Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 36
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Front Neurosci ; 17: 1172016, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37859764

RESUMO

Neurons forming the human brain are generated during embryonic development by neural stem and progenitor cells via a process called neurogenesis. A crucial feature contributing to neural stem cell morphological and functional heterogeneity is cell polarity, defined as asymmetric distribution of cellular components. Cell polarity is built and maintained thanks to the interplay between polarity proteins and polarity-generating organelles, such as the endoplasmic reticulum (ER) and the Golgi apparatus (GA). ER and GA affect the distribution of membrane components and work as a hub where glycans are added to nascent proteins and lipids. In the last decades our knowledge on the role of polarity in neural stem and progenitor cells have increased tremendously. However, the role of traffic and associated glycosylation in neural stem and progenitor cells is still relatively underexplored. In this review, we discuss the link between cell polarity, architecture, identity and intracellular traffic, and highlight how studies on neurons have shaped our knowledge and conceptual framework on traffic and polarity. We will then conclude by discussing how a group of rare diseases, called congenital disorders of glycosylation (CDG) offers the unique opportunity to study the contribution of traffic and glycosylation in the context of neurodevelopment.

2.
EMBO Rep ; 24(8): e56233, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37382163

RESUMO

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.


Assuntos
Aspartato-Amônia Ligase , Células-Tronco Neurais , Aspartato-Amônia Ligase/metabolismo , Diferenciação Celular/genética , Células-Tronco Neurais/metabolismo , Neurogênese/genética
4.
FEBS J ; 289(6): 1524-1535, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33638923

RESUMO

Comparing the biology of humans to that of other primates, and notably other hominids, is a useful path to learn more about what makes us human. Some of the most interesting differences among hominids are closely related to brain development and function, for example behaviour and cognition. This makes it particularly interesting to compare the hominid neural cells of the neocortex, a part of the brain that plays central roles in those processes. However, well-preserved tissue from great apes is usually extremely difficult to obtain. A variety of new alternative tools, for example brain organoids, are now beginning to make it possible to search for such differences and analyse their potential biological and biomedical meaning. Here, we present an overview of recent findings from comparisons of the neural stem and progenitor cells (NSPCs) and neurons of hominids. In addition to differences in proliferation and differentiation of NSPCs, and maturation of neurons, we highlight that the regulation of the timing of these processes is emerging as a general foundational difference in the development of the neocortex of hominids.


Assuntos
Hominidae , Neocórtex , Células-Tronco Neurais , Animais , Neocórtex/fisiologia , Neurogênese , Neurônios
5.
Stem Cell Reports ; 16(9): 2118-2127, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34358451

RESUMO

Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Células Cultivadas , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , RNA-Seq , Transcriptoma
6.
Front Cell Dev Biol ; 9: 661113, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968936

RESUMO

The cellular, molecular and functional comparison of neurons from closely related species is crucial in evolutionary neurobiology. The access to living tissue and post-mortem brains of humans and non-human primates is limited and the state of the tissue might not allow recapitulating important species-specific differences. A valid alternative is offered by neurons derived from induced pluripotent stem cells (iPSCs) obtained from humans and non-human apes and primates. We will review herein the contribution of iPSCs-derived neuronal models to the field of evolutionary neurobiology, focusing on species-specific aspects of neuron's cell biology and timing of maturation. In addition, we will discuss the use of iPSCs for the study of ancient human traits.

7.
J Vis Exp ; (167)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33554975

RESUMO

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.


Assuntos
Encéfalo/citologia , Microinjeções , Células-Tronco Neurais/citologia , Neurônios/citologia , Robótica , Análise de Célula Única , Animais , Automação , Imunofluorescência , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Tecidos
8.
Elife ; 102021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33470930

RESUMO

We generated induced excitatory neurons (iNeurons, iNs) from chimpanzee, bonobo, and human stem cells by expressing the transcription factor neurogenin-2 (NGN2). Single-cell RNA sequencing showed that genes involved in dendrite and synapse development are expressed earlier during iNs maturation in the chimpanzee and bonobo than the human cells. In accordance, during the first 2 weeks of differentiation, chimpanzee and bonobo iNs showed repetitive action potentials and more spontaneous excitatory activity than human iNs, and extended neurites of higher total length. However, the axons of human iNs were slightly longer at 5 weeks of differentiation. The timing of the establishment of neuronal polarity did not differ between the species. Chimpanzee, bonobo, and human neurites eventually reached the same level of structural complexity. Thus, human iNs develop slower than chimpanzee and bonobo iNs, and this difference in timing likely depends on functions downstream of NGN2.


