Acetaminophen induces an antioxidative response in lettuce plants

Contaminants of environmental concern, like pharmaceuticals, are being detected in increasing amounts in soils and irrigation waters and can thus be taken up by plants. In this work, the uptake of acetaminophen (ACT) by lettuce plants was evaluated through a hydroponic experiment at different concentrations (0, 0.1, 1 and 5 mg L1 ACT). The pathways related to oxidative stress induced by ACT were studied in lettuce leaves and roots at 1, 8 and 15 days after exposure. Stress indicators such as hydrogen peroxide and malondialdehyde (MDA) contents were analyzed, revealing increases in plants contaminated with ACT in comparison to control, confirming the occurrence of oxidative stress, with the exception of MDA in leaves. The enzymatic activities of catalase, superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and glutathione peroxidase, directly involved in the antioxidative system, showed significant differences when compared to control plants, and, depending on the enzyme and the tissue, different trends were observed. Glutathione reductase revealed a decrease in contaminated leaves, which may imply a specific impact of ACT in the glutathione cycle. Significant increases were found in the anthocyanin content of leaves, both with exposure time and ACT concentration, indicating an antioxidative response induced by ACT contaminationinfo:eu-repo/semantics/publishedVersio

An integrated in vitro approach unveils the biocompetence and glutathiolomic profile of a human hepatocyte-like cell 3d model

Funding: This work was supported by FCT (Portugal) through the research grant PTDC/MED-TOX/29183/2017. Acknowledgments: The authors thank ECBio S.A. for providing the hnMSCs and F.A. Beland (NCTR, Jefferson, AR, USA) for the kind donation of nevirapine. FCT (UID/DTP/04138/2019, UID/QUI/00100/2019, RECI/QEQ-MED/0330/2012, SFRH/BD/144130/2019 to J.S.R., SFRH/BD/110945/2015 to P.F.P. and CEECIND/02001/2017 to A.M.M.A) are also acknowledged.The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7. Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.publishersversionpublishe

Activation pathway to amino acid adducts

Funding: This work was supported in part by Fundação para a Ciência e a Tecnologia (FCT), Portugal (PTDC/QUI-QUI/113910/2009, RECI/QEQ-MED/0330/2012, UID/QUI/00100/2013 and IF/ 01091/2013/CP1163/CT0001), and by Interagency Agreement Y1ES1027 between the National Center for Toxicological Research/Food and Drug Administration and the National Institute of Environmental Health Sciences/National Toxicology Program. The opinions expressed in this paper do not necessarily represent those of the U.S. Food and Drug Administration. RW, ALG, ILM and SGH thank FCT for postdoctoral and doctoral fellowships (SFRH/BPD/70953/2010, SFRH/BD/72301/2010, SFRH/BD/75426/2010 and SFRH/BD/ 80690/2011, respectively). AMM also acknowledges Programa Operacional Potencial Humano from FCT and the European Social Fund (IF/01091/2013), and the LRI Innovative Science Award. We thank the Portuguese NMR and MS networks (IST nodes) for providing access to the facilities.Nevirapine (NVP) is the non-nucleoside HIV-1 reverse transcriptase inhibitor most commonly used in developing countries, both as a component of combined antiretroviral therapy and to prevent mother-to-child transmission of the virus; however, severe hepatotoxicity and serious adverse cutaneous effects raise concerns about its safety. NVP metabolism yields several phenolic derivatives conceivably capable of undergoing further metabolic oxidation to electrophilic quinoid derivatives prone to react with bionucleophiles and initiate toxic responses. We investigated the ability of two phenolic NVP metabolites, 2-hydroxy-NVP and 3-hydroxy-NVP, to undergo oxidation and subsequent reaction with bionucleophiles. Both metabolites yielded the same ring-contraction product upon oxidation with Frémy's salt in aqueous medium. This is consistent with the formation of a 2,3-NVP-quinone intermediate, which upon stabilization by reduction was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Additionally, we established that the oxidative activation of 2-hydroxy-NVP involved the transient formation of both the quinone and a quinone-imine, whereas 3-hydroxy-NVP was selectively converted into 2,3-NVP-quinone. The oxidations of 2-hydroxy-NVP and 3-hydroxy-NVP in the presence of the model amino acids ethyl valinate (to mimic the highly reactive N-terminal valine of hemoglobin) and N-acetylcysteine were also investigated. Ethyl valinate reacted with both 2,3-NVP-quinone and NVP-quinone-imine, yielding covalent adducts. By contrast, neither 2,3-NVP-quinone nor NVP-derived quinone-imine reacted with N-acetylcysteine. The product profile observed upon Frémy's salt oxidation of 2-hydroxy-NVP in the presence of ethyl valinate was replicated with myeloperoxidase-mediated oxidation. Additionally, tyrosinase-mediated oxidations selectively yielded 2,3-NVP-quinone-derived products, while quinone-imine-derived products were obtained upon lactoperoxidase catalysis. These observations suggest that the metabolic conversion of phenolic NVP metabolites into quinoid electrophiles is biologically plausible. Moreover, the lack of reaction with sulfhydryl groups might hamper the in vivo detoxification of NVP-derived quinone and quinone-imine metabolites via glutathione conjugation. As a result, these metabolites could be available for reaction with nitrogen-based bionucleophiles (e.g., lysine residues of proteins) ultimately eliciting toxic events.publishersversionpublishe

