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1.
Pestic Biochem Physiol ; 159: 136-143, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400775

RESUMO

Cytochrome P450s are part of a super-gene family that has undergone gene duplication, divergence, over-expression and, in some cases, loss of function. One such case is the 91-R and 91-C strains of common origin, in Drosophila melanogaster, whereby 91-R (DDT resistant strain) overexpresses Cyp4p1 and Cyp4p2 and both genes are lost in 91-C (DDT susceptible strain). In this study, we used a comparative approach to demonstrate that transcription of Cyp4p1 and Cyp4p2 were constitutively up-regulated in the Drosophila melanogaster strain 91-R as compared to another DDT susceptible strain Canton-S which does not have a loss of function of these genes. Furthermore, significantly increased expression of Cyp4p1 and Cyp4p2 was induced in 91-R in response to sublethal DDT exposure, however, such induction did not occur in the DDT treated Canton-S. Additionally, fixed nucleotide variation within putative transcription factor binding sites of Cyp4p1 and Cyp4p2 promoters were observed between 91-R and Canton-S, however, their impact on transcription remains to be determined. Two GAL4/UAS transgenic strains with integrated heat shock-inducible Cyp4p1- or Cyp4p2-RNAi constructs within wild-type genetic backgrounds were developed. Following heat shock induction of Cyp4p1 and Cyp4p2 knockdown, these transgenic lines showed increased DDT mortality as compared to their corresponding non-heat shock controls. These results provide a functional link of Cyp4p1 and Cyp4p2 in conferring tolerance to DDT exposure.


Assuntos
DDT/farmacologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Inseticidas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/genética , Resistência a Inseticidas/genética
2.
Dokl Biochem Biophys ; 486(1): 224-228, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367827

RESUMO

TRF2 protein (TBP-related factor 2) can substitute for TBP forming alternative transcription initiation complexes on TATA-less promoters, including the promoters of histone H1 and piRNA clusters required for transposon repression. The Drosophilatrf2 gene codes for two isoforms: a "short" and a "long" one, in which the same short TRF2 sequence is preceded by a long N-terminal domain. Here, we demonstrated that the long TFR2 isoform has a greater functional activity than the short isoform by expressing each of them at a reduced rate under the endogenous promoters. Expression of the long isoform alone affects neither the flies' viability nor the sex ratio. Expression of the short isoform alone leads to the phenotype described for the trf2 gene insufficiency and derepression of transposable elements, that is, decreased viability, disturbance of homologous chromosome pairing and segregation, and apparent female-biased sex ratio.


Assuntos
Drosophila melanogaster/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Animais , Drosophila melanogaster/genética , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética
3.
Nat Commun ; 10(1): 2951, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273212

RESUMO

Epithelial-mesenchymal transition (EMT) is an essential process both in physiological and pathological contexts. Intriguingly, EMT is often associated with tissue invagination during development; however, the impact of EMT on tissue remodeling remain unexplored. Here, we show that at the initiation of the EMT process, cells produce an apico-basal force, orthogonal to the surface of the epithelium, that constitutes an important driving force for tissue invagination in Drosophila. When EMT is ectopically induced, cells starting their delamination generate an orthogonal force and induce ectopic folding. Similarly, during mesoderm invagination, cells undergoing EMT generate an apico-basal force through the formation of apico-basal structures of myosin II. Using both laser microdissection and in silico physical modelling, we show that mesoderm invagination does not proceed if apico-basal forces are impaired, indicating that they constitute driving forces in the folding process. Altogether, these data reveal the mechanical impact of EMT on morphogenesis.


Assuntos
Drosophila melanogaster/embriologia , Transição Epitelial-Mesenquimal , Epitélio/embriologia , Morfogênese , Animais , Polaridade Celular , Simulação por Computador , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Epitélio/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Modelos Moleculares , Miosina Tipo II/metabolismo
4.
Nat Cell Biol ; 21(6): 710-720, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160709

