better use of existing alternative methods, such as mechanistically relevant in vitro assays. Information on repeated dose toxicity might also be useful in supporting classification and labelling for acute systemic toxicity. One clear target is minimising animal use for satisfying information requirements for acute systemic toxicity in relation to the 2018 REACH registration deadline. The aims and objectives underpinning the EURL ECVAM strategy can only be achieved through the coordinated and concerted efforts of all stakeholders.JRC.I.5-Systems Toxicolog

Hypoxia-induced transcriptional stress is mediated by ROS-induced R-loops

Hypoxia is a common feature of solid tumors and is associated with poor patient prognosis, therapy resistance and metastasis. Radiobiological hypoxia (<0.1% O2) is one of the few physiologically relevant stresses that activates both the replication stress/DNA damage response and the unfolded protein response. Recently, we found that hypoxia also leads to the robust accumulation of R-loops, which led us to question here both the mechanism and consequence of hypoxia-induced R-loops. Interestingly, we found that the mechanism of R-loop accumulation in hypoxia is dependent on non-DNA damaging levels of reactive oxygen species. We show that hypoxia-induced R-loops play a critical role in the transcriptional stress response, evidenced by the repression of ribosomal RNA synthesis and the translocation of nucleolin from the nucleolus into the nucleoplasm. Upon depletion of R-loops, we observed a rescue of both rRNA transcription and nucleolin translocation in hypoxia. Mechanistically, R-loops accumulate on the rDNA in hypoxia and promote the deposition of heterochromatic H3K9me2 which leads to the inhibition of Pol I-mediated transcription of rRNA. These data highlight a novel mechanistic insight into the hypoxia-induced transcriptional stress response through the ROS–R-loop–H3K9me2 axis. Overall, this study highlights the contribution of transcriptional stress to hypoxia-mediated tumorigenesis

Pleistocene divergence of two disjunct conifers in the eastern Australian temperate zone

The eastern Australian temperate biota harbours many plants with fragmented geographic ranges distributed over 1000s of kilometres, yet the spatial genetic structure of their populations remains largely unstudied. In this study, we investigated genetic variation of the nuclear internal transcribed spacer (ITS) and chloroplast DNA sequences to disentangle the phylogeography of two widely distributed but highly fragmented eastern Australian fire-sensitive temperate conifers: Callitris oblonga (12 populations and 121 individuals) and C. rhomboidea (22 populations and 263 individuals). The three highly disjunct populations of C. oblonga all had unique chloroplast and ITS haplotypes consistent with the classification of these three populations as distinct subspecies. Molecular dating indicates that divergences of these populations occurred pre- to mid- Pleistocene (2.66 to 1.08 mya). Callitris rhomboidea showed greater diversity of chloroplast haplotypes which was strongly phylogeographically structured (Gst = 0.972), with haplotypes unique to specific geographic regions. ITS haplotype diversity was far higher than in C. oblonga with 38 haplotypes displaying high geographic structuring (Gst = 0.387) with many population-specific haplotypes. A phylogeographic break was identified between populations north and south of eastern Victoria dated at 0.43–0.47 mya. In both species, the strong genetic structuring of both chloroplast and ITS haplotypes provides evidence that their widespread ranges have resulted from long term persistence in low fire frequency refugia combined with poor dispersal. Any loss of populations due to increasing fire frequency or habitat loss is likely to result in a reduction of genetic diversity

Use of serial qualitative interviews to understand patients’ evolving experiences and needs

Interviewing patients over the course of their illness can give a much better picture of their experience than single interviews, but the approach is rarely used. Scott Murray and colleagues explain how to get the most from it

Use of multiperspective qualitative interviews to understand patients’ and carers’ beliefs, experiences, and needs

A better understanding of the needs of patients and their carers can help improve services. Marilyn Kendall and colleagues describe how to conduct multiperspective studies

Observational study to characterise 24-hour COPD symptoms and their relationship with patient-reported outcomes : results from the ASSESS study