Marine sponge and octocoral-associated bacteria show versatile secondary metabolite biosynthesis potential and antimicrobial activities against human pathogens

Marine microbiomes are prolific sources of bioactive natural products of potential pharmaceutical value. This study inspected two culture collections comprising 919 host-associated marine bacteria belonging to 55 genera and several thus-far unclassified lineages to identify isolates with potentially rich secondary metabolism and antimicrobial activities. Seventy representative isolates had their genomes mined for secondary metabolite biosynthetic gene clusters (SM-BGCs) and were screened for antimicrobial activities against four pathogenic bacteria and five pathogenic Candida strains. In total, 466 SM-BGCs were identified, with antimicrobial peptide- and polyketide synthase-related SM-BGCs being frequently detected. Only 38 SM-BGCs had similarities greater than 70% to SM-BGCs encoding known compounds, highlighting the potential biosynthetic novelty encoded by these genomes. Cross-streak assays showed that 33 of the 70 genome-sequenced isolates were active against at least one Candida species, while 44 isolates showed activity against at least one bacterial pathogen. Taxon-specific differences in antimicrobial activity among isolates suggested distinct molecules involved in antagonism against bacterial versus Candida pathogens. The here reported culture collections and genome-sequenced isolates constitute a valuable resource of understudied marine bacteria displaying antimicrobial activities and potential for the biosynthesis of novel secondary metabolites, holding promise for a future sustainable production of marine drug leads.info:eu-repo/semantics/publishedVersio

Unrevealing the interactive effects of climate change and oil contamination on lab-simulated estuarine benthic communities

There is growing concern that modifications to the global environment such as ocean acidification and increased ultraviolet radiation may interact with anthropogenic pollutants to adversely affect the future marine environment. Despite this, little is known about the nature of the potential risks posed by such interactions. Here, we performed a multifactorial microcosm experiment to assess the impact of ocean acidification, ultraviolet radiation B (UV-B) and oil hydrocarbon contamination on sediment chemistry, the microbial community (composition and function) and biochemical marker response of selected indicator species. We found that increased ocean acidification and oil contamination in the absence of UV-B will significantly alter bacterial composition by, among other changes, greatly reducing the relative abundance of Desulfobacterales, known to be important oil hydrocarbon degraders. Along with changes in bacterial composition, we identified concomitant shifts in the composition of aromatic hydrocarbons in the sediment and an increase in oxidative stress effects on our indicator species. Interestingly, our study identifies UV-B as a critical component in the interaction between these factors, since its presence alleviates harmful effects caused by the combination of reduced pH and oil pollution. The model system used here shows that the interactive effect of reduced pH and oil contamination can adversely affect the structure and functioning of sediment benthic communities, with the potential to exacerbate the toxicity of oil hydrocarbons in marine ecosystems

Functional dissection of the chickpea (Cicer arietinum l.) stay-green phenotype associated with molecular variation at an ortholog of mendel’s i gene for cotyledon color: Implications for crop production and carotenoid biofortification

