RESUMO
The potential to regenerate a damaged body part is expressed to a different extent in animals. Echinoderms, in particular starfish, are known for their outstanding regenerating potential. Differently, humans have restricted abilities to restore organ systems being dependent on limited sources of stem cells. In particular, the potential to regenerate the central nervous system is extremely limited, explaining the lack of natural mechanisms that could overcome the development of neurodegenerative diseases and the occurrence of trauma. Therefore, understanding the molecular and cellular mechanisms of regeneration in starfish could help the development of new therapeutic approaches in humans. In this study, we tackle the problem of starfish central nervous system regeneration by examining the external and internal anatomical and behavioral traits, the dynamics of coelomocyte populations, and neuronal tissue architecture after radial nerve cord (RNC) partial ablation. We noticed that the removal of part of RNC generated several anatomic anomalies and induced behavioral modifications (injured arm could not be used anymore to lead the starfish movement). Those alterations seem to be related to defense mechanisms and protection of the wound. In particular, histology showed that tissue patterns during regeneration resemble those described in holothurians and in starfish arm tip regeneration. Flow cytometry coupled with imaging flow cytometry unveiled a new coelomocyte population during the late phase of the regeneration process. Morphotypes of these and previously characterized coelomocyte populations were described based on IFC data. Further studies of this new coelomocyte population might provide insights on their involvement in radial nerve cord regeneration.
Assuntos
Nervo Radial , Pepinos-do-Mar , Animais , Humanos , Nervo Radial/fisiologia , Estrelas-do-Mar/fisiologia , Regeneração Nervosa/fisiologiaRESUMO
Tuberculosis (TB) is a transmissible disease listed as one of the 10 leading causes of death worldwide (10 million infected in 2019). A swift and precise diagnosis is essential to forestall its transmission, for which the discovery of effective diagnostic biomarkers is crucial. In this study, we aimed to discover molecular biomarkers for the early diagnosis of tuberculosis. Two independent cohorts comprising 29 and 34 subjects were assayed by proteomics, and 49 were included for metabolomic analysis. All subjects were arranged into three experimental groupshealthy controls (controls), latent TB infection (LTBI), and TB patients. LC-MS/MS blood serum protein and metabolite levels were submitted to univariate, multivariate, and ROC analysis. From the 149 proteins quantified in the discovery set, 25 were found to be differentially abundant between controls and TB patients. The AUC, specificity, and sensitivity, determined by ROC statistical analysis of the model composed of four of these proteins considering both proteomic sets, were 0.96, 93%, and 91%, respectively. The five metabolites (9-methyluric acid, indole-3-lactic acid, trans-3-indoleacrylic acid, hexanoylglycine, and N-acetyl-L-leucine) that better discriminate the control and TB patient groups (VIP > 1.75) from a total of 92 metabolites quantified in both ionization modes were submitted to ROC analysis. An AUC = 1 was determined, with all samples being correctly assigned to the respective experimental group. An integrated ROC analysis enrolling one protein and four metabolites was also performed for the common control and TB patients in the proteomic and metabolomic groups. This combined signature correctly assigned the 12 controls and 12 patients used only for prediction (AUC = 1, specificity = 100%, and sensitivity = 100%). This multiomics approach revealed a biomarker signature for tuberculosis diagnosis that could be potentially used for developing a point-of-care diagnosis clinical test.