RESUMO

The capacity of stem cells to self-renew or differentiate has been attributed to distinct metabolic states. A genetic screen targeting regulators of mitochondrial dynamics revealed that mitochondrial fusion is required for the maintenance of male germline stem cells (GSCs) in Drosophila melanogaster. Depletion of Mitofusin (dMfn) or Opa1 led to dysfunctional mitochondria, activation of Target of rapamycin (TOR) and a marked accumulation of lipid droplets. Enhancement of lipid utilization by the mitochondria attenuated TOR activation and rescued the loss of GSCs that was caused by inhibition of mitochondrial fusion. Moreover, constitutive activation of the TOR-pathway target and lipogenesis factor Sterol regulatory element binding protein (SREBP) also resulted in GSC loss, whereas inhibition of SREBP rescued GSC loss triggered by depletion of dMfn. Our findings highlight a critical role for mitochondrial fusion and lipid homeostasis in GSC maintenance, providing insight into the potential impact of mitochondrial and metabolic diseases on the function of stem and/or germ cells.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Membrana/genética , Dinâmica Mitocondrial/genética , Células-Tronco/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Animais , Diferenciação Celular/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Homeostase , Metabolismo dos Lipídeos/genética , Masculino , Mitocôndrias/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Nicho de Células-Tronco/genética , Células-Tronco/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
5.
Genes Dev ; 33(13-14): 844-856, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31123065

RESUMO

The Piwi-interacting RNA (piRNA) pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialized machinery that converts long single-stranded precursors into small RNAs of ∼25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and the correct localization of the indispensable piRNA biogenesis factor Armitage (Armi). We found that Gasz and Daed interact with each other and likely provide a mitochondrial "anchoring platform" to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb bodies in a manner that depends on Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing; namely, how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Linhagem Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismo
6.
J Mass Spectrom ; 54(6): 557-566, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31038251

RESUMO

Matrix-assisted laser/desorption ionization imaging mass spectrometry (MALDI IMS) is an analytical technique for understanding the spatial distribution of biomolecules across a sample surface. Originally employed for mammalian tissues, this technology has been adapted to study specimens as diverse as microbes and cell cultures, food such as strawberries, and invertebrates including the vinegar fly Drosophila melanogaster. As an ideal model organism, Drosophila has brought greater understanding about conserved biological processes, organism development, and diseased states and even informed management practices of agriculturally and environmentally important species. Drosophila displays anatomically separated renal (Malpighian) tubules that are the physiological equivalent to the vertebrate nephron. Insect Malpighian tubules are also responsible for pesticide detoxification. In this article, we first describe an effective workflow and sample preparation method to study the phospholipid distribution of the Malpighian tubules that initially involves the manual microdissection of the tubules in saline buffer followed by a series of washes to remove excess salt and enhances the phospholipid signals prior to matrix deposition and IMS at 25-µm spatial resolution. We also established a complementary methodology for lipid IMS analysis of whole-body fly sections using a dual-polarity data acquisition approach at the same spatial resolution after matrix deposition by sublimation. Both procedures yield rich signal profiles from the major phospholipid classes. The reproducibility and high-quality results offered by these methodologies enable cohort studies of Drosophila through MALDI IMS.


Assuntos
Drosophila melanogaster/metabolismo , Túbulos de Malpighi/metabolismo , Fosfolipídeos/metabolismo , Animais , Imagem Molecular/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
7.
BMC Genomics ; 20(1): 356, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072326

RESUMO

BACKGROUND: Cadmium (Cd)-containing chemicals can cause serious damage to biological systems. In animals and plants, Cd exposure can lead to metabolic disorders or death. However, for the most part the effects of Cd on specific biological processes are not known. DNA methylation is an important mechanism for the regulation of gene expression. In this study we examined the effects of Cd exposure on global DNA methylation in a living organism by whole-genome bisulfite sequencing (WGBS) using Drosophila melanogaster as model. RESULTS: A total of 71 differentially methylated regions and 63 differentially methylated genes (DMGs) were identified by WGBS. A total of 39 genes were demethylated in the Cd treatment group but not in the control group, whereas 24 showed increased methylation in the former relative to the latter. In most cases, demethylation activated gene expression: genes such as Cdc42 and Mekk1 were upregulated as a result of demethylation. There were 37 DMGs that overlapped with differentially expressed genes from the digital expression library including baz, Act5C, and ss, which are associated with development, reproduction, and energy metabolism. CONCLUSIONS: DNA methylation actively regulates the physiological response to heavy metal stress in Drosophila in part via activation of apoptosis.


