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1.
Anticancer Res ; 39(8): 4171-4177, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366502

RESUMO

BACKGROUND/AIM: Identification of prostatic stem cells in primary prostate tissue sections, organ cultures of prostate and cell lines requires a range of techniques that allows characterization of stem cells for their potential use in the treatment of patients. Isolated cells usually round-up and develop changes in shape, size and cellular characteristics. The aim of this study was to provide a range of methods for identifying prostatic stem cells and characterizing them with regard to ultrastructure, nuclear morphology, cytoplasmic organelles, and/or expression stem cell marker CD133. MATERIALS AND METHODS: Prostate biopsy and prostatectomy specimens were used for studying prostatic stem cells and their known marker CD133 in tissue sections by light and/or electron microscopy. Inverted capsule embedding was used to study archival metastatic prostate in pelvic nodes and Du145 cell line in a monolayer culture. RESULTS: Staining for CD133 positively identified stem cells that were found in benign prostatic hyperplasia, benign prostate, and prostate cancer cells. Paraffin embedded sections showed a single type of stem cells, whereas methylene blue-stained Epon sections showed both light and dark stem cells. Electron microscopy showed that both basal and stem cells were closely associated with the basement membrane (basal lamina). Stem cells had smooth plasma and nuclear membranes, a prominent nucleolus, small mitochondria, and few ribosomes. Du145 cells were separated by intercellular spaces in monolayer culture. CONCLUSION: The inverted capsule embedding method allowed the study of metastasized prostate cancer in pelvic lymph nodes. Our approach enabled the assessment of stem cells in tissue sections by light and electron microscopy.


Assuntos
Antígeno AC133/genética , Membrana Basal/ultraestrutura , Hiperplasia Prostática/genética , Neoplasias da Próstata/genética , Membrana Basal/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Microscopia Eletrônica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/ultraestrutura , Próstata/metabolismo , Próstata/patologia , Próstata/ultraestrutura , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/ultraestrutura
2.
Nature ; 571(7763): 63-71, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270481

RESUMO

Knowledge of connectivity in the nervous system is essential to understanding its function. Here we describe connectomes for both adult sexes of the nematode Caenorhabditis elegans, an important model organism for neuroscience research. We present quantitative connectivity matrices that encompass all connections from sensory input to end-organ output across the entire animal, information that is necessary to model behaviour. Serial electron microscopy reconstructions that are based on the analysis of both new and previously published electron micrographs update previous results and include data on the male head. The nervous system differs between sexes at multiple levels. Several sex-shared neurons that function in circuits for sexual behaviour are sexually dimorphic in structure and connectivity. Inputs from sex-specific circuitry to central circuitry reveal points at which sexual and non-sexual pathways converge. In sex-shared central pathways, a substantial number of connections differ in strength between the sexes. Quantitative connectomes that include all connections serve as the basis for understanding how complex, adaptive behavior is generated.


Assuntos
Caenorhabditis elegans/metabolismo , Conectoma , Sistema Nervoso/anatomia & histologia , Sistema Nervoso/metabolismo , Caracteres Sexuais , Animais , Comportamento Animal , Caenorhabditis elegans/citologia , Feminino , Cabeça/anatomia & histologia , Cabeça/inervação , Organismos Hermafroditas , Masculino , Microscopia Eletrônica , Atividade Motora , Movimento , Sistema Nervoso/citologia , Vias Neurais
3.
Medicine (Baltimore) ; 98(29): e16390, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31335688

