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1.
Parasitol Res ; 120(3): 899-910, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33432440

RESUMO

During their different life stages, parasites undergo remarkable morphological, physiological, and behavioral "metamorphoses" to meet the needs of their changing habitats. This is even true for ectoparasites, such as the monogeneans, which typically have a free-swimming larval stage (oncomiracidium) that seeks out and attaches to the external surfaces of fish where they mature. Before any obvious changes occur, there are ultrastructural differences in the oncomiracidium's outer surface that prepare it for a parasitic existence. The present findings suggest a distinct variation in timing of the switch from oncomiracidia epidermis to the syncytial structure of the adult tegument and so, to date, there are three such categories within the Monogenea: (1) Nuclei of both ciliated cells and interciliary cytoplasm are shed from the surface layer and the epidermis becomes a syncytial layer during the later stages of embryogenesis; (2) nuclei of both ciliated cells and interciliary syncytium remain distinct and the switch occurs later after the oncomiracidia hatch (as in the present study); and (3) the nuclei remain distinct in the ciliated epidermis but those of the interciliary epidermis are lost during embryonic development. Here we describe how the epidermis of the oncomiracidium of Discocotyle sagittata is differentiated into two regions, a ciliated cell layer and an interciliary, syncytial cytoplasm, both of which are nucleated. The interciliary syncytium extends in-between and underneath the ciliated cells and sometimes covers part of their apical surfaces, possibly the start of their shedding process. The presence of membranous whorls and pyknotic nuclei over the surface are indicative of membrane turnover suggesting that the switch in epidermis morphology is already initiated at this stage. The body tegument and associated putative sensory receptors of subadult and adult D. sagittata are similar to those in other monogeneans.


Assuntos
Epiderme/ultraestrutura , Doenças dos Peixes/parasitologia , Salmonidae/parasitologia , Trematódeos/ultraestrutura , Infecções por Trematódeos/veterinária , Animais , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Epiderme/crescimento & desenvolvimento , Brânquias/parasitologia , Larva/ultraestrutura , Trematódeos/crescimento & desenvolvimento , Infecções por Trematódeos/parasitologia
2.
Dev Biol ; 469: 111-124, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33141038

RESUMO

Although somatic cells play an integral role in animal gametogenesis, their organization and function are usually poorly characterized, especially in non-model systems. One such example is a peculiar cell found in leech ovaries - the apical cell (AC). A single AC can be found at the apical tip of each ovary cord, the functional unit of leech ovaries, where it is surrounded by other somatic and germline cells. The AC is easily distinguished due to its enormous size and its numerous long cytoplasmic projections that penetrate the space between neighboring cells. It is also characterized by a prominent accumulation of mitochondria, Golgi complexes and electron-dense vesicles. ACs are also enriched in cytoskeleton, mainly in form of intermediate filaments. Additionally, the AC is connected to neighboring cells via junctions that structurally resemble hemidesmosomes. In spite of numerous descriptive data about the AC, its functions remain poorly understood. Its suggested functions include a role in forming skeleton for the germline cells, and a role in defining a niche for germline stem cells. The latter is more speculative, since germline stem cells have not been identified in leech ovaries. Somatic cells with similar morphological properties to those of the AC have been found in gonads of nematodes - the distal tip cell - and in insects - Verson's cell, hub cells and cap cells. In the present article we summarize information about the AC structure and its putative functions. AC is compared with other well-described somatic cells with potentially similar roles in gametogenesis.


Assuntos
Sanguessugas/citologia , Ovário/citologia , Animais , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Feminino , Oogênese , Ovário/fisiologia , Ovário/ultraestrutura , Nicho de Células-Tronco
3.
BMC Bioinformatics ; 21(1): 399, 2020 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-32907544

