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1.
Methods Mol Biol ; 2255: 135-147, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033100

RESUMO

Transmission electron microscopy (TEM) is an all-in-one tool to visualize the complex systems of any specimen that is 1 nm in size or smaller. The current chapter provides detailed guidelines for imaging morphological changes during programmed cell necrosis using TEM as a single-step methodology. In this protocol, a novel aldehyde dehydrogenase inhibitor is used to induce cell programmed necrosis in ovarian cancer cell lines (A2780 and SKOV3). This process is followed by gradient dehydration with ethanol, chemical fixation, sampled grid preparation, and staining with 0.75% uranyl formate. Following fixation and grid preparation, cells are imaged using TEM. The resulting images reveal morphological changes consistent with necrotic morphology, including swelling of cells and organelles, appearance of vacuoles, and plasma membrane rupture followed by leakage of cellular contents. The current approach allows a single-step methodology for characterization of cell-programmed necrosis in cells based on morphology.


Assuntos
Adenocarcinoma/patologia , Microscopia Eletrônica de Transmissão/métodos , Necroptose , Neoplasias Ovarianas/patologia , Adenocarcinoma/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
2.
Molecules ; 26(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809519

RESUMO

Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.


Assuntos
DNA/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanoestruturas/química , Sequência de Aminoácidos , Apolipoproteína A-I/química , Microscopia Eletrônica de Transmissão/métodos , Fosfolipídeos/química
3.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925347

RESUMO

The development of multifunctional nanoscale systems that can mediate efficient tumor targeting, together with high cellular internalization, is crucial for the diagnosis of glioma. The combination of imaging agents into one platform provides dual imaging and allows further surface modification with targeting ligands for specific glioma detection. Herein, transferrin (Tf)-decorated niosomes with integrated magnetic iron oxide nanoparticles (MIONs) and quantum dots (QDs) were formulated (PEGNIO/QDs/MIONs/Tf) for efficient imaging of glioma, supported by magnetic and active targeting. Transmission electron microscopy confirmed the complete co-encapsulation of MIONs and QDs in the niosomes. Flow cytometry analysis demonstrated enhanced cellular uptake of the niosomal formulation by glioma cells. In vitro imaging studies showed that PEGNIO/QDs/MIONs/Tf produces an obvious negative-contrast enhancement effect on glioma cells by magnetic resonance imaging (MRI) and also improved fluorescence intensity under fluorescence microscopy. This novel platform represents the first niosome-based system which combines magnetic nanoparticles and QDs, and has application potential in dual-targeted imaging of glioma.


Assuntos
Glioma/diagnóstico por imagem , Lipossomos/química , Transferrina/química , Animais , Linhagem Celular Tumoral , Meios de Contraste , Compostos Férricos/química , Glioma/genética , Glioma/metabolismo , Humanos , Lipossomos/metabolismo , Nanopartículas Magnéticas de Óxido de Ferro/química , Imageamento por Ressonância Magnética/métodos , Magnetismo , Microscopia Eletrônica de Transmissão/métodos , Nanopartículas , Polietilenoglicóis , Pontos Quânticos/química
4.
Methods Mol Biol ; 2259: 3-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687705

RESUMO

In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.


Assuntos
Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestrutura , Células-Tronco Mesenquimais/citologia , Proteínas/análise , Células Cultivadas , Cromatografia Líquida/métodos , Meios de Cultura/química , Humanos , Células-Tronco Mesenquimais/química , Microscopia Eletrônica de Transmissão/métodos , Cultura Primária de Células/métodos , Proteínas/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cordão Umbilical/citologia
5.
Int J Mol Sci ; 22(4)2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33672065

RESUMO

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential "developmental hemostatic mismatch risk" associated with transfusions containing plasma exosomes from adults.