RESUMO

INTRODUCTION: Sjögren's syndrome (SS) often causes lymphoproliferative disorders such as malignant lymphoma and macroglobrinemia. Approximately 5% of long-term follow-up SS patients develop malignant lymphoma. Recently, the tumor necrosis factor receptor superfamily cluster of differentiation 30 (CD30) has been thought to be implicated in malignant cells in organs affected by Hodgikin lymphoma or in a prognostic marker of diffuse large B cell lymphoma. In this study, we investigated CD30 expression in lacrimal gland and conjunctiva in patients with SS. METHODS: We examined lacrimal gland and conjunctival tissues for the diagnosis from 3 female SS patients with a median age of 51 and 3 female chronic graft-versus-host disease (cGVHD) patients with a median age of 41. Histological analysis of these tissues of the remaining samples was conducted by methods including immunohistochemistry and electron microscopy (#20090277). We analyzed the expression and localization of cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), cluster of differentiation 20 (CD20), CD30, and Interferon-γ in tissue sections prepared from lacrimal glands and conjunctiva in 3 each of SS and cGVHD patients. RESULTS: There were more B cells and plasma cells in lobules of SS-affected lacrimal glands than in those of their cGVHD-affected counterparts. Interferon-γ was expressed on endothelia of capillaries in SS-affected lacrimal gland and conjunctival tissues whereas it was expressed on fibroblasts in their GVHD-affected equivalents. Furthermore, lacrimal glands and conjunctiva disordered by SS had a greater number of CD30 cells than those disordered by cGVHD. CONCLUSION: Our results suggest that CD30 cells are increased in lacrimal glands and conjunctiva affected by SS and that a subset of SS patients are thereby at risk of development malignant lymphoma.


Assuntos
Túnica Conjuntiva , Doença Enxerto-Hospedeiro , Antígeno Ki-1 , Aparelho Lacrimal , Linfoma/diagnóstico , Síndrome de Sjogren , Adulto , Linfócitos B/imunologia , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/patologia , Feminino , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Imuno-Histoquímica , Interferon gama/sangue , Antígeno Ki-1/análise , Antígeno Ki-1/imunologia , Aparelho Lacrimal/imunologia , Aparelho Lacrimal/patologia , Microscopia Eletrônica/métodos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/sangue , Plasmócitos/imunologia , Prognóstico , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/patologia
4.
Nat Commun ; 10(1): 2736, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227718

RESUMO

Reconstruction and annotation of volume electron microscopy data sets of brain tissue is challenging but can reveal invaluable information about neuronal circuits. Significant progress has recently been made in automated neuron reconstruction as well as automated detection of synapses. However, methods for automating the morphological analysis of nanometer-resolution reconstructions are less established, despite the diversity of possible applications. Here, we introduce cellular morphology neural networks (CMNs), based on multi-view projections sampled from automatically reconstructed cellular fragments of arbitrary size and shape. Using unsupervised training, we infer morphology embeddings (Neuron2vec) of neuron reconstructions and train CMNs to identify glia cells in a supervised classification paradigm, which are then used to resolve neuron reconstruction errors. Finally, we demonstrate that CMNs can be used to identify subcellular compartments and the cell types of neuron reconstructions.


Assuntos
Encéfalo/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Redes Neurais (Computação) , Neurônios/citologia , Sinapses , Algoritmos , Animais , Encéfalo/citologia , Conjuntos de Dados como Assunto , Estudos de Viabilidade , Masculino , Microscopia Eletrônica , Passeriformes
5.
Nat Commun ; 10(1): 2370, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147549

RESUMO

FAM134B/RETREG1 is a selective ER-phagy receptor that regulates the size and shape of the endoplasmic reticulum. The structure of its reticulon-homology domain (RHD), an element shared with other ER-shaping proteins, and the mechanism of membrane shaping remain poorly understood. Using molecular modeling and molecular dynamics (MD) simulations, we assemble a structural model for the RHD of FAM134B. Through MD simulations of FAM134B in flat and curved membranes, we relate the dynamic RHD structure with its two wedge-shaped transmembrane helical hairpins and two amphipathic helices to FAM134B functions in membrane-curvature induction and curvature-mediated protein sorting. FAM134B clustering, as expected to occur in autophagic puncta, amplifies the membrane-shaping effects. Electron microscopy of in vitro liposome remodeling experiments support the membrane remodeling functions of the different RHD structural elements. Disruption of the RHD structure affects selective autophagy flux and leads to disease states.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/genética , Forma das Organelas/genética , Autofagia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Humanos , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana/genética , Microscopia Eletrônica , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Transporte Proteico/genética
6.
Nat Commun ; 10(1): 2654, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201326