RESUMO

BACKGROUND: Cryo-electron tomography is an important and powerful technique to explore the structure, abundance, and location of ultrastructure in a near-native state. It contains detailed information of all macromolecular complexes in a sample cell. However, due to the compact and crowded status, the missing edge effect, and low signal to noise ratio (SNR), it is extremely challenging to recover such information with existing image processing methods. Cryo-electron tomogram simulation is an effective solution to test and optimize the performance of the above image processing methods. The simulated images could be regarded as the labeled data which covers a wide range of macromolecular complexes and ultrastructure. To approximate the crowded cellular environment, it is very important to pack these heterogeneous structures as tightly as possible. Besides, simulating non-deformable and deformable components under a unified framework also need to be achieved. RESULT: In this paper, we proposed a unified framework for simulating crowded cryo-electron tomogram images including non-deformable macromolecular complexes and deformable ultrastructures. A macromolecule was approximated using multiple balls with fixed relative positions to reduce the vacuum volume. A ultrastructure, such as membrane and filament, was approximated using multiple balls with flexible relative positions so that this structure could deform under force field. In the experiment, 400 macromolecules of 20 representative types were packed into simulated cytoplasm by our framework, and numerical verification proved that our method has a smaller volume and higher compression ratio than the baseline single-ball model. We also packed filaments, membranes and macromolecules together, to obtain a simulated cryo-electron tomogram image with deformable structures. The simulated results are closer to the real Cryo-ET, making the analysis more difficult. The DOG particle picking method and the image segmentation method are tested on our simulation data, and the experimental results show that these methods still have much room for improvement. CONCLUSION: The proposed multi-ball model can achieve more crowded packaging results and contains richer elements with different properties to obtain more realistic cryo-electron tomogram simulation. This enables users to simulate cryo-electron tomogram images with non-deformable macromolecular complexes and deformable ultrastructures under a unified framework. To illustrate the advantages of our framework in improving the compression ratio, we calculated the volume of simulated macromolecular under our multi-ball method and traditional single-ball method. We also performed the packing experiment of filaments and membranes to demonstrate the simulation ability of deformable structures. Our method can be used to do a benchmark by generating large labeled cryo-ET dataset and evaluating existing image processing methods. Since the content of the simulated cryo-ET is more complex and crowded compared with previous ones, it will pose a greater challenge to existing image processing methods.


Assuntos
Citoplasma/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Simulação de Dinâmica Molecular , Algoritmos , Análise por Conglomerados , Microscopia Crioeletrônica , Citoplasma/metabolismo , Processamento de Imagem Assistida por Computador , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Razão Sinal-Ruído
5.
Nat Struct Mol Biol ; 27(4): 382-391, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251414

RESUMO

The bestrophin family of calcium (Ca2+)-activated chloride (Cl-) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca2+, comprises four members in mammals (bestrophin 1-4). Here we report cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca2+ at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis reveals that, although the channel is sensitive to Ca2+, it has substantial Ca2+-independent activity for Cl-, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway even when Ca2+ is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating.


Assuntos
Bestrofinas/ultraestrutura , Canais de Cloreto/ultraestrutura , Conformação Proteica , Animais , Bestrofinas/química , Bestrofinas/genética , Cálcio/química , Bovinos , Canais de Cloreto/química , Canais de Cloreto/genética , Microscopia Crioeletrônica , Citoplasma/química , Citoplasma/genética , Citoplasma/ultraestrutura , Humanos , Ativação do Canal Iônico/genética , Ligação Proteica/genética , Transdução de Sinais
6.
Sci Rep ; 10(1): 4621, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165642

RESUMO

Histones are typically located within the intracellular compartment, and more specifically, within the nucleus. When histones are located within the extracellular compartment, they change roles and become damage-associated molecular patterns (DAMPs), promoting inflammation and coagulation. Patients with sepsis have increased levels of extracellular histones, which have been shown to correlate with poor prognosis and the development of sepsis-related sequelae, such as end-organ damage. Until now, neutrophils were assumed to be the primary source of circulating histones during sepsis. In this paper, we show that megakaryocytes contain extranuclear histones and transfer histones to their platelet progeny. Upon examination of isolated platelets from patients with sepsis, we identified that patients with sepsis have increased amounts of platelet-associated histones (PAHs), which appear to be correlated with the type of infection. Taken together, these results suggest that megakaryocytes and platelets may be a source of circulating histones during sepsis and should be further explored.


