RESUMO
Epigenetic mechanisms play diverse roles in the regulation of genome stability in eukaryotes. In Arabidopsis thaliana, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. The loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the transcriptional co-activator and conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires the GCN5-interacting protein ADA2b and the chromatin remodeler CHR6 to mediate the heterochromatic defects in atxr5 atxr6 mutants. Our results also indicate that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants, but H3.1K27 and H3.1K36 play essential functions in inducing genomic instability in the absence of H3.1K27me1. Finally, we show that H3.1K36 acetylation by GCN5 is negatively regulated by H3.1K27me1 in vitro. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining transcriptional silencing and genome stability in heterochromatin by restricting GCN5-mediated histone acetylation in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Instabilidade Genômica , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Acetilação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Inativação Gênica , Genoma de Planta , Heterocromatina/genética , Heterocromatina/metabolismo , Histona Acetiltransferases/genética , Histonas/genética , Lisina/genética , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
AIM: To investigate the opinion and personal experience of parents of children born with Hypoplastic Left Heart Syndrome and what advice they would give to other parents who have to decide between treatment options. METHODS: We conducted a qualitative, descriptive and retrospective study by means of a survey directed to parents of children born with Hypoplastic Left Heart Syndrome in a tertiary hospital in Buenos Aires (Argentina). Their answers and data regarding medical procedures were analysed. RESULTS: Parents of thirteen out of sixteen patients with Hypoplastic Left Heart Syndrome were surveyed. Norwood surgery had been performed in all the patients, many had received other procedures, and five had died. In relation to the decision-making process, sixty-one percent of parents would recommend other parents to remain at peace after having done everything possible and 54% would suggest to not feel guilt despite the final result. None of the parents would recommend rejecting surgical treatment and choosing comfort care. CONCLUSION: The majority of parents of children with Hypoplastic Left Heart Syndrome would recommend continuing with the therapeutic effort in order to feel at peace and reduce feelings of guilt.
Assuntos
Síndrome do Coração Esquerdo Hipoplásico , Procedimentos de Norwood , Criança , Humanos , Síndrome do Coração Esquerdo Hipoplásico/cirurgia , Estudos Retrospectivos , Pais , EmoçõesRESUMO
Direct-acting antiviral drugs (DAAs) have recently changed the paradigm of hepatitis C therapy, significantly improving treatment response rates, patient life expectancy and quality of life. In Portugal, sofosbuvir (SOF) and SOF/ledipasvir (SOF/LDV) were fully reimbursed by the National Health System since early 2015 and generalized use of interferon-free DAA based regimens became current practice. During 2016, the remaining DAAs were sequentially added and covered by the same health access policy. The Portuguese Study Group of Hepatitis and HIV Co-infection (GEPCOI) collected data from 15 clinical centres in Portugal, pertaining to the HCV treatment experience with DAA regimens. A cohort of 2133 patients was analysed, representing one of the largest DAA treated HCV/HIV co-infected individuals. The global sustained virologic response (SVR) achieved was 95% in this real-life cohort setting. Linear regression analysis showed significant differences in treatment response rates when using SOF plus ribavirin (RBV) combination in genotype 2 or 3 infected individuals (P < .002) and in those with liver cirrhosis (P < .002). These findings corroborate that early treatment is mandatory in HIV/HCV co-infected patients, as response rates may be negatively influenced by higher fibrosis stages and suboptimal DAA regimens. The current national Portuguese health policy should continue to promote wider treatment access and individualized therapy strategies, aiming at the elimination of HCV infection in this high-risk co-infected population.
Assuntos
Antivirais , Coinfecção , Infecções por HIV , Hepatite C Crônica , Adulto , Idoso , Antivirais/uso terapêutico , Coinfecção/tratamento farmacológico , Quimioterapia Combinada , Feminino , Genótipo , Infecções por HIV/tratamento farmacológico , Hepacivirus , Hepatite C Crônica/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Nigéria , Portugal , Qualidade de Vida , Sofosbuvir/uso terapêutico , Resultado do Tratamento , Adulto JovemRESUMO
The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.
Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Genoma de Planta/genética , Streptococcus pyogenes/enzimologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Temperatura Alta , Mutagênese Sítio-Dirigida , Mutação , Plantas Geneticamente Modificadas , Estresse FisiológicoRESUMO
BACKGROUND: The whole repeat unit of 5S rDNA and the internal transcribed spacer (ITS) of four European Donax species were analysed. After amplifying, cloning and sequencing several 5S and ITS units, their basic features and their variation were described. The phylogenetic usefulness of 5S and ITS sequences in the inference of evolutionary relationships among these wedge clams was also investigated. RESULTS: The length of the 5S repeat presented little variation among species, except D. trunculus that differed from the rest of the Donax species in 170-210 bp. The deduced coding region covered 120 bp, and showed recognizable internal control regions (ICRs) involved in the transcription. The length of non-transcribed spacer region (NTS) ranged from 157 bp to 165 bp in Donax trunculus and from 335 bp to 367 bp in the other three species. The conservation degree of transcriptional regulatory regions was analysed revealing a conserved TATA-like box in the upstream region. Regarding ITS sequences, the four Donax species showed slight size differences among clones due to the variation occurring in the ITS1 and ITS2, except Donax variegatus did not display size differences in the ITS2. The total length of the ITS sequence ranged between 814 and 1014 bp. Resulting phylogenetic trees display that the two ribosomal DNA regions provide well-resolved phylogenies where the four European Donax species form a single clade receiving high support in nodes. The topology obtained with 5S sequences was in agreement with Donax evolutionary relationships inferred from several sequences of different nature in previous studies. CONCLUSIONS: This is not only a basic research work, where new data and new knowledge is provided about Donax species, but also have allowed the authentication of these wedge clams and offers future applications to provide other genetic resources.
Assuntos
Bivalves/genética , RNA Ribossômico 5S/genética , Animais , Bivalves/classificação , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Europa (Continente) , Fases de Leitura Aberta/genética , Filogenia , RNA Ribossômico 5S/classificação , Análise de Sequência de DNARESUMO
Histones are the fundamental constituents of the eukaryotic chromatin, facilitating the physical organization of DNA in chromosomes and participating in the regulation of its metabolism. The H2A family displays the largest number of variants among core histones, including the renowned H2A.X, macroH2A, H2A.B (Bbd), and H2A.Z. This latter variant is especially interesting because of its regulatory role and its differentiation into 2 functionally divergent variants (H2A.Z.1 and H2A.Z.2), further specializing the structure and function of vertebrate chromatin. In the present work we describe, for the first time, the presence of a second H2A.Z variant (H2A.Z.2) in the genome of a non-vertebrate animal, the mussel Mytilus. The molecular and evolutionary characterization of mussel H2A.Z.1 and H2A.Z.2 histones is consistent with their functional specialization, supported on sequence divergence at promoter and coding regions as well as on varying gene expression patterns. More precisely, the expression of H2A.Z.2 transcripts in gonadal tissue and its potential upregulation in response to genotoxic stress might be mirroring the specialization of this variant in DNA repair. Overall, the findings presented in this work complement recent reports describing the widespread presence of other histone variants across eukaryotes, supporting an ancestral origin and conserved role for histone variants in chromatin.
