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Nanoplastics can cause severe malformations in chicken embryos. To improve our understanding of the toxicity of nanoplastics to embryos, we have studied their biodistribution in living chicken embryos. We injected the embryos in the vitelline vein at stages 18-19. We injected polystyrene nanoparticles (PS-NPs) tagged with europium- or fluorescence. Their biodistribution was tracked using inductively-coupled plasma mass spectrometry on tissue lysates, paraffin histology, and vibratome sections analysed by machine learning algorithms. PS-NPs were found at high levels in the heart, liver and kidneys. Furthermore, PS-NPs crossed the endocardium of the heart at sites of epithelial-mesenchymal transformation; they also crossed the liver endothelium. Finally, we detected PS-NPs in the allantoic fluid, consistent with their being excreted by the kidneys. Our study shows the power of the chicken embryo model for analysing the biodistribution of nanoplastics in embryos. Such experiments are difficult or impossible in mammalian embryos. These findings are a major advance in our understanding of the biodistribution and tissue-specific accumulation of PS-NPs in developing animals.
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Nanopartículas , Poliestirenos , Animais , Poliestirenos/farmacocinética , Embrião de Galinha , Distribuição Tecidual , Rim/metabolismo , Fígado/metabolismo , Espectrometria de MassasRESUMO
Zebrafish hearts can regenerate by replacing damaged tissue with new cardiomyocytes. Although the steps leading up to the proliferation of surviving cardiomyocytes have been extensively studied, little is known about the mechanisms that control proliferation and redifferentiation to a mature state. We found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat-containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. We found that its function was conserved in mammalian cardiomyocytes. This study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes.
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Cálcio , Coração , Miócitos Cardíacos , Regeneração , Sarcômeros , Peixe-Zebra , Animais , Cálcio/fisiologia , Proliferação de Células , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Sarcômeros/fisiologia , Peixe-Zebra/fisiologiaRESUMO
Nanomaterials are widespread in the human environment as pollutants, and are being actively developed for use in human medicine. We have investigated how the size and dose of polystyrene nanoparticles affects malformations in chicken embryos, and have characterized the mechanisms by which they interfere with normal development. We find that nanoplastics can cross the embryonic gut wall. When injected into the vitelline vein, nanoplastics become distributed in the circulation to multiple organs. We find that the exposure of embryos to polystyrene nanoparticles produces malformations that are far more serious and extensive than has been previously reported. These malformations include major congenital heart defects that impair cardiac function. We show that the mechanism of toxicity is the selective binding of polystyrene nanoplastics nanoparticles to neural crest cells, leading to the death and impaired migration of those cells. Consistent with our new model, most of the malformations seen in this study are in organs that depend for their normal development on neural crest cells. These results are a matter of concern given the large and growing burden of nanoplastics in the environment. Our findings suggest that nanoplastics may pose a health risk to the developing embryo.
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Cardiopatias Congênitas , Crista Neural , Animais , Gravidez , Feminino , Embrião de Galinha , Humanos , Crista Neural/metabolismo , Microplásticos , Poliestirenos/toxicidade , Desenvolvimento EmbrionárioRESUMO
Gold nanoparticles (GNPs) can be manufactured in various shapes, and their size is programmable, which permits the study of the effects imposed by these parameters on biological processes. However, there is currently no clear evidence that a certain shape or size is beneficial. To address this issue, we have utilised GNPs and gold nanorods (GNRs) functionalised with model epitopes derived from chicken ovalbumin (OVA257-264 and OVA323-339). By using two distinct epitopes, it was possible to draw conclusions regarding the impact of nanoparticle shape and size on different aspects of the immune response. Our findings indicate that the peptide amphiphile-coated GNPs and GNRs are a safe and versatile epitope-presenting system. Smaller GNPs (â¼15 nm in diameter) induce significantly less intense T-cell responses. Furthermore, effective antigen presentation via MHC-I was observed for larger spherical particles (â¼40 nm in diameter), and to a lesser extent for rod-like particles (40 by 15 nm). At the same time, antigen presentation via MHC-II strongly correlated with the cellular uptake, with smaller GNPs being the least efficient. We believe these findings will have implications for vaccine development, and lead to a better understanding of cellular uptake and antigen egress from lysosomes into the cytosol.