Assuntos
Tuberculose Latente , Tuberculose , Humanos , Proteômica , Cromatografia Líquida , Espectrometria de Massas em Tandem , Tuberculose/diagnóstico , Tuberculose Latente/diagnóstico , BiomarcadoresRESUMO
An increased knowledge on the real impacts of ectomycorrhizal symbiosis in forest species is needed to optimize forest sustainable productivity and thus to improve forest services and their capacity to act as carbon sinks. In this study, we investigated the response of an oak species to ectomycorrhizae formation using a proteomics approach complemented by biochemical analysis of carbohydrate levels. Comparative proteome analysis between mycorrhizal and nonmycorrhizal cork oak plants revealed no differences at the foliar level. However, the protein profile of 34 unique oak proteins was altered in the roots. Consistent with the results of the biochemical analysis, the proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose for the metabolic activity of mycorrhizal roots which is consistent with an increased allocation of carbohydrates from the plant to the fungus in order to sustain the symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of defense reactions, increased nutrient mobilization from the plant-fungus interface (N and P), as well as cytoskeleton rearrangements and induction of plant cell wall loosening for fungal root accommodation in colonized roots are also suggested by the results. The suggested improvement in root capacity to take up nutrients accompanied by an increase of root biomass without apparent changes in aboveground biomass strongly re-enforces the potential of mycorrhizal inoculation to improve cork oak forest resistance capacity to cope with coming climate change.
Assuntos
Basidiomycota/fisiologia , Micorrizas/fisiologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Quercus/metabolismo , Quercus/microbiologia , Biomassa , Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Metabolismo dos Lipídeos/fisiologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Estresse Fisiológico , SimbioseRESUMO
Mechanisms underpinning chronic sustained hypoxia (CH)-induced structural and functional adaptations in respiratory muscles are unclear despite the clinical relevance to respiratory diseases. The objectives of the present study were to thoroughly assess the putative role of CH-induced redox remodeling in murine diaphragm muscle over time and the subsequent effects on metabolic enzyme activities, catabolic signaling and catabolic processes, and diaphragm muscle contractile function. C57Bl6/J mice were exposed to normoxia or normobaric CH (fraction of inspired oxygen = 0.1) for 1, 3, or 6 weeks. A second cohort was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine). After CH exposure, we performed two-dimensional redox proteomics with mass spectrometry, enzyme activity assays, and cell-signaling assays on diaphragm homogenates. We also assessed diaphragm isotonic contractile and endurance properties ex vivo. Global protein redox changes in the diaphragm after CH are indicative of oxidation. Remodeling of proteins key to contractile, metabolic, and homeostatic functions was observed. Several oxidative and glycolytic enzyme activities were decreased by CH. Redox-sensitive chymotrypsin-like proteasome activity of the diaphragm was increased. CH decreased phospho-forkhead box O3a (FOXO3a) and phospho-mammalian target of rapamycin content. Hypoxia-inducible factor-1α and phospho-p38 mitogen-activated protein kinase content was increased in CH diaphragm, and this was attenuated by antioxidant treatment. CH exposure decreased force- and power-generating capacity of the diaphragm, and this was prevented by antioxidant supplementation with N-acetyl cysteine but not tempol. Redox remodeling is pivotal for diaphragm adaptation to CH, affecting metabolic activity, atrophy signaling, and functional performance. Antioxidant supplementation may be useful as an adjunctive therapy in respiratory-related diseases characterized by hypoxic stress.
Assuntos
Adaptação Fisiológica , Diafragma/metabolismo , Diafragma/fisiopatologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Animais , Antioxidantes/metabolismo , Doença Crônica , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Contração Muscular , Oxirredução , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Carbonilação Proteica , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Compostos de Sulfidrila/metabolismoRESUMO
All bryophytes evolved desiccation tolerance (DT) mechanisms during the invasion of terrestrial habitats by early land plants. Are these DT mechanisms still present in bryophytes that colonize aquatic habitats? The aquatic bryophyte Fontinalis antipyreticaâ Hedw. was subjected to two drying regimes and alterations in protein profiles and sucrose accumulation during dehydration and rehydration were investigated. Results show that during fast dehydration, there is very little variation in protein profiles, and upon rehydration proteins are leaked. On the other hand, slow dehydration induces changes in both dehydration and rehydration protein profiles, being similar to the protein profiles displayed by the terrestrial bryophytes Physcomitrella patens (Hedw.) Bruch and Schimp. and, to what is comparable with Syntrichia ruralis (Hedw.) F. Weber and D. Mohr. During dehydration there was a reduction in proteins associated with photosynthesis and the cytoskeleton, and an associated accumulation of proteins involved in sugar metabolism and plant defence mechanisms. Upon rehydration, protein accumulation patterns return to control values for both photosynthesis and cytoskeleton whereas proteins associated with sugar metabolism and defence proteins remain high. The current results suggest that bryophytes from different ecological adaptations may share common DT mechanisms.
