Targeted genome-wide SNP genotyping in feral horses using non-invasive fecal swabs.

The development of high-throughput sequencing has prompted a transition in wildlife genetics from using microsatellites toward sets of single nucleotide polymorphisms (SNPs). However, genotyping large numbers of targeted SNPs using non-invasive samples remains challenging due to relatively large DNA input requirements. Recently, target enrichment has emerged as a promising approach requiring little template DNA. We assessed the efficacy of Tecan Genomics' Allegro Targeted Genotyping (ATG) for generating genome-wide SNP data in feral horses using DNA isolated from fecal swabs. Total and host-specific DNA were quantified for 989 samples collected as part of a long-term individual-based study of feral horses on Sable Island, Nova Scotia, Canada, using dsDNA fluorescence and a host-specific qPCR assay, respectively. Forty-eight samples representing 44 individuals containing at least 10 ng of host DNA (ATG's recommended minimum input) were genotyped using a custom multiplex panel targeting 279 SNPs. Genotyping accuracy and consistency were assessed by contrasting ATG genotypes with those obtained from the same individuals with SNP microarrays, and from multiple samples from the same horse, respectively. 62% of swabs yielded the minimum recommended amount of host DNA for ATG. Ignoring samples that failed to amplify, ATG recovered an average of 88.8% targeted sites per sample, while genotype concordance between ATG and SNP microarrays was 98.5%. The repeatability of genotypes from the same individual approached unity with an average of 99.9%. This study demonstrates the suitability of ATG for genome-wide, non-invasive targeted SNP genotyping, and will facilitate further ecological and conservation genetics research in equids and related species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12686-022-01259-2.

A practical toxicity bioassay for vicine and convicine levels in faba bean (Vicia faba).

BACKGROUND: Faba bean (Vicia faba) vicine and convicine (V-C) aglycones (divicine and isouramil respectively) provoke an acute hemolytic anemia called favism in individuals with a glucose-6-phosphate dehydrogenase (G6PD) enzyme defect in their red blood cells. Geneticists/plant breeders are working with faba bean to decrease V-C levels to improve public acceptance of this high-protein pulse crop. Here, we present a fast and simple ex vivo in vitro bioassay for V-C toxicity testing of faba bean or faba bean food products. RESULTS: We have shown that 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU)-treated (i.e., sensitized) normal red blood cells, like G6PD-defective blood, displayed (i) continuous glutathione (GSH) depletion with no regeneration as incubation time and the dose of aglycones increased, (ii) progressive accumulation of denatured hemoglobin products into high molecular weight (HMW) proteins with increased aglycone dose, (iii) both band 3 membrane proteins and hemichromes, in HMW protein aggregates. We have also demonstrated that sensitized red blood cells can effectively differentiate various levels of toxicity among faba bean varieties through the two hemolysis biomarkers: GSH depletion and HMW clumping. CONCLUSION: BCNU-sensitized red blood cells provide an ideal model for favism blood, to assess and compare the toxicity of faba bean varieties and their food products. © 2018 Society of Chemical Industry.

Detoxification, endocrine, and immune responses of tree swallow nestlings naturally exposed to air contaminants from the Alberta oil sands.

Changes in environmental and wildlife health from contaminants in tailings water on the Canadian oil sands have been well-studied; however, effects of air contaminants on wildlife health have not. A field study was conducted to assess biological costs of natural exposure to oil sands-related air emissions on birds. Nest boxes for tree swallows (Tachycineta bicolor) were erected at two sites; within 5 km of active oil sands mining and extraction, and ≥ 60 km south, at one reference site. Passive air monitors were deployed at the nest boxes to measure nitrogen dioxide, sulfur dioxide, ozone, volatile organic compounds, and polycyclic aromatic hydrocarbons (PAHs). Nestlings were examined at day 9 post hatching to assess T cell function and morphometry. At day 14 post hatching, a subset of nestlings was euthanized to measure detoxification enzymes, endocrine changes, and histological alterations of immune organs. Except for ozone, all air contaminants were higher at the two oil sands sites than the reference site (up to 5-fold). Adult birds had similar reproductive performance among sites (p>0.05). Nestlings from industrial sites showed higher hepatic ethoxyresorufin O-dealkylase (EROD) induction (p<0.0001) with lower relative hepatic mass (p=0.0001), a smaller T cell response to the phytohemagglutinin skin test (p=0.007), and smaller bursae of Fabricius (p<0.02); a low sample size for one site indicating lower body condition scores (p=0.01) at day 14 warrants cautious interpretation. There were no differences among nestlings for feather corticosterone (p>0.6), and no histological alterations in the spleen or bursa of Fabricius (p>0.05). This is the first report examining toxicological responses in wild birds exposed to air contaminants from industrial activity in the oil sands. It is also the first time that small, individual air contaminant monitors have been used to determine local contaminant levels in ambient air around nest boxes of wild birds.

Leishmania donovani mitochondrial iron superoxide dismutase A is released into the cytosol during miltefosine induced programmed cell death.

The oxidative phosphorylation process is the main source of endogenous reactive oxygen species (ROS) such as superoxide in mitochondria. In mammals, manganese superoxide dismutase plays an important role in detoxification of superoxide before it interferes with mitochondrial function and causes programmed cell death. Here, we investigated the role of Leishmania donovani mitochondrial iron superoxide dismutase-A (LdFeSODA) in protecting the parasite from oxidative stress and in the control of programmed cell death events. We have shown that overexpression of LdFeSODA protects Leishmania donovani from miltefosine induced cytotoxicity and reduced mitochondrial-derived superoxide generation. Furthermore, parasites overexpressing LdFeSODA showed (i) lower level of phosphatidylserine exposure as measured by flow cytometry and fluorescent microscopy; and (ii) reduced level of TUNEL staining of parasites compared to the control parasites. Finally, prolonged incubation of the parasites with miltefosine induced the release of both cytochrome C and LdFeSODA into the cytosol as demonstrated by Western blotting and fluorescence microscopy indicating programmed cell death. The results indicate that LdFeSODA protects the mitochondria of Leishmania from oxidative stress thereby inhibiting programmed cell death.

Leishmania donovani iron superoxide dismutase A is targeted to the mitochondria by its N-terminal positively charged amino acids.

Many reports have shown that Leishmania species are susceptible to reactive oxygen species (ROS) and reactive nitrogen species (RNS)-mediated killing. The superoxide dismutase (SOD) is one of the antioxidant defense enzymes important for parasite survival through its detoxification of superoxide into hydrogen peroxide and oxygen. The mitochondria produce numerous superoxide radicals as a by-product of cellular respiration and hence targeting of SODs to the mitochondria is critical in maintaining healthy mitochondria. This study examines the characteristic determinants for mitochondrial localization of Leishmania donovani FeSODA. We show that FeSODA is localized to the mitochondria and that the N-terminal 31 amino acid extension is important for its localization. Interestingly, further dissection of the 31 amino acid extension revealed that the first 8 amino acids of the FeSODA protein are sufficient for targeting to the mitochondria. In addition, we found that the four basic amino acid residues contained within the N-terminal extension are also important for targeting. These studies highlight important features of mitochondrial targeting sequences in kinetoplastids.