Assuntos
Plaquetas/metabolismo , Citoplasma/metabolismo , Histonas/metabolismo , Megacariócitos/metabolismo , Sepse/metabolismo , Biomarcadores , Coagulação Sanguínea , Plaquetas/ultraestrutura , Citoplasma/ultraestrutura , Imunofluorescência , Humanos , Megacariócitos/ultraestrutura , Modelos Biológicos , Sepse/sangue , Sepse/etiologia
7.
Eur J Pharmacol ; 870: 172912, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-31926992

RESUMO

Immunotoxin therapy is one of the immunotherapy strategies providing a new, effective and high potency treatment against various cancers. Breast cancer is the most common cancer among women in many countries. The EPH receptors are a large part of tyrosine kinase receptors family and play an effective role in tumor development and angiogenesis. Among EPH receptors, EPHA2 is more commonly well-known and widely expressed in many cancers like breast cancer. In this study, we evaluated the specification of a designed immunotoxin formed by EPHA2-specific scfv linked with PE38KDEL on EPHA2-overexpressing breast cancer cell line. This new scfv-based recombinant immunotoxin was studied in terms of features such as binding potency, cytotoxicity effects, apoptosis induction ability, and internalization. The flow cytometry results showed that the immunotoxin can significantly (approximately 99%) bind to EPHA2-overexpressing breast cancer cell line (MDA-MB-231) in a low concentration (2.5 ng/ul) while cannot significantly bind to the normal cell line (HEK-293) or even EPHA2-very low expressing cell line (MCF-7). Using the MTT assay and Annexin V/Propidium iodide (PI) double staining method by flow cytometry, we observed significant killing and apoptosis induction of the MDA-MB-231 cells at different concentrations. Immunotoxin tracking by confocal microscopy at 2 h and 6 h revealed a massive presence of immunotoxin in the cytoplasm. Finally, given the in vitro results, it seems that this immunotoxin is competent enough to serve as a good candidate for in vivo studies to further explore the possibility of breast cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/terapia , Imunotoxinas/genética , Receptor EphA2/genética , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/genética , Apoptose , Linhagem Celular , Citoplasma/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia , Imunotoxinas/farmacologia , Mutação , Metástase Neoplásica/prevenção & controle , Proteínas Recombinantes/farmacologia
8.
Theriogenology ; 143: 88-97, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31862672

RESUMO

The ultrastructural morphology of the bovine oocyte at different maturation stages has been previously analyzed but without detailed structural observations at the mature stage. The objective of the present study was thus to establish the ultrastructural characteristics of the mature bovine oocyte in full detail. Oocytes from Bos taurus (Holstein-Friesian) cows were aspirated from ovaries collected after being slaughtered at a local abattoir. After in vitro culture for 24 h, some of them were processed for electron microscopy. We described the ultrastructure of the zona pellucida, which presented three different regions, and novel cytoplasmic findings. There were two types of electron-lucent vesicles (heterogeneous and striated), which were suggested to give rise to lipid droplets, and presence of receptor-mediated endocytosis. In conclusion, our results indicate that although the mature bovine oocyte is devoid of evident yolk, it might be filled with an extensive lipid factory. In addition, even before fertilization, the mature oocyte seemed to absorb nutrients through receptor-mediated endocytosis, indicating active energy use or storage.


Assuntos
Bovinos/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Oócitos/ultraestrutura , Animais , Citoplasma/ultraestrutura , Feminino , Zona Pelúcida/ultraestrutura
9.
J Mol Biol ; 432(2): 585-596, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31518613

RESUMO

Aggregation of amyloidogenic proteins is an abnormal biological process implicated in neurodegenerative disorders. Whereas the aggregation process of amyloid-forming proteins has been studied extensively, the mechanism of aggregate removal is poorly understood. We recently demonstrated that proteasomes could fragment filamentous aggregates into smaller entities, restricting aggregate size [1]. Here, we show in vitro that UBE2W can modify the N-terminus of both α-synuclein and a tau tetra-repeat domain with a single ubiquitin. We demonstrate that an engineered N-terminal ubiquitin modification changes the aggregation process of both proteins, resulting in the formation of structurally distinct aggregates. Single-molecule approaches further reveal that the proteasome can target soluble oligomers assembled from ubiquitin-modified proteins independently of its peptidase activity, consistent with our recently reported fibril-fragmenting activity. Based on these results, we propose that proteasomes are able to target oligomers assembled from N-terminally ubiquitinated proteins. Our data suggest a possible disassembly mechanism by which N-terminal ubiquitination and the proteasome may together impede aggregate formation.


