An integrated strategy involving high-throughput sequencing to characterize an unknown GM wheat event in Canada.

Glyphosate-resistant wheat plants were discovered in southern Alberta in 2017, representing an unauthorized GM release in Canada. The Canadian Food Inspection Agency undertook a series of experiments to characterize and identify this unknown GM wheat, as well as to develop and validate construct-specific and event-specific qPCR assays. Results of PCR-based assays and Sanger sequencing indicated the presence of CaMV 35S promoter (p35S), Rice Actin 1 intron (RactInt1), CP4-EPSPS gene and nopaline synthase terminator (tNOS) elements in the unknown GM wheat. Genome walking and bead capture strategies, combined with high-throughput sequencing, were used to identify the 5' and 3' wheat junctions and the subsequent mapping of the insert to chromosome 3B of the wheat genome. A probable transformation vector, pMON25497, was recognized, and further testing identified the unknown GM wheat as MON71200 event, one of two events obtained with the pMON25497 vector. The two construct-specific assays targeted the junctions of the RactInt1 and the CP4-EPSPS elements and the CP4-EPSPS and tNOS elements, while the event-specific assay was located at the 3' junction into the wheat genome. Both construct-specific and event-specific assays had limits of detection of 0.10% of MON71200 in a seed pool. As expected, the two construct-specific assays cross-reacted with other wheat and corn events containing the same elements in the same order. No cross-reactivity was observed for the event-specific assay. The integrated strategy employed in this study can serve as a model for other cases when facing similar challenges involving unknown GM events.

Challenges and Recommendations in Assessing Potential Endocrine-Disrupting Properties of Metals in Aquatic Organisms.

New tools and refined frameworks for identifying and regulating endocrine-disrupting chemicals (EDCs) are being developed as our scientific understanding of how they work advances. Although focus has largely been on organic chemicals, the potential for metals to act as EDCs in aquatic systems is receiving increasing attention. Metal interactions with the endocrine system are complicated because some metals are essential to physiological systems, including the endocrine system, and nonessential metals can have similar physiochemical attributes that allow substitution into or interference with these systems. Consequently, elevated metal exposure could potentially cause endocrine disruption (ED) but can also cause indirect effects on the endocrine system via multiple pathways or elicit physiologically appropriate compensatory endocrine-mediated responses (endocrine modulation). These latter two effects can be confused with, but are clearly not, ED. In the present study, we provide several case studies that exemplify the challenges encountered in evaluating the endocrine-disrupting (ED) potential of metals, followed by recommendations on how to meet them. Given that metals have multiple modes of action (MOAs), we recommend that assessments use metal-specific adverse outcome pathway networks to ensure that accurate causal links are made between MOAs and effects on the endocrine system. We recommend more focus on establishing molecular initiating events for chronic metal toxicity because these are poorly understood and would reduce uncertainty regarding the potential for metals to be EDCs. Finally, more generalized MOAs such as oxidative stress could be involved in metal interactions with the endocrine system, and we suggest it may be experimentally efficient to evaluate these MOAs when ED is inferred. These experiments, however, must provide explicit linkage to the ED endpoints of interest. Environ Toxicol Chem 2023;42:2564-2579. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Evaluation of the use of CRISPR loci for discrimination of Salmonella enterica subsp. enterica serovar Enteritidis strains recovered in Canada and comparison with other subtyping methods.

Salmonella enterica subsp. enterica serovar Enteritidis remains one of the most important foodborne pathogens worldwide. To minimise its public health impact when outbreaks of the disease occur, timely investigation to identify and recall the contaminated food source is necessary. Central to this approach is the need for rapid and accurate identification of the bacterial subtype epidemiologically linked to the outbreak. While traditional methods of S. Enteritidis subtyping, such as pulsed field gel electrophoresis (PFGE) and phage typing (PT), have played an important role, the clonal nature of this organism has spurred efforts to improve subtyping resolution and timeliness through molecular based approaches. This study uses a cohort of 92 samples, recovered from a variety of sources, to compare these two traditional methods for S. Enteritidis subtyping with recently developed molecular techniques. These latter methods include the characterisation of two clustered regularly interspaced short palindromic repeats (CRISPR) loci, either in isolation or together with sequence analysis of virulence genes such as fimH. For comparison, another molecular technique developed in this laboratory involved the scoring of 60 informative single nucleotide polymorphisms (SNPs) distributed throughout the genome. Based on both the number of subtypes identified and Simpson's index of diversity, the CRISPR method was the least discriminatory and not significantly improved with the inclusion of fimH gene sequencing. While PT analysis identified the most subtypes, the SNP-PCR process generated the greatest index of diversity value. Combining methods consistently improved the number of subtypes identified, with the SNP/CRISPR typing scheme generating a level of diversity comparable with that of PT/PFGE. While these molecular methods, when combined, may have significant utility in real-world situations, this study suggests that CRISPR analysis alone lacks the discriminatory capability required to support investigations of foodborne disease outbreaks.