Assuntos
Cádmio/toxicidade , Metilação de DNA , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Genoma , Estresse Oxidativo , Animais , Drosophila melanogaster/efeitos dos fármacos , Feminino , Genômica , Sulfitos/química , Sequenciamento Completo do Genoma/métodos
8.
Nat Methods ; 16(6): 509-518, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133760

RESUMO

In mass-spectrometry-based proteomics, the identification and quantification of peptides and proteins heavily rely on sequence database searching or spectral library matching. The lack of accurate predictive models for fragment ion intensities impairs the realization of the full potential of these approaches. Here, we extended the ProteomeTools synthetic peptide library to 550,000 tryptic peptides and 21 million high-quality tandem mass spectra. We trained a deep neural network, termed Prosit, resulting in chromatographic retention time and fragment ion intensity predictions that exceed the quality of the experimental data. Integrating Prosit into database search pipelines led to more identifications at >10× lower false discovery rates. We show the general applicability of Prosit by predicting spectra for proteases other than trypsin, generating spectral libraries for data-independent acquisition and improving the analysis of metaproteomes. Prosit is integrated into ProteomicsDB, allowing search result re-scoring and custom spectral library generation for any organism on the basis of peptide sequence alone.


Assuntos
Aprendizado Profundo , Redes Neurais (Computação) , Fragmentos de Peptídeos/análise , Biblioteca de Peptídeos , Proteoma/análise , Software , Espectrometria de Massas em Tandem/métodos , Animais , Caenorhabditis elegans/metabolismo , Bases de Dados de Proteínas , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Fragmentos de Peptídeos/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo
9.
Molecules ; 24(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018591

RESUMO

Sonic hedgehog (Shh) signaling plays a tumor-promoting role in many epithelial cancers. Cancer cells produce soluble a Shh that signals to distant stromal cells that express the receptor Patched (Ptc). These receiving cells respond by producing other soluble factors that promote cancer cell growth, generating a positive feedback loop. To interfere with reinforced Shh signaling, we examined the potential of defined heparin and heparan sulfate (HS) polysaccharides to block Shh solubilization and Ptc receptor binding. We confirm in vitro and in vivo that proteolytic cleavage of the N-terminal Cardin-Weintraub (CW) amino acid motif is a prerequisite for Shh solubilization and function. Consistent with the established binding of soluble heparin or HS to the Shh CW target motif, both polysaccharides impaired proteolytic Shh processing and release from source cells. We also show that HS and heparin bind to, and block, another set of basic amino acids required for unimpaired Shh binding to Ptc receptors on receiving cells. Both modes of Shh activity downregulation depend more on HS size and overall charge than on specific HS sulfation modifications. We conclude that heparin oligosaccharide interference in the physiological roles of HS in Shh release and reception may be used to expand the field of investigation to pharmaceutical intervention of tumor-promoting Shh functions.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas Hedgehog/química , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Receptor Patched-1/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Heparina/química , Heparitina Sulfato/química , Humanos , Modelos Moleculares , Receptor Patched-1/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Solubilidade , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
10.
BMC Genomics ; 20(1): 309, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31014246

RESUMO

BACKGROUND: Trait loss is a pervasive phenomenon in evolution, yet the underlying molecular causes have been identified in only a handful of cases. Most of these cases involve loss-of-function mutations in one or more trait-specific genes. However, in parasitoid insects the evolutionary loss of a metabolic trait is not associated with gene decay. Parasitoids have lost the ability to convert dietary sugars into fatty acids. Earlier research suggests that lack of lipogenesis in the parasitoid wasp Nasonia vitripennis is caused by changes in gene regulation. RESULTS: We compared transcriptomic responses to sugar-feeding in the non-lipogenic parasitoid species Nasonia vitripennis and the lipogenic Drosophila melanogaster. Both species adjusted their metabolism within 4 hours after sugar-feeding, but there were sharp differences between the expression profiles of the two species, especially in the carbohydrate and lipid metabolic pathways. Several genes coding for key enzymes in acetyl-CoA metabolism, such as malonyl-CoA decarboxylase (mcd) and HMG-CoA synthase (hmgs) differed in expression between the two species. Their combined action likely blocks lipogenesis in the parasitoid species. Network-based analysis showed connectivity of genes to be negatively correlated to the fold change of gene expression. Furthermore, genes involved in the fatty acid metabolic pathway were more connected than the set of genes of all metabolic pathways combined. CONCLUSION: High connectivity of lipogenesis genes is indicative of pleiotropic effects and could explain the absence of gene degradation. We conclude that modification of expression levels of only a few little-connected genes, such as mcd, is sufficient to enable complete loss of lipogenesis in N. vitripennis.