Assuntos
Adaptação Fisiológica , Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteômica , Água/metabolismo , Sequência de Aminoácidos , Biomassa , Citoesqueleto/metabolismo , Desidratação , Dessecação , Eletroforese em Gel Bidimensional , Metabolismo Energético , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Plantas/química , Análise de Componente Principal , Biossíntese de Proteínas , Proteoma/química , Transdução de Sinais , Estresse Fisiológico , Sacarose/metabolismoRESUMO
BACKGROUND: Colostrum intake by newborn lambs plays a fundamental role in the perinatal period, ensuring lamb survival. In this study, blood plasma samples from two groups of newborn lambs (Colostrum group and Delayed Colostrum group) at 2 and 14 h after birth were treated to reduce the content of high abundance proteins and analyzed using Two-Dimensional Differential in Gel Electrophoresis and MALDI MS/MS for protein identification in order to investigate low abundance proteins with immune function in newborn lambs. RESULTS: The results showed that four proteins were increased in the blood plasma of lambs due to colostrum intake. These proteins have not been previously described as increased in blood plasma of newborn ruminants by colostrum intake. Moreover, these proteins have been described as having an immune function in other species, some of which were previously identified in colostrum and milk. CONCLUSIONS: In conclusion, colostrum intake modified the low abundance proteome profile of blood plasma from newborn lambs, increasing the concentration of apolipoprotein A-IV, plasminogen, serum amyloid A and fibrinogen, demonstrating that colostrum is essential, not only for the provision of immunoglobulins, but also because of increases in several low abundance proteins with immune function.
Assuntos
Animais Recém-Nascidos/sangue , Proteínas Sanguíneas/análise , Colostro/fisiologia , Proteoma/fisiologia , Ovinos/sangue , Animais , Animais Recém-Nascidos/fisiologia , Apolipoproteínas A/sangue , Proteínas Sanguíneas/fisiologia , Fibrinogênio , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Fragmentos de Peptídeos/sangue , Plasminogênio/análise , Proteína Amiloide A Sérica/análise , Ovinos/fisiologiaRESUMO
This study aims at identifying molecular biomarkers differentiating responders and non-responders to treatment with Tumor Necrosis Factor inhibitors (TNFi) among patients with axial spondyloarthritis (axSpA). Whole blood mRNA and plasma proteins were measured in a cohort of biologic-naïve axSpA patients (n = 35), pre and post (14 weeks) TNFi treatment with adalimumab. Differential expression analysis was used to identify the most enriched pathways and in predictive models to distinguish responses to TNFi. A treatment-associated signature suggests a reduction in inflammatory activity. We found transcripts and proteins robustly differentially expressed between baseline and week 14 in responders. C-reactive protein (CRP) and Haptoglobin (HP) proteins showed strong and early decrease in the plasma of axSpA patients, while a cluster of apolipoproteins (APOD, APOA2, APOA1) showed increased expression at week 14. Responders to TNFi treatment present higher levels of markers of innate immunity at baseline, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP, gender, and AFF3, the top differentially expressed gene at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC = 0.97). In conclusion, innate and adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axSpA patients. A model including clinical and gene expression variables should also be considered.