RESUMO

Animal locomotion requires spatiotemporally coordinated contraction of muscles throughout the body. Here, we investigate how contractions of antagonistic groups of muscles are intersegmentally coordinated during bidirectional crawling of Drosophila larvae. We identify two pairs of higher-order premotor excitatory interneurons present in each abdominal neuromere that intersegmentally provide feedback to the adjacent neuromere during motor propagation. The two feedback neuron pairs are differentially active during either forward or backward locomotion but commonly target a group of premotor interneurons that together provide excitatory inputs to transverse muscles and inhibitory inputs to the antagonistic longitudinal muscles. Inhibition of either feedback neuron pair compromises contraction of transverse muscles in a direction-specific manner. Our results suggest that the intersegmental feedback neurons coordinate contraction of synergistic muscles by acting as delay circuits representing the phase lag between segments. The identified circuit architecture also shows how bidirectional motor networks could be economically embedded in the nervous system.


Assuntos
Retroalimentação Fisiológica , Locomoção/fisiologia , Rede Nervosa/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Interneurônios/fisiologia , Larva/fisiologia , Microscopia Eletrônica , Modelos Animais , Contração Muscular/fisiologia , Músculos/inervação , Músculos/fisiologia , Optogenética
7.
Int. j. morphol ; 37(2): 694-700, June 2019. graf
Artigo em Espanhol | LILACS | ID: biblio-1002279

RESUMO

Spondylus limbatus es una especie bajo protección especial en México, de la que existe poca información biológica y nada sobre estudios histológicos o de ultraestructura del ovario. El objetivo de esta investigación fue caracterizar la morfología ultraestructural de los gametos femeninos maduros y en degeneración. La gónada femenina de S. limbatus en estado de madurez presentó ovocitos postvitelogénicos de 60-70 µm de diámetro, que presentan el aspecto característico de células metabólicamente activas y altamente sintetizadoras. La membrana citoplasmática posee especializaciones destinadas a aumentar la superficie de absorción de la célula, las microvellosidades; el citoplasma presenta numerosos sistemas membranosos relacionados con la síntesis de material de reserva y secreción; y el patrón de organización nuclear altamente lobulado, y por consiguiente con una gran superficie que asegura el intercambio núcleo-citoplasma, se incorpora de forma estructural al proceso de vitelogénesis. Finalmente, se describen los cambios ultraestructurales resultantes de la lisis de los ovocitos: colapso de las membranas nuclear y citoplásmica, y presencia de células hemocíticas macrófagas.


Spondylus limbatus is a species under special protection in Mexico, of which there is little or no information in the literature of biological, histological or ultrastructural studies of the ovary. The objective of this research was to characterize the ultrastructural morphology of mature and degenerating female gametes. The female gonad of S. limbatus in mature state presented post-vitellogenic oocytes 60-70 µm in diameter, which have characteristics of metabolically active and highly synthesizing cells. The cytoplasmic membrane has specializations designed to increase the absorption surface of the cell, the microvilli; the cytoplasm presents numerous membranous systems related to synthesis of reserve and secretion material as well as the highly lobed nuclear organization pattern; a large surface that ensures core-cytoplasm exchange, is structurally incorporated into the vitellogenesis process. Finally, ultrastructural changes resulting from the lysis of the oocytes are described: collapse of nuclear and cytoplasmic membranes, and the presence of macrophage hemocytic cells.


Assuntos
Animais , Feminino , Oócitos/ultraestrutura , Bivalves , Gônadas/ultraestrutura , Reprodução , Microscopia Eletrônica
8.
Nat Neurosci ; 22(7): 1099-1109, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235907

RESUMO

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.


