RESUMO
Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.
Assuntos
Análise Mutacional de DNA , Genoma Humano/genética , Meduloblastoma/classificação , Meduloblastoma/genética , Sequenciamento Completo do Genoma , Carcinogênese/genética , Proteínas de Transporte/genética , Estudos de Coortes , Metilação de DNA , Conjuntos de Dados como Assunto , Epistasia Genética , Genômica , Humanos , Terapia de Alvo Molecular , Proteínas Musculares/genética , Mutação , Oncogenes/genética , Fatores de Transcrição/genética , Proteínas Wnt/genéticaRESUMO
AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA) in multiple signaling contexts. Here, we demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF) mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, we demonstrate that AKAP200 interacts positively with Notch in both the eye and the thorax. We further show that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, our genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, we demonstrate that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, our data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Lisossomos/metabolismo , Proteínas de Membrana/fisiologia , Proteólise , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores Notch/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Larva , Proteínas de Membrana/genética , Estabilidade Proteica , Transdução de Sinais/genética , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
Planar cell polarity (PCP) instructs tissue patterning in a wide range of organisms from fruit flies to humans. PCP signaling coordinates cell behavior across tissues and is integrated by cells to couple cell fate identity with position in a developing tissue. In the fly eye, PCP signaling is required for the specification of R3 and R4 photoreceptors based upon their positioning relative to the dorso-ventral axis. The 'core' PCP pathway involves the asymmetric localization of two distinct membrane-bound complexes, one containing Frizzled (Fz, required in R3) and the other Van Gogh (Vang, required in R4). Inhibitory interactions between the cytosolic components of each complex reinforce asymmetric localization. Prickle (Pk) and Spiny-legs (Pk-Sple) are two antagonistic isoforms of the prickle (pk) gene and are cytoplasmic components of the Vang complex. The balance between their levels is critical for tissue patterning, with Pk-Sple being the major functional isoform in the eye. Here we uncover a post-translational role for Nemo kinase in limiting the amount of the minor isoform Pk. We identified Pk as a Nemo substrate in a genome-wide in vitro band-shift screen. In vivo, nemo genetically interacts with pkpk but not pksple and enhances PCP defects in the eye and leg. Nemo phosphorylation limits Pk levels and is required specifically in the R4 photoreceptor like the major isoform, Pk-Sple. Genetic interaction and biochemical data suggest that Nemo phosphorylation of Pk leads to its proteasomal degradation via the Cullin1/SkpA/Slmb complex. dTAK and Homeodomain interacting protein kinase (Hipk) may also act together with Nemo to target Pk for degradation, consistent with similar observations in mammalian studies. Our results therefore demonstrate a mechanism to maintain low levels of the minor Pk isoform, allowing PCP complexes to form correctly and specify cell fate.
Assuntos
Polaridade Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas com Domínio LIM/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Olho/citologia , Olho/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , Transdução de Sinais/genética , Especificidade por Substrato , Asas de Animais/citologia , Asas de Animais/metabolismoRESUMO
Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.
Assuntos
Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Meduloblastoma/genética , Análise de Sequência de DNA/métodos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Feminino , Genoma/genética , Histonas/metabolismo , Humanos , Meduloblastoma/patologia , Camundongos , Regiões Promotoras Genéticas/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Variação Estrutural do Genoma/genética , Meduloblastoma/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Criança , Cromossomos Humanos Par 9/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Meduloblastoma/classificação , Meduloblastoma/patologia , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
Assuntos
Neoplasias Cerebelares/genética , Genoma Humano/genética , Meduloblastoma/genética , Envelhecimento/genética , Sequência de Aminoácidos , Transformação Celular Neoplásica , Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/diagnóstico , Neoplasias Cerebelares/patologia , Criança , Cromatina/metabolismo , Cromossomos Humanos/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genômica , Proteínas Hedgehog/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Histona Desmetilases/genética , Humanos , Meduloblastoma/classificação , Meduloblastoma/diagnóstico , Meduloblastoma/patologia , Metilação , Mutação/genética , Taxa de Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Receptores Patched , Receptor Patched-1 , Fosfoproteínas Fosfatases/genética , Poliploidia , Receptores de Superfície Celular/genética , Análise de Sequência de RNA , Transdução de Sinais , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
OBJECTIVE: To demonstrate the effects of allogeneic islet cell matrix implants for glycaemic control in rats with induced diabetes. METHOD: Sprague-Dawley rats were used as allogeneic donors of islet cells. Cells were seeded on three-dimensional proprietary poly-(l-lactide) matrices. Animals were rendered diabetic and a week later a matrix seeded with islet cells (IMI group) or a control matrix (placebo group) was implanted in the small bowel mesentery. Blood glucose levels were measured weekly for 12 weeks. After sacrifice, implant sections were Gomori stained for beta-cells and immuno-stained for insulin 3, 4, 5, and 6 months post implantation. RESULTS: 82% of seeded islet cells attached to the matrices. In the IMI group blood glucose levels were significantly reduced after implantation compared with before implantation across several time points. In the IMI group beta-cells and insulin-positive cells were identified at the implant site. CONCLUSION: The islet cell matrix implant reduced the blood glucose levels although complete normo-glycaemia was not established. The islet cell matrix implant may serve as an additional option for islet cell transplantation using 3D scaffold platforms for better survival and function of the islet cells.