Assuntos
Antirreumáticos , Espondiloartrite Axial , Espondilite Anquilosante , Humanos , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Adalimumab/uso terapêutico , Antirreumáticos/uso terapêutico , Fator de Necrose Tumoral alfa , Resultado do TratamentoRESUMO
Regeneration is a complex cellular process that, rather than simply forming a scar following injury, the animal forms a new functional tissue. Regeneration is a widespread process among metazoa, although not uniformly. Planaria, starfish, and some worms can regenerate most of their body, whereas many other species can only regenerate parts of specific tissues or fail to accomplish a functional regrowth, as is the case of mammals CNS. Research in regenerative medicine will possibly culminate in the regeneration of organs/tissues originally not prone to this process. Despite the complexity of the interactions and regulatory systems involved, the variety of tissues and organs these cells differentiate into has so far impaired the success of direct transplantation to restore damaged tissues. For this reason, a study, at the molecular level of the regeneration mechanisms developed by different animal models is likely to provide answers to why these processes are not readily activated in mammals. Proteomic-based approaches are being recognized as extremely useful to study of regeneration events, also because there is a relevant contribution of posttranscriptional processes that involve frequently the occurrence of a broad range of PTMs. The present review focuses on the significant knowledge brought up by proteomics in diverse aspects of regeneration research on different animal models, tissues, and organs.
Assuntos
Regeneração Nervosa , Proteoma/metabolismo , Cicatrização , Animais , Humanos , Fígado/fisiologia , Músculo Esquelético/fisiologia , Proteômica , Regeneração , Medicina Regenerativa , Transplante de Células-TroncoRESUMO
Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Medicago truncatula/embriologia , Reguladores de Crescimento de Plantas/análise , Proteínas de Plantas/análise , Sementes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Variação Genética , Genótipo , Medicago truncatula/química , Medicago truncatula/genética , Proteínas de Plantas/metabolismo , Proteômica , Sementes/crescimento & desenvolvimento , Especificidade da EspécieRESUMO
Staphylococcus epidermidis is one of the most common bacteria of the human skin microbiota. Despite its role as a commensal, S. epidermidis has emerged as an opportunistic pathogen, associated with 80% of medical devices related infections. Moreover, these bacteria are extremely difficult to treat due to their ability to form biofilms and accumulate resistance to almost all classes of antimicrobials. Thus new preventive and therapeutic strategies are urgently needed. However, the molecular mechanisms associated with S. epidermidis colonisation and disease are still poorly understood. A deeper understanding of the metabolic and cellular processes associated with response to environmental factors characteristic of SE ecological niches in health and disease might provide new clues on colonisation and disease processes. Here we studied the impact of pH conditions, mimicking the skin pH (5.5) and blood pH (7.4), in a S. epidermidis commensal strain by means of next-generation proteomics and 1H NMR-based metabolomics. Moreover, we evaluated the metabolic changes occurring during a sudden pH change, simulating the skin barrier break produced by a catheter. We found that exposure of S. epidermidis to skin pH induced oxidative phosphorylation and biosynthesis of peptidoglycan, lipoteichoic acids and betaine. In contrast, at blood pH, the bacterial assimilation of monosaccharides and its oxidation by glycolysis and fermentation was promoted. Additionally, several proteins related to virulence and immune evasion, namely extracellular proteases and membrane iron transporters were more abundant at blood pH. In the situation of an abrupt skin-to-blood pH shift we observed the decrease in the osmolyte betaine and changes in the levels of several metabolites and proteins involved in cellular redoxl homeostasis. Our results suggest that at the skin pH S. epidermidis cells are metabolically more active and adhesion is promoted, while at blood pH, metabolism is tuned down and cells have a more virulent profile. pH increase during commensal-to-pathogen conversion appears to be a critical environmental signal to the remodelling of the S. epidermidis metabolism toward a more pathogenic state. Targeting S. epidermidis proteins induced by pH 7.4 and promoting the acidification of the medical device surface or surrounding environment might be new strategies to treat and prevent S. epidermidis infections.
