RESUMO
Neurofibromatosis type 1, a genetic disorder caused by pathogenic germline variations in NF1, predisposes individuals to the development of tumors, including cutaneous and plexiform neurofibromas (CNs and PNs), optic gliomas, astrocytomas, juvenile myelomonocytic leukemia, high-grade gliomas and malignant peripheral nerve sheath tumors (MPNSTs), which are chemotherapy- and radiation-resistant sarcomas with poor survival. Loss of NF1 also occurs in sporadic tumors, such as glioblastoma (GBM), melanoma, breast, ovarian and lung cancers. We performed a high-throughput screen for compounds that were synthetic lethal with NF1 loss, which identified several leads, including the small molecule Y102. Treatment of cells with Y102 perturbed autophagy, mitophagy and lysosome positioning in NF1-deficient cells. A dual proteomics approach identified BLOC-one-related complex (BORC), which is required for lysosome positioning and trafficking, as a potential target of Y102. Knockdown of a BORC subunit using siRNA recapitulated the phenotypes observed with Y102 treatment. Our findings demonstrate that BORC might be a promising therapeutic target for NF1-deficient tumors.
Assuntos
Lisossomos , Neurofibromina 1 , Humanos , Lisossomos/metabolismo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Neurofibromatose 1/metabolismo , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Autofagia/efeitos dos fármacos , Mutações Sintéticas Letais , Transporte Proteico/efeitos dos fármacosRESUMO
Numerous successful gene-targeted therapies are arising for the treatment of a variety of rare diseases. At the same time, current treatment options for neurofibromatosis 1 and schwannomatosis are limited and do not directly address loss of gene/protein function. In addition, treatments have mostly focused on symptomatic tumors, but have failed to address multisystem involvement in these conditions. Gene-targeted therapies hold promise to address these limitations. However, despite intense interest over decades, multiple preclinical and clinical issues need to be resolved before they become a reality. The optimal approaches to gene-, mRNA-, or protein restoration and to delivery to the appropriate cell types remain elusive. Preclinical models that recapitulate manifestations of neurofibromatosis 1 and schwannomatosis need to be refined. The development of validated assays for measuring neurofibromin and merlin activity in animal and human tissues will be critical for early-stage trials, as will the selection of appropriate patients, based on their individual genotypes and risk/benefit balance. Once the safety of gene-targeted therapy for symptomatic tumors has been established, the possibility of addressing a wide range of symptoms, including non-tumor manifestations, should be explored. As preclinical efforts are underway, it will be essential to educate both clinicians and those affected by neurofibromatosis 1/schwannomatosis about the risks and benefits of gene-targeted therapy for these conditions.
Assuntos
Neurilemoma , Neurofibromatoses , Neurofibromatose 1 , Neurofibromatose 2 , Neoplasias Cutâneas , Animais , Humanos , Neurofibromatose 1/genética , Neurofibromatose 1/terapia , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/genética , Neurofibromatose 2/patologia , Neurofibromatoses/genética , Neurofibromatoses/terapia , Neurofibromatoses/diagnóstico , Neurilemoma/genética , Neurilemoma/terapia , Neurilemoma/diagnósticoRESUMO
DICER1 syndrome is a cancer pre-disposition disorder caused by mutations that disrupt the function of DICER1 in miRNA processing. Studying the molecular, cellular and oncogenic effects of these mutations can reveal novel mechanisms that control cell homeostasis and tumor biology. Here, we conduct the first analysis of pathogenic DICER1 syndrome allele from the DICER1 3'UTR. We find that the DICER1 syndrome allele, rs1252940486, abolishes interaction with the PUMILIO RNA binding protein with the DICER1 3'UTR, resulting in the degradation of the DICER1 mRNA by AUF1. This single mutational event leads to diminished DICER1 mRNA and protein levels, and widespread reprogramming of miRNA networks. The in-depth characterization of the rs1252940486 DICER1 allele, reveals important post-transcriptional regulatory events that control DICER1 levels.
Assuntos
MicroRNAs , RNA Mensageiro , MicroRNAs/genéticaRESUMO
Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. We asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. Here, we find that htt mutations suppress axon outgrowth defects of αß neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αß neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. We find that Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth.