Assuntos
Proteínas Amiloidogênicas/genética , Doenças Neurodegenerativas/genética , Enzimas de Conjugação de Ubiquitina/genética , alfa-Sinucleína/genética , Proteínas tau/genética , Proteínas Amiloidogênicas/ultraestrutura , Citoplasma/genética , Citoplasma/ultraestrutura , Holoenzimas/genética , Holoenzimas/ultraestrutura , Humanos , Doenças Neurodegenerativas/patologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Agregação Patológica de Proteínas/genética , Domínios Proteicos , Multimerização Proteica , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Ubiquitinação/genética , alfa-Sinucleína/ultraestrutura , Proteínas tau/ultraestrutura
10.
Environ Geochem Health ; 42(1): 45-58, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30874936

RESUMO

Effects of Cu toxicity from contaminated soil were analysed in spring barley (Hordeum sativum distichum), a widely cultivated species in South Russia. In this study, H. sativum was planted outdoors in one of the most fertile soils-Haplic Chernozem spiked with high concentration of Cu and examined between the boot and head emergence phase of growth. Copper toxicity was observed to cause slow ontogenetic development of plants, changing their morphometric parameters (shape, size, colour). To the best of our knowledge, the ultrastructural changes in roots, stems and leaves of H. sativum induced by excess Cu were fully characterized for the first time using transmission electron microscopy. The plant roots were the most effected, showing degradation of the epidermis, reduced number of parenchyma cells, as well as a significant decrease in the diameter of the stele and a disruption and modification to its cell structure. The comparative analysis of the ultrastructure of control plants and plants exposed to the toxic effects of Cu has made it possible to reveal significant disruption of the integrity of the cell wall and cytoplasmic membranes in the root with deposition of electron-dense material. The changes in the ultrastructure of the main cytoplasmic organelles-endoplasmic reticulum, mitochondria, chloroplasts and peroxisomes-in the stem and leaves were found. The cellular Cu deposition, anatomical and ultrastructural modifications could mainly account for the primary impact points of metal toxicity. Therefore, this work extends the available knowledge of the mechanisms of the Cu effect tolerance of barley.


Assuntos
Cobre/toxicidade , Hordeum/efeitos dos fármacos , Poluentes do Solo/toxicidade , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Hordeum/anatomia & histologia , Hordeum/citologia , Hordeum/ultraestrutura , Microscopia Eletrônica de Transmissão , Células Vegetais/efeitos dos fármacos , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/ultraestrutura , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/ultraestrutura , Caules de Planta/citologia , Caules de Planta/efeitos dos fármacos , Caules de Planta/ultraestrutura , Federação Russa
11.
J Eukaryot Microbiol ; 67(1): 28-44, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31332877

RESUMO

All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed-sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5-6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.


Assuntos
Citoplasma/ultraestrutura , Microsporídios/ultraestrutura , Esporos Fúngicos/ultraestrutura , Microscopia Crioeletrônica , Microscopia , Microscopia Eletrônica de Transmissão , Microsporídios/citologia , Esporos Fúngicos/citologia
12.
Parasitol Res ; 119(1): 177-187, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31811425

RESUMO

The spermatozoon ultrastructure of the progenetic cestode Diplocotyle olrikii (Spathebothriidea) has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. The spermatozoon is a filiform cell, tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths. New for the Cestoda is a finding of three types of the mature spermatozoa with respect to different axonemal structure. The first type has both axonemes with standard 9 + '1' trepaxonematan pattern. The second type is represented by a spermatozoon having one axoneme with 9 + '1' structure and the second one with 9 + 0 pattern. The third type includes the two axonemes with 9 + 0 pattern. Microtubule doublets of the 9 + 0 axonemes contain either inner dynein arms or no dynein arms. In addition to the two axonemes, all three types of the mature sperm cells contain parallel nucleus, parallel cortical microtubules, four electron-dense plaques/attachment zones, and glycogen. The anterior extremity of the gamete exhibits a centriole surrounded by a semiarc of up to five electron-dense tubular structures. The distal end of the first type spermatozoa exhibits two morphological variants, represented either by (i) nucleus or (ii) remnants of the disorganized axoneme. Distal extremity of the spermatozoa of the second and third types contains doublets and singlets of disorganized axoneme. The ultrastructural characters of the spermatozoon of progenetic D. olrikii support the basal position of the Spathebothriidea within the Eucestoda.