Assuntos
Diabetes Mellitus Experimental/terapia , Hiperglicemia/terapia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas , Animais , Glicemia/metabolismo , Peso Corporal , Técnicas de Cultura de Células , Separação Celular , Sobrevivência Celular , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Hiperglicemia/etiologia , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Masculino , Poliésteres , Ratos , Ratos Sprague-DawleyRESUMO
Tropomyosins are widespread actin-binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin-binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1-mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half-life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin-deficient human MDA-MB 231 breast cancer cells and TPMα-depleted fibroblasts. Gene silencing of TPMα but not TPMß causes loss of stress fibres by promoting Smurf1-mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA-MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin-binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type-specific fashion.
Assuntos
Proteínas dos Microfilamentos/fisiologia , Neoplasias/genética , Tropomiosina/genética , Tropomiosina/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Humanos , Camundongos , Células NIH 3T3 , Neoplasias/patologiaRESUMO
The standard surgical procedure for abdominal hernia repair with conventional prosthetic mesh still results in a high recurrence rate. In the present study, we propose a fibroblast matrix implant (FMI), which is a three-dimensional (3D) poly-L-lactic acid scaffold coated with collagen (matrix) and seeded with fibroblasts, as an alternative mesh for hernia repair. The matrix was seeded with fibroblasts (cellularized) and treated with a conditioned medium (CM) of human Umbilical Cord Mesenchymal Stem Cells (hUC-MSC). Fibroblast proliferation and function were assessed and compared between treated with CM hUC-MSC and untreated group, 24 h after seeding onto the matrix (n= 3). To study the matricesin vivo,the hernia was surgically created on male Sprague Dawley rats and repaired with four different grafts (n= 3), including a commercial mesh (mesh group), a matrix without cells (cell-free group), a matrix seeded with fibroblasts (FMI group), and a matrix seeded with fibroblasts and cultured in medium treated with 1% CM hUC-MSC (FMI-CM group).In vitroexamination showed that the fibroblasts' proliferation on the matrices (treated group) did not differ significantly compared to the untreated group. CM hUC-MSC was able to promote the collagen synthesis of the fibroblasts, resulting in a higher collagen concentration compared to the untreated group. Furthermore, thein vivostudy showed that the matrices allowed fibroblast growth and supported cell functionality for at least 1 month after implantation. The highest number of fibroblasts was observed in the FMI group at the 14 d endpoint, but at the 28 d endpoint, the FMI-CM group had the highest. Collagen deposition area and neovascularization at the implantation site were observed in all groups without any significant difference between the groups. FMI combined with CM hUC-MSC may serve as a better option for hernia repair, providing additional reinforcement which in turn should reduce hernia recurrence.