Assuntos
Evolução Molecular , Lipogênese/genética , Vespas/genética , Vespas/metabolismo , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Pleiotropia Genética , Transcrição Genética , Vespas/fisiologia
11.
Genome Biol Evol ; 11(5): 1463-1482, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31028390

RESUMO

Previous studies of the evolution of genes expressed at different life-cycle stages of Drosophila melanogaster have not been able to disentangle adaptive from nonadaptive substitutions when using nonsynonymous sites. Here, we overcome this limitation by combining whole-genome polymorphism data from D. melanogaster and divergence data between D. melanogaster and Drosophila yakuba. For the set of genes expressed at different life-cycle stages of D. melanogaster, as reported in modENCODE, we estimate the ratio of substitutions relative to polymorphism between nonsynonymous and synonymous sites (α) and then α is discomposed into the ratio of adaptive (ωa) and nonadaptive (ωna) substitutions to synonymous substitutions. We find that the genes expressed in mid- and late-embryonic development are the most conserved, whereas those expressed in early development and postembryonic stages are the least conserved. Importantly, we found that low conservation in early development is due to high rates of nonadaptive substitutions (high ωna), whereas in postembryonic stages it is due, instead, to high rates of adaptive substitutions (high ωa). By using estimates of different genomic features (codon bias, average intron length, exon number, recombination rate, among others), we also find that genes expressed in mid- and late-embryonic development show the most complex architecture: they are larger, have more exons, more transcripts, and longer introns. In addition, these genes are broadly expressed among all stages. We suggest that all these genomic features are related to the conservation of mid- and late-embryonic development. Globally, our study supports the hourglass pattern of conservation and adaptation over the life-cycle.


Assuntos
Adaptação Biológica , Drosophila melanogaster/genética , Evolução Molecular , Estágios do Ciclo de Vida , Seleção Genética , Animais , Sequência de Bases , Sequência Conservada , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Perfilação da Expressão Gênica , Genoma de Inseto , Masculino
12.
Mol Genet Genomics ; 294(4): 1073-1083, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31006039

RESUMO

Nopp140, often called the nucleolar and Cajal body phosphoprotein (NOLC1), is an evolutionarily conserved chaperone for the transcription and processing of rRNA during ribosome subunit assembly. Metazoan Nopp140 contains an amino terminal LisH dimerization domain and a highly conserved carboxyl domain. A large central domain consists of alternating basic and acidic motifs of low sequence complexity. Orthologous versions of Nopp140 contain variable numbers of repeating basic-acidic units. While vertebrate Nopp140 genes use multiple exons to encode the central domain, the Nopp140 gene in Drosophila uses exclusively exon 2 to encode the central domain. Here, we define three overlapping repeat sequence patterns (P, P', and P″) within the central domain of D. melanogaster Nopp140. These repeat patterns are poorly conserved in other Drosophila species. We also describe a length polymorphism in exon 2 that pertains specifically to the P' pattern in D. melanogaster. The pattern displays either two or three 96 base pair repeats, respectively, referred to as Nopp140-Short and Nopp140-Long. Fly lines homozygous for one or the other allele, or heterozygous for both alleles, show no discernible phenotypes. PCR characterization of the long and short alleles shows a poorly defined, artifactual bias toward amplifying the long allele over the short allele. The significance of this polymorphism will be in discerning the largely unknown properties of Nopp140's large central domain in rDNA transcription and ribosome biogenesis.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Polimorfismo Genético , Animais , Drosophila melanogaster/genética , Éxons , Fenótipo , Domínios Proteicos , RNA Ribossômico/genética , Sequências Repetitivas de Ácido Nucleico
13.
Int J Mol Sci ; 20(8)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010135

RESUMO

The Drosophila homeodomain-interacting protein kinase (Hipk) is the fly representative of the well-conserved group of HIPKs in vertebrates. It was initially found through its characteristic interactions with homeodomain proteins. Hipk is involved in a variety of important developmental processes, such as the development of the eye or the nervous system. In the present study, we set Hipk and the Drosophila homeodomain proteins Homeobrain (Hbn), Empty spiracles (Ems), and Muscle segment homeobox (Msh) in an enzyme-substrate relationship. These homeoproteins are transcription factors that function during Drosophila neurogenesis and are, at least in part, conserved in vertebrates. We reveal a physical interaction between Hipk and the three homeodomain proteins in vivo using bimolecular fluorescence complementation (BiFC). In the course of in vitro phosphorylation analysis and subsequent mutational analysis we mapped several Hipk phosphorylation sites of Hbn, Ems, and Msh. The phosphorylation of Hbn, Ems, and Msh may provide further insight into the function of Hipk during development of the Drosophila nervous system.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/química , Proteínas de Homeodomínio/química , Fosforilação , Ligação Proteica
14.
Nat Commun ; 10(1): 1897, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015486

