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Viruses Dec 2022Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular...
Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular membranes, promoting viral replication and virion assembly. A full understanding of this drastic remodeling and the process of virion morphogenesis remains lacking. In this study, we applied room temperature transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to visualize the SARS-CoV-2 replication factory in Vero cells, and present our results in comparison with published cryo-EM studies. We obtained cryo-EM-like clarity of the ultrastructure by employing high-pressure freezing, freeze substitution (HPF-FS) and embedding, allowing room temperature visualization of double-membrane vesicles (DMVs) in a near-native state. In addition, our data illustrate the consecutive stages of virion morphogenesis and reveal that SARS-CoV-2 ribonucleoprotein assembly and membrane curvature occur simultaneously. Finally, we show the tethering of virions to the plasma membrane in 3D, and that accumulations of virus particles lacking spike protein in large vesicles are most likely not a result of defective virion assembly at their membrane. In conclusion, this study puts forward a room-temperature EM technique providing near-native ultrastructural information about SARS-CoV-2 replication, adding to our understanding of the interaction of this pandemic virus with its host cell.
Topics: Animals; Chlorocebus aethiops; Humans; Vero Cells; SARS-CoV-2; Pandemics; COVID-19; Virion
PubMed: 36560790
DOI: 10.3390/v14122786 -
Materials (Basel, Switzerland) May 2022Lanthanum aluminate-based perovskite (LaAlO) has excellent stability at high temperatures, low toxicity, and high chemical resistance and also offers wide versatility to... (Review)
Review
Lanthanum aluminate-based perovskite (LaAlO) has excellent stability at high temperatures, low toxicity, and high chemical resistance and also offers wide versatility to the substitution of La and Al, thus, allowing it to be applied as a catalyst, nano-adsorbent, sensor, and microwave dielectric resonator, amongst other equally important uses. As such, LaAlO perovskites have gained importance in recent years. This review considers the extensive literature of the past 10 years on the synthesis and catalytic applications of perovskites based on lanthanum and aluminum (LaAlO). The aim is, first, to provide an overview of the structure, properties, and classification of perovskites. Secondly, the most recent advances in synthetic methods, such as solid-state methods, solution-mediated methods (co-precipitation, sol-gel, and Pechini synthesis), thermal treatments (combustion, microwave, and freeze drying), and hydrothermal and solvothermal methods, are also discussed. The most recent energetic catalytic applications (the dry and steam reforming of methane; steam reforming of toluene, glycerol, and ethanol; and oxidative coupling of methane, amongst others) using these functional materials are also addressed. Finally, the synthetic challenges, advantages, and limitations associated with the preparation methods and catalytic applications are discussed.
PubMed: 35591622
DOI: 10.3390/ma15093288 -
The Journal of Biophysical and... Sep 1958Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O degrees C. A 1 to 3 mm. specimen was...
Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O degrees C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175 degrees C. (with precautions). The frozen tissue was placed in substituting fluid at -70 degrees C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances.
Topics: Acetone; Freeze Substitution; Freezing; Histological Techniques; Histology; Microscopy, Electron; Osmium Tetroxide; Preservation, Biological; Solvents; Temperature
PubMed: 13587555
DOI: 10.1083/jcb.4.5.593 -
Journal of Bacteriology Oct 1992The cell envelope architectures and cytoplasmic structures of Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were... (Comparative Study)
Comparative Study
The cell envelope architectures and cytoplasmic structures of Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-14C]acetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (approximately 25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures.
Topics: Cell Membrane; Cell Wall; Cytoplasm; Freeze Substitution; Microscopy, Electron; Mycobacterium; Mycolic Acids; Nontuberculous Mycobacteria; Plastic Embedding
PubMed: 1400203
DOI: 10.1128/jb.174.20.6508-6517.1992 -
Journal of Structural Biology Dec 2023The advent of volume electron microscopy (vEM) has provided unprecedented insights into cellular and subcellular organization, revolutionizing our understanding of...
The advent of volume electron microscopy (vEM) has provided unprecedented insights into cellular and subcellular organization, revolutionizing our understanding of cancer biology. This study presents a previously unexplored comparative analysis of the ultrastructural disparities between cancer cells cultured as monolayers and tumorspheres. By integrating a robust workflow that incorporates high-pressure freezing followed by freeze substitution (HPF/FS), serial block face scanning electron microscopy (SBF-SEM), manual and deep learning-based segmentation, and statistical analysis, we have successfully generated three-dimensional (3D) reconstructions of monolayer and tumorsphere cells, including their subcellular organelles. Our findings reveal a significant degree of variation in cellular morphology in tumorspheres. We observed the increased prevalence of nuclear envelope invaginations in tumorsphere cells compared to monolayers. Furthermore, we detected a diverse range of mitochondrial morphologies exclusively in tumorsphere cells, as well as intricate cellular interconnectivity within the tumorsphere architecture. These remarkable ultrastructural differences emphasize the use of tumorspheres as a superior model for cancer research due to their relevance to in vivo conditions. Our results strongly advocate for the utilization of tumorsphere cells in cancer research studies, enhancing the precision and relevance of experimental outcomes, and ultimately accelerating therapeutic advancements.