Assuntos
Plaquetas/citologia , Exossomos/metabolismo , Exossomos/ultraestrutura , Sangue Fetal/citologia , Plasma/citologia , Adulto , Coagulação Sanguínea , Grânulos Citoplasmáticos/metabolismo , Humanos , Imunoglobulinas/sangue , Recém-Nascido , Microscopia Eletrônica de Transmissão/métodos , Ativação Plaquetária , Proteína S/análise , Proteína S/metabolismo , Proteoma , Proteômica/métodos , Pesquisa Qualitativa , Ultracentrifugação , Fator de von Willebrand/análise , Fator de von Willebrand/metabolismo
6.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672911

RESUMO

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Flavobacterium/fisiologia , Locomoção/fisiologia , Proteínas de Bactérias/genética , Flavobacterium/genética , Flavobacterium/ultraestrutura , Locomoção/genética , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Mutação , Imagem com Lapso de Tempo/métodos
7.
Methods Mol Biol ; 2273: 251-262, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604859

RESUMO

Oviduct and uterus are key female reproductive organs lined by ciliated simple columnar epithelia, which are the first line of maternal contact with gametes and the developing embryo during reproduction and which warrant the optimal developmental environment for the conceptus. A major challenge for modeling these epithelia in vitro is the preservation of apical-basal polarization and cilia formation. The air-liquid interface (ALI) culture approach is a technology originally invented for modeling epidermal and airway epithelia. It has recently been shown that it also allows the establishment of highly differentiated in vitro models of epithelia that do not have access to ambient air in vivo. In this chapter, we present a comprehensive ALI procedure to model female reproductive tract (FRT) epithelia of different mammalian species in vitro over extended time periods. As a working example, the protocol focuses on primary oviductal epithelial cells (OEC) isolated from domestic pig. Hints on protocol variations for the culture of OEC from other species are provided in the Subheading 4.


Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Tubas Uterinas/citologia , Animais , Diferenciação Celular , Separação Celular/métodos , Células Cultivadas , Feminino , Humanos , Microscopia Eletrônica de Transmissão/métodos , Suínos
8.
Life Sci ; 270: 119122, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33508294

RESUMO

The adrenal glands have striking morpho-biochemical features that render them vulnerable to the effects of toxins. AIMS: This study was conducted to explore the therapeutic utility of extracellular vesicles derived from bone marrow mesenchymal stem cells (BMSC-EVs) against fluoride-induced adrenal toxicity. MATERIALS AND METHODS: The work included isolation and further identification of BMSC-EVs by transmission electron microscopy and flow cytometric analysis. Adrenal toxicity in rats was induced by oral administration of 300 ppm of sodium fluoride (NaF) in drinking water for 60 days followed by a single dose injection of BMSC-EVs. The effects of BMSC-EVs against NaF was evaluated by adrenal oxidant/antioxidant biomarkers, hormonal assay of plasma adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) and mRNA gene expression quantitation for adrenal cortical steroidogenic pathway-encoding genes. Histopathological examination of the adrenal tissue was performed. KEY FINDINGS: BMSC-EVs were effectively isolated and characterized. NaF exposure decreased adrenal superoxide dismutase and catalase activities, increased adrenal malondialdehyde levels, elevated plasma ACTH, diminished CORT concentrations and downregulated the adrenal cortical steroidogenic pathway-encoding genes. In addition, NaF-induced marked adrenal histopathological lesions. SIGNIFICANCE: BMSC-EVs treatment repaired damaged adrenal tissue and recovered its function greatly following NaF consumption. BMSC-EVs reversed the toxic effects of NaF and reprogramed injured adrenal cells by activating regenerative processes.


Assuntos
Glândulas Suprarrenais/metabolismo , Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Feminino , Fluoretos/efeitos adversos , Fluoretos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Microscopia Eletrônica de Transmissão/métodos , Ratos
9.
Nat Commun ; 12(1): 573, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495475

RESUMO

The biological identity of nanoparticles (NPs) is established by their interactions with a wide range of biomolecules around their surfaces after exposure to biological media. Understanding the true nature of the biomolecular corona (BC) in its native state is, therefore, essential for its safe and efficient application in clinical settings. The fundamental challenge is to visualize the biomolecules within the corona and their relationship/association to the surface of the NPs. Using a synergistic application of cryo-electron microscopy, cryo-electron tomography, and three-dimensional reconstruction, we revealed the unique morphological details of the biomolecules and their distribution/association with the surface of polystyrene NPs at a nanoscale resolution. The analysis of the BC at a single NP level and its variability among NPs in the same sample, and the discovery of the presence of nonspecific biomolecules in plasma residues, enable more precise characterization of NPs, improving predictions of their safety and efficacies.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Nanopartículas/química , Nanopartículas/ultraestrutura , Plasma/química , Poliestirenos/química , Simulação por Computador , Humanos , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Transmissão/métodos , Coroa de Proteína/química , Reprodutibilidade dos Testes
10.
Methods Mol Biol ; 2183: 499-511, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959263

