RESUMO
Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. Here we present evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Homeostase/genética , Insulina/metabolismo , Mucosa Intestinal/metabolismo , MicroRNAs/metabolismo , Receptores Notch/metabolismo , Células-Tronco/citologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Diferenciação Celular , Proliferação de Células , Drosophila/citologia , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , MicroRNAs/genética , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
Post-transcriptional regulation of stem cell self-renewal by microRNAs is emerging as an important mechanism controlling tissue homeostasis. Here, we provide evidence that bantam microRNA controls neuroblast number and proliferation in the Drosophila central brain. Bantam also supports proliferation of transit-amplifying intermediate neural progenitor cells in type II neuroblast lineages. The stem cell factors brat and prospero are identified as bantam targets acting on different aspects of these processes. Thus, bantam appears to act in multiple regulatory steps in the maintenance and proliferation of neuroblasts and their progeny to regulate growth of the central brain.
Assuntos
Drosophila/citologia , Drosophila/fisiologia , MicroRNAs/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Larva/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
MicroRNAs (miRNAs) have been implicated as regulators of central nervous system (CNS) development and function. miR-124 is an evolutionarily ancient, CNS-specific miRNA. On the basis of the evolutionary conservation of its expression in the CNS, miR-124 is expected to have an ancient conserved function. Intriguingly, investigation of miR-124 function using antisense-mediated miRNA depletion has produced divergent and in some cases contradictory findings in a variety of model systems. Here we investigated miR-124 function using a targeted knockout mutant and present evidence for a role during central brain neurogenesis in Drosophila melanogaster. miR-124 activity in the larval neuroblast lineage is required to support normal levels of neuronal progenitor proliferation. We identify anachronism (ana), which encodes a secreted inhibitor of neuroblast proliferation, as a functionally important target of miR-124 acting in the neuroblast lineage. ana has previously been thought to be glial specific in its expression and to act from the cortex glia to control the exit of neuroblasts from quiescence into the proliferative phase that generates the neurons of the adult CNS during larval development. We provide evidence that ana is expressed in miR-124-expressing neuroblast lineages and that ana activity must be limited by the action of miR-124 during neuronal progenitor proliferation. We discuss the possibility that the apparent divergence of function of miR-124 in different model systems might reflect functional divergence through target site evolution.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/fisiologia , Neurônios/citologia , Animais , Linhagem da Célula , Proliferação de Células , Sistema Nervoso Central/citologia , Cruzamentos Genéticos , Drosophila melanogaster , Hibridização in Situ Fluorescente , MicroRNAs/genética , MicroRNAs/metabolismo , Microscopia de Fluorescência/métodos , Mutação , Células-Tronco/citologia , TransgenesRESUMO
OBJECTIVES: To investigate multi-dose and timings of COVID-19 vaccines in preventing antenatal infection. DESIGN: Prospective observational study investigating primary vaccinations, boosters, antenatal COVID-19 infections, neutralizing antibody (Nab) durability, and cross-reactivity to Delta and Omicron variants of concern (VOCs). RESULTS: Ninety-eight patients completed primary vaccination prepregnancy (29.6%) and antenatally (63.3%), 24.2% of whom had antenatal COVID-19, while 7.1% were unvaccinated (28.6% had antenatal COVID-19). None had severe COVID-19. Prepregnancy vaccination resulted in vaccination-to-infection delay of 23.3 weeks, which extended to 45.2 weeks with a booster, compared to 16.9 weeks following antenatal vaccination (P < 0.001). Infections occurred at 26.2 weeks gestation in women vaccinated prepregnancy compared to 36.2 weeks gestation in those vaccinated during pregnancy (P < 0.007). The risk of COVID-19 infection was higher without antenatal vaccination (hazard ratio [HR] 14.6, P = 0.05) and after prepregnancy vaccination without a booster (HR 10.4, P = 0.002). Antenatal vaccinations initially led to high Nab levels, with mild waning but subsequent rebound. Significant Nab enhancement occurred with a third-trimester booster. Maternal-neonatal Nab transfer was efficient (transfer ratio >1), and cross-reactivity to VOCs was observed. CONCLUSION: Completing vaccination during any trimester delays COVID-19 infection and maintains effective neutralizing activity throughout pregnancy, with robust cross-reactivity to VOCs and efficient maternal-neonatal transfer.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Imunização Secundária , Complicações Infecciosas na Gravidez , SARS-CoV-2 , Vacinação , Humanos , Gravidez , Feminino , COVID-19/prevenção & controle , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , Adulto , Estudos Prospectivos , Complicações Infecciosas na Gravidez/prevenção & controle , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Adulto JovemRESUMO