Assuntos
Proliferação de Células , Colágeno , Fibroblastos , Herniorrafia , Hérnia Incisional , Células-Tronco Mesenquimais , Ratos Sprague-Dawley , Telas Cirúrgicas , Alicerces Teciduais , Animais , Fibroblastos/metabolismo , Ratos , Masculino , Humanos , Células-Tronco Mesenquimais/citologia , Herniorrafia/métodos , Herniorrafia/instrumentação , Colágeno/química , Alicerces Teciduais/química , Hérnia Incisional/cirurgia , Poliésteres/química , Teste de Materiais , Meios de Cultivo Condicionados/farmacologia , Materiais Biocompatíveis/química , Células Cultivadas , Hérnia Abdominal/cirurgia , Cordão Umbilical/citologiaRESUMO
The establishment of polarity in many cell types depends on Lgl, the tumour suppressor product of lethal giant larvae, which is involved in basolateral protein targeting. The conserved complex of Par3, Par6 and atypical protein kinase C phosphorylates and inactivates Lgl at the apical surface; however, the signalling mechanisms that coordinate cell polarization in development are not well defined. Here we show that a vertebrate homologue of Lgl associates with Dishevelled, an essential mediator of Wnt signalling, and that Dishevelled regulates the localization of Lgl in Xenopus ectoderm and Drosophila follicular epithelium. We show that both Lgl and Dsh are required for normal apical-basal polarity of Xenopus ectodermal cells. In addition, we show that the Wnt receptor Frizzled 8, but not Frizzled 7, causes Lgl to dissociate from the cortex with the concomitant loss of its activity in vivo. These findings suggest a molecular basis for the regulation of cell polarity by Frizzled and Dishevelled.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fosfoproteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Membrana Celular/metabolismo , Polaridade Celular , Proteínas Desgrenhadas , Ectoderma/citologia , Ectoderma/metabolismo , Epitélio/metabolismo , Feminino , Imunoprecipitação , Masculino , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Ligação Proteica , Proteína Quinase C/metabolismo , Transporte Proteico , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriologiaRESUMO
The phenomenon of tissue fluidity-cells' ability to rearrange relative to each other in confluent tissues-has been linked to several morphogenetic processes and diseases, yet few molecular regulators of tissue fluidity are known. Ommatidial rotation (OR), directed by planar cell polarity signaling, occurs during Drosophila eye morphogenesis and shares many features with polarized cellular migration in vertebrates. We utilize in vivo live imaging analysis tools to quantify dynamic cellular morphologies during OR, revealing that OR is driven autonomously by ommatidial cell clusters rotating in successive pulses within a permissive substrate. Through analysis of a rotation-specific nemo mutant, we demonstrate that precise regulation of junctional E-cadherin levels is critical for modulating the mechanical properties of the tissue to allow rotation to progress. Our study defines Nemo as a molecular tool to induce a transition from solid-like tissues to more viscoelastic tissues broadening our molecular understanding of tissue fluidity.
Assuntos
Junções Aderentes , Polaridade Celular , Líquido Extracelular , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Caderinas , Polaridade Celular/genética , Polaridade Celular/fisiologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ectoderma , Olho/citologia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfogênese , Asas de Animais/citologiaRESUMO
Orthotopic liver transplantation is presently the most effectual method for the treatment of end-stage liver diseases. Though, one major issue is the restricted number of donor organs that are accessible. Hence, liver tissue engineering is under investigation with the goal of restoring liver functions. In this study, we investigated 3D porous scaffolds made of PLLA coated with a nano thick collagen layer (matrices). Primary rat dermal fibroblasts were used in a first study phase to check matrices' cytocompatibility. More than 70% of seeded cells could adhere and remain viable 24 and 48 hours after the seeding. To test the suitability of the matrices for human primary hepatocytes, HepaRG cells were seeded and analyzed for viability, adhesion rate, and functionality such as albumin secretion. About 80% of seeded HepaRG adhered to the scaffolds remaining viable up to 72 hours. Cells were homogeneously distributed in the entire scaffold with albumin secretion increasing with time. Our results indicate that PLLA collagen-coated matrices allow hepatocytes attachment and distribution throughout the 3D structure, as well as support cell functionality. Such matrices have been applied in our clinical phase II trial. Functional hepatocytes were successfully implanted in patients suffering from liver-cirrhosis with higher cell numbers and adhesions rate compared to our previous trial with the first matrix type and a general improvement in clinical condition.
Assuntos
Colágeno/química , Fibroblastos/citologia , Hepatócitos/citologia , Nanoestruturas/química , Poliésteres/química , Alicerces Teciduais/química , Animais , Linhagem Celular , Matriz Extracelular , Masculino , Microscopia Eletrônica de Varredura , Poliésteres/metabolismo , Porosidade , Ratos , Ratos Sprague-Dawley , Suínos , Sais de Tetrazólio/química , Tiazóis/química , Engenharia Tecidual/métodosRESUMO
The human autologous hepatocyte matrix implant is a promising alternative procedure to counter liver damage. We assessed the outcome of human hepatocytes isolation from cirrhotic liver compared to the clinical and histological scores of disease severity. A total of 11 patients with various clinical scores (CTP and MELD) and histological score (Metavir, fibrosis) of liver cirrhosis were included in the hepatocyte matrix implant clinical phase I study. The liver segment and pancreatic tissue were harvested from each patient, and hepatocytes and cells of islets of Langerhans were isolated. The freshly isolated human hepatocytes were coseeded with the islet cells onto poly(l-lactic acid) (PLLA) scaffolds, cultured, and transplanted back into the patient. Human hepatocytes were isolated from 11 cirrhotic liver specimens with a resulting yield of 1.4 ± 0.5 × 106 cells per gram of the liver specimen and a viability rate of 52 ± 13%. It was found that the yield and viability of the liver cells were not correlated with the clinical and histological scores of the liver cirrhosis. A correlation was found between the hepatocyte yield obtained and the average number of hepatocytes counted in 10 microscopic fields of view. More viable cells were obtained from cirrhotic livers caused by chronic hepatitis B as compared to chronic hepatitis C in the same MELD score range. There was no correlation between the clinical and histological disease severity scores of liver cirrhosis and the outcome of hepatocytes isolation. It seems that the yield could depend on the type of hepatitis underlying the cirrhotic tissue. The study was registered at www.clinicaltrial.gov with the study identifier: NCT01335568.