Assuntos
Centro Germinativo/metabolismo , Mytilus/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Histonas/metabolismo , Mutação/genética , Mytilus/genética , Filogenia , Conformação Proteica , Proteínas/química , Homologia de Sequência do Ácido NucleicoRESUMO
Okadaic acid (OA) is the predominant biotoxin responsible for diarrhetic shellfish poisoning (DSP) syndrome in humans. While its harmful effects have been extensively studied in mammalian cell lines, the impact on marine organisms routinely exposed to OA is still not fully known. Few investigations available on bivalve molluscs suggest less genotoxic and cytotoxic effects of OA at high concentrations during long exposure times. In contrast, no apparent information is available on how sublethal concentrations of OA affect these organisms over short exposure times. In order to fill this gap, this study addressed for the first time in vitro analysis of early genotoxic and cytotoxic effects attributed to OA in two cell types of the mussel Mytilus galloprovincialis. Accordingly, hemocytes and gill cells were exposed to low OA concentrations (10, 50, 100, 200, or 500 nM) for short periods of time (1 or 2 h). The resulting DNA damage, as apoptosis and necrosis, was subsequently quantified using the comet assay and flow cytometry, respectively. Data demonstrated that (1) mussel hemocytes seem to display a resistance mechanism against early genotoxic and cytotoxic OA-induced effects, (2) mussel gill cells display higher sensitivity to early OA-mediated genotoxicity than hemocytes, and (3) mussel gill cells constitute more suitable systems to evaluate the genotoxic effect of low OA concentrations in short exposure studies. Taken together, this investigation provides evidence supporting the more reliable suitability of mussel gill cells compared to hemocytes to evaluate the genotoxic effect of low short-duration exposure to OA.
Assuntos
Citotoxinas/toxicidade , Dano ao DNA/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Mytilus/efeitos dos fármacos , Ácido Okadáico/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Monitoramento Ambiental , Citometria de Fluxo , Brânquias/efeitos dos fármacos , Brânquias/patologia , Hemócitos/citologia , Hemócitos/efeitos dos fármacos , Necrose/induzido quimicamente , Fatores de TempoRESUMO
The wedge clam Donax trunculus is an Atlantic-Mediterranean warm-temperate species found from Senegal to the northern coast of France, including the Mediterranean and Black Sea. It is commercially exploited in several European countries and constitutes an important fishing resource due to its high economical value. To contribute to its conservation and management, nineteen microsatellite markers were isolated from two enriched genomic libraries. These loci were characterized in 30 clams from a single population from northwest Spain. The number of alleles per locus ranged from 2 to 17 and observed and expected heterozygosity varied from zero to 0.714 and from 0.078 to 0.950, respectively. Linkage disequilibrium was not detected and nine loci were in agreement with Hardy-Weinberg equilibrium. Fifteen polymorphic markers were arranged into three multiplex PCR sets to reduce both time and cost of microsatellite genotyping. This is the first time that polymorphic microsatellite markers have been reported for D. trunculus. These new markers provide a valuable resource for future population genetics studies and management and culture of this species.
Assuntos
Bivalves/genética , Repetições de Microssatélites , Reação em Cadeia da Polimerase Multiplex/métodos , Alelos , Animais , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Biblioteca Gênica , Loci Gênicos , Técnicas de Genotipagem , Heterozigoto , Desequilíbrio de Ligação , Filogeografia , Polimorfismo Genético , Senegal , Análise de Sequência de DNA , EspanhaRESUMO
Cow's milk allergy (CMA) is an immune-based disease that has become an increasing problem. The diagnosis and management of CMA varies from one clinical setting to another and represents a challenge in pediatric practice. In addition, because nonallergic food reactions can be confused with CMA symptoms, there is an overdiagnosis of the disease. In response to these situations, pediatric specialties from recognized institutions throughout Latin America decided to develop a clinical guideline for diagnosis and management of cow's milk allergy. These guidelines include definitions, epidemiology, pathophysiology overview, clinical and evidencebased recommendations for the diagnosis and treatment of CMA. They also include prevention and prognosis sections and identify gaps in the current knowledge to be addressed through future research.
