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1.
J Vis Exp ; (175)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34605807

RESUMO

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. Here, the in-nucleus Hi-C method has been described in detail, which provides important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements.


Assuntos
Cromatina , Drosophila , Animais , Núcleo Celular , Drosophila/genética , Drosophila melanogaster/genética , Genômica
2.
Lab Chip ; 21(18): 3420-3428, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34486609

RESUMO

Volumetric imaging of dynamic signals in a large, moving, and light-scattering specimen is extremely challenging, owing to the requirement on high spatiotemporal resolution and difficulty in obtaining high-contrast signals. Here we report that through combining a microfluidic chip-enabled digital scanning light-sheet illumination strategy with deep-learning based image restoration, we can realize isotropic 3D imaging of a whole crawling Drosophila larva on an ordinary inverted microscope at a single-cell resolution and a high volumetric imaging rate up to 20 Hz. Enabled with high performances even unmet by current standard light-sheet fluorescence microscopes, we in toto record the neural activities during the forward and backward crawling of a 1st instar larva, and successfully correlate the calcium spiking of motor neurons with the locomotion patterns.


Assuntos
Aprendizado Profundo , Microscopia , Animais , Drosophila , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Larva
3.
G3 (Bethesda) ; 11(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34544118

RESUMO

DNA topoisomerase I (Top1) maintains chromatin conformation during transcription. While Top1 is not essential in simple eukaryotic organisms such as yeast, it is required for the development of multicellular organisms. In fact, tissue and cell-type-specific functions of Top1 have been suggested in the fruit fly Drosophila. A better understanding of Top1's function in the context of development is important as Top1 inhibitors are among the most widely used anticancer drugs. As a step toward such a better understanding, we studied its localization in live cells of Drosophila. Consistent with prior results, Top1 is highly enriched at the nucleolus in transcriptionally active polyploid cells, and this enrichment responds to perturbation of transcription. In diploid cells, we uncovered evidence for Top1 foci formation at genomic regions not limited to the active rDNA locus, suggestive of novel regulation of Top1 recruitment. In the male germline, Top1 is highly enriched at the paired rDNA loci on sex chromosomes suggesting that it might participate in regulating their segregation during meiosis. Results from RNAi-mediated Top1 knockdown lend support to this hypothesis. Our study has provided one of the most comprehensive descriptions of Top1 localization during animal development.


Assuntos
DNA Topoisomerases Tipo I , Drosophila , Animais , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Ribossômico/genética , Drosophila/genética , Drosophila/metabolismo , Meiose , Saccharomyces cerevisiae/genética , Inibidores da Topoisomerase I
4.
G3 (Bethesda) ; 11(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34544125

RESUMO

During oogenesis, a group of specialized follicle cells, known as stretched cells (StCs), flatten drastically from cuboidal to squamous shape. While morphogenesis of epithelia is critical for organogenesis, genes and signaling pathways involved in this process remain to be revealed. In addition to formation of gap junctions for intercellular exchange of small molecules, gap junction proteins form channels or act as adaptor proteins to regulate various cellular behaviors. In invertebrates, gap junction proteins are Innexins. Knockdown of Innexin 2 but not other Innexins expressed in follicle cells attenuates StC morphogenesis. Interestingly, blocking of gap junctions with an inhibitor carbenoxolone does not affect StC morphogenesis, suggesting that Innexin 2 might control StCs flattening in a gap-junction-independent manner. An excessive level of ßPS-Integrin encoded by myospheroid is detected in Innexin 2 mutant cells specifically during StC morphogenesis. Simultaneous knockdown of Innexin 2 and myospheroid partially rescues the morphogenetic defect resulted from Innexin 2 knockdown. Furthermore, reduction of ßPS-Integrin is sufficient to induce early StCs flattening. Taken together, our data suggest that ßPS-Integrin acts downstream of Innexin 2 in modulating StCs morphogenesis.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Conexinas/genética , Drosophila/genética , Proteínas de Drosophila/genética , Feminino , Integrinas , Morfogênese/genética , Ovário
5.
Nat Commun ; 12(1): 5660, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580289

RESUMO

Small Open Reading Frames (smORFs) coding for peptides of less than 100 amino-acids are an enigmatic and pervasive gene class, found in the tens of thousands in metazoan genomes. Here we reveal a short 80 amino-acid peptide (Pegasus) which enhances Wingless/Wnt1 protein short-range diffusion and signalling. During Drosophila wing development, Wingless has sequential functions, including late induction of proneural gene expression and wing margin development. Pegasus mutants produce wing margin defects and proneural expression loss similar to those of Wingless. Pegasus is secreted, and co-localizes and co-immunoprecipitates with Wingless, suggesting their physical interaction. Finally, measurements of fixed and in-vivo Wingless gradients support that Pegasus increases Wingless diffusion in order to enhance its signalling. Our results unveil a new element in Wingless signalling and clarify the patterning role of Wingless diffusion, while corroborating the link between small open reading frame peptides, and regulation of known proteins with membrane-related functions.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos/metabolismo , Asas de Animais/crescimento & desenvolvimento , Proteína Wnt1/metabolismo , Animais , Animais Geneticamente Modificados , Microscopia Intravital , Peptídeos/genética , Imagem com Lapso de Tempo
6.
J Vis Exp ; (174)2021 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-34487109

RESUMO

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.