OBJECTIVES: We studied the immunogenicity after primary and booster vaccinations of the Abdala COVID-19 vaccine, a receptor-binding domain protein subunit vaccine, in Vietnamese people by determining the level of neutralization and cross-neutralization activities against the ancestral SARS-CoV-2 and its variants and SARS-CoV-1. METHODS: We performed a prospective observational study, enrolling adults aged 19-59 years in Dong Thap province, southern Vietnam, and collected blood samples from baseline until 4 weeks after the booster dose. We measured anti-nucleocapsid, anti-spike, and neutralizing antibodies against SARS-CoV-2 and assessed the cross-neutralization against 14 SARS-CoV-2 variants and SARS-CoV-1. Complementary antibody data came from Vietnamese health care workers fully vaccinated with ChAdOx1-S. RESULTS: After primary vaccination, anti-spike antibody and neutralizing antibodies were detectable in 98.4% and 87% of 251 study participants, respectively, with neutralizing antibody titers similar to that induced by ChAdOx1-S vaccine. Antibody responses after a homologous (Abdala COVID-19) or heterologous (messenger RNA BNT162b2) booster could neutralize 14 SARS-CoV-2 variants (including Omicron) and SARS-CoV-1. CONCLUSIONS: Abdala COVID-19 vaccine is immunogenic in Vietnamese people. Enhanced antibody response after a booster dose could cross-neutralize 14 SARS-CoV-2 variants and SARS-CoV-1. Our results have added to the growing body of knowledge about the contribution of protein subunit vaccine platforms to pandemic control.
RESUMO
Introduction: A well-validated diagnostic assay with curated biomarkers complements clinicopathological factors to facilitate early diagnosis and ensure timely treatment delivery. This study focuses on an Asian-centric cancer diagnostic assay designed and thoroughly validated against commercially available standard references and a cohort of over 200 clinical specimens spanning 12 diverse Asian-centric cancer types. Methods: The assay uses hybrid-capture probes capable of profiling DNA aberrations from 572 cancer-related genes and 91 RNA fusion partners. The panel can detect clinically-tractable biomarkers such as microsatellite instability (MSI) and tumor mutation burden (TMB). Results: Analytical evaluation demonstrated 100% specificity and 99.9% sensitivity within a ≥5% VAF limit of detection (LoD) for SNV/Indels. RNA-based fusion features an LoD of ≥5 copies per nanogram input when evaluated against commercial references. Excellent linearity and concordance were observed when benchmarking against orthogonal methods in identifying MSI status, TMB scores and RNA fusions. Actionable genetic alterations were identified in 65% of the clinical samples. Conclusion: These results demonstrate a molecular diagnostic assay that accurately detects genomic alterations and complex biomarkers. The data also supports an excellent performance of this assay for making critical diagnoses and well-informed therapeutic decisions in Asian prevalent cancers.
Assuntos
Antivirais/administração & dosagem , Ácido Ascórbico/administração & dosagem , Tratamento Farmacológico da COVID-19 , Fatores Imunológicos/administração & dosagem , Zinco/administração & dosagem , Adulto , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , COVID-19/imunologia , COVID-19/virologia , Feminino , Humanos , Masculino , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/imunologiaRESUMO
Gustatory pheromones play an essential role in shaping the behavior of many organisms. However, little is known about the processing of taste pheromones in higher order brain centers. Here, we describe a male-specific gustatory circuit in Drosophila that underlies the detection of the anti-aphrodisiac pheromone (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503). Using behavioral analysis, genetic manipulation, and live calcium imaging, we show that Gr68a-expressing neurons on the forelegs of male flies exhibit a sexually dimorphic physiological response to the pheromone and relay information to the central brain via peptidergic neurons. The release of tachykinin from 8 to 10 cells within the subesophageal zone is required for the pheromone-triggered courtship suppression. Taken together, this work describes a neuropeptide-modulated central brain circuit that underlies the programmed behavioral response to a gustatory sex pheromone. These results will allow further examination of the molecular basis by which innate behaviors are modulated by gustatory cues and physiological state.