RESUMO

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Cromatina/química , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Animais , Butadienos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nitrilos/farmacologia , Piperazinas/farmacologia , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
15.
Parasitol Res ; 118(6): 1993-1998, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31001677

RESUMO

Antimicrobial peptides (AMPs) are important components of the vertebrate and invertebrate innate immune systems. Although AMPs are widely recognized for their broad-spectrum activity against bacteria, fungi, and viruses, their activity against protozoan parasites has not been investigated in detail. In this study, we tested 10 AMPs from three different insect species: the greater wax moth Galleria mellonella (cecropin A-D), the fruit fly Drosophila melanogaster (drosocin, Mtk-1 and Mtk-2), and the blow fly Lucilia sericata (LSerPRP-2, LSerPRP-3 and stomoxyn). We tested each AMP against the protozoan parasite Plasmodium falciparum which is responsible for the most severe form of malaria in humans. We also evaluated the impact of these insect AMPs on mouse and pig erythrocytes. Whereas all AMPs showed low hemolytic effects towards mouse and pig erythrocytes, only D. melanogaster Mtk-1 and Mtk-2 significantly inhibited the growth of P. falciparum at low concentrations. Mtk-1 and Mtk-2 could therefore be considered as leads for the development of antiparasitic drugs targeting the clinically important asexual blood stage of P. falciparum.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiparasitários/farmacologia , Proteínas de Drosophila/farmacologia , Drosophila melanogaster/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Animais , Anti-Infecciosos/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Glicopeptídeos/farmacologia , Humanos , Malária Falciparum/tratamento farmacológico , Camundongos , Mariposas/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Suínos
16.
BMC Bioinformatics ; 20(1): 174, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953451

RESUMO

BACKGROUND: Identifying transcriptional enhancers and other cis-regulatory modules (CRMs) is an important goal of post-sequencing genome annotation. Computational approaches provide a useful complement to empirical methods for CRM discovery, but it is critical that we develop effective means to evaluate their performance in terms of estimating their sensitivity and specificity. RESULTS: We introduce here pCRMeval, a pipeline for in silico evaluation of any enhancer prediction tools that are flexible enough to be applied to the Drosophila melanogaster genome. pCRMeval compares the result of predictions with the extensive existing knowledge of experimentally-validated Drosophila CRMs in order to estimate the precision and relative sensitivity of the prediction method. In the case of supervised prediction methods-when training data composed of validated CRMs are used-pCRMeval can also assess the sensitivity of specific training sets. We demonstrate the utility of pCRMeval through evaluation of our SCRMshaw CRM prediction method and training data. By measuring the impact of different parameters on SCRMshaw performance, as assessed by pCRMeval, we develop a more robust version of SCRMshaw, SCRMshaw_HD, that improves the number of predictions while maintaining sensitivity and specificity. Our analysis also demonstrates that SCRMshaw_HD, when applied to increasingly less well-assembled genomes, maintains its strong predictive power with only a minor drop-off in performance. CONCLUSION: Our pCRMeval pipeline provides a general framework for evaluation that can be applied to any CRM prediction method, particularly a supervised method. While we make use of it here primarily to test and improve a particular method for CRM prediction, SCRMshaw, pCRMeval should provide a valuable platform to the research community not only for evaluating individual methods, but also for comparing between competing methods.


Assuntos
Biologia Computacional , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Animais , Sequência de Bases , Mapeamento Cromossômico , Drosophila melanogaster/metabolismo , Feminino , Genoma de Inseto , Masculino , Especificidade da Espécie , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Toxicol Lett ; 310: 92-98, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30999038

RESUMO

Fullerenes have attracted attention since their discovery as structural units of complex carbon nanostructures capable of transporting drugs and macromolecules. As such artificial nanomaterials are applied in biology and medicine, they are routinely scrutinized for their effects on living organisms. The results of such studies range from direct destabilizing effects on DNA molecules to amelioration of the toxic effects of known genotoxic agents. We tested the effect of buckminsterfullerene (C60) on Drosophila melanogaster at DNA, tissue and organism levels. The water-soluble pristine C60 fullerene at the concentration of 20 µg/ml and 40 µg/ml leads to the activation of the mus209 gene in D. melanogaster larvae salivary glands, which can indicate higher levels of DNA damage. However, the absence of effects at the cell and organismal level could be explained by the activation of repair systems or by active elimination of damaged cells.