Assuntos
Proteínas de Drosophila , Heterocromatina , Animais , Cromossomos , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Glândulas Salivares , Coloração e Rotulagem
7.
J Neurodev Disord ; 13(1): 37, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34525941

RESUMO

The idea that alterations in gut-microbiome-brain axis (GUMBA)-mediated communication play a crucial role in human brain disorders like autism remains a topic of intensive research in various labs. Gastrointestinal issues are a common comorbidity in patients with autism spectrum disorder (ASD). Although gut microbiome and microbial metabolites have been implicated in the etiology of ASD, the underlying molecular mechanism remains largely unknown. In this review, we have summarized recent findings in human and animal models highlighting the role of the gut-brain axis in ASD. We have discussed genetic and neurobehavioral characteristics of Drosophila as an animal model to study the role of GUMBA in ASD. The utility of Drosophila fruit flies as an amenable genetic tool, combined with axenic and gnotobiotic approaches, and availability of transgenic flies may reveal mechanistic insight into gut-microbiota-brain interactions and the impact of its alteration on behaviors relevant to neurological disorders like ASD.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Microbioma Gastrointestinal , Animais , Encéfalo , Drosophila , Humanos
8.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34531300

RESUMO

The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. Here we combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.


Assuntos
Moléculas de Adesão Celular/química , Adesão Celular , Síndrome de Down/patologia , Proteínas de Drosophila/química , Neurogênese , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Camundongos , Neuritos
9.
eNeuro ; 8(5)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34475263

RESUMO

Mutations in the voltage-gated sodium channel gene SCN1A are associated with human epilepsy disorders, but how most of these mutations alter channel properties and result in seizures is unknown. This study focuses on two different mutations occurring at one position within SCN1A R1648C (R-C) is associated with the severe disorder Dravet syndrome, and R1648H (R-H), with the milder disorder GEFS+. To explore how these different mutations contribute to distinct seizure disorders, Drosophila lines with the R-C or R-H mutation, or R1648R (R-R) control substitution in the fly sodium channel gene para were generated by CRISPR-Cas9 gene editing. The R-C and R-H mutations are homozygous lethal. Animals heterozygous for R-C or R-H mutations displayed reduced life spans and spontaneous and temperature-induced seizures not observed in R-R controls. Electrophysiological recordings from adult GABAergic neurons in R-C and R-H mutants revealed the appearance of sustained neuronal depolarizations and altered firing frequency that were exacerbated at elevated temperature. The only significant change observed in underlying sodium currents in both R-C and R-H mutants was a hyperpolarized deactivation threshold at room and elevated temperature compared with R-R controls. Since this change is constitutive, it is likely to interact with heat-induced changes in other cellular properties to result in the heat-induced increase in sustained depolarizations and seizure activity. Further, the similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines, suggests that disease symptoms of different severity associated with these mutations in humans could be due in large part to differences in genetic background.


Assuntos
Epilepsias Mioclônicas , Epilepsia , Animais , Drosophila , Epilepsia/genética , Neurônios GABAérgicos , Humanos , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Fenótipo , Convulsões/genética
10.
Prog Mol Subcell Biol ; 60: 27-56, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386871

RESUMO

The fact that satellite DNAs (satDNAs) in eukaryotes are abundant genomic components, can perform functional roles, but can also change rapidly across species while being homogenous within a species, makes them an intriguing and fascinating genomic component to study. It is also becoming clear that satDNAs represent an important piece in genome architecture and that changes in their structure, organization, and abundance can affect the evolution of genomes and species in many ways. Since the discovery of satDNAs more than 50 years ago, species from the Drosophila genus have continuously been used as models to study several aspects of satDNA biology. These studies have been largely concentrated in D. melanogaster and closely related species from the Sophophora subgenus, even though the vast majority of all Drosophila species belong to the Drosophila subgenus. This chapter highlights some studies on the satDNA structure, organization, and evolution in two species groups from the Drosophila subgenus: the repleta and virilis groups. We also discuss and review the classification of other abundant tandem repeats found in these species in the light of the current information available.