Assuntos
Neurônios/fisiologia , Pan paniscus/fisiologia , Pan troglodytes/fisiologia , Animais , Diferenciação Celular , Humanos , Neuritos/metabolismo , Neurogênese , Especificidade da Espécie
9.
Results Probl Cell Differ ; 67: 359-375, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435803

RESUMO

Neurons forming the central nervous system are generated by neural stem and progenitor cells, via a process called neurogenesis (Götz and Huttner, Nat Rev Mol Cell Biol, 6:777-788, 2005). In this book chapter, we focus on neurogenesis in the dorsolateral telencephalon, the rostral-most region of the neural tube, which contains the part of the central nervous system that is most expanded in mammals (Borrell and Reillo, Dev Neurobiol, 72:955-971, 2012; Wilsch-Bräuninger et al., Curr Opin Neurobiol 39:122-132, 2016). We will discuss recent advances in the dissection of the cell biological mechanisms of neurogenesis, with particular attention to the organization and function of the Golgi apparatus and its relationship to the centrosome.


Assuntos
Polaridade Celular , Complexo de Golgi/metabolismo , Células-Tronco Neurais/citologia , Células Neuroepiteliais/citologia , Neurogênese , Neurônios/citologia , Animais , Centrossomo
10.
EMBO Rep ; 20(10): e47880, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31469223

RESUMO

Microinjection into single cells in brain tissue is a powerful technique to study and manipulate neural stem cells. However, such microinjection requires expertise and is a low-throughput process. We developed the "Autoinjector", a robot that utilizes images from a microscope to guide a microinjection needle into tissue to deliver femtoliter volumes of liquids into single cells. The Autoinjector enables microinjection of hundreds of cells within a single organotypic slice, resulting in an overall yield that is an order of magnitude greater than manual microinjection. The Autoinjector successfully targets both apical progenitors (APs) and newborn neurons in the embryonic mouse and human fetal telencephalon. We used the Autoinjector to systematically study gap-junctional communication between neural progenitors in the embryonic mouse telencephalon and found that apical contact is a characteristic feature of the cells that are part of a gap junction-coupled cluster. The throughput and versatility of the Autoinjector will render microinjection an accessible high-performance single-cell manipulation technique and will provide a powerful new platform for performing single-cell analyses in tissue for bioengineering and biophysics applications.


Assuntos
Encéfalo/citologia , Microinjeções , Robótica , Análise de Célula Única , Animais , Automação , Comunicação Celular , Linhagem da Célula , Humanos , Processamento de Imagem Assistida por Computador , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Neurogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telencéfalo/citologia , Telencéfalo/embriologia
11.
Curr Mol Pharmacol ; 12(2): 147-159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30714537

RESUMO

OBJECTIVE: Marycin is a porphyrin-type compound synthetically modified to spontaneously release fluorescence. This study is aimed at understanding possible mechanisms that could account for the antiproliferative effects observed in marycin. A proteomic approach was used to identify molecular effects. The proteome of proliferating MDA-MB-231 breast cancer cells was compared with that of marycin-treated cells. METHODS: Label-free proteomic analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to reveal changes in protein expression and fluorescence microscopy and flow cytometry were used to detect subcellular organelle dysfunctions. RESULTS: The bioinformatic analysis indicated an enhancement of the expression of proteins remodeling RNA splicing and more in general, of RNA metabolism. Marycin did not localize into the mitochondria and did not produce a dramatic increase of ROS levels in MDA-MB-231 cells. Marycin stained organelles probably peroxisomes. CONCLUSIONS: The results could support the possibility that the peroxisomes are involved in cell response to marycin.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hematoporfirinas/farmacologia , Porfirinas/farmacologia , Proteômica/métodos , RNA/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Feminino , Hematoporfirinas/química , Humanos , Porfirinas/química , Splicing de RNA/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas em Tandem
12.
J Exp Clin Cancer Res ; 37(1): 75, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29615075

