Interlaboratory Evaluation of in Vitro Cytotoxicity and Inflammatory Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells. Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β. Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity

Interlaboratory Evaluation of in Vitro Cytotoxicity and Inflammatory Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells. Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β. Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity

De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis

<p>Abstract</p> <p>Background</p> <p>Arthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod <it>Daphnia pulex </it>is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod <it>Parhyale hawaiensis</it>. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models.</p> <p>Results</p> <p>To generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of <it>P. hawaiensis</it>. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them <it>de novo </it>to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid <it>Acyrthosiphon pisum</it>. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against <b>nr </b>(E-value cutoff e-10). These included over 400 predicted transcripts with significant similarity to <it>D. pulex </it>sequences but not to sequences of any other animal. Annotation of several hundred genes revealed <it>P. hawaiensis </it>homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways.</p> <p>Conclusions</p> <p>The amphipod <it>P. hawaiensis </it>has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as <it>D. pulex </it>and <it>P. hawaiensis</it>, as well as the need for development of further substantial crustacean genomic resources.</p

Molecular approach of genetic affinities between wild and ornamental Platanus

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Genetic differentiation in the olive complex (Olea europaea) revealed by RAPDs and RFLPs in the rRNA genes

We assessed the genetic differentiation of the Mediterranean olive from its wild relatives found in different geographic areas (Mediterranean, Asia, Africa) using eighty RAPDs revealed with eight primers. Variance analysis (AMOVA) enabled us to estimate the overall genetic differentiation parameters between wild populations. Oleasters from the Near East and Turkey were discriminated from the other Mediterranean populations. #Olea laperrinei#, #0. maroccana# and #0. cerasiformis# were the taxa the most related to the Mediterranean olive. In contrast, #0. africana# was shown to be the most genetically distant taxa from the Mediterranean olive. However, we characterized hybrid trees between these two taxa. Significant trends between genetic and geographic distances were met within the subspecies #cuspidata# and within the Mediterranean olive. A genetic diversity gradient was observed in both subspecies #europaea# and #cuspidata#. These results are in agreement with a mechanism of differentiation by distance in the #0. europaea# complex, but another non-exclusive mechanism could also be gene flow between differentiated taxa. Furthermore, we characterised the discriminating power of each RAPD to recognise the different taxa using intraclass correlation coefficients. Lastly, IGS-RFLPs enabled us to assess rDNA polymorphisms on a sub-sample of individuals. On the basis of these data, a low interspecific differentiation was found. This suggests a recent genetic divergence between the different taxa of the #0. europaea# complex or the occurrence of gene flow during favourable periods or because human displacements. All the olive cultivars were genetically related to the oleaster populations supporting that Mediterranean is the olive domestication area. (Résumé d'auteur

Variation and phylogeny of the ribosomal DNA unit types and 5 s DNA in Petunia Jussieu. P.hybrida hort. rDNA units originated in several wild Petunia species

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Variation de l’ADN ribosomique et hérédité du polymorphisme dans 6 lignées de Petunia hybrida Hort

International audienceRibosomal DNA polymorphisms were studied in 6 lines of Petunia hybrida using EcoRI, BamHI, HindIII, KpnI, SacI or XhoI. Each line carries several unit types, and 13 types were found in lines, which was not expected. We characterized the unit types and we determined the number of loci. Two kinds of unit types carrying no or several HindIII sites were revealed. The longest EcoRI and BamHI fragments in St43 correspond to a 11.4 kb unit type. Moreover, a 2.6 kb EcoRI fragment cannot be mapped in the 11.4 kb unit. It was found to be equivalent to the 2.45 kb EcoRI fragment carrying the 25 S rRNA coding sequence. Consequently, it was mapped in another unit 11.7 kb long. In TIh1 the corresponding EcoRI and BamHI fragments enabled us to construct 8.8, 9.2 and 10.8 kb segments. These fragments are therefore considered to be part of the 11.4 kb unit length. Other lines display combinations of these length units. The inheritance of polymorphic fragments of lines (St43 and TIh1) for 50 individuals of the 2 possible backcrosses [(St43 x TIh1) x St43] and [(St43 x TIh1) x TIh1] indicated at least 2 loci. The presence in Sk176 of 6.2, 5.7 and 5.4 EcoRI fragments suggested 3 loci. The haploid plants obtained from the hybrid (St43 x TIh1) display 1 individual carrying the 3 unit types present in the hybrid which proves the presence of 3 rDNA loci in TIh1. Moreover, the segregation in the backcrosses corresponds to only 2 loci in St43. It carries a nulli-allele. Evidence for such hypotheses were obtained by in situ hybridization with a biotinylated probe. The TIh1 and TIh7 dihaploid lines display more unit types and, consequently, more polymorphisms than other lines.Dans 6 lignées de Petunia hybrida dont l’ADN a été hydrolysé par EcoRI, BamHI, HindIII, KpnI, SacI ou XhoI, l’ADN ribosomique est apparu très polymorphe. Chaque lignée porte plusieurs types d’unités ; ainsi 13 types différents sont révélés dans les lignées, ce qui est surprenant. Nous avons caractérisé les différents types et déterminé le nombre de loci. Deux types d’unités avec et sans sites HindIII sont révélés. Pour la lignée St43 les fragments EcoRI et BamHI permettent de construire une unité de 11,4 kb. En outre un fragment EcoRI de 2,6 kb ne peut être placé dans l’unité de 11,4 kb. II est en effet équivalent au fragment de 2,4 kb portant la séquence codante du gène 25 S. Il est donc placé dans une unité de 11,7 kb. Dans la lignée TIh1 les fragments correspondants ne permettent de construire que des unités de 8,8 kb, 9,2 kb, et 10,8 kb, donc considérées comme une partie d’unités de 11,4 kb. Les autres lignées montrent une combinaison des fragments précédents. L’hérédité du polymorphisme dans 50 descendants du couple de lignées (St43 et TIh1) et les 2 rétrocroisements possibles [(St43 x TIh1) x St43] et [(St43 x TIh1) x TIh1] indique au moins 2 loci. La présence dans Sk176 des fragments EcoRI 6,2, 5,7 et 5,4 suggère 3 loci. Parmi les plantes haploïdes obtenues de l’hybride F1 (St43 x TIh1), un descendant porte les 3 types d’unité présents dans l’hybride, ce qui ne peut s’expliquer que s’ils sont répartis sur 3 loci. De plus la ségrégation dans les rétrocroisements correspond à 2 loci pour St43, il porte donc un nuiliallèle. La confirmation des hypothèses est apportée par hybridation in situ avec une sonde ribosomique biotinylée. Les 2 lignées dihaploïdes TIh1 et TIh7 montrent le plus d’unités et par conséquent le polymorphisme le plus élevé