Assuntos
Membranas Intracelulares/ultraestrutura , Corpos de Lewy/ultraestrutura , Doença por Corpos de Lewy/patologia , Lipídeos de Membrana/análise , Organelas/ultraestrutura , Doença de Parkinson/patologia , alfa-Sinucleína/análise , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Hipocampo/química , Hipocampo/ultraestrutura , Humanos , Imagem Tridimensional , Corpos de Lewy/química , Doença por Corpos de Lewy/metabolismo , Mesencéfalo/química , Mesencéfalo/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica/métodos , Microscopia de Fluorescência , Doença de Parkinson/metabolismo , Substância Negra/química , Substância Negra/ultraestrutura , Sequenciamento Completo do Exoma
9.
Histochem Cell Biol ; 152(2): 133-143, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154480

RESUMO

Actin fulfills important cytoplasmic but also nuclear functions in eukaryotic cells. In the nucleus, actin modulates gene expression and chromatin remodeling. Monomeric (G-actin) and polymerized actin (F-actin) have been analyzed by fluorescence microscopy in the nucleus; however, the resolution at the ultrastructural level has not been investigated in great detail. We provide a first documentation of nuclear actin in mouse fibroblasts by electron microscopy (EM). For this, we employed correlative light and electron microscopy on the same section using actin-directed nanobodies recognizing endogenous monomeric and polymeric actin proteins (so-called nuclear Actin-chromobody-GFP; nAC-GFP). Indeed, using this strategy, we could identify actin proteins present in the nucleus. Here, immunogold-labeled actin proteins were spread throughout the entire nucleoplasm. Of note, nuclear actin was complementarily localized to DAPI-positive areas, the latter marking preferentially transcriptionally inactive heterochromatin. Since actin aggregates in rod structures upon cell stress including neurodegeneration, we analyzed nuclear actin at the ultrastructural level after DMSO or UV-mediated cell damage. In those cells the ratio between cytoplasmic and nuclear gold-labeled actin proteins was altered compared to untreated control cells. In summary, this EM analysis (i) confirmed the presence of endogenous nuclear actin at ultrastructural resolution, (ii) revealed the actin abundance in less chromatin-dense regions potentially reflecting more transcriptionally active euchromatin rather than transcriptionally inactive heterochromatin and (iii) showed an altered abundance of actin-associated gold particles upon cell stress.


Assuntos
Actinas/análise , Núcleo Celular/química , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Actinas/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Fibroblastos/química , Fibroblastos/citologia , Camundongos , Células NIH 3T3 , Tamanho da Partícula , Conformação Proteica
10.
Virol J ; 16(1): 55, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036009

RESUMO

BACKGROUND: Nucleorhabdoviruses possess bacilliform particles which contain a single-stranded negative-sense RNA genome. They replicate and mature in the nucleus of infected cells. Together with viruses of three other genera of the family Rhabdoviridae, they are known to infect plants and can be transmitted by arthropod vectors, during vegetative propagation, or by mechanical means. In 2010, an alfalfa (Medicago sativa) plant showing virus-like symptoms was collected from Stadl-Paura, Austria and sent to Julius Kühn Institute for analysis. METHODS: Electron microscopy (EM) of leaf extracts from infected plants revealed the presence of rhabdovirus-like particles and was further used for ultrastructural analyses of infected plant tissue. Partially-purified preparations of rhabdovirus nucleocapsids were used for raising an antiserum. To determine the virus genome sequence, high throughput sequencing (HTS) was performed. RT-PCR primers were designed to confirm virus infection and to be used as a diagnostic tool. RESULTS: EM revealed bacilliform virions resembling those of plant-infecting rhabdoviruses. HTS of ribosomal RNA-depleted total RNA extracts revealed a consensus sequence consisting of 13,875 nucleotides (nt) and containing seven open reading frames (ORFs). Homology and phylogenetic analyses suggest that this virus isolate represents a new species of the genus Nucleorhabdovirus (family Rhabdoviridae). Since the virus originated from an alfalfa plant in Austria, the name alfalfa-associated nucleorhabdovirus (AaNV) is proposed. Viroplasms (Vp) and budding virions were observed in the nuclei of infected cells by EM, thus confirming its taxonomic assignment based on sequence data. CONCLUSIONS: In this study, we identified and characterised a new nucleorhabdovirus from alfalfa. It shared only 39.8% nucleotide sequence identity with its closest known relative, black currant-associated rhabdovirus 1. The virus contains an additional open reading frame (accessory gene) with unknown function, located between the matrix protein and the glycoprotein genes. Serological and molecular diagnostic assays were designed for future screening of field samples. Further studies are needed to identify other natural hosts and potential vectors.