RESUMO

BACKGROUND: Current insights into the effects of iron deficiency in tumour cells are not commensurate with the importance of iron in cell metabolism. Studies have predominantly focused on the effects of oxygen or glucose scarcity in tumour cells, while attributing insufficient emphasis to the inadequate supply of iron in hypoxic regions. Cellular responses to iron deficiency and hypoxia are interlinked and may strongly affect tumour metabolism. METHODS: We examined the morphological, proteomic, and metabolic effects induced by two iron chelators-deferoxamine (DFO) and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT)-on MDA-MB-231 and MDA-MB-157 breast cancer cells. RESULTS: These chelators induced a cytoplasmic massive vacuolation and accumulation of lipid droplets (LDs), eventually followed by implosive, non-autophagic, and non-apoptotic death similar to methuosis. Vacuoles and LDs are generated by expansion of the endoplasmic reticulum (ER) based on extracellular fluid import, which includes unsaturated fatty acids that accumulate in LDs. Typical physiological phenomena associated with hypoxia are observed, such as inhibition of translation, mitochondrial dysfunction, and metabolic remodelling. These survival-oriented changes are associated with a greater expression of epithelial/mesenchymal transcription markers. CONCLUSIONS: Iron starvation induces a hypoxia-like program able to scavenge nutrients from the extracellular environment, and cells assume a hypertrophic phenotype. Such survival strategy is accompanied by the ER-dependent massive cytoplasmic vacuolization, mitochondrial dysfunctions, and LD accumulation and then evolves into cell death. LDs containing a greater proportion of unsaturated lipids are released as a consequence of cell death. The consequence of the disruption of iron metabolism in tumour tissue and the effects of LDs on intercellular communication, cancer-inflammation axis, and immunity remain to be explored. Considering the potential benefits, these are crucial subjects for future mechanistic and clinical studies.


Assuntos
Neoplasias da Mama/metabolismo , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desferroxamina/farmacologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteoma , Proteômica/métodos , Vacúolos/metabolismo
13.
Neuron ; 97(6): 1299-1314.e8, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29503187

RESUMO

Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.


Assuntos
Junções Aderentes/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Ligação a DNA/biossíntese , Regulação para Baixo/fisiologia , Neocórtex/metabolismo , Células-Tronco Neurais/metabolismo , Fatores de Transcrição/biossíntese , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Técnicas de Cultura de Órgãos , Gravidez , Proteínas Repressoras
14.
Front Cell Neurosci ; 11: 384, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29259543

RESUMO

Neurons populating the cerebral cortex are generated during embryonic development from neural stem and progenitor cells in a process called neurogenesis. Neural stem and progenitor cells are classified into several classes based on the different location of mitosis (apical or basal) and polarity features (bipolar, monopolar and non-polar). The polarized architecture of stem cells is linked to the asymmetric localization of proteins, mRNAs and organelles, such as the centrosome and the Golgi apparatus (GA). Polarity affects stem cell function and allows stem cells to integrate environmental cues from distinct niches in the developing cerebral cortex. The crucial role of polarity in neural stem and progenitor cells is highlighted by the fact that impairment of cell polarity is linked to neurodevelopmental disorders such as Down syndrome, Fragile X syndrome, autism spectrum disorders (ASD) and schizophrenia.

15.
Oncotarget ; 8(28): 46177-46190, 2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28526811

RESUMO

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.


Assuntos
Biomarcadores Tumorais/líquido cefalorraquidiano , Neoplasias Encefálicas/metabolismo , Neoplasias do Sistema Nervoso Central/metabolismo , Proteínas do Líquido Cefalorraquidiano/metabolismo , Carcinomatose Meníngea/metabolismo , Proteoma/metabolismo , Adolescente , Adulto , Neoplasias Encefálicas/patologia , Neoplasias do Sistema Nervoso Central/patologia , Criança , Pré-Escolar , Conjuntos de Dados como Assunto , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Nanotecnologia , Adulto Jovem
16.
Int J Mol Sci ; 18(2)2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28216608

RESUMO

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Ferro/metabolismo , Proteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Sanguíneas , Neoplasias da Mama/genética , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Eritropoetina/sangue , Feminino , Ferritinas/metabolismo , Hepcidinas/metabolismo , Humanos , Interleucina-6/sangue , Pessoa de Meia-Idade , Análise Serial de Proteínas , Proteínas/genética , Receptores da Transferrina/metabolismo , Adulto Jovem
17.
Biopreserv Biobank ; 14(6): 480-490, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27403896

