Workshop summary: Top concentration for in vitro mammalian cell genotoxicity assays; and report from working group on toxicity measures and top concentration for in vitro cytogenetics assays (chromosome aberrations and micronucleus)

The selection of maximum concentrations for in vitro mammalian cell genotoxicity assays was reviewed at the 5th International Workshop on Genotoxicity Testing (IWGT), 2009. Currently, the top concentration recommended when toxicity is not limiting is 10mM or 5mg/ml, whichever is lower. The discussion was whether to reduce the limit, and if so whether the 1mM limit proposed for human pharmaceuticals was appropriate for testing other chemicals. The consensus was that there was reason to consider reducing the 10mM limit, and many, but not all, attendees favored a reduction to 1mM. Several proposals are described here for the concentration limit. The in vitro cytogenetics expert working group also discussed appropriate measures and level of cytotoxicity. Data were reviewed from a multi-laboratory trial of the in vitro micronucleus (MN) assay with multiple cell types and several types of toxicity measurements. The group agreed on a preference for toxicity measures that take cell proliferation after the beginning of treatment into account (relative increase in cell counts, relative population doubling, cytokinesis block proliferation index or replicative index), and that this applies both to in vitro MN assays and to in vitro chromosome aberration assays. Since relative cell counts (RCC) underestimate toxicity, many group members favored making a recommendation against the use of RCC as a toxicity measure for concentration selection. All 14 chemicals assayed for MN induction in the multi-laboratory trial were detected without exceeding 50% toxicity by any measure, but some were positive only at concentrations with toxicity quite close to 50%. The expert working group agreed to accept the cytotoxicity range recommended by OECD guideline 487 (55±5% toxicity at the top concentration scored). This also reinforces the original intent of the guidance for the in vitro chromosome aberration assay, where ">50%" was intended to target the range close to 50% toxicity.     

Waddlia chondrophila Infects and Multiplies in Ovine Trophoblast Cells Stimulating an Inflammatory Immune Response

Background

Waddlia chondrophila (W. chondrophila) is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus). This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity.

Methodology/Principal Findings

Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i.) and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1β were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1β could be observed following active infection with W. chondrophila. However a significant increase in IL-1β expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways.

Conclusions/Significance

W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms.     

Multisite phosphorylation of the NDC80 complex gradually tunes its microtubule-binding affinity

Microtubule (MT) attachment to kinetochores is vitally important for cell division, but how these interactions are controlled by phosphorylation is not well known. We used quantitative approaches in vitro combined with molecular dynamics simulations to examine phosphoregulation of the NDC80 complex, a core kinetochore component. We show that the outputs from multiple phosphorylation events on the unstructured tail of its Hec1 subunit are additively integrated to elicit gradual tuning of NDC80-MT binding both in vitro and in silico. Conformational plasticity of the Hec1 tail enables it to serve as a phosphorylation-controlled rheostat, providing a new paradigm for regulating the affinity of MT binders. We also show that cooperativity of NDC80 interactions is weak and is unaffected by NDC80 phosphorylation. This in vitro finding strongly supports our model that independent molecular binding events to MTs by individual NDC80 complexes, rather than their structured oligomers, regulate the dynamics and stability of kinetochore-MT attachments in dividing cells.     

Inhibition of Nitric Oxide Synthase Blocks N-Methyl-D-Aspartate-, Quisqualate-, Kainate-, Harmaline-, and Pentylenetetrazole-Dependent Increases in Cerebellar Cyclic GMP In Vivo

The synthesis of nitric oxide by brain slices has been demonstrated in several laboratories. In addition, in vitro studies have demonstrated stimulation of nitric oxide synthesis by excitatory amino acid receptor agonists. These data have led to the hypothesis that this readily diffusible "intercellular messenger molecule" acts to generate a cascade effect by activating guanylate cyclase in several cell types and thereby augment levels of the second messenger cyclic GMP (cGMP). Therefore, we evaluated this hypothesis in vivo, by testing the actions of the nitric oxide synthase inhibitor N-mono-methyl-L-arginine (NMMA) on elevations in level of mouse cerebellar cGMP generated by excitatory amino acid receptor agonists. The stimulatory effects of D-serine, quisqualate, and kainate were all found to be antagonized by this enzyme inhibitor. In addition, NMMA antagonized the increases in cerebellar cGMP level elicited by harmaline and pentylenetetrazole, pharmacological agents that augment endogenous excitatory amino acid transmission. Our data are, therefore, the first in vivo demonstration that nitric oxide is an important "messenger molecule" in the cerebellum, mediating the actions of kainate, quisqualate, and N-methyl-D-aspartate receptor agonists on guanylate cyclase. These data are consistent with previous in vitro findings with kainate and N-methyl-D-aspartate.     

Detection of chromosome 17- and X-bearing human spermatozoa using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with DNA probes specific to chromosomes 17 and the X has been applied to human ejaculated sperm. After sperm nuclei were decondensed with EDTA and DTT, biotinylated alpha satellite DNA probes TR17 and TRX were separately used on preparations from thirteen healthy donors. After hybridization 96% of sperm were labelled with the TR17 probe and 48% of sperm were labelled with the TRX probe. Frequencies of 0.33% disomic 17 and 0.29% disomic X sperm were found. The frequencies of diploid sperm were assessed as 0.37% using the TR17 probe and 0.20% using the TRX probe which labelled only one half of the sperm; after correcting the result from the X-probe to 0.40% the two frequencies are very similar.     

GO‐FAANG meeting: a Gathering On Functional Annotation of Animal Genomes

The Functional Annotation of Animal Genomes (FAANG) Consortium recently held a Gathering On FAANG (GO‐FAANG) Workshop in Washington, DC on October 7–8, 2015. This consortium is a grass‐roots organization formed to advance the annotation of newly assembled genomes of domesticated and non‐model organisms ( www.faang.org ). The workshop gathered together from around the world a group of 100+ genome scientists, administrators, representatives of funding agencies and commodity groups to discuss the latest advancements of the consortium, new perspectives, next steps and implementation plans. The workshop was streamed live and recorded, and all talks, along with speaker slide presentations, are available at www.faang.org . In this report, we describe the major activities and outcomes of this meeting. We also provide updates on ongoing efforts to implement discussions and decisions taken at GO‐FAANG to guide future FAANG activities. In summary, reference datasets are being established under pilot projects; plans for tissue sets, morphological classification and methods of sample collection for different tissues were organized; and core assays and data and meta‐data analysis standards were established.