RESUMO
The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. We therefore systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. Our studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.
Assuntos
Biomimética , Montagem e Desmontagem da Cromatina , Ciclo Celular , Cromatina , DNA , Replicação do DNA , Complexo de Reconhecimento de Origem/metabolismo , Proteoma , Animais , Drosophila , Embrião não Mamífero/química , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismoRESUMO
Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are ubiquitous contaminants that can be formed through oxidation of parent PAHs. Our previous studies found 2-hydroxychrysene (2-OHCHR) to be significantly more toxic to Japanese medaka embryos than 6-hydroxychrysene (6-OHCHR), an example of regioselective toxicity. We have also previously identified a sensitive developmental window to 2-OHCHR toxicity that closely coincided with liver development, leading us to hypothesize that differences in metabolism may play a role in the regioselective toxicity. To test this hypothesis, Japanese medaka embryos were treated with each isomer for 24 h during liver development (52-76 hpf). Although 6-OHCHR was absorbed 97.2 ± 0.18% faster than 2-OHCHR, it was eliminated 57.7 ± 0.36% faster as a glucuronide conjugate. Pretreatment with cytochrome P450 inhibitor, ketoconazole, reduced anemia by 96.8 ± 3.19% and mortality by 95.2 ± 4.76% in 2-OHCHR treatments. Formation of chrysene-1,2-diol (1,2-CAT) was also reduced by 64.4 ± 2.14% by ketoconazole pretreatment. While pretreatment with UDP-glucuronosyltransferase inhibitor, nilotinib, reduced glucuronidation of 2-OHCHR by 52.4 ± 2.55% and of 6-OHCHR by 63.7 ± 3.19%, it did not alter toxicity for either compound. These results indicate that CYP-mediated activation, potentially to 1,2-CAT, may explain the isomeric differences in developmental toxicity of 2-OHCHR.
Assuntos
Oryzias , Hidrocarbonetos Policíclicos Aromáticos , Poluentes Químicos da Água , Animais , Oryzias/fisiologia , Cetoconazol/metabolismo , Poluentes Químicos da Água/toxicidade , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidadeRESUMO
Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.
Assuntos
Trifosfato de Adenosina/metabolismo , Bombyx/metabolismo , Metabolismo Energético , Homeostase , Animais , Bombyx/embriologia , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Regulação para CimaRESUMO
Isotopic values of two Caribbean sharpnose shark Rhizoprionodon porosus litters (Poey, 1861) with two and three embryos and one litter of 11 smalltail shark Carcharhinus porosus embryos showed enriched 15 N and 13 C compared to their mothers. In R. porosus, embryonic isotope values were 3.06 ± 0.07 and 0.69 ± 0.15 greater than their mothers' for δ15 N and δ13 C, respectively, whereas in C. porosus, δ15 N and δ13 C were 1.79 ± 0.09 and 1.31 ± 0.17 greater in embryos than their mothers.
Assuntos
Isótopos de Carbono/análise , Embrião não Mamífero/metabolismo , Isótopos de Nitrogênio/análise , Tubarões/embriologia , Animais , Região do Caribe , Embrião não Mamífero/química , Tubarões/metabolismoRESUMO
Controllably activating the bio-reactivity of metal complexes in living systems is challenging but highly desirable because it can minimize off-target bindings and improve spatiotemporal specificity. Herein, we report a new bioorthogonal activation approach by employing Pd(II)-triggered transmetallation reactions to conditionally activate the bio-reactivity of NHC-Au(I)-phenylacetylide complexes (1 a) inâ vitro and inâ vivo. A combination of 1 Hâ NMR, LC-MS, DFT calculation and fluorescence screening assays reveals that 1 a displays a reasonable stability against biological thiols, but its phenylacetylide ligand can be efficiently transferred to Pd(II), leading to inâ situ formation of labile NHC-Au(I) species that is catalytically active inside living cells and zebrafish, and can meanwhile effectively suppress the activity of thioredoxin reductase, potently inhibit the proliferation of cancer cells and efficiently suppress angiogenesis in zebrafish models.
