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Urine cytology is non-invasive, easy to collect, with medium sensitivity and a high specificity. It is an effective way to detect high-grade bladder cancer (BC), but it is less effective on low-grade BC because the rate of equivocal results is much higher. Recently, the fluorescent properties of plasma membranes of urothelial tumor cells (UTC) found in urine cytology have been shown to be useful in improving the early detection of BC. This phenomenon is called peri-membrane fluorescence (PMF). Based on previous studies that have identified the PMF on UTCs, the main objective was to characterize this phenomenon. For this study, a software was specially created to quantify the PMF of all tested cells and different treatments performed. PMF was not found to be a morphological and discriminating feature of UTCs, all cells in shape and not from urine show PMF. We were able to highlight the crucial role of plasma membrane integrity in the maintenance of PMF. Finally, it was found that the induction of a strong cellular stress induced a decrease in PMF, mimicking what was observed in non-tumor cells collected from urine. These results suggest that PMF is found in cells able to resist this stress, such as tumor cells.
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Liquid biopsies are increasingly used for the diagnosis and follow-up of cancer patients. Urine is a body fluid that can be used to detect cancers and others diseases. It is noninvasive and easy to collect. To detect Bladder Cancer (BC), cytology is the first assay used. It is an effective way to detect high grade BC but has a high rate of equivocal results, especially for low grade BC. Furthermore, cystoscopy is used to confirm cytology results and to determine cancer status. Cystoscopy is also effective but highly invasive, and not well accepted by patients, especially for BC follow-up. In this review we survey the numerous assays recently developed in order to diagnose BC at an early stage, and to facilitate the follow-up of patients. We discuss their effectiveness, ease of use, and applications. Finally, we discuss assays that, in the future, could improve the diagnosis and management of BC patients.
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Facioscapulohumeral dystrophy (FSHD, OMIM: 158900, 158901) is the most common dystrophy in adults and so far, there is no treatment. Different loci of the disease have been characterized and they all lead to the aberrant expression of the DUX4 protein, which impairs the function of the muscle, ultimately leading to cell death. Here, we used gene editing to try to permanently shut down DUX4 expression by targeting its poly(A) sequence. We used transcription activator-like effector nucleases (TALEN) and CRISPR-Cas9 nucleases in vitro on FSHD myoblasts. More than 150 TOPO clones were sequenced and only indels were observed in 4%. Importantly, in 2 of them, the DUX4 poly(A) signal was eliminated at the genomic level but DUX4 mRNA was still produced thanks to the use of a non-canonical upstream poly(A) signal sequence. These experiments show that targeting DUX4 PAS at the genomic level might not be an appropriate gene editing strategy for FSHD therapy.
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CEP55 regulates the final critical step of cell division termed cytokinetic abscission. We report herein that CEP55 contains two NEMO-like ubiquitin-binding domains (UBDs), NOA and ZF, which regulate its function in a different manner. In vitro studies of isolated domains showed that NOA adopts a dimeric coiled-coil structure, whereas ZF is based on a UBZ scaffold. Strikingly, CEP55 knocked-down HeLa cells reconstituted with the full-length CEP55 ubiquitin-binding defective mutants, containing structure-guided mutations either in NOACEP55 or ZFCEP55 domains, display severe abscission defects. In addition, the ZFCEP55 can be functionally replaced by some ZF-based UBDs belonging to the UBZ family, indicating that the essential function of ZFCEP55 is to act as ubiquitin receptor. Our work reveals an unexpected role of CEP55 in non-degradative ubiquitin signaling during cytokinetic abscission and provides a molecular basis as to how CEP55 mutations can lead to neurological disorders such as the MARCH syndrome.
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The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.
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Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Mitocondrial/genética , Fibroblastos/fisiologia , Dosagem de Genes/genética , Mitocôndrias/genética , Modelos Animais , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Pitx2c, a homeodomain transcription factor, is classically known for its left-right patterning role. However, an early wave of pitx2 expression occurs at the onset of gastrulation in several species, indicating a possible earlier role that remains relatively unexplored. Here we show that in zebrafish, maternal-zygotic (MZ) pitx2c mutants exhibit a shortened body axis indicative of convergence and extension (CE) defects. Live imaging reveals that MZpitx2c mutants display less persistent mesendodermal migration during late stages of gastrulation. Transplant data indicate that Pitx2c functions cell non-autonomously to regulate this cell behavior by modulating cell shape and protrusive activity. Using transcriptomic analyses and candidate gene approaches, we identify transcriptional changes in components of the chemokine-ECM-integrin dependent mesendodermal migration network. Together, our results define pathways downstream of Pitx2c that are required during early embryogenesis and reveal novel functions for Pitx2c as a regulator of morphogenesis.