An Unusual Salmonella Enteritidis Strain Carrying a Modified Virulence Plasmid Lacking the prot6e Gene Represents a Geographically Widely Distributed Lineage.

This study identifies a strain of Salmonella enterica subspecies enterica serovar Enteritidis that harbors a highly unusual virulence plasmid. During the characterisation of a group of S. Enteritidis isolates, 10 isolates recovered from Canadian duck production facilities, of which seven were phage type 9b and three were closely related atypical phage types, failed detection by a PCR targeting the prot6e gene, a marker located on the virulence plasmid often employed for identification of this serovar. Comparison to prot6e+ isolates by several standard genetic typing tools, further revealed their distinctive genomic makeup. Both short read and long read whole genome sequencing were completed on six of these isolates. In addition to loss of the prot6e gene, the virulence plasmid of each isolate was found to be exceptionally large (86.5 Kb) due to a 28 Kb insertion of S. Typhimurium plasmid sequence that encodes multiple genes of the incF operon. Interrogation of the chromosome sequence data of these isolates using a SNP-based typing tool and MLST both indicated their close genetic relatedness. One additional isolate carrying this plasmid was identified in an in-house collection of S. Enteritidis isolates. Finally, the identification of this unusual plasmid sequence in additional isolates submitted to public repositories of Salmonella sequence data was explored. All these analyses indicated that a very distinctive but rarely reported strain of S. Enteritidis was widely distributed across North America and the United Kingdom with one additional report involving a case from Brazil. With increased use of genetic methods for Salmonella identification, the loss of the prot6e sequence may confound correct identification of this serovar while also potentially altering the mode of transmission to humans given the gene's role in facilitating propagation of this bacterium in eggs. Accordingly, this strain may present certain challenges with respect to public health investigations. Our studies also suggest this strain is often associated with duck hosts thereby providing a possible mechanism by which this strain has spread over an extensive geographical area.

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples.

A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella -specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non- Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

Bisphenol A increases aP2 expression in 3T3L1 by enhancing the transcriptional activity of nuclear receptors at the promoter.

Environmental pollutants, such as bisphenol A (BPA), have the potential to affect the differentiation processes and the biology of the adipose tissue. The 3T3-L1 model is one of the murine cell models used extensively for the investigation of the molecular events that govern the differentiation of adipocytes from a committed preadipocyte to a mature, lipid laden adipocyte. Most of the studies investigating the effects of BPA on preadipocyte differentiation have investigated the effects of this chemical in the presence of an optimal differentiation cocktail containing high concentrations of the synthetic glucocorticoid dexamethasone, conditions that result in 90% to 100% of differentiated adipocytes. Our studies employed the 3T3-L1 cell model in the absence of exogenous glucocorticoids. We show that BPA is able to increase the differentiation of the 3T3-L1 cells under these conditions. Furthermore, the effect of BPA was observed in the absence of the synthetic glucocorticoid (dexamethasone), a hormone known to be required for the differentiation of the 3T3-L1 cells. In addition, BPA upregulated the mRNA expression and protein levels of the terminal marker of adipogenesis the fatty acid binding protein (aP2) in these cells. Interestingly, the known modulators of adipogenesis such as the peroxisome proliferator-activated receptor (PPAR) γ or CCAAT enhancer binding protein (C/EBP) α were not elevated at the mRNA or protein level in response to BPA. Furthermore, BPA upregulated the expression levels of the marker of adipogenesis aP2, through an effect on the transcriptional activity of C/EBPδ and the glucocorticoid receptor (GR) at its promoter.

Fate and effects of ivermectin on soil invertebrates in terrestrial model ecosystems.

The effect of ivermectin on soil organisms was assessed in Terrestrial Model Ecosystems (TMEs). Intact soil cores were extracted from a pasture in England and kept for up to 14 weeks in the laboratory. Ivermectin was applied to the soil surface via spiked cow dung slurry at seven concentration rates ranging from 0.25 to 180 mg/TME, referring to concentrations of 0.19-227 mg ivermectin/kg soil dry weight in the uppermost (0-1 cm) soil layer. After 7, 28 and 96 days following the application soil cores were destructively sampled to determine ivermectin residues in soil and to assess possible effects on microbial biomass, nematodes, enchytraeids, earthworms, micro-arthropods, and bait-lamina feeding activity. No significant effect of ivermectin was found for microbial respiration and numbers of nematodes and mites. Due to a lack of dose-response patterns no effect concentrations could be determined for the endpoints enchytraeid and collembolan numbers as well as total earthworm biomass. In contrast, EC50 values for the endpoint feeding rate could be calculated as 0.46, 4.31 and 15.1 mg ivermectin/kg soil dry weight in three soil layers (0-1, 0-5 and 0-8 cm, respectively). The multivariate Principal Response Curve (PRC) was used to calculate the NOEC(community), based on earthworm, enchytraeid and collembolan abundance data, as 0.33 and 0.78 mg ivermectin/kg soil dw for day 7 and day 96, respectively. The results shown here are in line with laboratory data, indicating in general low to moderate effects of ivermectin on soil organisms. As shown by the results of the bait-lamina tests, semi-field methods such as TMEs are useful extensions of the battery of potential test methods since complex and ecologically relevant endpoints can be included.