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Mycobacterium avium is the most common nontuberculous mycobacterium (NTM) species causing infectious disease. Here, we characterized a M. avium infection model in zebrafish larvae, and compared it to M. marinum infection, a model of tuberculosis. M. avium bacteria are efficiently phagocytosed and frequently induce granuloma-like structures in zebrafish larvae. Although macrophages can respond to both mycobacterial infections, their migration speed is faster in infections caused by M. marinum. Tlr2 is conservatively involved in most aspects of the defense against both mycobacterial infections. However, Tlr2 has a function in the migration speed of macrophages and neutrophils to infection sites with M. marinum that is not observed with M. avium. Using RNAseq analysis, we found a distinct transcriptome response in cytokine-cytokine receptor interaction for M. avium and M. marinum infection. In addition, we found differences in gene expression in metabolic pathways, phagosome formation, matrix remodeling, and apoptosis in response to these mycobacterial infections. In conclusion, we characterized a new M. avium infection model in zebrafish that can be further used in studying pathological mechanisms for NTM-caused diseases.
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Infecções por Mycobacterium não Tuberculosas , Micobactérias não Tuberculosas , Animais , Peixe-Zebra , Receptor 2 Toll-Like , Imunidade Inata , LarvaRESUMO
Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.
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Granuloma/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/crescimento & desenvolvimento , Fator 88 de Diferenciação Mieloide/metabolismo , Tuberculose/microbiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/microbiologia , Animais , Animais Geneticamente Modificados , Carga Bacteriana , Modelos Animais de Doenças , Granuloma/genética , Granuloma/metabolismo , Granuloma/patologia , Concentração de Íons de Hidrogênio , Leucócitos/metabolismo , Leucócitos/microbiologia , Leucócitos/ultraestrutura , Lisossomos/metabolismo , Lisossomos/microbiologia , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/metabolismo , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/ultraestrutura , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Teleost fish embryos are protected by two acellular membranes against particulate pollutants that are present in the water column. These membranes provide an effective barrier preventing particle uptake. In this study, we tested the hypothesis that the adsorption of antimicrobial titanium dioxide nanoparticles onto zebrafish eggs nevertheless harms the developing embryo by disturbing early microbial colonization. Zebrafish eggs were exposed during their first day of development to 2, 5 and 10 mg TiO2â¯L-1 (NM-105). Additionally, eggs were exposed to gold nanorods to assess the effectiveness of the eggs' membranes in preventing particle uptake, localizing these particles by way of two-photon microscopy. This confirmed that particles accumulate onto zebrafish eggs, without any detectable amounts of particles crossing the protective membranes. By way of particle-induced X-ray emission analysis, we inferred that the titanium dioxide particles could cover 25-45 % of the zebrafish egg surface, where the concentrations of sorbed titanium correlated positively with concentrations of potassium and correlated negatively with concentrations of silicon. A combination of imaging and culture-based microbial identification techniques revealed that the adsorbed particles exerted antimicrobial effects, but resulted in an overall increase of microbial abundance, without any change in heterotrophic microbial activity, as inferred based on carbon substrate utilization. This effect persisted upon hatching, since larvae from particle-exposed eggs still comprised higher microbial abundance than larvae that hatched from control eggs. Notably, pathogenic aeromonads tolerated the antimicrobial properties of the nanoparticles. Overall, our results show that the adsorption of suspended antimicrobial nanoparticles on aquatic eggs can have cascading effects across different life stages of oviparous animals. Our study furthermore suggests that aggregation dynamics may occur that could facilitate the dispersal of pathogenic bacteria through aquatic ecosystems.