Assuntos
DNA Satélite , Drosophila , Animais , DNA Satélite/genética , Drosophila/genética , Drosophila melanogaster/genética , Evolução Molecular , Filogenia
11.
Prog Mol Subcell Biol ; 60: 1-26, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386870

RESUMO

Satellite repeats make up a large fraction of the genomes of many higher eukaryotes. Until recently these sequences were viewed as molecular parasites with few functions. Drosophila melanogaster and related species have a wealth of diverse satellite repeats. Comparative studies of Drosophilids have been instrumental in understanding how these rapidly evolving sequences change and move. Remarkably, satellite repeats have been found to modulate gene expression and mediate genetic conflicts between chromosomes and between closely related fly species. This suggests that satellites play a key role in speciation. We have taken advantage of the depth of research on satellite repeats in flies to review the known functions of these sequences and consider their central role in evolution and gene expression.


Assuntos
Drosophila melanogaster , Drosophila , Animais , Cromatina/genética , Cromossomos , DNA Satélite/genética , Drosophila/genética , Drosophila melanogaster/genética , Evolução Molecular
12.
J Biol Rhythms ; 36(5): 442-460, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34428956

RESUMO

Circadian clocks schedule biological functions at a specific time of the day. Full comprehension of the clock function requires precise understanding of their entrainment to the environment. The phase of entrained clock is plastic, which depends on different factors such as the period of endogenous oscillator, the period of the zeitgeber cycle (T), and the proportion of light and darkness (day length). The circadian clock of fruit fly Drosophila melanogaster is able to entrain to a wide range of T-cycles and day lengths. Here, we investigated the importance of the neuropeptide Pigment-Dispersing Factor (PDF) for entrainment by systematically studying locomotor activity rhythms of Pdf 0 mutants and wild-type flies under different T-cycles (T22 to T32) and different day lengths (8, 12, and 16 hour [h]). Furthermore, we analysed PERIOD protein oscillations in selected groups of clock neurons in both genotypes under T24 and T32 at a day length of 16 h. As expected, we found that the phase of Drosophila's evening activity and evening neurons advanced with increasing T in all the day lengths. This advance was much larger in Pdf 0 mutants (~7 h) than in wild-type flies causing (1) pronounced desynchrony between morning and evening neurons and (2) evening activity to move in the morning instead of the evening. Most interestingly, we found that the lights-off transition determines the phase of evening neurons in both genotypes and that PDF appears necessary to delay the evening neurons by ~3 h to their wild-type phase. Thus, in T32, PDF first delays the molecular cycling in the evening neurons, and then, as shown in previous studies, delays their neuronal firing rhythms to produce a total delay of ~7 h necessary for a wild-type evening activity phase. We conclude that PDF is crucial for appropriate phasing of Drosophila activity rhythm.


Assuntos
Relógios Circadianos , Proteínas de Drosophila , Neuropeptídeos , Animais , Ritmo Circadiano , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Atividade Motora , Neurônios , Neuropeptídeos/genética
13.
EMBO Rep ; 22(10): e52679, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34338441

RESUMO

It has long been thought that microtubule disassembly, one of the earliest cellular events, contributes to neuronal pruning and neurodegeneration in development and disease. However, how microtubule disassembly drives neuronal pruning remains poorly understood. Here, we conduct a systematic investigation of various microtubule-destabilizing factors and identify exchange factor for Arf6 (Efa6) and Stathmin (Stai) as new regulators of dendrite pruning in ddaC sensory neurons during Drosophila metamorphosis. We show that Efa6 is both necessary and sufficient to regulate dendrite pruning. Interestingly, Efa6 and Stai facilitate microtubule turnover and disassembly prior to dendrite pruning without compromising the minus-end-out microtubule orientation in dendrites. Moreover, our pharmacological and genetic manipulations strongly support a key role of microtubule disassembly in promoting dendrite pruning. Thus, this systematic study highlights the importance of two selective microtubule destabilizers in dendrite pruning and substantiates a causal link between microtubule disassembly and neuronal pruning.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Dendritos , Drosophila/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Microtúbulos , Plasticidade Neuronal
14.
J Vis Exp ; (174)2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34424253

RESUMO

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700µÎ´6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700µÎ´6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700µÎ´6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.


Assuntos
Esclerose Amiotrófica Lateral , Proteinopatias TDP-43 , Esclerose Amiotrófica Lateral/genética , Animais , Modelos Animais de Doenças , Drosophila , Glucose
15.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34355743

RESUMO

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B, H2A and H2Av accumulate on lipid droplets (LDs), which are cytoplasmic fat storage organelles. Without LD binding, maternally provided H2B, H2A and H2Av are absent; however, how LDs ensure histone storage is unclear. Using quantitative imaging, we uncover when during oogenesis these histones accumulate, and which step of accumulation is LD dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. Our findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites.