Assuntos
Complexos de Coordenação/química , Ouro/química , Alcinos/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Teoria da Densidade Funcional , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Humanos , Metano/análogos & derivados , Metano/química , Imagem Óptica , Paládio/química , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
The multifunctional insect cuticle serves as the exoskeleton, determines body shape, restricts water loss, provides attachment sites for muscles and internal organs and is a formidable barrier to invaders. It is morphologically divided into three layers, including envelope, epicuticle, and procuticle and is composed of chitin and cuticular proteins (CPs). Annotation of CPs and their cognate genes may help understand the structure and functions of insect cuticles. In this paper, we interrogated the genome of Pteromalus puparum, an endoparasitoid wasp that parasitizes Pieris rapae and Papilio xuthus pupae, and identified 82 genes encoding CPs belonging to six CP families, including 62 in the CPR family, 8 in CPAP3, 5 in CPF/CPFL, 2 low complexity proteins, 2 in TWDL, and 3 in Apidermin. We used six RNA-seq libraries to determine CP gene expression profiles through development and compared the cuticle hydrophobicity between the P. puparum and the ectoparasitoid Nasonia vitripennis based on GRAVY values of CPR sequences. In the Nasonia-Pteromalus comparison, we found in both N. vitripennis and P. puparum, the peak of their CPR hydrophobicity displayed at their pupal stage, whereas their adult stage showed the lowest level. Except at the adult stage, the CPR hydrophobicity in N. vitripennis is always higher than P. puparum. Finally, we identified three novel Apidermin genes, a family found solely in Hymenoptera and revealed a new sequence feature of this family. This new information contributes to a broader understanding of insect CPs generally.
Assuntos
Proteínas de Insetos/genética , Vespas/genética , Sequência de Aminoácidos , Animais , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Filogenia , Pupa/genética , Pupa/metabolismo , Transcriptoma , Vespas/crescimento & desenvolvimento , Vespas/metabolismoRESUMO
Pteromalus puparum is a gregarious pupal endoparasitoid with a wide host range. It deposits eggs into pierid and papilionid butterfly pupae. Glutathione S-transferases (GSTs) are a family of multifunctional detoxification enzymes that act in xenobiotic metabolism in insects. Insect genome projects have facilitated identification and characterization of GST family members. We identified 20 putative GSTs in the P. puparum genome, including 19 cytosolic and one microsomal. Phylogenetic analysis showed that P. puparum GSTs are clustered into Hymenoptera-specific branches. Transcriptomic data of embryos, larvae, female pupae, male pupae, female adults, male adults, venom glands, carcass, salivary glands, and ovaries revealed stage-, sex-, and tissue-specific expression patterns of GSTs in P. puparum. This is the most comprehensive study of genome-wide identification, characterization, and expression profiling of GST family in hymenopterans. Our results provide valuable information for understanding the metabolic adaptation of this wasp.
Assuntos
Glutationa Transferase/genética , Proteínas de Insetos/genética , Vespas/genética , Sequência de Aminoácidos , Animais , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Feminino , Perfilação da Expressão Gênica , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Filogenia , Pupa/genética , Pupa/metabolismo , Alinhamento de Sequência , Vespas/crescimento & desenvolvimento , Vespas/metabolismoRESUMO
Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT7 genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.
Assuntos
Aminas Biogênicas/metabolismo , Borboletas/genética , Proteínas de Insetos/genética , Vespas/genética , Sequência de Aminoácidos , Animais , Borboletas/química , Borboletas/metabolismo , Borboletas/parasitologia , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Feminino , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Filogenia , Pupa/genética , Pupa/metabolismo , Alinhamento de Sequência , Vespas/enzimologia , Vespas/crescimento & desenvolvimento , Vespas/metabolismoRESUMO
In insects, neuropeptides constitute a group of signaling molecules that act in regulation of multiple physiological and behavioral processes by binding to their corresponding receptors. On the basis of the bioinformatic approaches, we screened the genomic and transcriptomic data of the parasitoid wasp, Pteromalus puparum, and annotated 36 neuropeptide precursor genes and 33 neuropeptide receptor genes. Compared to the number of precursor genes in Bombyx mori (Lepidoptera), Chilo suppressalis (Lepidoptera), Drosophila melanogaster (Diptera), Nilaparvata lugens (Hemiptera), Apis mellifera (Hymenoptera), and Tribolium castaneum (Coleoptera), P. puparum (Hymenoptera) has the lowest number of neuropeptide precursor genes. This lower number may relate to its parasitic life cycle. Transcriptomic data of embryos, larvae, pupae, adults, venom glands, salivary glands, ovaries, and the remaining carcass revealed stage-, sex-, and tissue-specific expression patterns of the neuropeptides, and their receptors. These data provided basic information about the identity and expression profiles of neuropeptides and their receptors that are required to functionally address their biological significance in an endoparasitoid wasp.