Assuntos
Hipersensibilidade a Leite/diagnóstico , Proteínas do Leite/efeitos adversos , Guias de Prática Clínica como Assunto , Medicina Baseada em Evidências , Humanos , América Latina , Hipersensibilidade a Leite/epidemiologia , Hipersensibilidade a Leite/terapia , Proteínas do Leite/imunologia , PrognósticoRESUMO
Cellular signal transduction comprises a complex series of biochemical reactions by which extracellular signals such as growth factors, hormones, cytokines, and neurotransmitters are translated into specific intracellular responses. Signal transduction is mediated by protein kinase phosphorylation cascades that culminate in the regulation of numerous cellular responses, including division, differentiation, migration, and survival. Importantly, signal relay pathways are dysregulated in human diseases, making the study of signal transduction important for both uncovering basic biology and understanding pathophysiology. Established laboratory cell culture models are useful for studying signal transduction mechanisms, but differences in sample handling procedures can introduce unwanted variability in experimental outcomes and conclusions. One such potential source of experimental variability is the introduction of fluid shear stress upon handling of tissue culture cells. Fluid shear stress triggers a wide range of cellular responses in adherent cell culture, including stimulating the production of cyclic AMP, potentiating the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and ultimately inducing changes in the gene expression of growth and remodeling factors. Further, mechanical stress on cells is physiologically relevant to the development of many pathologies. Here, we describe a detailed protocol for cell lysis and protein extraction that minimizes shear stress induced by classical cell harvest protocols. We also highlight the impact of fluid shear stress by using immunoblotting to assess ERK pathway activation as a readout for this protocol. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Gentle cell lysis and protein extraction Basic Protocol 2: Immunoblotting for cell signaling readouts by SDS-PAGE.
Assuntos
Sistema de Sinalização das MAP Quinases , Transdução de Sinais , Humanos , Estresse Mecânico , Transdução de Sinais/fisiologia , Fosforilação , Diferenciação CelularRESUMO
Due to their extensive use, the release of zinc oxide nanoparticles (ZnO NP) into the environment is increasing and may lead to unintended risk to both human health and ecosystems. Access of ZnO NP to the brain has been demonstrated, so their potential toxicity on the nervous system is a matter of particular concern. Although evaluation of ZnO NP toxicity has been reported in several previous studies, the specific effects on the nervous system are not completely understood and, particularly, effects on genetic material and on organism behaviour are poorly addressed. We evaluated the potential toxic effects of ZnO NP in vitro and in vivo, and the role of zinc ions (Zn2+) in these effects. In vitro, the ability of ZnO NP to be internalized by A172 glial cells was verified, and the cytotoxic and genotoxic effects of ZnO NP or the released Zn2+ ions were addressed by means of vital dye exclusion and comet assay, respectively. In vivo, behavioural alterations were evaluated in zebrafish embryos using a total locomotion assay. ZnO NP induced decreases in viability of A172 cells after 24 h of exposure and genetic damage after 3 and 24 h. The involvement of the Zn2+ ions released from the NP in genotoxicity was confirmed. ZnO NP exposure also resulted in decreased locomotor activity of zebrafish embryos, with a clear role of released Zn2+ ions in this effect. These findings support the toxic potential of ZnO NP showing, for the first time, genetic effects on glial cells and proving the intervention of Zn2+ ions.
RESUMO
Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms (HAB), belongs to the Diarrheic Shellfish Poisoning toxins that cause gastrointestinal symptoms in humans after consumption. In the present work, Ruditapes decussatus haemocytes were selected to evaluate the effect of OA on cell viability, enzymatic status and immune capacity through the measure by flow cytometry of apoptosis-cell death, non-specific esterase activity and phagocytosis. In order to compare different exposure conditions, two experiments were developed: in vitro exposure to OA and HAB simulation by feeding clams with the OA producer, Prorocentrum lima. Apoptosis was not OA dose-dependent and cell death increased in both assays. Phagocytosis of latex beads and esterase activity decreased in haemocytes incubated with OA. In contrast, esterases increased during the feeding with P. lima. Our results showed that OA and the simulated HAB caused damages on haemocyte functions and viability.