Assuntos
Axonema/ultraestrutura , Núcleo Celular/ultraestrutura , Centríolos/ultraestrutura , Cestoides/ultraestrutura , Espermatozoides/ultraestrutura , Animais , Citoplasma/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Espermatogênese/fisiologia
13.
Int J Mol Sci ; 20(21)2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31694312

RESUMO

Cytoplasmic male sterility (CMS) is a maternally inherited trait used for hybrid production in plants, a novel kenaf CMS line 722HA was derived from the thermo-sensitive male-sterile mutant 'HMS' by recurrent backcrossing with 722HB. The line 722HA has great potential for hybrid breeding in kenaf. However, the underlying molecular mechanism that controls pollen abortion in 722HA remains unclear, thus limiting the full utilization of this line. To understand the possible mechanism governing pollen abortion in 722HA, cytological, transcriptomic, and biochemical analyses were carried out to compare the CMS line 722HA and its maintainer line 722HB. Cytological observations of the microspore development revealed premature degradation of the tapetum at the mononuclear stage, which resulted in pollen dysfunction. The k-means clustering analysis of differentially expressed genes (DEGs) revealed that these genes are related to processes associated with the accumulation of reactive oxygen species (ROS), including electron transport chain, F1F0-ATPase proton transport, positive regulation of superoxide dismutase (SOD), hydrogen peroxide catabolic, and oxidation-reduction. Biochemical analysis indicated that ROS-scavenging capability was lower in 722HA than in 722HB, resulting in an accumulation of excess ROS, which is consistent with the transcriptome results. Taken together, these results demonstrate that excessive ROS accumulation may affect the normal development of microspores. Our study provides new insight into the molecular mechanism of pollen abortion in 722HA and will promote further studies of kenaf hybrids.


Assuntos
Regulação da Expressão Gênica de Plantas , Hibiscus/genética , Infertilidade das Plantas/genética , Pólen/genética , Transcriptoma , Citoplasma/genética , Citoplasma/ultraestrutura , Hibiscus/crescimento & desenvolvimento , Hibiscus/ultraestrutura , Melhoramento Vegetal , Pólen/crescimento & desenvolvimento , Pólen/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo
14.
Genes Cells ; 24(11): 731-745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31554018

RESUMO

Cluap1/IFT38 is a ciliary protein that belongs to the IFT-B complex and is required for ciliogenesis. In this study, we have examined the behaviors of Cluap1 protein in nonciliated and ciliated cells. In proliferating cells, Cluap1 is located at the distal appendage of the mother centriole. When cells are induced to form cilia, Cluap1 is found in a novel noncentriolar compartment, the cytoplasmic IFT spot, which mainly exists once in a cell. Other IFT-B proteins such as IFT46 and IFT88 are colocalized in this spot. The cytoplasmic IFT spot is present in mouse embryonic fibroblasts (MEFs) but is absent in ciliogenesis-defective MEFs lacking Cluap1, Kif3a or Odf2. The cytoplasmic IFT spot is also found in mouse embryos but is absent in the Cluap1 mutant embryo. When MEFs are induced to form cilia, the cytoplasmic IFT spot appears at an early step of ciliogenesis but starts to disappear when ciliogenesis is mostly completed. These results suggest that IFT-B proteins such as Cluap1 accumulate in a previously undescribed cytoplasmic compartment during ciliogenesis.


Assuntos
Cílios/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Cílios/ultraestrutura , Citoplasma/ultraestrutura , Fibroblastos , Proteínas de Choque Térmico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesina , Camundongos , Camundongos Knockout , Proteínas Supressoras de Tumor
15.
Physiol Int ; 106(3): 225-235, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31560236