RESUMO
BACKGROUND: Chronic rhinosinusitis may be associated with nasal polyposis. Recurrence of disease is often observed and may be due to an intolerance of acetylsalicylic acid. Sex hormones are known to modulate allergic reactions and inflammation. Whether they may be involved in the development and progression of nasal polyposis has not been investigated yet. AIM: Examine the relationship between levels of sex hormones and nasal polyposis. METHODS: Hormonal levels (estradiol, testosterone and progesterone) in patients with nasal polyposis (n = 26) with or without acetylsalicylic acid-intolerance were determined and compared to hormonal levels in patients with septal deviation (n = 35). Cone-beam computed tomography scans were analysed by using scores as defined by Lund and Mackay and by Kennedy. RESULTS: Our results show a 5 times greater odds (p = 0.01) for developing nasal polyposis in the presence of lowered estradiol plasma levels than in the presence of normal / elevated levels. When analyzing females and males separately, a 6 times greater odds for females to develop nasal polyposis in the presence of lowered estradiol plasma levels was calculated (p = 0.02). Thus, females are more likely to develop nasal polyposis when they have lowered estradiol levels than males. In addition, female patients showed an increased risk for developing ASA intolerance (p = 0.01). CONCLUSION: Variation of sex hormones may be involved in nasal polyposis. Further studies including more patients to validate the presented results are required. SIGNIFICANCE: Retrospective clinical investigation suggesting a correlation between varying sex hormones and nasal polyposis.
Assuntos
Aspirina/efeitos adversos , Hipersensibilidade a Drogas/epidemiologia , Estradiol/sangue , Pólipos Nasais/imunologia , Progesterona/sangue , Testosterona/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Tomografia Computadorizada de Feixe Cônico , Hipersensibilidade a Drogas/sangue , Hipersensibilidade a Drogas/imunologia , Estradiol/imunologia , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/diagnóstico por imagem , Pólipos Nasais/sangue , Pólipos Nasais/diagnóstico , Progesterona/imunologia , Recidiva , Estudos Retrospectivos , Rinite/sangue , Rinite/induzido quimicamente , Rinite/epidemiologia , Rinite/imunologia , Sinusite/sangue , Sinusite/induzido quimicamente , Sinusite/epidemiologia , Sinusite/imunologia , Testosterona/imunologia , Adulto JovemRESUMO
Frizzled (Fz)/PCP signaling regulates planar, vectorial orientation of cells or groups of cells within whole tissues. Although Fz/PCP signaling has been analyzed in several contexts, little is known about nuclear events acting downstream of Fz/PCP signaling in the R3/R4 cell fate decision in the Drosophila eye or in other contexts. Here we demonstrate a specific requirement for Egfr-signaling and the transcription factors Fos (AP-1), Yan and Pnt in PCP dependent R3/R4 specification. Loss and gain-of-function assays suggest that the transcription factors integrate input from Fz/PCP and Egfr-signaling and that the ETS factors Pnt and Yan cooperate with Fos (and Jun) in the PCP-specific R3/R4 determination. Our data indicate that Fos (either downstream of Fz/PCP signaling or parallel to it) and Yan are required in R3 to specify its fate (Fos) or inhibit R4 fate (Yan) and that Egfr-signaling is required in R4 via Pnt for its fate specification. Taken together with previous work establishing a Notch-dependent Su(H) function in R4, we conclude that Fos, Yan, Pnt, and Su(H) integrate Egfr, Fz, and Notch signaling input in R3 or R4 to establish cell fate and ommatidial polarity.
Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Receptores ErbB/metabolismo , Olho/crescimento & desenvolvimento , Receptores Frizzled/metabolismo , Proteínas Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos de Invertebrados/metabolismo , Fatores de Transcrição/metabolismo , Animais , Polaridade Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/citologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Receptores ErbB/genética , Olho/citologia , Olho/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Receptores Frizzled/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos de Invertebrados/genética , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The role of phospholipids in the regulation of membrane trafficking and signaling is largely unknown. Phosphatidylcholine (PC) is a main component of the plasma membrane. Mutants in the Drosophila phosphocholine cytidylyltransferase 1 (CCT1), the rate-limiting enzyme in PC biosynthesis, show an altered phospholipid composition with reduced PC and increased phosphatidylinositol (PI) levels. Phenotypic features of dCCT1 indicate that the enzyme is not required for cell survival, but serves a role in endocytic regulation. CCT1- cells show an increase in endocytosis and enlarged endosomal compartments, whereas lysosomal delivery is unchanged. As a consequence, an increase in endocytic localization of EGF receptor (Egfr) and Notch is observed, and this correlates with a reduction in signaling strength and leads to patterning defects. A further link between PC/PI content, endocytosis, and signaling is supported by genetic interactions of dCCT1 with Egfr, Notch, and genes affecting endosomal traffic.
Assuntos
Drosophila/crescimento & desenvolvimento , Endocitose/fisiologia , Receptores ErbB/fisiologia , Proteínas de Membrana/fisiologia , Fosfolipídeos/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Sobrevivência Celular , Cromatografia em Camada Fina , Clatrina/metabolismo , Clonagem Molecular , Invaginações Revestidas da Membrana Celular/ultraestrutura , Proteínas de Drosophila , Anormalidades do Olho/genética , Anormalidades do Olho/ultraestrutura , Proteínas de Choque Térmico/genética , Imuno-Histoquímica , Proteínas de Insetos/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Chaperonas Moleculares/genética , Mutação , Fenótipo , Fosfolipídeos/química , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Notch , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , TransgenesRESUMO
Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.
Assuntos
Membrana Celular/química , Núcleo Celular/química , Colina-Fosfato Citidililtransferase/metabolismo , Elasticidade , Membrana Nuclear/química , Fosfatidilcolinas/metabolismo , Estresse Fisiológico , Animais , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Colina-Fosfato Citidililtransferase/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase, well known for its role in cell-cycle progression. However, it has been linked to additional functions, mainly in neuronal contexts, when using the co-activator Cdh1/Fzr. Here, our data indicate a post-mitotic requirement for the APC/CFzr/Cdh1 in epithelial cell patterning and planar cell polarity (PCP) in Drosophila. PCP signaling is critical for development by establishing cellular asymmetries and orientation within the plane of an epithelium, via differential localization of distinct complexes of core PCP factors. Loss of APC/C function leads to reduced levels of Dishevelled (Dsh), a core PCP factor. The effect of APC/C on Dsh is mediated by Nek2 kinase, which can phosphorylate Dsh and is a direct APC/CFzr/Cdh1 substrate. We have thus uncovered a pathway of regulation whereby APC/CFzr/Cdh1 negatively regulates Nek2, which negatively regulates Dsh, to ensure its proper stoichiometric requirement and localization during PCP establishment.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Polaridade Celular , Proteínas Desgrenhadas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Ciclo Celular , Olho/citologia , Olho/enzimologia , Técnicas de Silenciamento de Genes , Complexo de Endopeptidases do Proteassoma/metabolismo , Asas de Animais/citologia , Asas de Animais/enzimologiaRESUMO
A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo.
Assuntos
Fator 1 de Ribosilação do ADP/genética , Complexo 1 de Proteínas Adaptadoras/genética , Polaridade Celular/genética , Proteínas de Drosophila/genética , Drosophila/embriologia , Asas de Animais/embriologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Desgrenhadas , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fosfoproteínas/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Krüppel like factors (KLFs) are conserved transcription factors that have been implicated in many developmental processes including differentiation, organ patterning, or regulation of stem cell pluripotency. We report the generation and analysis of loss-of-function mutants of Drosophila Klf6/7, the luna gene. We demonstrate that luna mutants are associated with very early embryonic defects prior to cellularization at the syncytial stage and cause DNA separation defects during the rapid mitotic cycles resulting in un-coupled DNA and centrosome cycles. These defects manifest themselves, both in animals that are maternally homozygous and heterozygous mutant. Surprisingly, luna is only required during the syncytial stages and not later in development, suggesting that the DNA segregation defect is linked to centrosomes, since centrosomes are dispensable for later cell divisions.