Assuntos
Aciltransferases/genética , Axônios/metabolismo , Proteínas de Drosophila/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Transporte Axonal/genética , Axônios/patologia , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Humanos , Doença de Huntington/patologia , Aprendizagem/fisiologia , Memória/fisiologia , Corpos Pedunculados/crescimento & desenvolvimento , Corpos Pedunculados/patologia , Mutação/genética , Degeneração Neural/genética , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais/genéticaRESUMO
Neurofibromatosis type 1 is a monogenetic disorder that predisposes individuals to tumor formation and cognitive and behavioral symptoms. The neuronal circuitry and developmental events underlying these neurological symptoms are unknown. To better understand how mutations of the underlying gene (NF1) drive behavioral alterations, we have examined grooming in the Drosophila neurofibromatosis 1 model. Mutations of the fly NF1 ortholog drive excessive grooming, and increased grooming was observed in adults when Nf1 was knocked down during development. Furthermore, intact Nf1 Ras GAP-related domain signaling was required to maintain normal grooming. The requirement for Nf1 was distributed across neuronal circuits, which were additive when targeted in parallel, rather than mapping to discrete microcircuits. Overall, these data suggest that broadly-distributed alterations in neuronal function during development, requiring intact Ras signaling, drive key Nf1-mediated behavioral alterations. Thus, global developmental alterations in brain circuits/systems function may contribute to behavioral phenotypes in neurofibromatosis type 1.
Assuntos
Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Proteínas do Tecido Nervoso/genética , Neurofibromatose 1/genética , Neurônios/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Animais , Cognição/fisiologia , Modelos Animais de Doenças , Drosophila melanogaster/genética , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Asseio Animal/fisiologia , Humanos , Mutação/genética , Neurofibromatose 1/patologia , Neurônios/patologiaRESUMO
Flapping flight is the most energetically demanding form of sustained forwards locomotion that vertebrates perform. Flock dynamics therefore have significant implications for energy expenditure. Despite this, no studies have quantified the biomechanical consequences of flying in a cluster flock or pair relative to flying solo. Here, we compared the flight characteristics of homing pigeons (Columba livia) flying solo and in pairs released from a site 7 km from home, using high-precision 5 Hz global positioning system (GPS) and 200 Hz tri-axial accelerometer bio-loggers. As expected, paired individuals benefitted from improved homing route accuracy, which reduced flight distance by 7% and time by 9%. However, realising these navigational gains involved substantial changes in flight kinematics and energetics. Both individuals in a pair increased their wingbeat frequency by 18% by decreasing the duration of their upstroke. This sharp increase in wingbeat frequency caused just a 3% increase in airspeed but reduced the oscillatory displacement of the body by 22%, which we hypothesise relates to an increased requirement for visual stability and manoeuvrability when flying in a flock or pair. The combination of the increase in airspeed and a higher wingbeat frequency would result in a minimum 2.2% increase in the total aerodynamic power requirements if the wingbeats were fully optimised. Overall, the enhanced navigational performance will offset any additional energetic costs as long as the metabolic power requirements are not increased above 9%. Our results demonstrate that the increases in wingbeat frequency when flying together have previously been underestimated by an order of magnitude and force reinterpretation of their mechanistic origin. We show that, for pigeons flying in pairs, two heads are better than one but keeping a steady head necessitates energetically costly kinematics.
Assuntos
Fenômenos Biomecânicos/fisiologia , Columbidae/fisiologia , Voo Animal/fisiologia , Animais , Aves/fisiologia , Metabolismo Energético/fisiologia , Asas de Animais/fisiologiaRESUMO
Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais/fisiologia , Mucosa Intestinal/fisiologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Animais , Drosophila , Regulação da Expressão Gênica , Humanos , Camundongos , Via de Sinalização WntRESUMO
African elephants (Loxodonta africana) use many sensory modes to gather information about their environment, including the detection of seismic, or ground-based, vibrations. Seismic information is known to include elephant-generated signals, but also potentially encompasses biotic cues that are commonly referred to as 'noise'. To investigate seismic information transfer in elephants beyond communication, here we tested the hypothesis that wild elephants detect and discriminate between seismic vibrations that differ in their noise types, whether elephant- or human-generated. We played three types of seismic vibrations to elephants: seismic recordings of elephants (elephant-generated), white noise (human-generated) and a combined track (elephant- and human-generated). We found evidence of both detection of seismic noise and discrimination between the two treatments containing human-generated noise. In particular, we found evidence of retreat behaviour, where seismic tracks with human-generated noise caused elephants to move further away from the trial location. We conclude that seismic noise are cues that contain biologically relevant information for elephants that they can associate with risk. This expands our understanding of how elephants use seismic information, with implications for elephant sensory ecology and conservation management.