Assuntos
Núcleo Celular/virologia , Genoma Viral , Medicago sativa/virologia , Rhabdoviridae/genética , Áustria , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia Eletrônica , Fases de Leitura Aberta , Doenças das Plantas/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Rhabdoviridae/ultraestrutura , Análise de Sequência de DNA , Proteínas Virais/genética , Vírion/genética
11.
Nat Methods ; 16(5): 361, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31040433
12.
Plant Sci ; 283: 95-115, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128719

RESUMO

The green oleaginous microalga Lobosphaera incisa accumulates storage lipids triacylglycerols (TAG) enriched in the long-chain polyunsaturated fatty acid arachidonic acid under nitrogen (N) deprivation. In contrast, under phosphorous (P) deprivation, the production of the monounsaturated oleic acid prevails. We compared physiological responses, ultrastructural, and metabolic consequences of L. incisa acclimation to N and P deficiency to provide novel insights into the key determinants of ARA accumulation. Differential responses to nutrient deprivation on growth performance, carbon-to-nitrogen stoichiometry, membrane lipid composition and TAG accumulation were demonstrated. Ultrastructural analyses suggested a dynamic role for vacuoles in sustaining cell homeostasis under conditions of different nutrient availability and their involvement in autophagy in L. incisa. Paralleling ARA-rich TAG accumulation in lipid droplets, N deprivation triggered intensive chloroplast dismantling and promoted catabolic processes. Metabolome analysis revealed depletion of amino acids and pyrimidines, and repression of numerous biosynthetic hubs to favour TAG biosynthesis under N deprivation. Under P deprivation, despite the relatively low growth penalties, the presence of the endogenous P reserves and the characteristic lipid remodelling, metabolic signatures of energy deficiency were revealed. Metabolome adjustments to P deprivation included depletion in ATP and phosphorylated nucleotides, increased levels of TCA-cycle intermediates and osmoprotectants. We conclude that characteristic cellular and metabolome adjustments tailor the adaptive responses of L. incisa to N and P deprivation modulating its LC-PUFA production.


Assuntos
Ácido Araquidônico/metabolismo , Clorófitas/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Microalgas/efeitos dos fármacos , Nitrogênio/deficiência , Fósforo/deficiência , Clorófitas/metabolismo , Clorófitas/ultraestrutura , Metabolômica , Microalgas/metabolismo , Microalgas/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Triglicerídeos/metabolismo
13.
Gene ; 707: 65-77, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31059736

RESUMO

The genic male sterility (MS) plays a major role in melon hybrids production, it could reduce the cost of pollination and increase the yield and quality. However, the molecular mechanism underlying genetic male sterility is yet poorly understood. The morphological differences of flower buds of melon were observed showed that the flower buds were tetrad when they were 1 mm stage and monocyte microspore when they were 2 mm stage. Electron microscopy showed that there was significant difference between MS lines and MF (male fertility) lines. In order to detect the global expression of the genes during the melon anther development and association with MS, 12 DEGs (differentially expressed genes) libraries were constructed from the anther of MS and MF in the bud stage with 1 and 2 mm diameter, respectively. A total of 765 DEGs expressed in anther during different developmental stage (MS 1 mm vs. MS 2 mm), 148 and 309 DEGs were found to be related to MS as compared to MF (MS 1 mm vs. MF 1 mm, and MS 2 mm vs. MF 2 mm) at a false discovery rate FDR <0.01. Among these, 10 DEGs were expressed in all the three comparisons, including transcription factor bHLH genes. Among the DEGs in RNA-seq analysis, 28 were validated by qRT-PCR. Of these, a number of genes were involved in ABC transfactor B family, cytochrome-related genes, hormone-related genes (auxin transporter, gibberellin-regulated protein), MADS-box protein genes, F-box protein genes, peroxidase-related, and Zinc finger protein genes. These genes are involved in many biological pathways, including starch and sucrose metabolism, signal transduction mechanisms and transcription factors, etc. Compared to the same developmental stage of MS and MF, the different developmental stages of MS indicated diverse gene regulation pathways involved in the anther development in MS. These results would provide novel insight into the global network to male sterility in melon.