Assuntos
Proteínas de Insetos/genética , Neuropeptídeos/genética , Receptores de Neuropeptídeos/genética , Vespas/genética , Sequência de Aminoácidos , Animais , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Filogenia , Pupa/genética , Pupa/metabolismo , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/metabolismo , Alinhamento de Sequência , Vespas/crescimento & desenvolvimento , Vespas/metabolismoRESUMO
Parasitoids serve as effective biocontrol agents for agricultural pests. However, they face constant challenges from host immune defense and numerous pathogens and must develop potent immune defense against these threats. Despite the recent advances in innate immunity, little is known about the immunological mechanisms of parasitoids. Here, we identified and characterized potential immune-related genes of the endoparasitoid, Pteromalus puparum, which act in regulating populations of some members of the Pieridae. We identified 216 immune-related genes based on interrogating the P. puparum genome and transcriptome databases. We categorized the cognate gene products into recognition molecules, signal moieties and effector proteins operating in four pathways, Toll, IMD, JAK/STAT, and JNK. Comparative analyses of immune-related genes from seven insect species indicate that recognition molecules and effector proteins are more expanded and diversified than signaling genes in these signal pathways. There are common 1:1 orthologs between the endoparasitoid P. puparum and its relative, the ectoparasitoid Nasonia vitripennis. The developmental expression profiles of immune genes randomly selected from the transcriptome analysis were verified by a quantitative polymerase chain reaction. Our work provides comprehensive analyses of P. puparum immune genes, some of which may be exploited in advancing parasitoid-based biocontrol technologies.
Assuntos
Proteínas de Transporte/genética , Imunidade Inata/genética , Proteínas de Insetos/genética , Transdução de Sinais/imunologia , Vespas/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Filogenia , Pupa/genética , Pupa/metabolismo , Alinhamento de Sequência , Vespas/crescimento & desenvolvimento , Vespas/metabolismo , Vespas/fisiologiaRESUMO
Many aquatic animals rely on chemicals released by injured individuals of the same species to assess predation risk. Among these chemical cues, alarm substances released from the injured skin of ostariophysan fishes have been extensively examined. In most fish species examined, these cues appear to be released by all injured individuals (including larvae, juveniles and adults) and elicit alarm responses in conspecifics. Adult alarm cues also affect development and physiology of embryos. Nonetheless, whether embryos produce alarm cues that affect adults is not known. This study reports that extracts of zebrafish (Danio rerio) embryos at 36 h post-fertilization or later induce antipredator behaviours reminiscent of those induced by skin alarm substances. At an equivalent of 10-6 g embryo per millilitre, the extract induced bottom-dwelling and freezing in adults. These behaviours are consistent with those induced by adult alarm substances. This study concludes that zebrafish embryos produce alarm substances.
Assuntos
Misturas Complexas/química , Sinais (Psicologia) , Embrião não Mamífero/química , Peixe-Zebra/fisiologia , AnimaisRESUMO
Stable nitrogen (δ15 N) and carbon (δ13 C) isotope ratios from muscle, liver and yolk were analysed from the mother and embryos of an ovoviviparous shark, Hexanchus griseus. Embryonic liver and muscle had similar δ15 N and δ13 C ratios or were depleted in heavy isotopes, compared to the same maternal somatic and reproductive yolk tissues, but no relationship existed between δ15 N or δ13 C and embryo length, as expected, because a switch to placental nourishment is lacking in this species. This study expands the understanding of maternal nourishment and embryonic stable isotope differences in ovoviviparous sharks.
