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.     

The E6 oncoproteins from human betapapillomaviruses differentially activate telomerase through an E6AP-dependent mechanism and prolong the lifespan of primary keratinocytes

Human papillomaviruses (HPVs) belonging to the Betapapillomavirus genus have recently been implicated in squamous cell carcinomas of the skin, though the mechanisms by which they initiate carcinogenesis are unclear. We show that human foreskin keratinocytes (HFKs) expressing several betapapillomavirus E6 (beta-E6) proteins display life span extension, but not to the extent seen in HFKs expressing HPV type 16 E6 (16E6). Additionally, we demonstrate that beta-E6 proteins can differentially activate telomerase. HFKs expressing 38E6 exhibit significant telomerase activity but to a lesser degree than that observed with 16E6; however, other beta-E6 proteins, including 5E6, 8E6, 20E6, and 22E6, exhibit low or background levels of telomerase activity. Utilizing glutathione S-transferase pull-down and coimmunoprecipitation experiments, the beta-E6 proteins were shown to interact with the cellular proteins E6-associated protein (E6AP) and NFX1-91, two proteins known to be important for telomerase activation by 16E6. Interestingly, the relative strength of the interaction between E6 and E6AP or NFX1-91 was proportionate to the activation of telomerase by each beta-E6 protein. To address the requirement for E6AP in telomerase activation by beta-E6 proteins, we utilized a shRNA to knock down endogenous levels of E6AP. Lysates with decreased levels of E6AP showed a reduced ability to activate telomerase, suggesting that E6AP is a necessary component. These data suggest that complex formation between E6, E6AP, and NFX1-91 is a critical step in mediating telomerase activation, which may be one contributing factor to cellular life span extension during human betapapillomavirus infection.     

Whole-genome sequence analysis reveals differences in population management and selection of European low-input pig breeds

Background

A major concern in conservation genetics is to maintain the genetic diversity of populations. Genetic variation in livestock species is threatened by the progressive marginalisation of local breeds in benefit of high-output pigs worldwide. We used high-density SNP and re-sequencing data to assess genetic diversity of local pig breeds from Europe. In addition, we re-sequenced pigs from commercial breeds to identify potential candidate mutations responsible for phenotypic divergence among these groups of breeds.

Results

Our results point out some local breeds with low genetic diversity, whose genome shows a high proportion of regions of homozygosis (>50%) and that harbour a large number of potentially damaging mutations. We also observed a high correlation between genetic diversity estimates using high-density SNP data and Next Generation Sequencing data (r = 0.96 at individual level). The study of non-synonymous SNPs that were fixed in commercial breeds and also in any local breed, but with different allele, revealed 99 non-synonymous SNPs affecting 65 genes. Candidate mutations that may underlie differences in the adaptation to the environment were exemplified by the genes AZGP1 and TAS2R40. We also observed that highly productive breeds may have lost advantageous genotypes within genes involve in immune response – e.g. IL12RB2 and STAB1–, probably as a result of strong artificial in the intensive production systems in pig.

Conclusions

The high correlation between genetic diversity computed with the 60K SNP and whole genome re-sequence data indicates that the Porcine 60K SNP Beadchip provides reliable estimates of genomic diversity in European pig populations despite the expected bias. Moreover, this analysis gave insights for strategies to the genetic characterization of local breeds. The comparison between re-sequenced local pigs and re-sequenced commercial pigs made it possible to report candidate mutations to be responsible for phenotypic divergence among those groups of breeds. This study highlights the importance of low input breeds as a valuable genetic reservoir for the pig production industry. However, the high levels of ROHs, inbreeding and potentially damaging mutations emphasize the importance of the genetic characterization of local breeds to preserve their genomic variability.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-601) contains supplementary material, which is available to authorized users.     

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Background

The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.

Results

Here we describe mitotic slippage in yeast bub2?? mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.

Conclusions

The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.     

The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

Background  

Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages.     

Early Indicators of Fatal Leptospirosis during the 2010 Epidemic in Puerto Rico

Background

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.

Methodology/Principal Findings

During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).

Conclusions/Significance

Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.     

The genus Teliocrinus (Crinoidea,Echinodermata): a key taxon among pentacrinid stalked crinoids

The main characters of the stalked crinoids of the family Pentacrinitidae attributed to the genus Teliocrinus are re‐evaluated from a quantitative study of phenotype variation, new observations on arm and stalk articulations, and observation of ontogenetic trends. All of the specimens collected in the northern Indian Ocean belong to the same species, i.e. Teliocrinus springeri (Clark, 1909). However, two phenotypes living at different depths remain valid as subspecies: Teliocrinus springeri springeri (Clark, 1909) and Teliocrinus springeri liliaceus (Clark, 1909). Teliocrinus shares several ontogenetic trends with Endoxocrinus, especially in nonfunctional brachial articulations and stalk symplexies. Its assignment to the Diplocrininae is confirmed. A discussion of its affinities with pentacrinid fossil genera in which the crown is well preserved suggests that Diplocrininae could have first appeared during the Lower Cretaceous. A shortening of brachitaxes and a paedomorphic trend of stalk symplexies are the main other evolutionary traits. Nonfunctional articulations are frequently found at the paedomorphic pole of the heterochronic gradient, without clear derived characters. Classification of pentacrinids mainly based on such symplesiomorphy or paedomorphic characters must be definitively abandoned. However, in post‐Palaeozoic stalked crinoids the scarcity of well‐preserved fossils, the high frequency of paedomorphy, and convergent adaptive characters makes phylogenetic reconstruction only based on morphological characters very difficult and speculative. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 22–39.     

Nitrogen and phosphorus budgets of the North Atlantic Ocean and its watershed

Anthropogenic food and energy production extensively mobilize reactive nitrogen (N) in the watershed of the North Atlantic Ocean (NAO). There is wide spread N distribution by both hydrologic and atmospheric processes within the watershed of the NAO, resulting in reactive N accumulation in terrestrial systems. Net denitrification in most estuaries and continental shelves exceeds the amount of N supplied to the shelves by rivers and requires a supply of nitrate from the open ocean. Thus riverine N is only transported to the open ocean in a few areas with the flow from a few major rivers (e.g., Amazon). Atmospheric N deposition to the open ocean has increased and may increase the productivity of the surface ocean. In addition, as a consequence of increased Fe deposition to the open ocean (due in part to anthropogenic processes), the rate of biological N-fixation may have increased resulting in N accumulation in the ocean. Phosphorus (P) is also mobilized by anthropogenic processes (primarily food production). Relative to N, more of the P is transported across the shelf to the open ocean from both estuaries and major rivers. There are several consequences of the increased availability of N and P that are unique to each element. However, the control on primary productivity in both coastal and open ocean ecosystems is dependent on a complex and poorly understood interaction between N and P mobilization and availability.     

Binding of NAD+ by cholera toxin.

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.