Assuntos
Cucumis melo/fisiologia , Perfilação da Expressão Gênica/métodos , Infertilidade das Plantas , Proteínas de Plantas/genética , Quimera/genética , Quimera/fisiologia , Cucumis melo/genética , Cucumis melo/ultraestrutura , Flores/genética , Flores/fisiologia , Flores/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Microscopia Eletrônica , Análise de Sequência de RNA
14.
Nat Commun ; 10(1): 2129, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086189

RESUMO

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.


Assuntos
Malformações do Desenvolvimento Cortical/genética , Microtúbulos/metabolismo , Neurogênese/genética , Neurônios/fisiologia , Tubulina (Proteína)/genética , Animais , Comportamento Animal , Movimento Celular/genética , Centrossomo/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/citologia , Córtex Cerebral/diagnóstico por imagem , Modelos Animais de Doenças , Embrião de Mamíferos , Epilepsia/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Células HeLa , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Microtúbulos/genética , Mutação de Sentido Incorreto
15.
MBio ; 10(3)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064826

RESUMO

Bacteria and archaea exhibit tactical behavior and can move up and down chemical gradients. This tactical behavior relies on a motility structure, which is guided by a chemosensory system. Environmental signals are sensed by membrane-inserted chemosensory receptors that are organized in large ordered arrays. While the cellular positioning of the chemotaxis machinery and that of the flagellum have been studied in detail in bacteria, we have little knowledge about the localization of such macromolecular assemblies in archaea. Although the archaeal motility structure, the archaellum, is fundamentally different from the flagellum, archaea have received the chemosensory machinery from bacteria and have connected this system with the archaellum. Here, we applied a combination of time-lapse imaging and fluorescence and electron microscopy using the model euryarchaeon Haloferax volcanii and found that archaella were specifically present at the cell poles of actively dividing rod-shaped cells. The chemosensory arrays also had a polar preference, but in addition, several smaller arrays moved freely in the lateral membranes. In the stationary phase, rod-shaped cells became round and chemosensory arrays were disassembled. The positioning of archaella and that of chemosensory arrays are not interdependent and likely require an independent form of positioning machinery. This work showed that, in the rod-shaped haloarchaeal cells, the positioning of the archaellum and of the chemosensory arrays is regulated in time and in space. These insights into the cellular organization of H. volcanii suggest the presence of an active mechanism responsible for the positioning of macromolecular protein complexes in archaea.IMPORTANCE Archaea are ubiquitous single cellular microorganisms that play important ecological roles in nature. The intracellular organization of archaeal cells is among the unresolved mysteries of archaeal biology. With this work, we show that cells of haloarchaea are polarized. The cellular positioning of proteins involved in chemotaxis and motility is spatially and temporally organized in these cells. This suggests the presence of a specific mechanism responsible for the positioning of macromolecular protein complexes in archaea.


Assuntos
Proteínas Arqueais/química , Polaridade Celular , Quimiotaxia , Haloferax volcanii/fisiologia , Citoplasma/química , Flagelos/fisiologia , Haloferax volcanii/ultraestrutura , Microscopia Eletrônica , Imagem com Lapso de Tempo
16.
Wiad Lek ; 72(3): 362-367, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050981

RESUMO

OBJECTIVE: Introduction: Cyclophosphamide has wide spectrum usage as first-line drug in cancer chemotherapy that is why a detailed study of its effect on individual cell populations is of great interest for science and practice. The interaction of the nervous, immune and endocrine systems plays essential role in the homeostasis maintaining. The aim: This study aimed to investigate the ultramicroscopic changes that occur in the parathyroid glands and thymus of male rats after cyclophosphamide administration. PATIENTS AND METHODS: Materials and methods: Twenty-four WAG matured male rats were divided randomly into two groups. The first group served as control and was provided 0.9% soluble sodium chloride. The second group received cyclophosphamide in a dosage 200 mg/kg of body weight of animal by intramuscular single injection. All animals were sacrificed on the 7th and 30th day after injection. Parathyroid gland and thymus specimens were dissected out and processed for electron microscopy. RESULTS: Results: The results showed that cyclophosphamide exposure caused marked ultramicroscopic changes in rats parathyroid glands and thymus. On the 7th day after immunosuppression, the nuclei of parathyrocytes have deep wavy invaginations, amount of the organelles that participate in the protein synthesis is reduced to a minimum in the cytoplasm of the chief cells. Characteristic feature is the appearance of numerous plasma cells and active macrophages in thymus. There is a tendency to normalization of the parathyroid structure on the 30th day after administration of cyclophosphamide and reduction of mitotic activity of lymphocytes in thymus, which points to the development of involution process. CONCLUSION: Conclusions: This data can be successfully extrapolated from experimental animals to humans.