Assuntos
Drosophila/fisiologia , Neurônios/fisiologia , Feromônios/metabolismo , Taquicininas/metabolismo , Animais , Proteínas de Drosophila/análise , Neurônios/química , Receptores de Superfície Celular/análise , Percepção GustatóriaRESUMO
BACKGROUND: Cancers develop in a complex mutational landscape. Interaction of genetically abnormal cancer cells with normal stromal cells can modify the local microenvironment to promote disease progression for some tumor types. Genetic models of tumorigenesis provide the opportunity to explore how combinations of cancer driver mutations confer distinct properties on tumors. Previous Drosophila models of EGFR-driven cancer have focused on epithelial neoplasia. RESULTS: Here, we report a Drosophila genetic model of EGFR-driven tumorigenesis in which the neoplastic transformation depends on interaction between epithelial and mesenchymal cells. We provide evidence that the secreted proteoglycan Perlecan can act as a context-dependent oncogene cooperating with EGFR to promote tumorigenesis. Coexpression of Perlecan in the EGFR-expressing epithelial cells potentiates endogenous Wg/Wnt and Dpp/BMP signals from the epithelial cells to support expansion of a mesenchymal compartment. Wg activity is required in the epithelial compartment, whereas Dpp activity is required in the mesenchymal compartment. This genetically normal mesenchymal compartment is required to support growth and neoplastic transformation of the genetically modified epithelial population. CONCLUSIONS: We report a genetic model of tumor formation that depends on crosstalk between a genetically modified epithelial cell population and normal host mesenchymal cells. Tumorigenesis in this model co-opts a regulatory mechanism that is normally involved in controlling growth of the imaginal disc during development.
Assuntos
Carcinogênese/genética , Transformação Celular Neoplásica/metabolismo , Epitélio/metabolismo , Discos Imaginais/crescimento & desenvolvimento , Mesoderma/metabolismo , Modelos Biológicos , Animais , Carcinogênese/metabolismo , Primers do DNA , Drosophila , Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Discos Imaginais/metabolismo , Microscopia Confocal , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor Cross-Talk/fisiologia , Receptores de Peptídeos de Invertebrados/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt1/metabolismoRESUMO
MicroRNAs are abundant in animal genomes, yet little is known about their functions in vivo. Here, we report the production of 80 new Drosophila miRNA mutants by targeted homologous recombination. These mutants remove 104 miRNAs. Together with 15 previously reported mutants, this collection includes 95 mutants deleting 130 miRNAs. Collectively, these genes produce over 99% of all Drosophila miRNAs, measured by miRNA sequence reads. We present a survey of developmental and adult miRNA phenotypes. Over 80% of the mutants showed at least one phenotype using a p < 0.01 significance threshold. We observed a significant correlation between miRNA abundance and phenotypes related to survival and lifespan, but not to most other phenotypes. miRNA cluster mutants were no more likely than single miRNA mutants to produce significant phenotypes. This mutant collection will provide a resource for future analysis of the biological roles of Drosophila miRNAs.
Assuntos
Drosophila/genética , MicroRNAs/genética , Mutação , Alelos , Animais , Biologia Computacional , Drosophila melanogaster/genética , Feminino , Vetores Genéticos , Masculino , MicroRNAs/metabolismo , Família Multigênica , Fenótipo , Recombinação GenéticaRESUMO
MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression that may act as buffering agents to stabilize gene-regulatory networks. Here, we identify two miRNAs that are maternally required for normal embryonic primordial germ cell development in Drosophila melanogaster. Embryos derived from miR-969 and miR-9c mutant mothers had, on average, reduced germ cell numbers. Intriguingly, this reduction correlated with an increase in the variance of this quantitative phenotypic trait. Analysis of an independent set of maternal mutant genotypes suggests that reduction of germ cell number need not lead to increased variance. Our observations are consistent with the hypothesis that miR-969 and miR-9c contribute to stabilizing the processes that control germ number, supporting phenotypic robustness.