Assuntos
Elefantes , Animais , Aprendizagem da Esquiva , Sinais (Psicologia) , Humanos , Ruído , VibraçãoRESUMO
Dynamic regulation of histone modifications is critical during development, and aberrant activity of chromatin-modifying enzymes has been associated with diseases such as cancer. Histone demethylases have been shown to play a key role in eukaryotic gene transcription; however, little is known about how their activities are coordinated in vivo to regulate specific biological processes. In Drosophila, two enzymes, dLsd1 (Drosophila ortholog of lysine-specific demethylase 1) and Lid (little imaginal discs), demethylate histone H3 at Lys 4 (H3K4), a residue whose methylation is associated with actively transcribed genes. Our studies show that compound mutation of Lid and dLsd1 results in increased H3K4 methylation levels. However, unexpectedly, Lid mutations strongly suppress dLsd1 mutant phenotypes. Investigation of the basis for this antagonism revealed that Lid opposes the functions of dLsd1 and the histone methyltransferase Su(var)3-9 in promoting heterochromatin spreading at heterochromatin-euchromatin boundaries. Moreover, our data reveal a novel role for dLsd1 in Notch signaling in Drosophila, and a complex network of interactions between dLsd1, Lid, and Notch signaling at euchromatic genes. These findings illustrate the complexity of functional interplay between histone demethylases in vivo, providing insights into the epigenetic regulation of heterochromatin/euchromatin boundaries by Lid and dLsd1 and showing their involvement in Notch pathway-specific control of gene expression in euchromatin.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Metilação , Mutação/genética , Oxirredutases N-Desmetilantes/genética , Fenótipo , Receptores Notch/genética , Transdução de SinaisRESUMO
The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.
Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Biossíntese de Proteínas , Proteínas Repressoras/metabolismo , Animais , Cromatografia de Afinidade , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/genética , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Frações Subcelulares/metabolismoRESUMO
INTRODUCTION: Current protocols for venous thromboembolism (VTE) prophylaxis after craniofacial surgery (CFS) vary widely with substantial disagreements in both indications and managements. An evidence-based approach to this issue requires the following: the incidence of postoperative VTE, comorbidities associated with coagulopathy, risk reduction after VTE prophylaxis, and complications attributable to prophylaxis. This study addresses the first two. DESIGN: Retrospective cross-sectional study. METHODS: Discharge data from 64,170 patients undergoing CFS between 2008 and 2013 extracted from the Healthcare Cost and Utilization Project Nationwide Inpatient Sample were analyzed. The outcome measures extracted were: deep venous thrombosis, pulmonary embolism, demographic data, common comorbidities, length of stay, total cost, and discharge outcome. RESULTS: Diagnoses of deep venous thrombosis or pulmonary embolism, collectively classified as VTE, were observed in 355 (0.55%) of 64,170 patients discharged after CFS. Other surgeries exhibited a VTE rate of 1.17%. Men exhibited nearly double the incidence of VTE relative to women (0.69% compared with 0.37% respectively, P < 0.001), and the risk factors of adulthood, advanced age, cardiovascular disease, obesity, and malignancy were associated with increased VTE incidence with odds ratios of 9.93, 3.66, 1.80, 2.02, and 2.02, respectively (P < 0.005). Tobacco use did not exhibit any significant association (odds ratio, 0.94; P = 0.679). Afflicted patients experienced 4.60 times longer hospital stays averaging 23.8 days (95% confidence interval, 21.4-26.2; P < 0.001) compared the average of 5.2 days experienced by CFS patients without VTE. They incurred an average cost of US $298,228 (95% confidence interval, 262,726 to 333,731; P < 0.001) which was 4.17 times the US $72,376 expense of treating other CFS patients. The likelihood for a CFS patient to experience a poor outcome at the time of discharge was 54.6% higher after VTE. CONCLUSIONS: The risk of postoperative VTE after CFS is significantly increased in adults, patients with advanced age, cardiovascular disease, obesity, and malignancy. However even in those high-risk cases, postoperative VTE incidence remains relatively low after CFS. These findings in conjunction with further study regarding the risk associated with the addition of VTE chemoprophylaxis compared against mechanical VTE prophylaxis, such as sequential pneumatic compression stockings, may determine whether routine use of VTE chemoprophylaxis is appropriate.