Assuntos
Ciclofosfamida/administração & dosagem , Glândulas Paratireoides , Timo , Animais , Humanos , Imunossupressão , Masculino , Microscopia Eletrônica , Glândulas Paratireoides/efeitos dos fármacos , Ratos , Timo/efeitos dos fármacos
17.
Indian J Ophthalmol ; 67(6): 801-805, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31124490

RESUMO

Purpose: To evaluate the effect of cyanidin-3-glucoside (C3G) in oxygen-induced retinopathy (OIR) mouse model. Methods: In this experimental study, 10 C57BL / 6J type mice exposed to room air comprised two control groups (n = 5 each; a negative control and a group receiving intravitreal sterile dimethyl sulfoxide [IVS DMSO]). Thirty C57BL / 6J type mice exposed to 75% ± 2% oxygen from postnatal day 7 to postnatal day 12 comprised the OIR groups. On postnatal day 12, these mice were randomized into six groups (n = 5 each): two OIR control groups (negative control and IVS DMSO), two intravitreal C3G groups (300 and 600 ng/µL), and two intraperitoneal C3G groups (0.05 and 0.1 mg/kg). We quantified neovascularization by counting endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina and examined histological and ultrastructural changes via light and electron microscopy and apoptosis by terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling. Results: The intravitreal C3G groups yielded lower endothelial cell counts compared with the intravitreal DMSO group. The intraperitoneal high-dose group had lower cell counts compared with the OIR control groups. Electron microscopy revealed significantly less mitochondrial dysmorphology in intravitreal groups and the high-dose intraperitoneal mice. We noted no difference in apoptotic cell count between the controls, low-dose intravitreal, and both intraperitoneal groups. However, apoptotic cell count was significantly higher in the high-dose intravitreal group. Conclusion: C3G suppresses endothelial cell proliferation in an OIR mouse model, leads to a reduced hyperoxia-induced mitochondrial dysmorphology, but increases apoptotic cell death in high concentrations.


Assuntos
Antocianinas/administração & dosagem , Glicosídeos/administração & dosagem , Retina/patologia , Doenças Retinianas/tratamento farmacológico , Animais , Animais Recém-Nascidos , Apoptose , Proliferação de Células , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Oxigênio/toxicidade , Retina/efeitos dos fármacos , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/patologia
18.
Transplant Proc ; 51(5): 1348-1352, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31076147

RESUMO

BACKGROUND: Although an association between body weight mismatch and impaired graft function has been reported, few histologic studies have evaluated this issue, especially using electric microscopic analysis. During routine observations, we have noted a thin glomerular basement membrane (GBM) in the 1-hour biopsy specimen in cases with an overweight recipient and a lightweight donor. Therefore, we hypothesized that donor-recipient body weight mismatch affects the GBM thickness in the 1-hour biopsy specimen. The aim of the present study was to clarify the effect of donor-recipient body weight mismatch on the GBM thickness of the 1-hour biopsy specimen measured using electron microscopy. METHODS: We used an electron microscope to measure the GBM thickness of specimens at 1-hour post-transplantation (n = 24) and at 1 year post-transplantation (n = 17). The GBM thickness of cases with donor-recipient body weight mismatch was compared with those without mismatch. In accordance with a previous study, we defined a donor/recipient body weight ratio of less than 0.9 as donor-recipient body weight mismatch and a ratio of more than 0.9 as no mismatch. RESULTS: At 1-hour post-transplantation, the mean GBM was significantly thinner in the mismatch group than in the nonmismatch group. However, at 1-year post-transplantation, the mean GBM thickness did not significantly differ between the 2 groups. CONCLUSIONS: The GBM thickness at 1-hour post-transplantation is thinner in cases with donor-recipient body weight mismatch than in cases without mismatch. This implies that donor-recipient body weight mismatch may have to be considered when assessing donor-derived thin GBM disease using the 1-hour biopsy specimen.