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Certain Aspergillus species such as Aspergillus flavus and A. parasiticus are well known for the formation of sclerotia. These developmental structures are thought to act as survival structures during adverse environmental conditions but are also a prerequisite for sexual reproduction. We previously described an A. niger mutant (scl-2) which formed sclerotium-like structures, suggesting a possible first stage of sexual development in this species. Several lines of evidence presented in this study support the previous conclusion that the sclerotium-like structures of scl-2 are indeed sclerotia. These included the observations that: (i) safranin staining of the sclerotia-like structures produced by the scl-2 mutant showed the typical cellular structure of a sclerotium; (ii) metabolite analysis revealed specific production of indoloterpenes, which have previously been connected to sclerotium formation; (iii) formation of the sclerotium-like structures is dependent on a functional NADPH complex, as shown for other fungi forming sclerotia. The mutation in scl-2 responsible for sclerotium formation was identified using parasexual crossing and bulk segregant analysis followed by high throughput sequencing and subsequent complementation analysis. The scl-2 strain contains a mutation that introduces a stop codon in the putative DNA binding domain of a previously uncharacterized Zn(II)2Cys6 type transcription factor (An08g07710). Targeted deletion of this transcription factor (sclB) confirmed its role as a repressor of sclerotial formation and in the promotion of asexual reproduction in A. niger. Finally, a genome-wide transcriptomic comparison of RNA extracted from sclerotia versus mycelia revealed major differences in gene expression. Induction of genes related to indoloterpene synthesis was confirmed and also let to the identification of a gene cluster essential for the production of aurasperones during sclerotium formation. Expression analysis of genes encoding other secondary metabolites, cell wall related genes, transcription factors, and genes related to reproductive processes identified many interesting candidate genes to further understand the regulation and biosynthesis of sclerotia in A. niger. The newly identified SclB transcription factor acts as a repressor of sclerotium formation and manipulation of sclB may represent a first prerequisite step towards engineering A. niger strains capable of sexual reproduction. This will provide exciting opportunities for further strain improvement in relation to protein or metabolite production in A. niger.
Assuntos
Aspergillus niger/genética , Proteínas Fúngicas/genética , Micélio/genética , Fatores de Transcrição/genética , Aspergillus niger/patogenicidade , Mutação/genética , Micélio/crescimento & desenvolvimento , Domínios Proteicos/genética , Reprodução Assexuada/genética , Esporos Fúngicos/genética , Zinco/químicaRESUMO
Background: We previously described that adenoid tissue in children with chronic otitis media (COM) contained more mucosal biofilms than adenoid tissue removed for hypertrophy.Aims/objectives: The aim of the second part was to characterize nasopharyngeal microbiota and explore virulence of the most common middle ear pathogens.Material and methods: Bacteriological analysis was performed following a culture-based approach on the samples recovered from 30 patients of COM group (15 biofilm-positive and 15 biofilm-negative) and from 30 patients of a control group (15 biofilm-positive and 15 biofilm-negative). Virulence factors of Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae were investigated.Results: The most frequent species were Firmicutes followed by Proteobacteria and Actinobacteria. The presence of biofilm was statistically associated with an increase of the number of bacterial species and Firmicutes phylum regardless of the condition (case/control). No virulence factors associated with invasive isolates were found for the most common middle ear pathogens.Conclusions and significance: This case-control study demonstrated that the presence of COM plus biofilm was associated with a given microbiota which contained more Firmicutes. Our study allows a better understanding of physiopathological mechanisms involved in chronic otitis media and paves the way for further investigations.
Assuntos
Tonsila Faríngea/microbiologia , Bactérias/isolamento & purificação , Otite Média/microbiologia , Análise de Variância , Biofilmes , Estudos de Casos e Controles , Criança , Doença Crônica , Farmacorresistência Bacteriana , Firmicutes/isolamento & purificação , Haemophilus influenzae/isolamento & purificação , Haemophilus influenzae/virologia , Humanos , Testes de Sensibilidade Microbiana , Nasofaringe/microbiologia , Staphylococcus aureus/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/virologia , Streptococcus pyogenes/isolamento & purificação , Streptococcus pyogenes/virologiaRESUMO
BACKGROUND: Biofilms are communities of bacteria embedded in a self-produced glycocalyx matrix. Adenoids have been shown to harbor bacterial biofilms. Aim/objectives: To compare the prevalence of biofilms in adenoid of children with chronic otitis media (COM) (group1) versus a control group without any COM (group 2) having adenoids removed because of hypertrophy. MATERIAL AND METHODS: One hundred and three children were prospectively enrolled in this case-control study, group 1 (n = 52) and group 2 (n = 51). The main outcome measurement was the prevalence of biofilm in adenoidectomy specimens analyzed using confocal laser scanning microscopy. Children in group 1 who had middle ear (ME) effusion and requiring the insertion of a tympanostomy tube underwent biopsy of the ME mucosa and effusion sampling. RESULTS: Biofilms were found in adenoids' specimens of both groups and in the ME biopsy and effusion. The biofilm prevalence in adenoids was 63.5% (33/52) in group 1 and 47.1% (24/51) in group 2. Day nursery and previous antibiotics intake were significantly more frequent in group 1 than in group 2. CONCLUSIONS AND SIGNIFICANCE: This case-control study demonstrates that adenoid tissue in children with COM contains more mucosal biofilms than adenoid tissue removed for hypertrophy. Biofilm was seen in ME biopsies and effusion.