Environmental risk assessment of ivermectin: A case study.

The veterinary parasiticide ivermectin was selected as a case study compound within the project ERAPharm (Environmental Risk Assessment of Pharmaceuticals). Based on experimental data generated within ERAPharm and additional literature data, an environmental risk assessment (ERA) was performed mainly according to international and European guidelines. For the environmental compartments surface water, sediment, and dung, a risk was indicated at all levels of the tiered assessment approach. Only for soil was no risk indicated after the lower tier assessment. However, the use of effects data from additional 2-species and multispecies studies resulted in a risk indication for collembolans. Although previously performed ERAs for ivermectin revealed no concern for the aquatic compartment, and transient effects on dung-insect populations were not considered as relevant, the present ERA clearly demonstrates unacceptable risks for all investigated environmental compartments and hence suggests the necessity of reassessing ivermectin-containing products. Based on this case study, several gaps in the existing guidelines for ERA of pharmaceuticals were shown and improvements have been suggested. The action limit at the start of the ERA, for example, is not protective for substances such as ivermectin when used on intensively reared animals. Furthermore, initial predicted environmental concentrations (PECs) of ivermectin in soil were estimated to be lower than refined PECs, indicating that the currently used tiered approach for exposure assessment is not appropriate for substances with potential for accumulation in soil. In addition, guidance is lacking for the assessment of effects at higher tiers of the ERA, e.g., for field studies or a tiered effects assessment in the dung compartment.

Topoisomerase IIalpha-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.

Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25muM etoposide was highly toxic and initiated a double-stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA-PKcs to gammaH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and gammaH2AX response to correlate with the inability of the cell to correct topoIIalpha-initiated DNA damage. gammaH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre-treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIalpha-dependent DNA defects within topologically open chromatin domains.

Assessment of the environmental fate and effects of ivermectin in aquatic mesocosms.

Pharmaceuticals in the environment have been subject to increasing public concern and scientific investigation over the past years. More than 100 active pharmaceutical ingredients have been detected in surface waters worldwide at the ng to microg L(-1) range. At these low levels it is commonly assumed that only chronic and/or mixture toxic effects will be discernible in aquatic ecosystems and that there are orders of magnitude between exposure and effect concentrations. Assessment of potential ecosystem risk of pharmaceuticals are recommended but rarely performed in mesocosms, so for most risk assessments the final tier to reduce extrapolation uncertainty is missing. This paper describes the fate and effects of the anthelmintic drug ivermectin for a 265-day period following treatment (nominal concentration levels of 0, 30, 100, 1000 ng L(-1) (or parts per trillion (ppt)) in fifteen 12,000 L outdoor aquatic mesocosms. Although it is established that ivermectin is highly toxic towards invertebrates, it has been believed that ivermectin does not present notable risks to aquatic systems due to the rapid dissipation of the compound and binding to the sediment. Hence, fate and exchange of ivermectin between water and sediment were evaluated in this study. The ivermectin DT(50aqueous) in water was found to be 3-5 days, but concentrations increased and appeared to be stabile in the sediment at 20-30 ng kg(-1) with no assessable DT(50sed). Acute effects (first week) following ivermectin exposure were identified and cladocerans were particularly sensitive (nom. 100 ppt). Chronic responses (229 days) were identified for more sediment-active organisms (e.g. Chydoriae and Ephemeroptera) (nom. 1000 ppt). This is the first study to demonstrate the potential environmental risk of ivermectin at or below the predicted environmental concentration using a standardized test methodology (mesocosm) with minimal extrapolation uncertainty.

A screening level index for assessing the impacts of veterinary medicines on dung flies.