Assuntos
Peso Corporal , Membrana Basal Glomerular/patologia , Transplante de Rim , Doadores de Tecidos , Adulto , Biópsia , Feminino , Membrana Basal Glomerular/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Transplante Homólogo
19.
N Engl J Med ; 380(22): 2116-2125, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31141633

RESUMO

BACKGROUND: In 2017, surveillance for tickborne diseases in China led to the identification of a patient who presented to a hospital in Inner Mongolia with a febrile illness that had an unknown cause. The clinical manifestation of the illness was similar to that of tickborne encephalitis virus (TBEV) infection, but neither TBEV RNA nor antibodies against the virus were detected. METHODS: We obtained a blood specimen from the index patient and attempted to isolate and identify a causative pathogen, using genome sequence analysis and electron microscopy. We also initiated a heightened surveillance program in the same hospital to screen for other patients who presented with fever, headache, and a history of tick bites. We used reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cell-culture assays to detect the pathogen and immunofluorescence and neutralization assays to determine the levels of virus-specific antibodies in serum specimens from the patients. RESULTS: We found that the index patient was infected with a previously unknown segmented RNA virus, which we designated Alongshan virus (ALSV) and which belongs to the jingmenvirus group of the family Flaviviridae. ALSV infection was confirmed by RT-PCR assay in 86 patients from Inner Mongolia and Heilongjiang who presented with fever, headache, and a history of tick bites. Serologic assays showed that seroconversion had occurred in all 19 patients for whom specimens were available from the acute phase and the convalescent phase of the illness. CONCLUSIONS: A newly discovered segmented virus was found to be associated with a febrile illness in northeastern China. (Funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China.).


Assuntos
Doenças Transmissíveis Emergentes/virologia , Flaviviridae/isolamento & purificação , Doenças Transmitidas por Carrapatos/virologia , Adulto , Idoso , Animais , China/epidemiologia , Doenças Transmissíveis Emergentes/epidemiologia , Fadiga/etiologia , Feminino , Febre/etiologia , Flaviviridae/classificação , Flaviviridae/genética , Flaviviridae/ultraestrutura , Cefaleia/etiologia , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Avaliação de Sintomas , Doenças Transmitidas por Carrapatos/complicações , Doenças Transmitidas por Carrapatos/epidemiologia , Carrapatos/virologia
20.
Molecules ; 24(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052152

RESUMO

Layered lithium-rich manganese oxide (LLO) cathode materials have attracted much attention for the development of high-performance lithium-ion batteries. However, they have suffered seriously from disadvantages, such as large irreversible capacity loss during the first cycle, discharge capacity decaying, and poor rate performance. Here, a novel method was developed to coat the surface of 0.4Li2MnO3∙0.6LiNi1/3Co1/3Mn1/3O2 cathode material with reduced graphene-oxide (rGO) in order to address these drawbacks, where a surfactant was used to facilitate the well-wrapping of rGO. As a result, the modified LLO (LLO@rGO) cathode exhibits superior electrochemical performance including cycling stability and rate capability compared to the pristine LLO cathode. In particular, the LLO@rGO with a 0.5% rGO content can deliver a high discharge capacity of 166.3 mAh g-1 at a 5C rate. The novel strategy developed here can provide a vital approach to inhibit the undesired side reactions and structural deterioration of Li-rich cathode materials, and should be greatly useful for other cathode materials to improve their electrochemical performance.


Assuntos
Fontes de Energia Elétrica , Eletrodos , Grafite/química , Lítio/química , Óxidos/química , Íons , Manganês , Microscopia Eletrônica , Pós/química , Análise Espectral
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