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Tonsila Faríngea/microbiologia , Biofilmes , Orelha Média/microbiologia , Otite Média/microbiologia , Adenoidectomia , Estudos de Casos e Controles , Criança , Pré-Escolar , Doença Crônica , Humanos , Lactente , Otite Média/cirurgia , Estudos Prospectivos , Fatores de RiscoRESUMO
Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection. Abbreviations: ATG: autophagy related gene; BECN1: Beclin 1; CFU: colony forming units; CYBA/P22PHOX: cytochrome b-245, alpha chain; CYBB/NOX2: cytochrome b-245 beta chain; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hfp: hours post fertilization; hpi: hours post infection; IRF8: interferon regulatory factor 8; Lcp1/L-plastin: lymphocyte cytosolic protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1A/1B-light chain 3; mCherry: red fluorescent protein; mpeg1: macrophage expressed gene 1; mpx: myeloid specific peroxidase; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX: neutrophil cytosolic factor 4; NTR-mCherry: nitroreductase-mCherry fusion; PTU: phenylthiourea; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB-1 inducible coiled coin 1; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; RUBCN/RUBICON: RUN and cysteine rich domain containing BECN1-interacting protein; SCV: Salmonella-containing vacuole; S. Typhimurium/S.T: Salmonella enterica serovar Typhimurium; TEM: transmission electron microscopy; Tg: transgenic; TSA: tyramide signal amplification; ULK1/2: unc-51-like autophagy activating kinase 1/2; UVRAG: UVRAG: UV radiation resistance associated; wt: wild type.
Assuntos
Modelos Animais de Doenças , Macrófagos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Fagocitose/genética , Salmonelose Animal , Salmonella typhimurium/imunologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Autofagia/fisiologia , Bacteriemia/genética , Bacteriemia/imunologia , Bacteriemia/microbiologia , Bacteriemia/patologia , Embrião não Mamífero , Proteínas Associadas aos Microtúbulos/genética , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismo , Salmonelose Animal/genética , Salmonelose Animal/imunologia , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The original version of this Article contained an error in the spelling of the author Shuxin Yang, which was incorrectly given as Shuxing Yang. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMO
Host pathways mediating changes in immune states elicited by intestinal microbial colonization are incompletely characterized. Here we describe alterations of the host immune state induced by colonization of germ-free zebrafish larvae with an intestinal microbial community or single bacterial species. We show that microbiota-induced changes in intestinal leukocyte subsets and whole-body host gene expression are dependent on the innate immune adaptor gene myd88. Similar patterns of gene expression are elicited by colonization with conventional microbiome, as well as mono-colonization with two different zebrafish commensal bacterial strains. By studying loss-of-function myd88 mutants, we find that colonization suppresses Myd88 at the mRNA level. Tlr2 is essential for microbiota-induced effects on myd88 transcription and intestinal immune cell composition.
Assuntos
Microbioma Gastrointestinal/imunologia , Imunidade Inata , Mucosa Intestinal/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Genes Reporter , Fator 88 de Diferenciação Mieloide/genética , Receptor 2 Toll-Like/metabolismo , Transcriptoma , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Protein delivery into the cytosol of cells is a challenging topic in the field of nanomedicine, because cellular uptake and endosomal escape are typically inefficient, hampering clinical applications. In this contribution cuboidal mesoporous silica nanoparticles (MSNs) containing disk-shaped cavities with a large pore diameter (10 nm) are studied as a protein delivery vehicle using cytochrome-c (cytC) as a model membrane-impermeable protein. To ensure colloidal stability, the MSNs are coated with a fusogenic lipid bilayer (LB) and cellular uptake is induced by a complementary pair of coiled-coil (CC) lipopeptides. Coiled-coil induced membrane fusion leads to the efficient cytosolic delivery of cytC and triggers apoptosis of cells. Delivery of these LB coated MSNs in the presence of various endocytosis inhibitors strongly suggests that membrane fusion is the dominant mechanism of cellular uptake. This method is potentially a universal way for the efficient delivery of any type of inorganic nanoparticle or protein into cells mediated by CC induced membrane fusion.