Assuntos
Procedimentos Cirúrgicos Bucais , Complicações Pós-Operatórias/epidemiologia , Embolia Pulmonar/epidemiologia , Tromboembolia Venosa/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Comorbidade , Custos e Análise de Custo , Estudos Transversais , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/prevenção & controle , Estudos Retrospectivos , Fatores de Risco , Estados Unidos/epidemiologia , Tromboembolia Venosa/prevenção & controleRESUMO
We report Ni-catalyzed formal carboacylation of o-allylbenzamides with arylboronic acid pinacol esters. The reaction is triggered by oxidative addition of an activated amide C-N bond to a Ni(0) catalyst and proceeds via alkene insertion into a Ni(II)-acyl bond. The exo-selective carboacylation reaction generates 2-benzyl-2,3-dihydro-1H-inden-1-ones in moderate to high yields (46-99%) from a variety of arylboronic acid pinacol esters and substituted o-allylbenzamides. These results show that amides are practical substrates for alkene carboacylation via amide C-N bond activation, and this approach bypasses challenges associated with alkene carboacylation triggered by C-C bond activation.
RESUMO
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by a CAG expansion mutation in HTT, the gene encoding huntingtin. Evidence from both human genotype-phenotype relationships and mouse model systems suggests that the mutation acts by dysregulating some normal activity of huntingtin. Recent work in the mouse has revealed a role for huntingtin in epigenetic regulation during development. Here, we examine the role of the Drosophila huntingtin ortholog (dhtt) in chromatin regulation in the development of the fly. Although null dhtt mutants display no overt phenotype, we found that dhtt acts as a suppressor of position-effect variegation (PEV), suggesting that it influences chromatin organization. We demonstrate that dhtt affects heterochromatin spreading in a PEV model by modulating histone H3K9 methylation levels at the heterochromatin-euchromatin boundary. To gain mechanistic insights into how dhtt influences chromatin function, we conducted a candidate genetic screen using RNAi lines targeting known PEV modifier genes. We found that dhtt modifies phenotypes caused by knockdown of a number of key epigenetic regulators, including chromatin-associated proteins, histone demethylases (HDMs) and methyltransferases. Notably, dhtt strongly modifies phenotypes resulting from loss of the HDM dLsd1, in both the ovary and wing, and we demonstrate that dhtt appears to act as a facilitator of dLsd1 function in regulating global histone H3K4 methylation levels. These findings suggest that a fundamental aspect of huntingtin function in heterochromatin/euchromatin organization is evolutionarily conserved across phyla.
Assuntos
Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Heterocromatina/metabolismo , Doença de Huntington/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Drosophila/genética , Proteínas de Drosophila , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/genética , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Masculino , Metilação , Proteínas Associadas aos Microtúbulos/genética , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
We report catalytic, intramolecular hydroacylations of N-allylimidazole-2-carboxaldehydes and N-allylbenzimidazole-2-carboxaldehydes. These exo-selective hydroacylations occur in the presence of a N-heterocyclic carbene catalyst to generate 5,6-dihydro-7H-pyrrolo[1,2-α]imidazol-7-ones and 1,2-dihydro-3H-benzo[d]pyrrolo[1,2-α]imidazol-2-ones in high yields (66-99%). In addition, hydroacylations of N-allylimidazole-2-carboxaldehydes in the presence of a chiral, non-racemic NHC catalyst occur, forming 5,6-dihydro-7H-pyrrolo[1,2-α]imidazol-7-ones in moderate-to-high yields (39-98%) with modest enantioselectivities (56-79% ee).
RESUMO
Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. Among Drosophila melanogaster Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3'-5' cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue we screened 486 1(st) and 2(nd) chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, we show that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting our previous contention that ALK might be a therapeutic target in NF1, we report that human ALK is expressed in cells that give rise to NF1 tumors and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man.
Assuntos
Drosophila melanogaster/genética , Transtornos da Memória/genética , Neurofibromatose 1/genética , Quinase do Linfoma Anaplásico , Animais , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Transtornos da Memória/patologia , Mutação , Neurofibromatose 1/metabolismo , Neurofibromatose 1/fisiopatologia , Junção Neuromuscular/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes.