Assuntos
Isótopos de Carbono/análise , Isótopos de Nitrogênio/análise , Tubarões/fisiologia , Animais , Embrião não Mamífero/química , Feminino , Fígado/química , Músculos/química , OvoviviparidadeRESUMO
Studies conducted on Zebrafish embryos in our laboratory have allowed for the identification of precise moments of organogenesis in which a lot of genes are switched on and off, a sign that the genome is undergoing substantial changes in gene expression. Stem cell growth and differentiation stage-factors present in different moments of organogenesis have proven to have different specific functions in gene regulation. The substances present in the first stages of cell differentiation in Zebrafish embryos have demonstrated an ability to counteract the senescence of stem cells, reducing the expression of the beta-galactosidase marker, enhancing the genes Oct-4, Sox-2, c-Myc, TERT, and the transcription of Bmi-1, which act as key telomerase-independent repressors of cell aging. The molecules present in the intermediate to late stages of cell differentiation have proven to be able to reprogram pathological human cells, such as cancer cells and those of the basal layer of the epidermis in psoriasis, which present a higher multiplication rate than normal cells. The factors present in all the stages of cell differentiation are able to counteract neurodegeneration, and to regenerate tissues: It has been possible to regenerate hair follicles in many patients with androgenetic alopecia through transdermal administration of stem cell differentiation stage factors (SCDSFs) by means of cryopass-laser.
Assuntos
Reprogramação Celular/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Organogênese/genética , Células-Tronco/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Tecido Adiposo/citologia , Administração Cutânea , Alopecia/tratamento farmacológico , Alopecia/patologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Pulmonar de Lewis/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Reprogramação Celular , Embrião não Mamífero/química , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Neoplasias Hepáticas/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Ensaios Clínicos Controlados Aleatórios como Assunto , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Resultado do Tratamento , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/isolamento & purificação , Proteínas de Peixe-Zebra/farmacologiaRESUMO
Label-free single-cell proteomics by mass spectrometry (MS) is currently incompatible with complex tissues without requiring cell culturing, single-cell dissection, or tissue dissociation. We here report the first example of label-free single-cell MS-based proteomics directly in single cells in live vertebrate embryos. Our approach integrates optically guided in situ subcellular capillary microsampling, one-pot extraction-digestion of the collected proteins, peptide separation by capillary electrophoresis, ionization by an ultrasensitive electrokinetically pumped nanoelectrospray, and detection by high-resolution MS (Orbitrap). With a 700 zmol (420â¯000 copies) lower limit of detection, this trace-sensitive technology confidently identified and quantified â¼750-800 protein groups (<1% false-discovery rate) by analyzing just â¼5 ng of protein digest, viz. <0.05% of the total protein content from individual cells in a 16-cell Xenopus laevis (frog) embryo. After validating the approach by recovering animal-vegetal-pole proteomic asymmetry in the frog zygote, the technology was applied to uncover proteomic reorganization as the animal-dorsal (D11) cell of the 16-cell embryo gave rise to its neural-tissue-fated clone in the embryo developing to the 32-, 64-, and 128-cell stages. In addition to enabling proteomics on smaller cells in X. laevis, we also demonstrated this technology to be scalable to single cells in live zebrafish embryos. Microsampling single-cell MS-based proteomics raises exciting opportunities to study cell and developmental processes directly in complex tissues and whole organisms at the level of the building block of life: the cell.
