RESUMO
BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.
Assuntos
COVID-19/patologia , Nasofaringe/ultraestrutura , SARS-CoV-2/ultraestrutura , Antígenos Virais/metabolismo , COVID-19/diagnóstico , COVID-19/virologia , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Humanos , Aumento da Imagem , Microscopia , Microvilosidades/ultraestrutura , Mucosa Nasal/ultraestrutura , Mucosa Nasal/virologia , Nasofaringe/virologia , SARS-CoV-2/isolamento & purificação , Vírion/ultraestruturaRESUMO
In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.
Assuntos
Membrana Celular/ultraestrutura , Extensões da Superfície Celular/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Algoritmos , Animais , Animais Geneticamente Modificados , Vesículas Citoplasmáticas/ultraestrutura , Embrião não Mamífero , Humanos , Microvilosidades/ultraestrutura , Pseudópodes/ultraestrutura , Peixe-Zebra/embriologiaRESUMO
Toxoplasma gondii crosses the intestinal barrier to spread into the body. We investigate the intestinal wall and epithelial cells of the duodenum of rats infected with T. gondii during different time points of acute infection. Male Wistar rats, 60 days of age, were assigned into groups that were orally inoculated with 5000 sporulated oocysts T. gondii for 6 h (G6), 12 h (G12), 24 h (G24), 48 h (G48), 72 h (G72), 7 days (G7d), and 10 days (G10d). The control group (CG) received saline. The rats were killed and the duodenum was processed to obtain histological sections stained with hematoxylin and eosin, Periodic Acid Schiff, and Alcian blue (pH 2.5 and 1.0). Morphometry was performed on the layers of the intestinal wall and enterocytes, and the number of goblet cells and intraepithelial lymphocytes was counted. The data were compared by ANOVA considering 5% as level of significance. The infection provoked an increase in the width of villi and crypts; decrease in enterocyte height; increase in the smaller-diameter and reduction in the larger-diameter of the enterocytes nuclei, increased number of goblet cells secreting neutral (G6, G12 and G7d) and acidic (G7d and G10d) mucus, and increase in the number of intraepithelial lymphocytes (G48). The infected groups showed atrophy of the submucosa and muscular layers and the total wall. Acute infection with T. gondii caused morphological changes in the intestinal wall and epithelial cells of the duodenum in rats.
Assuntos
Duodeno/patologia , Duodeno/parasitologia , Toxoplasma/fisiologia , Toxoplasmose Animal/parasitologia , Análise de Variância , Animais , Anticorpos Antiprotozoários/sangue , Contagem de Células , Enterócitos/patologia , Células Caliciformes/citologia , Imunoglobulina G/sangue , Mucosa Intestinal/citologia , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Cinética , Linfócitos/citologia , Masculino , Microvilosidades/patologia , Microvilosidades/ultraestrutura , Distribuição Aleatória , Ratos , Ratos Wistar , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Toxoplasmose Animal/patologiaRESUMO
BACKGROUND: With increase in the incidence of resistance to antibiotics, probiotics are emerging as a promising adjunctive periodontal therapy. The authors of this study evaluate the influence of probiotic (PROB) supplementation on ligature-induced periodontitis (LIP) and intestinal morphology in rats. METHODS: Thirty-two rats were randomly divided into four groups: control (C), LIP, PROB, and LIP/PROB. In groups PROB and LIP/PROB, the PROB was administered orally by addition to the drinking water of the animals for 44 days. In groups LIP and LIP/PROB, the mandibular right first molar of the animals received a cotton ligature that was left in the same position for 14 days. All animals were euthanized 44 days after the start of the PROB supplementation. The jaws were resected and histomorphometric analyses were performed. The measurements included evaluation of attachment loss (AL) and alveolar bone level (ABL) on the distal root of the mandibular first molar. Samples of the duodenum, jejunum, and ileum were also dissected from each animal to evaluate the villous height (VH) and crypt depth (CD). The data obtained were subjected to statistical analyses (analysis of variance, Tukey; P <0.05). RESULTS: Mean values of AL and ABL were significantly higher in group LIP compared with group LIP/PROB (AL: 3.05 ± 0.57 mm and 1.78 ± 0.63 mm, respectively; ABL: 4.21 ± 0.42 mm and 3.38 ± 0.17 mm, respectively). In group LIP/PROB, the mean values of VH and CD of the jejunum were significantly higher than the ones from group LIP (VH: 672.1 ± 83.3 µm and 528.0 ± 51.7 µm, respectively; CD: 463.8 ± 100.9 µm and 269.0 ± 48.4 µm, respectively). CONCLUSIONS: It can be concluded that PROB supplementation 1) reduces AL and alveolar bone loss in rats with LIP and 2) can protect the small intestine from reactive changes induced by LIP.