Assuntos
Adenosina Trifosfatases , Peptídeos Catiônicos Antimicrobianos , Proteínas de Drosophila , Drosophila melanogaster , Imunidade Inata , Fatores de Transcrição , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Corpo Adiposo/citologia , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica , Imunidade Inata/genética , Família Multigênica , Especificidade de Órgãos , Fagocitose/genética , Cromossomos Politênicos/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Proteína do Retinoblastoma , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Genetic changes in the SMARCB1 tumor suppressor gene have recently been reported in tumors and blood from families with schwannomatosis. Exon scanning of all nine SMARCB1 exons in genomic DNA from our cohort of families meeting the criteria for 'definite' or 'presumptive' schwannomatosis previously revealed constitutional alterations in 13 of 19 families (68%). Screening of four new familial schwannomatosis probands identified one additional constitutional alteration. We confirmed the presence of mRNA transcripts for two missense alterations, four mutations of conserved splice motifs and two additional mutations, in less conserved sequences, which also affect splicing. Furthermore, we found that transcripts for a rare 3'-untranslated region (c.*82C > T) alteration shared by four unrelated families did not produce splice variants but did show unequal allelic expression, suggesting that the alteration is either causative itself or linked to an unidentified causative mutation. Overexpression studies in cells lacking SMARCB1 suggest that mutant SMARCB1 proteins, like wild-type SMARCB1 protein, retain the ability to suppress cyclin D1 activity. These data, together with the expression of SMARCB1 protein in a proportion of cells from schwannomatosis-related schwannomas, suggest that these tumors develop through a mechanism that is distinct from that of rhabdoid tumors in which SMARCB1 protein is completely absent in tumor cells.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurilemoma/metabolismo , Neurofibromatoses/metabolismo , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas/genética , Alelos , Proteínas Cromossômicas não Histona/genética , Ciclina D1/genética , Ciclina D1/metabolismo , DNA Complementar , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa/genética , Humanos , Imuno-Histoquímica , Mutação , Mutação de Sentido Incorreto/genética , Neurilemoma/genética , Neurofibromatoses/genética , Splicing de RNA/genética , Estabilidade de RNA/genética , Proteína SMARCB1 , Neoplasias Cutâneas/genética , Fatores de Transcrição/genéticaRESUMO
Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. We report two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. We show that dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1.
Assuntos
Aprendizagem por Associação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Quinase do Linfoma Anaplásico , Animais , Tamanho Corporal/genética , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Humanos , Sistema de Sinalização das MAP Quinases/genética , Terapia de Alvo Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Neurofibromina 1/antagonistas & inibidores , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Neurônios/metabolismo , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
The three-dimensional swimming tracks of motile microorganisms can be used to identify their species, which holds promise for the rapid identification of bacterial pathogens. The tracks also provide detailed information on the cells' responses to external stimuli such as chemical gradients and physical objects. Digital holographic microscopy (DHM) is a well-established, but computationally intensive method for obtaining three-dimensional cell tracks from video microscopy data. We demonstrate that a common neural network (NN) accelerates the analysis of holographic data by an order of magnitude, enabling its use on single-board computers and in real time. We establish a heuristic relationship between the distance of a cell from the focal plane and the size of the bounding box assigned to it by the NN, allowing us to rapidly localise cells in three dimensions as they swim. This technique opens the possibility of providing real-time feedback in experiments, for example by monitoring and adapting the supply of nutrients to a microbial bioreactor in response to changes in the swimming phenotype of microbes, or for rapid identification of bacterial pathogens in drinking water or clinical samples.
Assuntos
Aprendizado Profundo , Holografia , Microscopia , Holografia/métodos , Microscopia/métodos , Imageamento Tridimensional/métodos , Bactérias , Imageamento Quantitativo de FaseRESUMO
Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutation of the NF1 gene that is associated with various symptoms, including the formation of benign tumors, called neurofibromas, within nerves. Drug treatments are currently limited. The mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib is used for a subset of plexiform neurofibromas (PNs) but is not always effective and can cause side effects. Therefore, there is a clear need to discover new drugs to target NF1-deficient tumor cells. Using a Drosophila cell model of NF1, we performed synthetic lethal screens to identify novel drug targets. We identified 54 gene candidates, which were validated with variable dose analysis as a secondary screen. Pathways associated with five candidates could be targeted using existing drugs. Among these, chloroquine (CQ) and bafilomycin A1, known to target the autophagy pathway, showed the greatest potential for selectively killing NF1-deficient Drosophila cells. When further investigating autophagy-related genes, we found that 14 out of 30 genes tested had a synthetic lethal interaction with NF1. These 14 genes are involved in multiple aspects of the autophagy pathway and can be targeted with additional drugs that mediate the autophagy pathway, although CQ was the most effective. The lethal effect of autophagy inhibitors was conserved in a panel of human NF1-deficient Schwann cell lines, highlighting their translational potential. The effect of CQ was also conserved in a Drosophila NF1 in vivo model and in a xenografted NF1-deficient tumor cell line grown in mice, with CQ treatment resulting in a more significant reduction in tumor growth than selumetinib treatment. Furthermore, combined treatment with CQ and selumetinib resulted in a further reduction in NF1-deficient cell viability. In conclusion, NF1-deficient cells are vulnerable to disruption of the autophagy pathway. This pathway represents a promising target for the treatment of NF1-associated tumors, and we identified CQ as a candidate drug for the treatment of NF1 tumors.