Acknowledgements The authors would like to thank the study investigators at each of the participating centres for their contribution to the study. The full list of study investigators is available in Additional file 1. We would also like to thank Deborah McGregor, PhD, of Complete Medical Communications, who provided medical writing support funded by Almirall, S.A. Barcelona, Spain. This study was funded by Almirall S.A., Barcelona, Spain. The study sponsor was involved in the design of the study, analysis of the data, review of the data, and review and approval of the manuscript. The sponsors placed no restrictions on statements made in the final version of the manuscript or on the decision to submit the manuscript for publication and all authors had full access to the data.Peer reviewedPublisher PD

Thymus transplantation for complete DiGeorge syndrome: European experience

Background: Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS). Methods: Twelve patients with cDGS were transplanted with allogeneic cultured thymus. Objective: To confirm and extend the results previously obtained in a single centre. Results: Two patients died of pre-existing viral infections without developing thymopoeisis and one late death occurred from autoimmune thrombocytopaenia. One infant suffered septic shock shortly after transplant resulting in graft loss and the need for a second transplant. Evidence of thymopoeisis developed from 5-6 months after transplantation in ten patients. The median (range) of circulating naïve CD4 counts (x10663 /L) were 44(11-440) and 200(5-310) at twelve and twenty-four months post-transplant and T-cell receptor excision circles were 2238 (320-8807) and 4184 (1582 -24596) per106 65 T-cells. Counts did not usually reach normal levels for age but patients were able to clear pre-existing and later acquired infections. At a median of 49 months (22-80), eight have ceased prophylactic antimicrobials and five immunoglobulin replacement. Histological confirmation of thymopoeisis was seen in seven of eleven patients undergoing biopsy of transplanted tissue including five showing full maturation through to the terminal stage of Hassall body formation. Autoimmune regulator (AIRE) expression was also demonstrated. Autoimmune complications were seen in 7/12 patients. In two, early transient autoimmune haemolysis settled after treatment and did not recur. The other five suffered ongoing autoimmune problems including: thyroiditis (3); haemolysis (1), thrombocytopaenia (4) and neutropenia (1). Conclusions: This study confirms the previous reports that thymus transplantation can reconstitute T cells in cDGS but with frequent autoimmune complications in survivors

Genome-wide association analysis reveals QTL and candidate mutations involved in white spotting in cattle

International audienceAbstractBackgroundWhite spotting of the coat is a characteristic trait of various domestic species including cattle and other mammals. It is a hallmark of Holstein–Friesian cattle, and several previous studies have detected genetic loci with major effects for white spotting in animals with Holstein–Friesian ancestry. Here, our aim was to better understand the underlying genetic and molecular mechanisms of white spotting, by conducting the largest mapping study for this trait in cattle, to date.ResultsUsing imputed whole-genome sequence data, we conducted a genome-wide association analysis in 2973 mixed-breed cows and bulls. Highly significant quantitative trait loci (QTL) were found on chromosomes 6 and 22, highlighting the well-established coat color genes KIT and MITF as likely responsible for these effects. These results are in broad agreement with previous studies, although we also report a third significant QTL on chromosome 2 that appears to be novel. This signal maps immediately adjacent to the PAX3 gene, which encodes a known transcription factor that controls MITF expression and is the causal locus for white spotting in horses. More detailed examination of these loci revealed a candidate causal mutation in PAX3 (p.Thr424Met), and another candidate mutation (rs209784468) within a conserved element in intron 2 of MITF transcripts expressed in the skin. These analyses also revealed a mechanistic ambiguity at the chromosome 6 locus, where highly dispersed association signals suggested multiple or multiallelic QTL involving KIT and/or other genes in this region.ConclusionsOur findings extend those of previous studies that reported KIT as a likely causal gene for white spotting, and report novel associations between candidate causal mutations in both the MITF and PAX3 genes. The sizes of the effects of these QTL are substantial, and could be used to select animals with darker, or conversely whiter, coats depending on the desired characteristics