Assuntos
Bivalves/efeitos dos fármacos , Dinoflagellida/metabolismo , Proliferação Nociva de Algas/fisiologia , Hemócitos/efeitos dos fármacos , Ácido Okadáico/toxicidade , Animais , Apoptose/efeitos dos fármacos , Bioensaio , Bivalves/citologia , Bivalves/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Esterases/antagonistas & inibidores , Esterases/metabolismo , Hemócitos/citologia , Humanos , Microesferas , Ácido Okadáico/metabolismo , Fagocitose/efeitos dos fármacosRESUMO
Venerupis rhomboides is a commercial clam whose production could be enhanced through effective management of natural and hatchery stocks. This study provides the first panel of microsatellite markers for the exploitation of this species according to genetic criteria. A total of 22 polymorphic microsatellite loci were isolated and characterized from two genomic libraries enriched for different motifs. The number of alleles per locus ranged from 2 to 14 in a sample of 20 clams from Spain, and the observed and expected heterozygosity from 0 to 0.95 and 0.05-0.901, respectively. Sixteen loci were in agreement with Hardy-Weinberg equilibrium after sequential Bonferroni correction and linkage disequilibrium between loci pairs was not detected. To reduce the cost of the genotyping process, tri- and pentaplex PCRs, amplifying a total of 13 microsatellites loci were optimized. The microsatellites developed here represent the first nuclear markers described in V. rhomboides and will be useful tools for genetic studies involving assessment of genetic variation and population structure of natural and cultivated populations, assignment testing, construction of genetic linkage maps and dissection of production traits.
Assuntos
Bivalves/genética , Repetições de Microssatélites , Reação em Cadeia da Polimerase Multiplex , Animais , Aquicultura , Sequência de Bases , Primers do DNA/genética , Tipagem Molecular , Polimorfismo Genético , Análise de Sequência de DNARESUMO
Harmful Algal Blooms (HABs) constitute one of the most important sources of contamination in the oceans, producing high concentrations of potentially harmful biotoxins that are accumulated across the food chains. One such biotoxin, Okadaic Acid (OA), is produced by marine dinoflagellates and subsequently accumulated within the tissues of filtering marine organisms feeding on HABs, rapidly spreading to their predators in the food chain and eventually reaching human consumers causing Diarrhetic Shellfish Poisoning (DSP) syndrome. While numerous studies have thoroughly evaluated the effects of OA in mammals, the attention drawn to marine organisms in this regard has been scarce, even though they constitute primary targets for this biotoxin. With this in mind, the present work aimed to provide a timely and comprehensive insight into the current literature on the effect of OA in marine invertebrates, along with the strategies developed by these organisms to respond to its toxic effect together with the most important methods and techniques used for OA detection and evaluation.
Assuntos
Toxinas Marinhas/toxicidade , Mutagênicos/toxicidade , Ácido Okadáico/toxicidade , Animais , Organismos Aquáticos/genética , Organismos Aquáticos/metabolismo , Dinoflagellida/metabolismo , Cadeia Alimentar , Contaminação de Alimentos , Proliferação Nociva de Algas , Humanos , Invertebrados/genética , Invertebrados/metabolismo , Toxinas Marinhas/isolamento & purificação , Mutagênicos/isolamento & purificação , Ácido Okadáico/isolamento & purificação , Intoxicação por Frutos do Mar/etiologiaRESUMO
Okadaic acid (OA) is one of the most frequent and worldwide distributed marine toxins. It is easily accumulated by shellfish, mainly bivalve mollusks and fish, and, subsequently, can be consumed by humans causing alimentary intoxications. OA is the main representative diarrheic shellfish poisoning (DSP) toxin and its ingestion induces gastrointestinal symptoms, although it is not considered lethal. At the molecular level, OA is a specific inhibitor of several types of serine/threonine protein phosphatases and a tumor promoter in animal carcinogenesis experiments. In the last few decades, the potential toxic effects of OA, beyond its role as a DSP toxin, have been investigated in a number of studies. Alterations in DNA and cellular components, as well as effects on immune and nervous system, and even on embryonic development, have been increasingly reported. In this manuscript, results from all these studies are compiled and reviewed to clarify the role of this toxin not only as a DSP inductor but also as cause of alterations at the cellular and molecular levels, and to highlight the relevance of biomonitoring its effects on human health. Despite further investigations are required to elucidate OA mechanisms of action, toxicokinetics, and harmful effects, there are enough evidences illustrating its toxicity, not related to DSP induction, and, consequently, supporting a revision of the current regulation on OA levels in food.