Veterinary parasiticides are administered to livestock to control a wide range of parasites. Following excretion, these substances may persist in the environment and impact nontarget organisms. This paper describes a simple screening-based index for predicting the effects of veterinary parasiticides on dung flies using data on parasiticide toxicity, animal husbandry, and parasiticide use. The utility of the index has been assessed, at the farm scale for a number of dipteran species, using data from a survey of farms in England and insect ecology and ecotoxicological data. The results indicate that a large proportion (35%) of parasiticide treatments in England will have no impact on dung fly populations. In terms of individual parasiticides, the macrocyclic lactone doramectin was predicted to have the highest impact on English dipteran populations with a maximum reduction in the population of horn flies on one farm of 28%. Ivermectin pour-on had the next highest impact (6.8%), followed by eprinomectin (6.4%), and ivermectin injection (4.1%). Due to a lack of data, it was not possible to assess the effects of the benzimidazole parasiticides (oxfendazole and fenbendazole), morantel and permethrin. The approach is simple, nondata-intensive and has the potential to be a valuable tool for use in environmental risk assessment or management of new and existing veterinary parasiticides.

Artemis phosphorylated by DNA-dependent protein kinase associates preferentially with discrete regions of chromatin.

Artemis is a nuclear phosphoprotein required for genomic integrity whose phosphorylation is increased subsequent to DNA damage. Artemis phosphorylation by the DNA-dependent protein kinase (DNA-PK) and the association of Artemis with DNA-PK catalytic subunit (DNA-PKcs) have been proposed to be crucial for the variable, diversity, joining (V(D)J) reaction, genomic stability and cell survival in response to double-stranded DNA breaks. The exact nature of the effectors of Artemis phosphorylation is presently being debated. Here, we have delimited the interface on Artemis required for its association with DNA-PKcs and present the characterization of six DNA-PK phosphorylation sites on Artemis whose phosphorylation shows dependence on its association with DNA-PKcs and is induced by double-stranded DNA damage. Surprisingly, DNA-PKcs Artemis association appeared to be dispensable in a V(D)J recombination assay with stably integrated DNA substrates. Phosphorylation at two of the sites on Artemis, S516 and S645, was verified in vivo using phosphospecific antibodies. Basal Artemis S516 and S645 phosphorylation in vivo showed a significant dependence on DNA-PKcs association. However, regardless of its association with DNA-PKcs, phosphorylation of Artemis at both S516 and S645 was stimulated in response to the double-stranded DNA-damaging agent bleomycin, albeit to a lesser extent. This suggests that additional factors contribute to promote DNA damage-induced Artemis phosphorylation. Intriguingly, pS516/pS645 Artemis was concentrated in chromatin-associated nuclear foci in naïve cells. These foci were maintained upon DNA damage but failed to overlap with the damage-induced gammaH2AX. These results provide the expectation of a specific role for DNA-PK-phosphorylated Artemis in both naïve and damaged cells.

Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex.

Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushing's syndrome and during prolonged steroid therapy. We show that glucocorticoids stimulate 3T3 L1 preadipocyte differentiation through a non-transcriptional mechanism mediated through the ligand-binding domain of the glucocorticoid receptor. This enhanced the onset of CCAAT/enhancer binding protein (C/EBPalpha) expression by potentiating its initial transcriptional activation by C/EBPbeta. In the absence of steroid, C/EBPbeta associated with a transcriptional corepressor complex containing mSin3A and histone deacetylase 1 (HDAC1), but lacking HDAC2 and RbAp46/48. HDAC1/mSin3A were recruited to the C/EBPalpha promoter with C/EBPbeta and promoted the deacetylation of histone H4. Steroid induced the specific depletion of this corepressor by targeting the HDAC1 within the complex for degradation through the 26S proteasome. Treatment with histone deacetylase inhibitors replaced the effects of steroid treatment on preadipocyte differentiation and C/EBPalpha expression, while overexpression of HDAC1 abrogated the stimulatory effects of steroid. Recapitulation of the glucocorticoid effect by progestin treatment in the presence of the progesterone receptor ligand-binding domain suggests a conserved mechanism relevant to many aspects of steroid-mediated differentiation.

Threonines 2638/2647 in DNA-PK are essential for cellular resistance to ionizing radiation.

DNA-dependent protein kinase (DNA-PK) is required for the repair of double-stranded DNA breaks through the nonhomologous DNA end joining pathway. DNA-PK activity is required for DNA repair, but kinase activity also appears to be attenuated through an autoregulatory feedback loop. We show that autophosphorylation of DNA-PK catalytic subunit occurs in trans at least three sites NH(2) terminal to the catalytic domain and that two sites, threonine 2638 and 2647, determine DNA-PK autophosphorylation in vitro. Thr2638/2647ala substitution in DNA-PK catalytic subunit compromised cellular resistance to ionizing radiation without affecting DNA end joining, suggesting a requirement for DNA-PK inactivation for cell survival at a step after the rejoining of double-stranded DNA breaks.