Assuntos
Mucosa Intestinal/anatomia & histologia , Intestino Delgado/anatomia & histologia , Periodontite/terapia , Probióticos/uso terapêutico , Perda do Osso Alveolar/patologia , Perda do Osso Alveolar/terapia , Animais , Bacillus subtilis , Duodeno/anatomia & histologia , Inserção Epitelial/patologia , Íleo/anatomia & histologia , Jejuno/anatomia & histologia , Masculino , Microvilosidades/ultraestrutura , Dente Molar/patologia , Perda da Inserção Periodontal/patologia , Perda da Inserção Periodontal/terapia , Periodontite/patologia , Distribuição Aleatória , Ratos , Ratos Wistar , Fatores de Tempo , Raiz Dentária/patologiaRESUMO
Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.
Assuntos
Fosfatase Alcalina/metabolismo , Bacillus thuringiensis/fisiologia , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Ligadas por GPI/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/metabolismo , Tenebrio/enzimologia , Vesículas Transportadoras/microbiologia , Fosfatase Alcalina/genética , Animais , Toxinas de Bacillus thuringiensis , Proteínas Ligadas por GPI/genética , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Interações Hospedeiro-Patógeno , Proteínas de Insetos/genética , Larva/enzimologia , Larva/microbiologia , Microvilosidades/enzimologia , Microvilosidades/microbiologia , Microvilosidades/ultraestrutura , Ligação Proteica , Tenebrio/microbiologia , Vesículas Transportadoras/enzimologiaRESUMO
The first-generation precursors producing adult-born neurons in the crayfish (Procambarus clarkii) brain reside in a specialized niche located on the ventral surface of the brain. In the present work, we have explored the organization and ultrastructure of this neurogenic niche, using light-level, confocal and electron microscopic approaches. Our goals were to define characteristics of the niche microenvironment, examine the morphological relationships between the niche and the vasculature and observe specializations at the boundary between the vascular cavity located centrally in the niche. Our results show that the niche is almost fully encapsulated by blood vessels, and that cells in the vasculature come into contact with the niche. This analysis also characterizes the ultrastructure of the cell types in the niche. The Type I niche cells are by far the most numerous, and are the only cell type present superficially in the most ventral cell layers of the niche. More dorsally, Type I cells are intermingled with Types II, III and IV cells, which are observed far less frequently. Type I cells have microvilli on their apical cell surfaces facing the vascular cavity, as well as junctional complexes between adjacent cells, suggesting a role in regulating transport from the blood into the niche cells. These studies demonstrate a close relationship between the neurogenic niche and vascular system in P. clarkii. Furthermore, the specializations of niche cells contacting the vascular cavity are also typical of the interface between the blood/cerebrospinal fluid (CSF)-brain barriers of vertebrates, including cells of the subventricular zone (SVZ) producing new olfactory interneurons in mammals. These data indicate that tissues involved in producing adult-born neurons in the crayfish brain use strategies that may reflect fundamental mechanisms preserved in an evolutionarily broad range of species, as proposed previously. The studies described here extend our understanding of neurovascular relationships in the brain of P. clarkii by characterizing the organization and ultrastructure of the neurogenic niche and associated vascular tissues.
Assuntos
Encéfalo/ultraestrutura , Microambiente Celular/fisiologia , Neurogênese/fisiologia , Neurônios/citologia , Citoesqueleto de Actina/ultraestrutura , Animais , Astacoidea/fisiologia , Astacoidea/ultraestrutura , Feminino , Masculino , Microvilosidades/ultraestruturaRESUMO
The effect of spironolactone (SL) pretreatment (200micromol/kg b.w./day, 3 consecutive days) on intestinal multidrug resistance-associated protein 2 (Mrp2) was evaluated in rats. A significant increase in protein levels in upper regions of small intestine, where Mrp2 is mainly present, was detected by western blotting. Real time PCR studies suggest a transcriptional regulation. The administration of ketoconazole, a pregnane X receptor (PXR) antagonist, was able to prevent the increase in Mrp2 mRNA levels induced by SL. The serosal to mucosal transport of dinitrophenyl S-glutathione, a model substrate of Mrp2 was evaluated in jejunal sac model. The data indicate that SL increased Mrp2 activity, well correlating with its up-regulation. We conclude that SL is able to induce intestinal Mrp2 transcriptionally, PXR being a potential mediator. We propose that SL could be of potential therapeutic application particularly in situations of down-regulation of intestinal Mrp2.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Espironolactona/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Polaridade Celular , Dinitroclorobenzeno/metabolismo , Dinitroclorobenzeno/farmacocinética , Glutationa/análogos & derivados , Glutationa/análise , Intestino Delgado/metabolismo , Masculino , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Esteroides/antagonistas & inibidoresRESUMO
The insect midgut has ultimately been the focus of researches tempting to control insect pests because alterations in the insect gut may affect not only its development, but also physiological events such as nutrient absorption and transformation. The objective of the present work was to describe morphologically, histochemically, and ultrastructurally the larva midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae), a cotton key pest in Brazil. Light and electronic transmission microscopy was used to obtain images from midgut sections of late fourth-instar larvae of A. argillacea. In general, the morphology, histochemistry, and ultrastructure characteristics of A. argillacea midgut follow that described in the literature for other lepidopteran species. However, the results showed a mitochondrial polymorphism and branched microvilli, which suggest an ultrastrucutural and physiological modification possibly associated with a high absorption and secretion activity by the columnar cells of this species. This intense activity may favor a faster response related to the action of ingested microbial agents and/or toxins, and can explain the high susceptibility of A. argillacea to the agents of control such as the toxin of Bacillus thuringiensis.