RESUMO
Proteomics combined to advanced bioinformatics tools is acquiring a pivotal role in the comprehensive understanding of living organism's biology, in particular for non-model organisms, which includes most marine and aquatic invertebrates. Depicting of protein composition in a whole organ/organism followed by their assembling in functional protein association networks promotes the understanding of key biological processes. Here, we provide a detailed description of the extraction procedure of cell-free coelomic fluid soluble proteins and the characterization of the proteome of the starfish Marthasterias glacialis. Due to coelomic fluid richness in glycoproteins, which complicates protein identification, extracts of soluble proteins are deglycosylated prior to tandem mass spectrometry. This experimental approach is useful at improving knowledge on the coelomic fluid physiological role and deciphering its involvement in regeneration of starfish body parts when comparing different regeneration conditions.
Assuntos
Proteoma , Estrelas-do-Mar , Animais , ProteômicaRESUMO
We describe the first proteomic characterization of the radial nerve cord (RNC) of an echinoderm, the sea star Marthasterias glacialis. The combination of 2-DE with MS (MALDI-TOF/TOF) resulted in the identification of 286 proteins in the RNC. Additionally, 158 proteins were identified in the synaptosomal membranes enriched fraction after 1-DE separation. The 2-DE RNC reference map is available via the WORLD-2DPAGE Portal (http://www.expasy.ch/world-2dpage/) along with the associated protein identification data which are also available in the PRIDE database. The identified proteins constitute the first high-throughput evidence that seems to indicate that echinoderms nervous transmission relies primarily on chemical synapses which is similar to the synaptic activity in adult mammal's spinal cord. Furthermore, several homologous proteins known to participate in the regeneration events of other organisms were also identified, and thus can be used as targets for future studies aiming to understand the poorly uncharacterized regeneration capability of echinoderms. This "echinoderm missing link" is also a contribution to unravel the mystery of deuterostomian CNS evolution.
Assuntos
Proteínas/metabolismo , Proteoma/metabolismo , Nervo Radial/metabolismo , Estrelas-do-Mar/metabolismo , Membranas Sinápticas/metabolismo , Sinaptossomos/metabolismo , Animais , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Expressão Gênica , Filogenia , Proteínas/genética , Proteoma/genética , Proteômica , Nervo Radial/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estrelas-do-Mar/genética , Membranas Sinápticas/genética , Sinaptossomos/químicaRESUMO
Hamadryas baboons possess salivary proline-rich proteins (PRP), as indicated by the presence of pink-staining protein bands using 1D SDS gel electrophoresis and Coomassie R250 staining. The ability of these protein bands to interact with tannic acid was further examined. In a tannin-binding assay using 5 µg tannic acid mixed with hamadryas whole saliva, we recently found four distinct protein bands of apparently 72, 55, 20, and 15 kDa that were precipitated during the experiments. In this work, we were able to identify these protein bands in a follow-up analysis using MS/MS mass spectrometry after excising such bands out of air-dried gels. Albumin and α-amylase were present in the tannic acid-protein complexes, with albumin already known to nonspecifically interact with a great diversity of chemical compounds. More interesting, we also identified a basic PRP and a cystatin precursor protein. This was the first successful attempt to identify a PRP from precipitated tannin-protein complexes in hamadryas baboons using MS/MS mass spectrometry. On the other hand, the role of cystatins in tannin binding is not yet well understood. However, there are recent reports on cystatin expression in saliva of rats responding to astringent dietary compounds. In conclusion, the follow-up data on tannin-binding proteins present in salivary secretions from hamadryas baboons adds important knowledge to primate physiology and feeding ecology, in order to shed light on the establishment and development of food adaptations in primates. It also demonstrates that tannin binding is characteristic for PRP, but might not be restricted to this particular group of proteins in primate species.
Assuntos
Papio hamadryas/metabolismo , Proteínas Salivares Ricas em Prolina/metabolismo , Taninos/metabolismo , Animais , Masculino , Proteínas Salivares Ricas em Prolina/química , Espectrometria de Massas em Tandem/veterináriaRESUMO
Sheep, cattle and goat are domestic ruminants of significant economic interest in the Mediterranean region. Although sharing the same pasture ranges, they ingest different plants and plant parts and, consequently different levels of tannins. This suggests an ability to detect and adapt ingestion according to animal physiological limits of tolerance for plant secondary metabolites. This review will detail the effects of dietary tannins on feeding behavior, and the role of the oral cavity in this process, with focus on such ruminant species. The role of salivary protein profile in tannin perception in the oral cavity, and as a defense mechanism, will be discussed.