RESUMO

Cryogenic transmission electron microscopy (Cryo-TEM) enables visualizing the physicochemical structure of nanocarriers in solution. Here, we demonstrate the typical applications of Cryo-TEM in characterizing archaeosome-based vesicles as antigen carriers, including the morphology and size of vaccine carriers. Cryo-TEM tomography, incorporated with immunogold labeling for identifying and localizing the antigens, reveals the antigen distribution within archaeosomes in three dimensions (3D).


Assuntos
Microscopia Crioeletrônica , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Microscopia Eletrônica de Transmissão , Vacinas/administração & dosagem , Microscopia Eletrônica de Transmissão/métodos , Software , Vacinas de Partículas Semelhantes a Vírus
11.
Methods Mol Biol ; 2233: 301-309, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222143

RESUMO

To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells. The exposed inner membrane surface can then be visualized with different electron-dense colorations, and specific proteins or lipids can be detected with gold-conjugated probes. Moreover, the membrane sheets are sufficiently resistant to support automated acquisition of multiple-tilt projections, and thus electron tomography allows to obtain three-dimensional (3D) ultrastructural images of secretory granule docked to the plasma membrane.


Assuntos
Tomografia com Microscopia Eletrônica/métodos , Exocitose/genética , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Transmissão/métodos , Animais , Transporte Biológico/genética , Membrana Celular/ultraestrutura , Camundongos , Vesículas Secretórias/ultraestrutura , Tomografia Computadorizada por Raios X
12.
Methods Mol Biol ; 2215: 309-319, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33368011

RESUMO

Microcrystal electron diffraction (MicroED) is a technique for structure determination that relies on the strong interaction of electrons with a minuscule, crystalline sample. While some of the electrons used to probe the crystal interact without altering the crystal, others deposit energy which changes the sample through a series of damage events. It follows that the sample cannot be observed without damaging it, and the frames obtained at the beginning of data collection reflect a crystal that differs from the one that yields the last frames of the dataset. Data acquisition at cryogenic temperatures has been found to reduce the rate of damage progression and is routinely used to increase the dose tolerance of the crystal, allowing more useful data to be obtained before the sample is destroyed. Low-dose data collection can further prolong the lifetime of the crystal, such that less damage is inflicted over the course of data acquisition. Ideally, lower doses increase the measurable volume of a single-crystal lattice by reducing the damage caused by probing electrons. However, the information that can be recovered from a diffraction image is directly related to the number of electrons used to probe the sample. The signal from a weakly exposed crystal runs the risk of being lost in the noise contributed by solvent, crystal disorder, and the electron detection process. This work focuses on obtaining the best possible data from a MicroED measurement, which requires considering several aspects such as sample, dose, and camera type.


Assuntos
Microscopia Crioeletrônica/métodos , Elétrons/efeitos adversos , Microscopia Eletrônica de Transmissão/métodos , Microscopia Crioeletrônica/normas , Radiação Eletromagnética , Microscopia Eletrônica de Transmissão/normas , Nanopartículas/química , Conformação Proteica
13.
Methods Mol Biol ; 2215: 321-327, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33368012

RESUMO

Automated coordination of microscope and camera functions for MicroED data collection simplifies the procedure for robust dataset acquisition and enables unattended sequential collection of many crystal targets. This chapter discusses the prerequisites for an algorithm of data collection automation for continuous-rotation MicroED and presents a practical protocol for achieving this goal using the popular TEM control software program SerialEM.


