Neural crest related gene transcript regulation by valproic acid analogues in the cardiac embryonic stem cell test.

In vivo, neural crest (NC) cells contribute critically to heart formation. The embryonic stem cells in the cardiac Embryonic Stem cell Test (ESTc) differentiate into a heterogeneous cell population including non-cardiomyocyte cells. The use of molecular biomarkers from different mechanistic pathways can refine quantitative embryotoxicity assessment. Gene expression levels representing different signalling pathways that could relate to beating cardiomyocyte formation were analysed at different time-points. Immunocytochemistry showed NC cells were present in the ESTc and RT-qPCR showed upregulation of NC related gene expression levels in a time-dependent manner. NC related genes were sensitive to VPA and its analogues 2-ethylhexanoic acid (EHA) and 2-ethylhexanol (EHOL) and indicated VPA as the most potent one. STITCH ('search tool for interactions of chemicals') analysis showed relationships between the examined signalling pathways and suggested additional candidate marker genes. Biomarkers from dedicated mechanistic pathways, e.g. NC differentiation, provide promising tools for monitoring specific effects in ESTc.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

This corrects the article DOI: 10.1038/mi.2017.81.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

Mucosal immunity is often required for protection against respiratory pathogens but the underlying cellular and molecular mechanisms of induction remain poorly understood. Here, systems vaccinology was used to identify immune signatures after pulmonary or subcutaneous immunization of mice with pertussis outer membrane vesicles. Pulmonary immunization led to improved protection, exclusively induced mucosal immunoglobulin A (IgA) and T helper type 17 (Th17) responses, and in addition evoked elevated systemic immunoglobulin G (IgG) antibody levels, IgG-producing plasma cells, memory B cells, and Th17 cells. These adaptive responses were preceded by unique local expression of genes of the innate immune response related to Th17 (e.g., Rorc) and IgA responses (e.g., Pigr) in addition to local and systemic secretion of Th1/Th17-promoting cytokines. This comprehensive systems approach identifies the effect of the administration route on the development of mucosal immunity, its importance in protection against Bordetella pertussis, and reveals potential molecular correlates of vaccine immunity to this reemerging pathogen.

A signature of renal stress resistance induced by short-term dietary restriction, fasting, and protein restriction.

During kidney transplantation, ischemia-reperfusion injury (IRI) induces oxidative stress. Short-term preoperative 30% dietary restriction (DR) and 3-day fasting protect against renal IRI. We investigated the contribution of macronutrients to this protection on both phenotypical and transcriptional levels. Male C57BL/6 mice were fed control food ad libitum, underwent two weeks of 30%DR, 3-day fasting, or received a protein-, carbohydrate- or fat-free diet for various periods of time. After completion of each diet, renal gene expression was investigated using microarrays. After induction of renal IRI by clamping the renal pedicles, animals were monitored seven days postoperatively for signs of IRI. In addition to 3-day fasting and two weeks 30%DR, three days of a protein-free diet protected against renal IRI as well, whereas the other diets did not. Gene expression patterns significantly overlapped between all diets except the fat-free diet. Detailed meta-analysis showed involvement of nuclear receptor signaling via transcription factors, including FOXO3, HNF4A and HMGA1. In conclusion, three days of a protein-free diet is sufficient to induce protection against renal IRI similar to 3-day fasting and two weeks of 30%DR. The elucidated network of common protective pathways and transcription factors further improves our mechanistic insight into the increased stress resistance induced by short-term DR.

Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Dried blood spot measurement of pregnancy-associated plasma protein A (PAPP-A) and free ß-subunit of human chorionic gonadotropin (ß-hCG) from a low-resource setting.