Assuntos
Comportamento Alimentar/efeitos dos fármacos , Ruminantes/fisiologia , Taninos/farmacologia , AnimaisRESUMO
Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.
Assuntos
Líquidos Corporais , Citometria de Fluxo , Fagócitos , Estrelas-do-Mar , Animais , Líquidos Corporais/citologia , Líquidos Corporais/imunologia , Fagócitos/citologia , Fagócitos/imunologia , Estrelas-do-Mar/citologia , Estrelas-do-Mar/imunologiaRESUMO
Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.
Assuntos
Ecotoxicologia , Poluentes Químicos da Água , Animais , Organismos Aquáticos , Humanos , Invertebrados , Reprodutibilidade dos Testes , Células-Tronco , Poluentes Químicos da Água/toxicidadeRESUMO
BACKGROUND: Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry. RESULTS: Fifty-seven protein spots were excised from control group gels, and 21 different proteins were identified. With tannin consumption, the expression levels of one α-amylase isoform and one unidentified protein increased, whereas acidic mammalian chitinase and Muc10 decreased. Additionally, two basic spots that stained pink with Coomassie Brilliant Blue R-250 were newly observed, suggesting that some induced PRPs may remain uncomplexed or form soluble complexes with tannins. CONCLUSION: This proteomic analysis provides evidence that other salivary proteins, in addition to tannin-precipitating proteins, are affected by tannin ingestion. Changes in the expression levels of the acidic mammalian chitinase precursor and in one of the 14 salivary α-amylase isoforms underscores the need to further investigate their role in tannin ingestion.
RESUMO
Grapevine downy mildew is an important disease affecting crop production leading to severe yield losses. This study aims to identify the grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry. In addition, we analysed ROS production, antioxidant capacity, lipid peroxidation and gene expression. Proteins related to photosynthesis and metabolism allowed the discrimination of resistant and susceptible grapevine cultivars prior to P. viticola inoculation. Following inoculation increase of hydrogen peroxide levels, cellular redox regulation, establishment of ROS signalling and plant cell death seem to be key points differentiating the resistant genotype. Lipid associated signalling events, particularly related to jasmonates appear also to play a major role in the establishment of resistance. The findings from this study contribute to a better understanding of genotype-specific differences that account for a successful establishment of a defence response to the downy mildew pathogen. BIOLOGICAL SIGNIFICANCE: Here, we present for the first time grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). We have highlighted that, following inoculation, the major factors differentiating the resistant from the susceptible grapevine cultivars are the establishment of effective ROS signalling together with lipid-associated signalling events, particularly related to jasmonates. It is believed that plants infected with biotrophic pathogens suppress JA-mediated responses, however recent evidences shown that jasmonic acid signalling pathway in grapevine resistance against Plasmopara viticola. Our results corroborate those evidences and highlight the importance of lipid- signalling for an effective resistance response against the downy mildew pathogen.