Assuntos
Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica de Transmissão/métodos , Automação Laboratorial/métodos , Conformação Proteica , Software
14.
Methods Mol Biol ; 2215: 329-348, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33368013

RESUMO

Structural elucidation of small macromolecules such as peptides has recently been facilitated by a growing number of technological advances to existing crystallographic methods. The emergence of electron micro-diffraction (MicroED) of protein nanocrystals under cryogenic conditions has enabled the interrogation of crystalline peptide assemblies only hundreds of nanometers thick. Collection of atomic or near-atomic resolution data by these methods has permitted the ab initio determination of structures of various amyloid-forming peptides, including segments derived from prions and ice-nucleating proteins. This chapter focuses on the process of ab initio structural determination from nano-scale peptide assemblies and other similar molecules.


Assuntos
Amiloide/química , Microscopia Eletrônica de Transmissão/métodos , Peptídeos/química , Microscopia Eletrônica de Transmissão/instrumentação , Nanopartículas/química
15.
Methods Mol Biol ; 2215: 299-307, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33368010

RESUMO

Here, we present a strategy to identify microcrystals from initial protein crystallization screen experiments and to optimize diffraction quality of those crystals using negative stain transmission electron microscopy (TEM) as a guiding technique. The use of negative stain TEM allows visualization along the process and thus enables optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nanocrystals bearing perfect lattices are seeded and can be used for MicroED as well as growing larger crystals for X-ray and free electron laser (FEL) data collection.


Assuntos
Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Nanopartículas/química , Conformação Proteica , Cristalização/métodos , Nanopartículas/ultraestrutura
16.
J Orthop Res ; 39(2): 376-388, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33377538

RESUMO

Recent breakthroughs in our understanding of orthopaedic infections have come from advances in transmission electron microscopy (TEM) imaging of murine models of bone infection, most notably Staphylococcus aureus invasion and colonization of osteocyte-lacuno canalicular networks of live cortical bone during the establishment of chronic osteomyelitis. To further elucidate this microbial pathogenesis and evaluate the mechanism of action of novel interventions, additional advances in TEM imaging are needed. Here we present detailed protocols for fixation, decalcification, and epoxy embedment of bone tissue for standard TEM imaging studies, as well as the application of immunoelectron microscopy to confirm S. aureus occupation within sub-micron canaliculi. We also describe the first application of the novel Automated-Tape-UltraMicrotome system with three-dimensional reconstruction and volumetric analyses to quantify S. aureus occupation within the osteocyte-lacuno canalicular networks. Reconstruction of the three-dimensional volume broadened our perspective of S. aureus colonization of the canalicular network and, surprisingly, revealed adjacent noninfected canaliculi. This observation has led us to hypothesize that viable osteocytes of the osteocyte-lacuno canalicular networks respond and resist infection, opening future research directions to explain the paradox of adjacent uninfected canaliculi and life-long deep bone infection in patients with chronic osteomyelitis.


Assuntos
Osso e Ossos/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Osteomielite/patologia , Infecções Estafilocócicas/patologia , Animais , Osso e Ossos/microbiologia , Camundongos , Osteomielite/microbiologia , Staphylococcus aureus
17.
PLoS One ; 15(9): e0238798, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941441

RESUMO

The mesothelium is a dynamic and specialized tissue layer that covers the somatic cavities (pleural, peritoneal, and pericardial) as well as the surface of the visceral organs such as the lung, heart, liver, bowel and tunica vaginalis testis. The potential therapeutic manipulation of visceral organs has been complicated by the carbohydrate surface layer-here, called the mesopolysaccharide (MPS)-that coats the outer layer of the mesothelium. The traditional understanding of MPS structure has relied upon fixation techniques known to degrade carbohydrates. The recent development of carbohydrate-preserving fixation for high resolution imaging techniques has provided an opportunity to re-examine the structure of both the MPS and the visceral mesothelium. In this report, we used high pressure freezing (HPF) as well as serial section transmission electron microscopy to redefine the structure of the MPS expressed on the murine lung, heart and liver surface. Tissue preserved by HPF and examined by transmission electron microscopy demonstrated a pleural MPS layer 13.01±1.1 um deep-a 100-fold increase in depth compared to previously reported data obtained with conventional fixation techniques. At the base of the MPS were microvilli 1.1±0.35 um long and 42±5 nm in diameter. Morphological evidence suggested that the MPS was anchored to the mesothelium by microvilli. In addition, membrane pits 97±17 nm in diameter were observed in the apical mesothelial membrane. The spatial proximity and surface density (29±4.5%) of the pits suggested an active process linked to the structural maintenance of the MPS. The striking magnitude and complex structure of the MPS indicates that it is an important consideration in studies of the visceral mesothelium.