“Stay-green” crop phenotypes have been shown to impact drought tolerance and nutritional content of several crops. We aimed to genetically describe and functionally dissect the particular stay-green phenomenon found in chickpeas with a green cotyledon color of mature dry seed and investigate its potential use for improvement of chickpea environmental adaptations and nutritional value. We examined 40 stay-green accessions and a set of 29 BC2F4-5 stay-green introgression lines using a stay-green donor parent ICC 16340 and two Indian elite cultivars (KAK2, JGK1) as recurrent parents. Genetic studies of segregating populations indicated that the green cotyledon trait is controlled by a single recessive gene that is invariantly associated with the delayed degreening (extended chlorophyll retention). We found that the chickpea ortholog of Mendel’s I locus of garden pea, encoding a SGR protein as very likely to underlie the persistently green cotyledon color phenotype of chickpea. Further sequence characterization of this chickpea ortholog CaStGR1 (CaStGR1, for carietinum stay-green gene 1) revealed the presence of five different molecular variants (alleles), each of which is likely a loss-of-function of the chickpea protein (CaStGR1) involved in chlorophyll catabolism. We tested the wild type and green cotyledon lines for components of adaptations to dry environments and traits linked to agronomic performance in different experimental systems and different levels of water availability. We found that the plant processes linked to disrupted CaStGR1 gene did not functionality affect transpiration efficiency or water usage. Photosynthetic pigments in grains, including provitaminogenic carotenoids important for human nutrition, were 2–3-fold higher in the stay-green type. Agronomic performance did not appear to be correlated with the presence/absence of the stay-green allele. We conclude that allelic variation in chickpea CaStGR1 does not compromise traits linked to environmental adaptation and agronomic performance, and is a promising genetic technology for biofortification of provitaminogenic carotenoids in chickpea

Adenosine A2A receptors contribute to the radial migration of cortical projection neurons through the regulation of neuronal polarization and axon formation

Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. Here we found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naïve mouse embryos exposed to an A2AR antagonist, we observed an accumulation of early-born migrating neurons in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar–bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5′-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks.European Regional Development Fund, through the Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000008; BrainHealth2020); COMPETE 2020-Operational Programme for Competitiveness and Internationalization and Portuguese national funds via FCT—Fundação para a Ciência e Tecnologia (PTDC/NEU-NMC/3567/2014 to R.J.R.; POCI-01-0145-FEDER-028160 to J.M.M.); Center for Neuroscience and Cell Biology (POCI-01-0145-FEDER-007440; UIDB/04539/2020); Spanish Ministry of Science, Innovation and Universities (PGC2018/096631-B-100 to G.L-B.).Peer reviewe

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome due to RFC1 repeat expansion.

Ataxia, causing imbalance, dizziness and falls, is a leading cause of neurological disability. We have recently identified a biallelic intronic AAGGG repeat expansion in replication factor complex subunit 1 (RFC1) as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and a major cause of late onset ataxia. Here we describe the full spectrum of the disease phenotype in our first 100 genetically confirmed carriers of biallelic repeat expansions in RFC1 and identify the sensory neuropathy as a common feature in all cases to date. All patients were Caucasian and half were sporadic. Patients typically reported progressive unsteadiness starting in the sixth decade. A dry spasmodic cough was also frequently associated and often preceded by decades the onset of walking difficulty. Sensory symptoms, oscillopsia, dysautonomia and dysarthria were also variably associated. The disease seems to follow a pattern of spatial progression from the early involvement of sensory neurons, to the later appearance of vestibular and cerebellar dysfunction. Half of the patients needed walking aids after 10 years of disease duration and a quarter were wheelchair dependent after 15 years. Overall, two-thirds of cases had full CANVAS. Sensory neuropathy was the only manifestation in 15 patients. Sixteen patients additionally showed cerebellar involvement, and six showed vestibular involvement. The disease is very likely to be underdiagnosed. Repeat expansion in RFC1 should be considered in all cases of sensory ataxic neuropathy, particularly, but not only, if cerebellar dysfunction, vestibular involvement and cough coexist

Ecology and genomics of an important crop wild relative as a prelude to agricultural innovation

Domesticated species are impacted in unintended ways during domestication and breeding. Changes in the nature and intensity of selection impart genetic drift, reduce diversity, and increase the frequency of deleterious alleles. Such outcomes constrain our ability to expand the cultivation of crops into environments that differ from those under which domestication occurred. We address this need in chickpea, an important pulse legume, by harnessing the diversity of wild crop relatives. We document an extreme domestication-related genetic bottleneck and decipher the genetic history of wild populations. We provide evidence of ancestral adaptations for seed coat color crypsis, estimate the impact of environment on genetic structure and trait values, and demonstrate variation between wild and cultivated accessions for agronomic properties. A resource of genotyped, association mapping progeny functionally links the wild and cultivated gene pools and is an essential resource chickpea for improvement, while our methods inform collection of other wild crop progenitor species