Assuntos
Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Peronospora/patogenicidade , Folhas de Planta/química , Proteoma/análise , Vitis/microbiologia , Ciclopentanos/farmacologia , Eletroforese em Gel Bidimensional , Genótipo , Metabolismo dos Lipídeos , Oxirredução , Oxilipinas/farmacologia , Doenças das Plantas/microbiologia , Transdução de SinaisRESUMO
UNLABELLED: A metal-resistant Rhodotorula mucilaginosa strain was isolated from an industrial wastewater. Effects on reduced/oxidized glutathione (GSSG/GSH), antioxidant enzymes and proteome were assessed on metal challenge (100mg/L). Increased GSH (mM/g) was found with CdCl2 (18.43±3.34), NaAsO2 (14.76±2.14), CuSO4 (14.73±2.49), and Pb(NO3)2 (15.74±5.3) versus control (7.67±0.95). GSH:GSSG ratio decreased with CdCl2, NaAsO2, and Pb(NO3)2 but not with CuSO4 and cysteine-containing protein levels increased with CdCl2 and NaAsO2. NaAsO2 exposure enhanced glutathione transferase activity but this decreased with CdCl2. Both metals significantly increased glutathione reductase and catalase activities. Metabolism-dependent uptake of Cd and As (12-day exposure) of approximately 65mg/g was observed in live cells with greater cell surface interaction for As compared to Cd. A particular role for arsenic oxidase in As resistance was identified. One dimensional electrophoresis revealed higher oxidation of protein thiols in response to NaAsO2 than to CdCl2. Two dimensional electrophoresis showed altered abundance of some proteins on metal treatment. Selected spots were excised for mass spectrometry and seven proteins identified. Under oxidative stress conditions, xylose reductase, putative chitin deacetylase, 20S proteasome subunit, eukaryotic translation elongation factor 2, valine-tRNA ligase and a metabolic enzyme F0F1 ATP synthase alpha subunit were all expressed as well as a unique hypothetical protein. These may comprise a protein expression signature for metal-induced oxidation in this yeast. SIGNIFICANCE: Fungi are of widespread importance in agriculture, biodegradation and often show extensive tolerance to heavy metals. This makes them of interest from the perspective of bioremediation. In this study an environmental isolate of R. mucilaginosa showing extensive tolerance of a panel of heavy metals, in particular cadmium and arsenic, was studied. Several biochemical parameters such as activity of antioxidant enzymes, status of reduced and oxidized glutathione and thiols associated with proteins were all found to be affected by metal exposure. A detailed analysis with arsenic and cadmium pointed to a particular role for arsenic oxidase in arsenic bioaccumulation and tolerance. This is the first time this has been reported in R. mucilaginosa, and suggests that this isolate may have potential in biosorption of these metals in the environment. Proteomic analysis revealed that seven proteins with a variety of roles - ATP synthesis, protein degradation/synthesis, and metabolism of xylose and chitin - were differentially affected by metal exposure in a manner consistent with oxidative stress. These may therefore represent a protein expression signature for exposure to cadmium and arsenic.
Assuntos
Arsênio/farmacologia , Biodegradação Ambiental , Cádmio/farmacologia , Proteômica/métodos , Rhodotorula/química , Catalase/efeitos dos fármacos , Catalase/metabolismo , Proteínas Fúngicas/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Glutationa Redutase/efeitos dos fármacos , Glutationa Redutase/metabolismo , Metais Pesados/farmacologia , Oxirredução/efeitos dos fármacos , Proteoma , Rhodotorula/efeitos dos fármacos , Rhodotorula/metabolismo , TranscriptomaRESUMO
Transthyretin (TTR) deposition in the peripheral nervous system is the hallmark of familial amyloidotic polyneuropathy (FAP). Currently, liver transplantation is the only available treatment to halt the progression of clinical symptoms; however, due to the limitations of this procedure, development of alternative therapeutic strategies is of utmost importance. In this regard, interference RNA (RNAi) targeting TTR is currently in phase III clinical development. To dissect molecular changes occurring in dorsal root ganglia (DRG) upon RNAi-mediated knockdown of TTR, we treated both chronically and acutely an FAP mouse model, in different stages of disease. Our data show that inhibition of TTR expression by the liver with RNAi reverse TTR deposition in DRG, decrease matrix metalloproteinase-2 (MMP-2) protein levels in plasma, inhibit Mmp-2 gene expression and downregulate MMP-9 activity in DRG, indicating extracellular matrix remodeling. Furthermore, protein levels of MMP-2 were found upregulated in plasma samples from FAP patients indicating that MMP-2 might be a novel potential biomarker for FAP diagnosis. Collectively, our data show that silencing TTR liver synthesis in vivo can modulate TTR-induced pathology in the peripheral nervous system and highlight the potential of MMP-2 as a novel disease biomarker.