Assuntos
Drosophila melanogaster/genética , Células Germinativas/citologia , MicroRNAs/genética , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Feminino , Genótipo , MicroRNAs/metabolismo , Mutação , FenótipoRESUMO
microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by destabilizing target transcripts and/or inhibiting their translation. miRNAs are thought to have roles in buffering gene expression to confer robustness. miRNAs have been shown to play important roles during tissue development to control cell proliferation, differentiation and morphogenesis. Many miRNAs are expressed in the germ line of Drosophila, and functions have been reported for a few miRNAs in maintenance of stem cell proliferation during oogenesis. Here, we analyse the function of Drosophila miR-989 in oogenesis. miR-989 is abundant in ovaries. Mutants lacking miR-989 did not display gross abnormalities affecting egg chamber formation or maturation. However, the migration of the border cell cluster was severely delayed in miR-989 mutant egg chambers. We demonstrate that miR-989 function is required in the somatic cells in the egg chamber, not in germ line cells for border cell migration. Loss of miR-989 from a fraction of the border cell cluster was sufficient to impair cluster migration as a whole, suggesting a role in border cells. Gene ontology analysis reveals that many predicted miR-989 target mRNAs are implicated in regulating cell migration, cell projection morphogenesis, cell adhesion as well as receptor tyrosine kinase and ecdysone signalling, consistent with an important regulatory role for miR-989 in border cell migration.
Assuntos
Movimento Celular/genética , Drosophila/genética , Drosophila/metabolismo , MicroRNAs/genética , Ovário/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese/genética , Mutação , Oogênese/genética , FenótipoRESUMO
Many aspects of social behavior are controlled by sex-specific pheromones. Gender-appropriate production of the sexually dimorphic transcription factors doublesex and fruitless controls sexual differentiation and sexual behavior. miR-124 mutant males exhibited increased male-male courtship and reduced reproductive success with females. Females showed a strong preference for wild-type males over miR-124 mutant males when given a choice of mates. These effects were traced to aberrant pheromone production. We identified the sex-specific splicing factor transformer as a functionally significant target of miR-124 in this context, suggesting a role for miR-124 in the control of male sexual differentiation and behavior, by limiting inappropriate expression of the female form of transformer. miR-124 is required to ensure fidelity of gender-appropriate pheromone production in males. Use of a microRNA provides a secondary means of controlling the cascade of sex-specific splicing events that controls sexual differentiation in Drosophila. DOI:http://dx.doi.org/10.7554/eLife.00640.001.
Assuntos
MicroRNAs/fisiologia , Reprodução/fisiologia , Animais , Feminino , Masculino , MicroRNAs/genética , Mutação , Reprodução/genética , Atrativos Sexuais/biossíntese , Comportamento Sexual AnimalRESUMO
We sequenced Drosophila head RNA to identify a small set of miRNAs that undergo robust circadian cycling. We concentrated on a cluster of six miRNAs, mir-959-964, all of which peak at about ZT12 or lights off. The cluster pri-miRNA is transcribed under bona fide circadian transcriptional control, and all six mature miRNAs have short half-lives, a requirement for cycling. A viable Gal4 knockin strain localizes prominent cluster miRNA expression to the adult head fat body. Analysis of cluster knockout and overexpression strains indicates that innate immunity, metabolism, and feeding behavior are under cluster miRNA regulation. Manipulation of food intake also affects the levels and timing of cluster miRNA transcription with no more than minor effects on the core circadian oscillator. These observations indicate a feedback circuit between feeding time and cluster miRNA expression function as well as a surprising role of posttranscriptional regulation in the circadian control of these phenotypes.
Assuntos
Drosophila melanogaster/metabolismo , MicroRNAs/metabolismo , Animais , Relógios Circadianos/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ingestão de Alimentos , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Imunidade Inata , MicroRNAs/genética , Família Multigênica , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
MicroRNAs (miRNAs) are noncoding RNA molecules that have come to attract considerable interest for their roles in animal and plant development and disease. One means to study miRNA function in animal development is to create mutations. Use of gene-targeting strategies based on ends-out homologous recombination is a useful approach to produce mutations of desired structure, and is gaining popularity for producing miRNA knockouts. Here we present a detailed protocol for miRNA gene targeting and for their subsequent molecular characterization as well as confirmation by rescue. The descriptions of a series of modified vectors designed to facilitate the analysis of miRNA function are included, and a method to manipulate the mutant genome using recombinase-mediated cassette exchange.
Assuntos
Drosophila melanogaster/genética , Marcação de Genes/métodos , MicroRNAs/genética , Animais , Organismos Geneticamente Modificados , Recombinação GenéticaRESUMO
A series of vectors has been designed to enhance the versatility of targeted homologous recombination. Recombinase-mediated cassette exchange permits sequential targeting at any locus and improves flexibility in making user-defined mutations. Application of RMCE to delete an intronic microRNA gene is described.