OBJECTIVES: The objectives of the article is to compare pregnancy-associated plasma protein A (PAPP-A) and free ß-subunit of human chorionic gonadotropin (ß-hCG) concentrations in dried blood spots (DBSs) with serum of samples obtained from a public hospital in a low-resource setting and to evaluate their stability. METHODS: Serum and DBS samples were obtained by venipuncture and finger prick from 50 pregnant participants in a cohort study in a public hospital in Accra, Ghana. PAPP-A and ß-hCG concentrations from serum and DBS were measured with an AutoDELFIA® (PerkinElmer, PerkinElmer, Turku, Finland) automatic immunoassay. Correlation and Passing-Bablok regression analyses were performed to compare marker levels. RESULTS: High correlation (>0.9) was observed for PAPP-A and ß-hCG levels between various sampling techniques. The ß-hCG concentration was stable between DBS and serum, PAPP-A concentration consistently lower in DBS. CONCLUSION: Our findings suggest that ß-hCG can be reliably collected from DBS in low-resource tropical settings. The exact conditions of the clinical workflow necessary for reliable PAPP-A measurement in these settings need to be further developed in the future. These findings could have implications for prenatal screening programs feasibility in low-income and middle-income countries, as DBS provides an alternative minimally invasive sampling method, with advantages in sampling technique, stability, logistics, and potential application in low-resource settings.

Liver DNA methylation analysis in adult female C57BL/6JxFVB mice following perinatal exposure to bisphenol A.

Bisphenol A (BPA) is a compound released from plastics and other consumer products used in everyday life. BPA exposure early in fetal development is proposed to contribute to programming of chronic diseases like obesity and diabetes, by affecting DNA methylation levels. Previously, we showed that in utero and lactational exposure of C57BL/6JxFVB hybrid mice via maternal feed using a dose range of 0-3000µg/kg body weight/day resulted in a sex-dependent altered metabolic phenotype in offspring at 23 weeks of age. The most univocal effects were observed in females, with reduced body weights and related metabolic effects associated with perinatal BPA exposure. To identify whether the effects of BPA in females are associated with changes in DNA methylation, this was analyzed in liver, which is important in energy homeostasis. Measurement of global DNA methylation did not show any changes. Genome-wide DNA methylation analysis at specific CpG sites in control and 3000µg/kg body weight/day females with the digital restriction enzyme analysis of methylation (DREAM) assay revealed potential differences, that could, however, not be confirmed by bisulfite pyrosequencing. Overall, we demonstrated that the observed altered metabolic phenotype in female offspring after maternal exposure to BPA was not detectably associated with liver DNA methylation changes. Still, other tissues may be more informative.

Identification of biomarkers to detect residual pertussis toxin using microarray analysis of dendritic cells.

In this study we aimed to identify genes that are responsive to pertussis toxin (PTx) and might eventually be used as biological markers in a testing strategy to detect residual PTx in vaccines. By microarray analysis we screened six human cell types (bronchial epithelial cell line BEAS-2B, fetal lung fibroblast cell line MRC-5, primary cardiac microvascular endothelial cells, primary pulmonary artery smooth muscle cells, hybrid cell line EA.Hy926 of umbilical vein endothelial cells and epithelial cell line A549 and immature monocyte-derived dendritic cells) for differential gene expression induced by PTx. Immature monocyte-derived dendritic cells (iMoDCs) were the only cells in which PTx induced significant differential expression of genes. Results were confirmed using different donors and further extended by showing specificity for PTx in comparison to Escherichia coli lipopolysaccharide (LPS) and Bordetella pertussis lipo-oligosaccharide (LOS). Statistical analysis indicated 6 genes, namely IFNG, IL2, XCL1, CD69, CSF2 and CXCL10, as significantly upregulated by PTx which was also demonstrated at the protein level for genes encoding secreted proteins. IL-2 and IFN-γ gave the strongest response. The minimal PTx concentrations that induced production of IL-2 and IFN-γ in iMoDCs were 12.5 and 25IU/ml, respectively. High concentrations of LPS slightly induced IFN-γ but not IL-2, while LOS and detoxified pertussis toxin did not induce production of either cytokine. In conclusion, using microarray analysis we evaluated six human cell lines/types for their responsiveness to PTx and found 6 PTx-responsive genes in iMoDCs of which IL2 is the most promising candidate to be used as a biomarker for the detection of residual PTx.