Assuntos
Drosophila melanogaster/efeitos dos fármacos , Fulerenos/toxicidade , Nanopartículas/toxicidade , Glândulas Salivares/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Masculino , Testes de Mutagenicidade , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Medição de Risco , Glândulas Salivares/embriologia , Glândulas Salivares/metabolismo , Ativação Transcricional/efeitos dos fármacos
18.
Genome Biol Evol ; 11(4): 1054-1065, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30859203

RESUMO

Codon usage patterns are affected by both mutational biases and translational selection. The frequency at which each codon is used in the genome is directly linked to the cellular concentrations of their corresponding tRNAs. Transfer RNA abundances-as well as the abundances of other potentially relevant factors, such as RNA-binding proteins-may vary across different tissues, making it possible that genes expressed in different tissues are subject to different translational selection regimes, and thus differ in their patterns of codon usage. These differences, however, are poorly understood, having been studied only in Arabidopsis, rice and human, with controversial results in human. Drosophila melanogaster is a suitable model organism to study tissue-specific codon adaptation given its large effective population size. Here, we compare 2,046 genes, each expressed specifically in one tissue of D. melanogaster. We show that genes expressed in different tissues exhibit significant differences in their patterns of codon usage, and that these differences are only partially due to differences in GC content, expression levels, or protein lengths. Remarkably, these differences are stronger when analyses are restricted to highly expressed genes. Our results strongly suggest that genes expressed in different tissues are subject to different regimes of translational selection.


Assuntos
Códon , Drosophila melanogaster/genética , Expressão Gênica , Adaptação Biológica , Animais , Composição de Bases , Drosophila melanogaster/metabolismo , Seleção Genética
19.
Dev Cell ; 48(6): 873-882.e4, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30827899

RESUMO

The kinetochore is a complex of proteins, broadly conserved from yeast to man, that resides at the centromere and is essential for chromosome segregation in dividing cells. There are no known functions of the core complex outside of the centromere. We now show that the proteins of the kinetochore have an essential post-mitotic function in neurodevelopment. At the embryonic neuromuscular junction of Drosophila melanogaster, mutation or knockdown of many kinetochore components cause neurites to overgrow and prevent formation of normal synaptic boutons. Kinetochore proteins were detected in synapses and axons in Drosophila. In post-mitotic cultured hippocampal neurons, knockdown of mis12 increased the filopodia-like protrusions in this region. We conclude that the proteins of the kinetochore are repurposed to sculpt developing synapses and dendrites and thereby contribute to the correct development of neuronal circuits in both invertebrates and mammals.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Cinetocoros/metabolismo , Mitose , Sistema Nervoso/citologia , Sistema Nervoso/embriologia , Animais , Axônios/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Células HEK293 , Humanos , Mutação/genética , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/metabolismo , Neurópilo/metabolismo , Fenótipo , Ratos , Sinapses/metabolismo
20.
PLoS Genet ; 15(3): e1008012, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30865627

RESUMO

orb is a founding member of the CPEB family of translational regulators and is required at multiple steps during Drosophila oogenesis. Previous studies showed that orb is required during mid-oogenesis for the translation of the posterior/germline determinant oskar mRNA and the dorsal-ventral determinant gurken mRNA. Here, we report that orb also functions upstream of these axes determinants in the polarization of the microtubule network (MT). Prior to oskar and gurken translational activation, the oocyte MT network is repolarized. The MT organizing center at the oocyte posterior is disassembled, and a new MT network is established at the oocyte anterior. Repolarization depends upon cross-regulatory interactions between anterior (apical) and posterior (basal) Par proteins. We show that repolarization of the oocyte also requires orb and that orb is needed for the proper functioning of the Par proteins. orb interacts genetically with aPKC and cdc42 and in egg chambers compromised for orb activity, Par-1 and aPKC protein and aPKC mRNA are mislocalized. Moreover, like cdc42-, the defects in Par protein localization appear to be connected to abnormalities in the cortical actin cytoskeleton. These abnormalities also disrupt the localization of the spectraplakin Shot and the microtubule minus-end binding protein Patronin. These two proteins play a critical role in the repolarization of the MT network.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Polaridade Celular/genética , Polaridade Celular/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Genes de Insetos , Quinase 3 da Glicogênio Sintase/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Oócitos/citologia , Oogênese/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo
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