Assuntos
Materiais Revestidos Biocompatíveis/química , Bicamadas Lipídicas/química , Nanopartículas/química , Dióxido de Silício/química , Apoptose/efeitos dos fármacos , Citocromos c/química , Citocromos c/metabolismo , Citocromos c/toxicidade , Citosol/metabolismo , Endocitose , Células HeLa , Humanos , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Fusão de Membrana , Microscopia Confocal , Tamanho da Partícula , PorosidadeRESUMO
Macrophages and neutrophils are the first responders to invading pathogens and contribute strongly to the host defense against intracellular pathogens. The collective interplay and dynamic interactions between these leukocytes are to a large extent not understood. In the present study, we have investigated their role using a combination of confocal laser-scanning and electron microscopy in a zebrafish model for tuberculosis, a local Mycobacterium marinum infection in the tissue of the larval tail fin. Our results show that neutrophils are efficient in phagocytosis of mycobacteria and that they contribute largely to their dissemination. Macrophages appear to play a major role in efferocytosis, phagocytosis of dead cells that contain bacterial content. Phagocytic cells with large bacterial aggregates are formed that can be extruded out of the tissue after cell death. Alternatively, these excessively infected cells can undergo necrosis leading to immediate recruitment of surrounding leukocytes and subsequent phagocytosis of released bacteria. Our data show that these necrotic burst events result in progression of the infection, whereas extrusion abates the infection.
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Leucócitos/microbiologia , Leucócitos/patologia , Mycobacterium/fisiologia , Fagocitose , Peixe-Zebra/microbiologia , Animais , Morte Celular , Movimento Celular , Humanos , Imageamento Tridimensional , Larva/microbiologia , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Modelos Biológicos , Mycobacterium/ultraestrutura , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Neutrófilos/ultraestruturaRESUMO
In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophila melanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects. Using immunohistochemistry, live imaging and transmission electron microscopy, we describe several striking differences to cellularization in Drosophila, including the formation of junctions between the forming basal membrane and the yolk plasmalemma. To identify the nature of this novel junction, we used the parental RNAi technique for a small-scale screen of junction proteins. We find that maternal knockdown of Tribolium innexin7a (Tc-inx7a), an ortholog of the Drosophila gap junction gene Innexin 7, leads to failure of cellularization. In Inx7a-depleted eggs, the invaginated plasma membrane retracts when basal cell closure normally begins. Furthermore, transiently expressed tagged Inx7a localizes to the nascent basal membrane of the forming cells in wild-type eggs. We propose that Inx7a forms the newly identified junctions that stabilize the forming basal membrane and enable basal cell closure. We put forward Tribolium as a model for studying a more ancestral mode of cellularization in insects.
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Blastoderma/embriologia , Conexinas/metabolismo , Proteínas de Drosophila/metabolismo , Junções Intercelulares/metabolismo , Tribolium/embriologia , Animais , Membrana Celular/fisiologia , Conexinas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Microtúbulos/metabolismo , Morfogênese/genética , Interferência de RNA , RNA Interferente PequenoRESUMO
Insects have been extraordinary successful in colonizing terrestrial habitats and this success is partly due to a protective cuticle that mainly contains chitin and proteins. The cuticle has been well studied in larvae and adults, but little attention has been paid to the cuticle of the egg. This cuticle is secreted by the serosa, an extraembryonic epithelium that surrounds the yolk and embryo in all insect eggs, but was lost in the Schizophoran flies to which Drosophila belongs. We therefore set out to investigate serosal cuticle formation and function in a beetle (Tribolium castaneum) using RNAi-mediated knockdown of three candidate genes known to structure chitin in the adult cuticle, and we aimed to identify other serosal cuticle genes using RNA sequencing. Knockdown of Knickkopf (TcKnk-1) or Retroactive (TcRtv) affects the laminar structure of the serosal cuticle, as revealed by Transmission Electron Microscopy in knockdown eggs. In the absence of this laminar structure, significantly fewer eggs survive at low humidity compared to wild-type eggs. Survival in dry conditions is also adversely affected when cross-linking among proteins and chitin is prevented by Laccase2 (TcLac-2) RNAi. Finally, we compare the transcriptomes of wild-type eggs to serosa-less eggs and find serosa-biased expression of 21 cuticle-related genes including structural components, chitin deacetylases and chitinases. Our data indicate that the serosal cuticle utilizes the same machinery for structuring the cuticle as adults. We demonstrate that the structure of the cuticle is crucial for desiccation resistance, and we put forward the serosal cuticle of Tribolium as an excellent model to study the ecological properties of the insect cuticle.