Assuntos
Epitélio/ultraestrutura , Polissacarídeos/ultraestrutura , Animais , Epitélio/química , Matriz Extracelular/ultraestrutura , Fígado/ultraestrutura , Pulmão/ultraestrutura , Glicoproteínas de Membrana/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Microvilosidades/ultraestrutura , Miocárdio/ultraestrutura
18.
Sci Rep ; 10(1): 12243, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699361

RESUMO

The development of an effective oral therapeutics is an immediate need for the control and elimination of visceral leishmaniasis (VL). We exemplify the preparation and optimization of 2-hydroxypropyl-ß-cyclodextrin (HPCD) modified solid lipid nanoparticles (SLNs) based oral combinational cargo system of Amphotericin B (AmB) and Paromomycin (PM) against murine VL. The emulsion solvent evaporation method was employed to prepare HPCD modified dual drug-loaded solid lipid nanoparticles (m-DDSLNs). The optimized formulations have a mean particle size of 141 ± 3.2 nm, a polydispersity index of 0.248 ± 0.11 and entrapment efficiency for AmB and PM was found to be 96% and 90% respectively. The morphology of m-DDSLNs was confirmed by scanning electron microscopy and transmission electron microscopy. The developed formulations revealed a sustained drug release profile upto 57% (AmB) and 21.5% (PM) within 72 h and were stable at both 4 °C and 25 °C during short term stability studies performed for 2 months. Confocal laser scanning microscopy confirmed complete cellular internalization of SLNs within 24 h of incubation. In vitro cytotoxicity study against J774A.1 macrophage cells confirmed the safety and biocompatibility of the developed formulations. Further, m-DDSLNs did not induce any hepatic/renal toxicities in Swiss albino mice. The in vitro simulated study was performed to check the stability in simulated gastric fluids and simulated intestinal fluids and the release was found almost negligible. The in vitro anti-leishmanial activity of m-DDSLNs (1 µg/ml) has shown a maximum percentage of inhibition (96.22%) on intra-cellular amastigote growth of L. donovani. m-DDSLNs (20 mg/kg × 5 days, p.o.) has significantly (P < 0.01) reduced the liver parasite burden as compared to miltefosine (3 mg/kg × 5 days, p.o.) in L. donovani-infected BALB/c mice. This work suggests that the superiority of as-prepared m-DDSLNs as a promising approach towards the oral delivery of anti-leishmanial drugs.


Assuntos
Anfotericina B/química , Anfotericina B/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Nanopartículas/química , Paromomicina/química , Paromomicina/farmacologia , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular , Emulsões/química , Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão/métodos , Tamanho da Partícula , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacologia
19.
Nat Commun ; 11(1): 2773, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32487987

RESUMO

Cryo-electron microscopy is an essential tool for high-resolution structural studies of biological systems. This method relies on the use of phase contrast imaging at high defocus to improve information transfer at low spatial frequencies at the expense of higher spatial frequencies. Here we demonstrate that electron ptychography can recover the phase of the specimen with continuous information transfer across a wide range of the spatial frequency spectrum, with improved transfer at lower spatial frequencies, and as such is more efficient for phase recovery than conventional phase contrast imaging. We further show that the method can be used to study frozen-hydrated specimens of rotavirus double-layered particles and HIV-1 virus-like particles under low-dose conditions (5.7 e/Å2) and heterogeneous objects in an Adenovirus-infected cell over large fields of view (1.14 × 1.14 µm), thus making it suitable for studies of many biologically important structures.


Assuntos
Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Crioeletrônica/instrumentação , Elétrons , HIV-1 , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Contraste de Fase/métodos , Modelos Teóricos , Vírion/ultraestrutura
20.
J Vis Exp ; (159)2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32478758

RESUMO

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.


Assuntos
Cílios/metabolismo , Imunofluorescência/métodos , Microscopia Eletrônica de Transmissão/métodos , Animais , Bovinos , Humanos
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