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Movimento Celular/genética , Desenvolvimento Embrionário/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Forma Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Embrião não Mamífero , Endoderma/citologia , Endoderma/metabolismo , Células Epiteliais/citologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Gastrulação/genética , Integrinas/genética , Integrinas/metabolismo , Mutação , Notocorda/citologia , Notocorda/metabolismo , Imagem com Lapso de Tempo , Fatores de Transcrição/metabolismo , Transcriptoma , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Zigoto/citologia , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismoRESUMO
A conserved organizational and functional principle of neural networks is the segregation of axon-dendritic synaptic connections into laminae. Here we report that targeting of synaptic laminae by retinal ganglion cell (RGC) arbors in the vertebrate visual system is regulated by a signaling system relying on target-derived Reelin and VLDLR/Dab1a on the projecting neurons. Furthermore, we find that Reelin is distributed as a gradient on the target tissue and stabilized by heparan sulfate proteoglycans (HSPGs) in the extracellular matrix (ECM). Through genetic manipulations, we show that this Reelin gradient is important for laminar targeting and that it is attractive for RGC axons. Finally, we suggest a comprehensive model of synaptic lamina formation in which attractive Reelin counter-balances repulsive Slit1, thereby guiding RGC axons toward single synaptic laminae. We establish a mechanism that may represent a general principle for neural network assembly in vertebrate species and across different brain areas.
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Moléculas de Adesão Celular Neuronais/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Células Ganglionares da Retina/metabolismo , Serina Endopeptidases/biossíntese , Sinapses/metabolismo , Vias Visuais/metabolismo , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular Neuronais/análise , Proteínas da Matriz Extracelular/análise , Rede Nervosa/química , Proteínas do Tecido Nervoso/análise , Proteína Reelina , Células Ganglionares da Retina/química , Serina Endopeptidases/análise , Sinapses/química , Vias Visuais/química , Peixe-ZebraRESUMO
Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington's disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction.
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Quebras de DNA de Cadeia Dupla , Reparo do DNA , Saccharomyces cerevisiae/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Modelos Biológicos , Mutação/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.
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Replicação do DNA , DNA Mitocondrial/metabolismo , Deleção de Sequência , Imagem Individual de Molécula/métodos , Envelhecimento/genética , DNA Helicases/genética , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.
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Processamento Alternativo , Proteínas Argonautas/metabolismo , HIV-1/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Sítios de Ligação , RNA Helicases DEAD-box/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Genoma Viral , Células HEK293 , HIV-1/fisiologia , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , Células Jurkat , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Viral/química , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Vírion/fisiologiaRESUMO
Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.
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DNA Mitocondrial/genética , Endonucleases/metabolismo , Mitocôndrias/metabolismo , Mutação/genética , Dedos de Zinco , Linhagem Celular Tumoral , Citometria de Fluxo , Dosagem de Genes , Humanos , RNA Catalítico/metabolismoRESUMO
Throughout vertebrates, cerebrospinal fluid-contacting neurons (CSF-cNs) are ciliated cells surrounding the central canal in the ventral spinal cord. Their contribution to modulate locomotion remains undetermined. Recently, we have shown CSF-cNs modulate locomotion by directly projecting onto the locomotor central pattern generators (CPGs), but the sensory modality these cells convey to spinal circuits and their relevance to innate locomotion remain elusive. Here, we demonstrate in vivo that CSF-cNs form an intraspinal mechanosensory organ that detects spinal bending. By performing calcium imaging in moving animals, we show that CSF-cNs respond to both passive and active bending of the spinal cord. In mutants for the channel Pkd2l1, CSF-cNs lose their response to bending and animals show a selective reduction of tail beat frequency, confirming the central role of this feedback loop for optimizing locomotion. Altogether, our study reveals that CSF-cNs constitute a mechanosensory organ operating during locomotion to modulate spinal CPGs.
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Líquido Cefalorraquidiano/citologia , Neurônios/citologia , Medula Espinal/citologia , Animais , Fenômenos Biomecânicos , Movimento Celular , Líquido Cefalorraquidiano/metabolismo , Feminino , Masculino , Mecanorreceptores/citologia , Mecanorreceptores/metabolismo , Neurônios/metabolismo , Medula Espinal/química , Medula Espinal/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.
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Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Marcação de Genes , Humanos , Mutação INDEL , Camundongos , Oligonucleotídeos/genética , Ratos , Peixe-ZebraRESUMO
The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.