Assuntos
Proteômica , Análise de Célula Única , Proteínas de Xenopus/análise , Proteínas de Peixe-Zebra/análise , Animais , Células Clonais/química , Células Clonais/citologia , Eletroforese Capilar , Embrião não Mamífero/química , Embrião não Mamífero/citologia , Espectrometria de Massas , Xenopus laevis , Peixe-ZebraRESUMO
Techniques that allow single cell analysis are gaining widespread attention, and most of these studies utilize genomics-based approaches. While nanofluidic technologies have enabled mass spectrometric analysis of single cells, these measurements have been limited to metabolomics and lipidomic studies. Single cell proteomics has the potential to improve our understanding of intercellular heterogeneity. However, this approach has faced challenges including limited sample availability, as well as a requirement of highly sensitive methods for sample collection, cleanup, and detection. We present a technique to overcome these limitations by combining a micropipette (pulled glass capillary) based sample collection strategy with offline sample preparation and nanoLC-MS/MS to analyze proteins through a bottom-up proteomic strategy. This study explores two types of proteomics data acquisition strategies namely data-dependent (DDA) and data-independent acquisition (DIA). Results from the study indicate DIA to be more sensitive enabling analysis of >1600 proteins from â¼130 µm Xenopus laevis embryonic cells containing <6 nL of cytoplasm. The method was found to be robust in obtaining reproducible protein quantifications from single cells spanning the 1-128-cell stages of development. Furthermore, we used micropipette sampling to study intercellular heterogeneity within cells in a single embryo and investigated embryonic asymmetry along both animal-vegetal and dorsal-ventral axes during early stages of development. Investigation of the animal-vegetal axis led to discovery of various asymmetrically distributed proteins along the animal-vegetal axis. We have further compared the hits found from our proteomic data sets with other studies and validated a few hits using an orthogonal imaging technique. This study forms the first report of vegetal enrichment of the germ plasm associated protein DDX4/VASA in Xenopus embyos. Overall, the method and data presented here holds promise to enable important leads in developmental biology.
Assuntos
Embrião não Mamífero/citologia , Proteômica/métodos , Análise de Célula Única/métodos , Proteínas de Xenopus/análise , Xenopus laevis/embriologia , Animais , Embrião não Mamífero/química , Espectrometria de Massas em Tandem/métodosRESUMO
BACKGROUND: The ability to efficiently visualize and manipulate chromosomes is fundamental to understanding the genome architecture of organisms. Conventional chromosome preparation protocols developed for mammalian cells and those relying on species-specific conditions are not suitable for many invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate species, is needed. RESULTS: We optimized a chromosome preparation protocol and applied it to several planarian species (phylum Platyhelminthes), the freshwater apple snail Pomacea canaliculata (phylum Mollusca), and the starlet sea anemone Nematostella vectensis (phylum Cnidaria). We demonstrated that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with deionized water was sufficient for karyotyping; growing cells in culture was not necessary. The obtained karyotypes allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla this protocol could be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization. CONCLUSIONS: Our simple and inexpensive chromosome preparation protocol can be readily adapted to new invertebrate research organisms to accelerate the discovery of novel genomic patterns across the branches of the tree of life.
Assuntos
Cromossomos/genética , Cariotipagem/métodos , Planárias/genética , Anêmonas-do-Mar/genética , Caramujos/genética , Animais , Cromossomos/química , Embrião não Mamífero/química , Embrião não Mamífero/fisiologia , Invertebrados , Moluscos/química , Moluscos/genética , Planárias/química , Platelmintos/química , Platelmintos/genética , Anêmonas-do-Mar/química , Caramujos/químicaRESUMO
Some yet unidentified factors released by both oocyte and embryonic microenvironments demonstrated to be non-permissive for tumor development and display the remarkable ability to foster cell/tissue reprogramming, thus ultimately reversing the malignant phenotype. In the present study we observed how molecular factors extracted from Zebrafish embryos during specific developmental phases (20 somites) significantly antagonize proliferation of breast cancer cells, while reversing a number of prominent aspects of malignancy. Embryo extracts reduce cell proliferation, enhance apoptosis, and dramatically inhibit both invasiveness and migrating capabilities of cancer cells. Counteracting the invasive phenotype is a relevant issue in controlling tumor spreading and metastasis. Moreover, such effect is not limited to cancerous cells as embryo extracts were also effective in inhibiting migration and invasiveness displayed by normal breast cells undergoing epithelial-mesenchymal transition upon TGF-ß1 stimulation. The reversion program involves the modulation of E-cadherin/ß-catenin pathway, cytoskeleton remodeling with dramatic reduction in vinculin, as well as downregulation of TCTP and the concomitant increase in p53 levels. Our findings highlight that-contrary to the prevailing current "dogma", which posits that neoplastic cells are irreversibly "committed"-the malignant phenotype can ultimately be "reversed", at least partially, in response to environmental morphogenetic influences.