RESUMO

OBJECTIVES: Impaired intestinal barrier function has been demonstrated in the pathophysiology of diarrhea-predominant irritable bowel syndrome (IBS-D). This study aimed to describe the intestinal ultrastructural findings in the intestinal mucosal layer of IBS-D patients. METHODS: In total, 10 healthy controls and 10 IBS-D patients were analyzed in this study. The mucosa of each patient's rectosigmoid colon was first assessed by confocal laser endomicroscopy (CLE); next, biopsied specimens of these sites were obtained. Intestinal tissues of IBS-D patients and healthy volunteers were examined to observe cellular changes by transmission electron microscopy (TEM). RESULTS: CLE showed no visible epithelial damage or inflammatory changes in the colonic mucosa of IBS-D compared with healthy volunteers. On transmission electron microscopic examination, patients with IBS-D displayed a larger apical intercellular distance with a higher proportion of dilated (>20 nm) intercellular junctional complexes, which was indicative of impaired mucosal integrity. In addition, microvillus exfoliation, extracellular vesicle as well as increased presence of multivesicular bodies were visible in IBS-D patients. Single epithelial cells appeared necrotic, as characterized by cytoplasmic vacuolization, cytoplasmic swelling, and presence of autolysosome. A significant association between bowel habit, frequency of abdominal pain, and enlarged intercellular distance was found. CONCLUSION: This study showed ultrastructural alterations in the architecture of intestinal epithelial cells and intercellular junctional complexes in IBS-D patients, potentially representing a pathophysiological mechanism in IBS-D.


Assuntos
Diarreia/patologia , Mucosa Intestinal/ultraestrutura , Síndrome do Intestino Irritável/patologia , Dor Abdominal/patologia , Colo Sigmoide/ultraestrutura , Citoplasma/patologia , Citoplasma/ultraestrutura , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Feminino , Humanos , Junções Intercelulares/ultraestrutura , Masculino , Pessoa de Meia-Idade , Reto/patologia , Reto/ultraestrutura
16.
J Virol ; 93(19)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292247

RESUMO

A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells and on monocytes/macrophages (Ms/Mϕs) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally "stitching" together contiguous SEM images. A single whole-cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the surfaces of AEC-IIs, Ms/Mϕs, and Neus; however, intranuclear dense tubules were found only in AEC-IIs. Computer-assisted processing of SSA-SEM images from each cell type enabled three-dimensional (3D) modeling of the distribution of virus particles within an ACE-II, a M/Mϕ, and a Neu.IMPORTANCE Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia.


Assuntos
Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/patologia , Influenza Humana/virologia , Pulmão/patologia , Adulto , Autopsia , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Citoplasma/ultraestrutura , Citoplasma/virologia , Vesículas Citoplasmáticas/ultraestrutura , Vesículas Citoplasmáticas/virologia , Humanos , Macrófagos/virologia , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Neutrófilos/virologia
17.
Nucleic Acids Res ; 47(18): e109, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31340014

RESUMO

Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.


Assuntos
Citoplasma/ultraestrutura , DNA/ultraestrutura , Hibridização in Situ Fluorescente/métodos , RNA/química , Núcleo Celular/ultraestrutura , DNA/isolamento & purificação , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico
18.
PLoS Genet ; 15(6): e1008061, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170156

RESUMO

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/genética , Proteínas de Schizosaccharomyces pombe/genética , Transporte Ativo do Núcleo Celular/genética , Ciclo Celular/genética , Divisão Celular/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Citoplasma/genética , Citoplasma/ultraestrutura , Humanos , Meiose/genética , Microscopia de Fluorescência , Membrana Nuclear/genética , Poro Nuclear/ultraestrutura , Ligação Proteica/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
19.
Methods Mol Biol ; 1992: 231-238, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148042

RESUMO

Optical tweezers allow for noninvasive manipulation of subcellular compartments to study their physical interactions and attachments. By measuring (delay of) displacements, (semi)quantitative force measurements within a living cell can be performed. In this chapter, we provide practical tips for setting up such experiments paying special attention to the technical considerations for integrating optical tweezers into a confocal microscope. Next, we describe experimental approaches we have taken to trap intracellular structures in plant cells.


Assuntos
Microscopia Confocal/instrumentação , Pinças Ópticas , Células Vegetais/ultraestrutura , Plantas/ultraestrutura , Citoplasma/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Desenho de Equipamento , Microscopia Confocal/métodos
20.
Proc Natl Acad Sci U S A ; 116(28): 14222-14227, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239340

RESUMO

Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or "mating" channel, the channel-pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.


Assuntos
Membrana Celular/ultraestrutura , Escherichia coli/ultraestrutura , Fator F/ultraestrutura , Fímbrias Bacterianas/ultraestrutura , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Membrana Celular/genética , Conjugação Genética/genética , Microscopia Crioeletrônica , Citoplasma/genética , Citoplasma/ultraestrutura , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fator F/genética , Fímbrias Bacterianas/genética , Sistemas de Secreção Tipo IV/genética
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