Assuntos
Trato Gastrointestinal/ultraestrutura , Mariposas/ultraestrutura , Animais , Bacillus thuringiensis/patogenicidade , Brasil , Células Enteroendócrinas/ultraestrutura , Controle de Insetos , Microvilosidades/ultraestrutura , Mitocôndrias/ultraestrutura , Mariposas/microbiologia , VirulênciaRESUMO
Perimicrovillar membranes (PMM) are structures present on the surface of midgut epithelial cells of the hematophagous insect, Rhodnius prolixus. They cover the microvilli and are especially evident 10 days after blood meal, providing the compartmentalization of the enzymatic processes in the intestinal microenvironment. Using an enzyme cytochemical approach, Mg2+-ATPase and ouabain-sensitive Na+K+-ATPase activities were observed in the plasma (or microvillar) membrane (MM) of midgut cells and in the PMM. In contrast, alkaline phosphatase was only detected in MM. Using cationized ferritin and colloidal iron hydroxide particles, anionic sites were found only on the luminal surface of the PMM. Using fluorescein isothiocyanate (FITC)-labeled lectins, residues of alpha-d-galactose, mannose, N-acetyl-neuraminic acid, N-acetyl-d-galactosamine and N-acetyl-galactosamine-alpha-1,3-galactose were detected on the apical surface of posterior midgut epithelial cells. On the other hand, using FITC-labeled neoglycoproteins (NGP) it was possible to detect the presence of carbohydrate binding molecules (CBM) recognizing N-acetyl-d-galactosamine, alpha-d-mannose, alpha-l-fucose and alpha-d-glucose in the posterior midgut epithelium. The use of digitonin showed the presence of sterols in the MM and PMM. These results have led the authors to suggest that for some components the PMM resembles the MM lining the midgut cells of R. prolixus, composing a system which covers the microvilli and stretches to the luminal space.
Assuntos
Mucosa Intestinal/citologia , Microvilosidades/ultraestrutura , Rhodnius/citologia , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Fluoresceína-5-Isotiocianato , Histocitoquímica , ATPase Trocadora de Sódio-Potássio/metabolismo , Esteróis/metabolismoRESUMO
BACKGROUND: Proximal tubule cells have specialized apical membranes with microvilli that provide an extensive surface area for unidirectional transport of solute from lumen to blood. The major structural solute component is F-actin, which interacts with transmembrane proteins, including ion transport molecules related to normal absorptive and secretory functions. Our study was to evaluate F-actin and fluid absorption (Jv) in proximal tubules after exposure to preservation solutions. METHODS: In vitro microperfusion technique and immunohistochemistry analysis. RESULTS: 1. Absorptions were similar in 1- and 24-hour-preserved tubules, as well as in fresh tubules. The exception was tubules for 24 hours in Euro-Collins solution, which did not show absorption, suggesting that it was affected. 2. Fluorescence intensity of actin tubules preserved for 1 hour in both solutions showed similar values to each other and to the control group; tubules preserved for 24 hours in both solutions were similar to each other, although statistically different than the control group and those preserved for 1 hour in Belzer (UW) solution. CONCLUSION: There were differences among groups in the distribution of F-actin; Jv values were different for 24-hour preservation in each solution, whereas fluorescence intensity was similar in both 24-hour solutions. Thus, actin cytoskeleton was not responsible for it, because 24-hour preservation in UW showed Jv results comparable to the control group.