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Proteínas de Insetos/fisiologia , Tribolium/fisiologia , Animais , Dessecação , Feminino , Expressão Gênica , Umidade , Masculino , Óvulo/fisiologia , Óvulo/ultraestrutura , Interferência de RNA , Análise de Sequência de RNA , Membrana Serosa/metabolismo , Tribolium/ultraestruturaRESUMO
Development and patterning of neural tissue in the vertebrate embryo involves a set of molecules and processes whose relationships are not fully understood. Classical embryology revealed a remarkable phenomenon known as vertical signalling, a gastrulation stage mechanism that copies anterior-posterior positional information from mesoderm to prospective neural tissue. Vertical signalling mediates unambiguous copying of complex information from one tissue layer to another. In this study, we report an investigation of this process in recombinates of mesoderm and ectoderm from gastrulae of Xenopus laevis. Our results show that copying of positional information involves non cell autonomous autoregulation of particular Hox genes whose expression is copied from mesoderm to neurectoderm in the gastrula. Furthermore, this information sharing mechanism involves unconventional translocation of the homeoproteins themselves. This conserved primitive mechanism has been known for three decades but has only recently been put into any developmental context. It provides a simple, robust way to pattern the neurectoderm using the Hox pattern already present in the mesoderm during gastrulation. We suggest that this mechanism was selected during evolution to enable unambiguous copying of rather complex information from cell to cell and that it is a key part of the original ancestral mechanism mediating axial patterning by the highly conserved Hox genes.
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Padronização Corporal/fisiologia , Comunicação Celular/fisiologia , Gástrula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/fisiologia , Placa Neural/fisiologia , Xenopus laevis/embriologia , Animais , Evolução Biológica , Primers do DNA/genética , Drosophila , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Discos Imaginais/crescimento & desenvolvimento , FaloidinaRESUMO
High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 µm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases.
Assuntos
Autofagia , Microscopia Eletrônica/métodos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/fisiologia , Peixe-Zebra/microbiologia , Nadadeiras de Animais/microbiologia , Nadadeiras de Animais/patologia , Nadadeiras de Animais/ultraestrutura , Animais , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/metabolismo , Larva/ultraestrutura , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Mycobacterium marinum/ultraestrutura , Proteínas de Peixe-Zebra/metabolismoRESUMO
Autophagy is an important defense mechanism against mycobacteria, the causative agents of tuberculosis. The molecular mechanisms that link mycobacterial recognition to autophagy remain unclear. Our analysis in zebrafish and human macrophage models of mycobacterial infection reveals that the DNA damage-regulated autophagy modulator DRAM1 functions downstream of pathogen recognition by the Toll-like receptor (TLR)/interleukin-1 receptor (IL1R)-MYD88-NF-κB innate immune sensing pathway to activate selective autophagy. Mycobacterial infection of human macrophages and zebrafish embryos induced DRAM1 expression in a MYD88 and NF-κB-dependent manner. DRAM1 knockdown increased mycobacterial infection, whereas overexpression lowered infection by hyperactivating autophagy. DRAM1-mediated selective autophagic defenses require the cytosolic DNA sensor STING and the selective autophagy receptor p62/SQSTM1. Contrary to its known role in autophagy-mediated cell death and cancer, this DRAM1 function is p53 independent. We propose that DRAM1 mediates autophagic defense against a broader range of intracellular pathogens, since DRAM1 expression was also induced by the common bacterial endotoxin lipopolysaccharide.