Assuntos
Toxinas Marinhas/intoxicação , Ácido Okadáico/intoxicação , Intoxicação por Frutos do Mar/fisiopatologia , Animais , Monitoramento Ambiental , HumanosRESUMO
Okadaic Acid (OA) constitutes the main active principle in Diarrhetic Shellfish Poisoning (DSP) toxins produced during Harmful Algal Blooms (HABs), representing a serious threat for human consumers of edible shellfish. Furthermore, OA conveys critical deleterious effects for marine organisms due to its genotoxic potential. Many efforts have been dedicated to OA biomonitoring during the last three decades. However, it is only now with the current availability of detailed molecular information on DNA organization and the mechanisms involved in the maintenance of genome integrity, that a new arena starts opening up for the study of OA contamination. In the present work we address the links between OA genotoxicity and chromatin by combining Next Generation Sequencing (NGS) technologies and bioinformatics. To this end, we introduce CHROMEVALOAdb, a public database containing the chromatin-associated transcriptome of the mussel Mytilus galloprovincialis (a sentinel model organism) in response to OA exposure. This resource constitutes a leap forward for the development of chromatin-based biomarkers, paving the road towards the generation of powerful and sensitive tests for the detection and evaluation of the genotoxic effects of OA in coastal areas.
Assuntos
Bases de Dados Factuais , Mutagênicos/análise , Mytilus/genética , Ácido Okadáico/análise , Animais , Carcinógenos/análise , Carcinógenos/isolamento & purificação , Carcinógenos/toxicidade , Cromatina/metabolismo , Monitoramento Ambiental/métodos , Humanos , Testes de Mutagenicidade/métodos , Mutagênicos/isolamento & purificação , Mutagênicos/toxicidade , Ácido Okadáico/toxicidade , Análise de Sequência de DNA , TranscriptomaRESUMO
Okadaic acid (OA) is one of the most common and highly distributed marine toxins. It can be accumulated in several molluscs and other marine organisms and cause acute gastrointestinal symptoms after oral consumption by humans, called diarrheic shellfish poisoning. However other toxic effects beyond these gastrointestinal symptoms were also reported. Thus, OA was found to induce important chromosomal abnormalities and other genetic injuries that can lead to severe pathologies, including cancer. Furthermore, the relationship between OA and carcinogenic processes has been previously demonstrated in in vivo studies with rodents, and also suggested in human epidemiological studies. In this context, further research is required to better understand the underlying mechanisms of OA-related tumourigenesis. In a previous study, we identified 247 genes differentially expressed in SHSY5Y neuroblastoma cells exposed to 100nM OA at different times (3, 24 and 48h) by means of suppression subtractive hybridization. These genes were involved in relevant cell functions such as signal transduction, cell cycle, metabolism, and transcription and translation processes. However, due to the high potential percentage of false positives that may be obtained by this approach, results from SSH are recommended to be analyzed by an independent method. In the present study, we selected ten genes related to cancer initiation or progression, directly or indirectly, for further quantitative PCR analysis (ANAPC13, PTTG1, CALM2, CLU, HN1, MALAT1, MAPRE2, MLLT11, SGA-81M and TAX1BP1). Results obtained showed important alterations in the expression patterns of all the genes evaluated at one or more treatment times, providing, for the first time, a possible explanation at the molecular level of the potential relationship between the consumption of OA-contaminated shellfish and the incidence of different cancers in humans. Nevertheless, given the complexity of this process, more exhaustive studies are required before drawing any final conclusion.