RESUMO

CONTEXT: Biobanks of frozen human normal and malignant tissues represent a valuable source for "omics" analysis in translational cancer research and molecular pathology. However, the success of molecular and cellular analysis strongly relies on the collection, handling, storage procedures, and quality control of fresh human tissue samples. OBJECTIVE: We tested whether under vacuum storage (UVS) effectively preserves tissues during the time between surgery and storage for "omics" analyses. DESIGN: Normal and matched tumor specimens, obtained from 16 breast, colon, or lung cancer patients and 5 independent mesenchymal tumors, were dissected within 20 minutes from surgical excision and divided in three to five aliquots; for each tissue sample, one aliquot was snap-frozen in liquid nitrogen (defined as baseline or T0 samples), and the other portions were sealed into plastic bags and kept at 4°C for 1, 24, 48, or 72 hours under vacuum and then frozen. The tissue and molecular preservation under vacuum was evaluated over time in terms of histomorphology, transcription (Illumina microarrays), protein (surface-enhanced laser desorption/ionization-time of flight/mass spectrometry and Western blot), and metabolic profile (nuclear magnetic resonance spectroscopy). RESULTS: Tissue morphology, Mib-1, and vimentin immunostaining were preserved over time without signs of tissue degradation. Principal variance component analysis showed that time of storage had a minimal effect on gene expression or the proteome, but affected the preservation of some metabolites to a greater extent. UVS did not impact the RNA and protein integrity or specific phosphorylation sites on mTOR and STAT3. Measurement of metabolites revealed pronounced changes after 1 hour of storage. CONCLUSIONS: Our results show that UVS can preserve tissue specimens for histological, transcriptomic, and proteomic examinations up to 48 hours and possibly longer, whereas it has limitations for metabolomic applications.


Assuntos
Neoplasias/patologia , Manejo de Espécimes/métodos , Bancos de Tecidos/normas , Feminino , Humanos , Masculino , Metabolômica , Proteômica , Manejo de Espécimes/instrumentação , Pesquisa Translacional Biomédica , Vácuo
18.
Sci Rep ; 6: 21206, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26879757

RESUMO

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change.


Assuntos
Complexo de Golgi/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Expressão Gênica , Genes Reporter , Complexo de Golgi/ultraestrutura , Camundongos , Camundongos Transgênicos , Mitose , Células-Tronco Neurais/ultraestrutura , Polissacarídeos/metabolismo , Transporte Proteico
19.
EMBO Rep ; 17(3): 338-48, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26758805

RESUMO

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈ 90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.


Assuntos
Sistemas CRISPR-Cas , Células-Tronco Embrionárias/metabolismo , Marcação de Genes/métodos , Células-Tronco Neurais/metabolismo , Telencéfalo/citologia , Animais , Células Cultivadas , Eletroporação/métodos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Guia de Cinetoplastídeos/genética , Análise de Célula Única/métodos , Telencéfalo/embriologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
20.
Expert Rev Proteomics ; 12(6): 695-701, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26496240

RESUMO

OBJECTIVE: Hepcidin-25 production is stimulated by systemic inflammation, and it interferes with iron utilization, leading to anemia. This study aimed to investigate the relationships between the plasma levels of hepcidin, interleukin-6 (IL-6), erythropoietin (EPO) and erythroferrone (ERFE) in patients with benign breast disease or cancer. METHODS: Plasma samples from a cohort of 131 patients (47 with benign breast disease and 84 with breast cancer) were subjected to the evaluation of hepcidin, IL-6, EPO and ERFE using SELDI-TOF-MS or immunoassays. RESULTS: An elevated hepcidin was observed in malignant breast tumors compared to benign ones. No correlation was observed between hepcidin and IL-6, EPO or ERFE. CONCLUSION: Since the study included a cohort of patients (87%) with breast cancers smaller than 2 cm, these results may support our previous evidence about the potential role of hepcidin in breast cancer disease.


Assuntos
Neoplasias da Mama/sangue , Eritropoetina/sangue , Hepcidinas/sangue , Interleucina-6/sangue , Hormônios Peptídicos/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...