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Embrião não Mamífero/química , Extratos de Tecidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo , Humanos , Fenótipo , Proteína Tumoral 1 Controlada por Tradução , Peixe-Zebra , beta Catenina/metabolismoRESUMO
Stem cells undergo senescence both in vivo, contributing to the progressive decline in self-healing mechanisms, and in vitro during prolonged expansion. Here, we show that an early developmental zebrafish embryo extract (ZF1) could act as a modulator of senescence in human mesenchymal stem cells (hMSCs) isolated from both adult tissues, including adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and a perinatal tissue such as the Wharton's Jelly (hWJ-MSCs). In all the investigated hMSCs, ZF1 decreased senescence-associated ß-galactosidase (SA ß-gal) activity and enhanced the transcription of TERT, encoding the catalytic telomerase core. In addition, it was associated, only in hASCs, with a transcriptional induction of BMI1, a pleiotropic repressor of senescence. In hBM-MSCs, hDP-MSCs, and hWJ-MSCs, TERT over-expression was concomitant with a down-regulation of two repressors of TERT, TP53 (p53), and CDKN1A (p21). Furthermore, ZF1 increased the natural ability of hASCs to perform adipogenesis. These results indicate the chance of using ZF1 to modulate stem cell senescence in a source-related manner, to be potentially used as a tool to affect stem cell senescence in vitro. In addition, its anti-senescence action could also set the basis for future in vivo approaches promoting tissue rejuvenation bypassing stem cell transplantation.
Assuntos
Senescência Celular , Embrião não Mamífero/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Extratos de Tecidos/farmacologia , Animais , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-ZebraRESUMO
Cadmium (Cd) is a toxic element widely distributed in the aquatic environment and producing a wide variety of harmful effects. In this study, the acute toxicity (96 h LC50) of Cd to rainbow trout Oncorhynchus mykiss embryos and larvae was determined. The obtained results showed that hatched larvae were the most sensitive to Cd exposure. After 4 days of exposure, embryos were found to have accumulated greater concentrations of Cd than larvae. Exposure to Cd at sublethal concentrations produced deleterious, exposure duration-related effects on biological parameters (mortality, heart rate and gill ventilation frequency) of larvae. Cd induced a significant elevation of all the studied geno- and cytotoxicity endpoints in larval erythroblasts.
Assuntos
Cádmio/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Larva/efeitos dos fármacos , Oncorhynchus mykiss/fisiologia , Poluentes Químicos da Água/toxicidade , Animais , Cádmio/análise , Embrião não Mamífero/química , Brânquias/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Larva/química , Dose Letal Mediana , Oncorhynchus mykiss/crescimento & desenvolvimento , Poluentes Químicos da Água/análiseRESUMO
De novo sequencing offers an alternative to database search methods for peptide identification from mass spectra. Since it does not rely on a predetermined database of expected or potential sequences in the sample, de novo sequencing is particularly appropriate for samples lacking a well-defined or comprehensive reference database. However, the low accuracy of many de novo sequence predictions has prevented the widespread use of the variety of sequencing tools currently available. Here, we present a new open-source tool, Postnovo, that postprocesses de novo sequence predictions to find high-accuracy results. Postnovo uses a predictive model to rescore and rerank candidate sequences in a manner akin to database search postprocessing tools such as Percolator. Postnovo leverages the output from multiple de novo sequencing tools in its own analyses, producing many times the length of amino acid sequence information (including both full- and partial-length peptide sequences) at an equivalent false discovery rate (FDR) compared to any individual tool. We present a methodology to reliably screen the sequence predictions to a desired FDR given the Postnovo sequence score. We validate Postnovo with multiple data sets and demonstrate its ability to identify proteins that are missed by database search even in samples with paired reference databases.