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn).

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO-BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO-BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO-BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO-BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

Genotoxic exposure: novel cause of selection for a functional ΔN-p53 isoform.

The p53 gene is frequently mutated in cancers and it is vital for cell cycle control, homeostasis and carcinogenesis. We describe a novel p53 mutational spectrum, different to those generally observed in human and murine tumors. Our study shows a high prevalence of nonsense mutations in the p53 N terminus of 2-acetylaminofluorene (2-AAF)-induced urinary bladder tumors. These nonsense mutations forced downstream translation initiation at codon 41 of Trp53, resulting in the aberrant expression of the p53 isoform ΔN-p53 (or p44). We propose a novel mechanism for the origination and the selection for this isoform. We show that chemical exposure can act as a novel cause of selection for this truncated protein. In addition, our data suggest that the occurrence of ΔN-p53 accounts, at least in mice, for a cancer phenotype. We also show that gene expression profiles of embryonic stem (ES) cells carrying the ΔN-p53 isoform in a p53-null background are divergent from p53 knockout ES cells, and therefore postulate that ΔN-p53 itself has functional transcriptional properties.

Proteomics and Down syndrome screening: a validation study.

OBJECTIVE: In a previous discovery study, we identified seven potential screening markers for Down syndrome (DS). Here, we report on an extended study to validate the discriminative potential of these markers. METHODS: Concentrations of the seven analytes were measured using bead-based multiplexed immunoassays in maternal serum from 27 DS pregnancies and 27 matched controls. Control samples were matched to the cases by gestational age (exact day), maternal weight ( ± 5 kg), and maternal age ( ± 1 year) and by closest sample date. Prediction values were obtained for current screening markers [pregnancy-associated plasma protein A (PAPP-A), free beta human chorionic gonadotrophin (fß-hCG) and nuchal translucency (NT)] and seven markers identified before based on concentration fold ratios between DS and controls. Models were fitted based on data of the discovery study or this study and also tested on both datasets. RESULTS: A significantly higher fold ratio was only found for epidermal growth factor (EGF) (-1.96; p = 0.006). In the prediction model for the current dataset, EGF improved the detection rate (DR) of DS by 5.7% [at a fixed 5% false-positive rate (FPR)] when added to the currently used screening markers. CONCLUSIONS: Validation of previously identified biomarkers only confirmed EGF for further consideration as a DS screening marker. This underlines the importance of validating biomarkers; in this study, limiting the range of plausible biomarkers to only one suitable biomarker.

Bead-based multiplexed immunoassays to identify new biomarkers in maternal serum to improve first trimester Down syndrome screening.

OBJECTIVES: To identify new discriminative biomarkers for Down syndrome (DS) pregnancies using a bead-based multiplexed immunoassay, and to use the newly identified biomarkers to construct a prediction model for non-invasive DS screening. METHODS: Maternal serum samples of 14 DS pregnancies and 15 matched controls were analyzed with a bead-based multiplexed immunoassay containing immunoassays for 90 different analytes. Potential biomarkers were selected on the basis of concentration fold ratios between DS and control samples. For these markers and the current screening markers (pregnancy-associated plasma protein-A, PAPP-A; free beta subunit of human chorion gonadotrophin (fbeta-hCG) and nuchal translucency) prediction values were obtained and used to calculate detection rates (DR) at a 5% false positive rate. RESULTS: Seven potential biomarkers of which the fold ratio exceeded 1.3 or -1.3 were selected for further analysis. All 14 DS cases in this study were detected using the combination of all currently used and newly identified markers. The modelled DR for all markers extrapolated to the general pregnant population was 82.5%, compared to a modelled DR of 56.2% for the current screening markers. CONCLUSION: This study demonstrates the possibility of improving the performance of the current first-trimester DS screening by addition of new biomarkers, which were identified using bead-based multiplexed immunoassays.