Assuntos
Aberrações Cromossômicas/induzido quimicamente , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Neurônios/efeitos dos fármacos , Ácido Okadáico/toxicidade , Carcinógenos/toxicidade , Ciclo Celular , Linhagem Celular , Transformação Celular Neoplásica/induzido quimicamente , Humanos , Neurônios/fisiologia , Hibridização de Ácido Nucleico , Intoxicação por Frutos do MarRESUMO
Patient Reported Outcomes (PROs) are an evidenced way of adding value to routine clinical care. As a source of unique information on the effect of a medical condition and its treatment from the patients' perspective (Mercieca-Bebber et al. in Patient Relat Outcome Meas 9: 353-367, https://doi.org/10.2147/PROM.S156279 , 2018), PROs allow for an improved assessment in routine clinical care of symptoms, side effects, functional outcomes (physical, sexual, social, emotional, cognitive functioning), and health-related quality of life (HRQoL). By helping to align healthcare providers' interventions with what matters most to the patient, PROs contribute to the individualized choice of Anti-Retroviral Therapy (Carfora et al. in PLoS ONE 17(4): e0267030, https://doi.org/10.1371/journal.pone.0267030 , 2022) as a paramount component of tailored and person-centred care management, in routine clinical practice. This article outlines a practical framework and process tested in Portugal to raise awareness of PROs' added value, and to help guide first steps in the implementation and seamless integration of PROs, in routine HIV care.
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Infecções por HIV , Qualidade de Vida , Humanos , Qualidade de Vida/psicologia , Medidas de Resultados Relatados pelo Paciente , Pessoal de Saúde , Infecções por HIV/diagnóstico , PortugalRESUMO
BACKGROUND: Okadaic acid (OA), a toxin produced by several dinoflagellate species is responsible for frequent food poisonings associated to shellfish consumption. Although several studies have documented the OA effects on different processes such as cell transformation, apoptosis, DNA repair or embryogenesis, the molecular mechanistic basis for these and other effects is not completely understood and the number of controversial data on OA is increasing in the literature. RESULTS: In this study, we used suppression subtractive hybridization in SHSY5Y cells to identify genes that are differentially expressed after OA exposure for different times (3, 24 and 48 h). A total of 247 subtracted clones which shared high homology with known genes were isolated. Among these, 5 specific genes associated with cytoskeleton and neurotransmission processes (NEFM, TUBB, SEPT7, SYT4 and NPY) were selected to confirm their expression levels by real-time PCR. Significant down-regulation of these genes was obtained at the short term (3 and 24 h OA exposure), excepting for NEFM, but their expression was similar to the controls at 48 h. CONCLUSIONS: From all the obtained genes, 114 genes were up-regulated and 133 were down-regulated. Based on the NCBI GenBank and Gene Ontology databases, most of these genes are involved in relevant cell functions such as metabolism, transport, translation, signal transduction and cell cycle. After quantitative PCR analysis, the observed underexpression of the selected genes could underlie the previously reported OA-induced cytoskeleton disruption, neurotransmission alterations and in vivo neurotoxic effects. The basal expression levels obtained at 48 h suggested that surviving cells were able to recover from OA-caused gene expression alterations.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Okadáico/farmacologia , Linhagem Celular Tumoral , Citoesqueleto/genética , Bases de Dados Genéticas , Regulação para Baixo/efeitos dos fármacos , Humanos , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Hibridização de Ácido Nucleico , Transmissão Sináptica/genética , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Okadaic acid (OA) is a widely distributed marine toxin produced by several phytoplanktonic species and responsible for diarrheic shellfish poisoning in humans. At the molecular level OA is a specific inhibitor of several types of serine/threonine protein phosphatases. Due to this enzymic inhibition, OA was reported to induce numerous alterations in relevant cellular physiological processes, including several metabolic pathways such as glucose uptake, lipolysis and glycolysis, heme metabolism, and glycogen and protein synthesis. In order to further understand the underlying mechanisms involved in OA-induced effects on cellular metabolism, the expression levels of six genes related to different catabolic and anabolic metabolism-related processes were analyzed by real-time polymerase chain reaction. Specifically, the expression patterns of GAPDH, TOMM5, SLC25A4, COII, QARS, and RGS5 genes were determined in SHSY5Y human neuroblastoma cells exposed to OA for 3, 24, or 48 h. All these genes showed alterations in their expression levels after at least one of the OA treatments tested. These alterations provide a basis to understand the mechanisms underlying the previously described OA-induced effects on different metabolic processes, mainly regarding glucose and mitochondrial metabolism. However, other OA-induced affected genes can not be ruled out, and further studies are required to more comprehensively characterize in the mechanisms of OA-induced interaction on cell metabolism.