Assuntos
Histonas/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Oócitos/metabolismo , Oogênese/fisiologia
16.
J Chem Ecol ; 47(8-9): 719-731, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34402994

RESUMO

Organisms depend on visual, auditory, and olfactory cues to signal the presence of danger that could impact survival and reproduction. Drosophila melanogaster emits an olfactory alarm signal, termed the Drosophila stress odorant (dSO), in response to mechanical agitation or electric shock. While it has been shown that conspecifics avoid areas previously occupied by stressed individuals, the contextual underpinnings of the emission of, and response to dSO, have received little attention. Using a binary choice assay, we determined that neither age and sex of emitters, nor the time of the day, affected the emission or avoidance of dSO. However, both sex and mating status affected the response to dSO. We also demonstrated that while D. melanogaster, D. simulans, and D. suzukii, have different dSO profiles, its avoidance was not species-specific. Thus, dSO should not be considered a pheromone but a general alarm signal for Drosophila. However, the response levels to both intra- and inter-specific cues differed between Drosophila species and possible reasons for these differences are discussed.


Assuntos
Drosophila/química , Odorantes/análise , Envelhecimento , Animais , Relógios Biológicos , Drosophila/fisiologia , Drosophila melanogaster/química , Drosophila melanogaster/fisiologia , Estimulação Elétrica , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Fatores Sexuais , Comportamento Sexual Animal , Especificidade da Espécie , Estresse Mecânico , Compostos Orgânicos Voláteis/análise
17.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445171

RESUMO

Tauopathy refers to a group of progressive neurodegenerative diseases, including frontotemporal lobar degeneration and Alzheimer's disease, which correlate with the malfunction of microtubule-associated protein Tau (MAPT) due to abnormal hyperphosphorylation, leading to the formation of intracellular aggregates in the brain. Despite extensive efforts to understand tauopathy and develop an efficient therapy, our knowledge is still far from complete. To find a solution for this group of devastating diseases, several animal models that mimic diverse disease phenotypes of tauopathy have been developed. Rodents are the dominating tauopathy models because of their similarity to humans and established disease lines, as well as experimental approaches. However, powerful genetic animal models using Drosophila, zebrafish, and C. elegans have also been developed for modeling tauopathy and have contributed to understanding the pathophysiology of tauopathy. The success of these models stems from the short lifespans, versatile genetic tools, real-time in-vivo imaging, low maintenance costs, and the capability for high-throughput screening. In this review, we summarize the main findings on mechanisms of tauopathy and discuss the current tauopathy models of these non-rodent genetic animals, highlighting their key advantages and limitations in tauopathy research.


Assuntos
Modelos Animais de Doenças , Tauopatias/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Drosophila/genética , Drosophila/fisiologia , Humanos , Tauopatias/fisiopatologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas tau/genética
18.
Immunity ; 54(8): 1745-1757.e7, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34348118

RESUMO

Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas contra Escherichia coli/imunologia , Gastroenteropatias/imunologia , Intestino Delgado/imunologia , Linfócitos T Reguladores/imunologia , Administração Oral , Animais , Linhagem Celular , Modelos Animais de Doenças , Drosophila , Escherichia coli/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Gastroenteropatias/microbiologia , Gastroenteropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Vacinação
19.
Nat Commun ; 12(1): 5044, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413298

RESUMO

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.


Assuntos
Mutação , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/genética , Ribossomos/patologia , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/patologia , Adolescente , Adulto , Animais , Fenômenos Biológicos , Células Cultivadas , Criança , Pré-Escolar , Dictyostelium , Drosophila , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células Germinativas , Humanos , Lactente , Simulação de Dinâmica Molecular , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Síndrome de Shwachman-Diamond/metabolismo , Adulto Jovem
20.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445633

RESUMO

Caspases, a family of cysteine-aspartic proteases, have an established role as critical components in the activation and initiation of apoptosis. Alongside this a variety of non-apoptotic caspase functions in proliferation, differentiation, cellular plasticity and cell migration have been reported. The activity level and context are important factors in determining caspase function. As a consequence of their critical role in apoptosis and beyond, caspases are uniquely situated to have pathological roles, including in cancer. Altered caspase function is a common trait in a variety of cancers, with apoptotic evasion defined as a "hallmark of cancer". However, the role that caspases play in cancer is much more complex, acting both to prevent and to promote tumourigenesis. This review focuses on the major findings in Drosophila on the dual role of caspases in tumourigenesis. This has major implications for cancer treatments, including chemotherapy and radiotherapy, with the activation of apoptosis being the end goal. However, such treatments may inadvertently have adverse effects on promoting tumour progression and acerbating the cancer. A comprehensive understanding of the dual role of caspases will aid in the development of successful cancer therapeutic approaches.


Assuntos
Apoptose , Carcinogênese , Caspases/metabolismo , Neoplasias/patologia , Animais , Drosophila , Humanos , Neoplasias/enzimologia , Neoplasias/etiologia , Neoplasias/prevenção & controle
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