Topics: Microscopy, Electron, Scanning; Imaging, Three-Dimensional; Volume Electron Microscopy; Nuclear Envelope
PubMed: 37805154
DOI: 10.1016/j.jsb.2023.108035 -
Histochemistry and Cell Biology Sep 2009The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex... (Review)
Review
The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be 'docked' or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as 'high pressure freezing' with 'freeze-substitution followed by room temperature plastic sectioning' or 'cryo-sectioning of unperturbed vitreous fully hydrated samples' have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported.
Topics: Cryoelectron Microscopy; Nanostructures; Organelles; Saccharomyces cerevisiae; Tomography
PubMed: 19649648
DOI: 10.1007/s00418-009-0622-0 -
Food Science & Nutrition Mar 2022The physicochemical properties of starch acetates with an equal degree of substitution prepared from pea, corn, and wheat starch and their effects on frozen cooked...
The physicochemical properties of starch acetates with an equal degree of substitution prepared from pea, corn, and wheat starch and their effects on frozen cooked noodle (FCN) quality were investigated. The result showed that the three kinds of starch acetates had different effects on the quality of FCN due to their different blue values, freeze-thaw stability, and crystalline morphology analyzed by XRD ( < .05). The FCN with the addition of 20% CAS exhibited slow deterioration of textural properties during holding for 30 min. The analysis of the changes in the content of free SH group and glutenin macropolymer (GMP) demonstrated that the addition of CAS promoted protein disulfide cross-linking and decreased protein mobility during holding. Fourier transform infrared spectroscopy (FT-IR) revealed that FCN with the addition of CAS had low decrement in α-helix and β-sheet during holding, indicating that starch acetates contributed to the maintenance of the gluten network structure.
PubMed: 35282008
DOI: 10.1002/fsn3.2692 -
Molecules (Basel, Switzerland) Mar 2022A new type of hydroxyalkyl starch, γ-hydroxypropyl starch (γ-HPS), was prepared by etherification of alkali-activated starch with 3-chloropropanol. The reaction...
A new type of hydroxyalkyl starch, γ-hydroxypropyl starch (γ-HPS), was prepared by etherification of alkali-activated starch with 3-chloropropanol. The reaction efficiency, morphological change, thermodynamic and apparent viscosity properties, and other physicochemical characteristics were described. The molar substitution (MS) of modified whole starch was determined to be 0.008, 0.017, 0.053, 0.106, and 0.178, with a ratio of 5%, 15%, 25%, 35%, and 45% 3-chloropropanol to starch (/), respectively. Compared to native starch, the granular size and shape and the X-ray diffraction pattern of γ-HPS are not very different. For low-substituted γ-HPS, the implications may be less evident. Thermal stability measurements by means of thermogravimetric analyses and differential scanning calorimetry (TGA-DSC) proved that thermal stability was reduced and water retaining capacity was increased after hydroxypropylation. Furthermore, the findings also showed that the solubility, light transmittance, and retrogradation of γ-HPS pastes could be improved by etherification. The greater the MS of the γ-HPS, the more its freeze-thaw stability and acid resistivity increased. In this study, we provide relevant information for the application of γ-HPS in food and non-food industries.
Topics: Calorimetry, Differential Scanning; Hypromellose Derivatives; Solubility; Starch; Viscosity; X-Ray Diffraction
PubMed: 35408517
DOI: 10.3390/molecules27072119 -
Advances in Experimental Medicine and... 2018The phototransductive membrane disks of a vertebrate photoreceptor outer segment (OS) are highly susceptible to perturbations during preservation for electron...
The phototransductive membrane disks of a vertebrate photoreceptor outer segment (OS) are highly susceptible to perturbations during preservation for electron microscopy. To optimize their preservation for nanostructural studies, such as with electron tomography (ET), we developed a protocol, using a combination of chemical and physical fixation approaches, including transcardiac perfusion, high-pressure freezing, and freeze-substitution.
Topics: Animals; Cryoultramicrotomy; Electron Microscope Tomography; Fixatives; Freeze Substitution; Heart; Imaging, Three-Dimensional; Intracellular Membranes; Mice; Mice, Inbred C57BL; Nanostructures; Perfusion; Photoreceptor Cells, Vertebrate; Pressure; Specimen Handling
PubMed: 29721993
DOI: 10.1007/978-3-319-75402-4_73 -
Foods (Basel, Switzerland) Jun 2022The present study investigated the effect of incorporating 10, 20, and 30% apple powder obtained by freeze-drying, and apple powder produced with osmotic pre-treatment...
The present study investigated the effect of incorporating 10, 20, and 30% apple powder obtained by freeze-drying, and apple powder produced with osmotic pre-treatment in sugar beet molasses solution, into muffins. The powder was freeze-dried and introduced as a whole spelt wheat flour replacement in muffins. The obtained products were investigated for their chemical composition and technological properties, and were subjected to a sensory analysis as well as a consumer acceptance test. Increasing the substitution level from 0 to 30% apple powder lowered the protein, starch, and fat content, while moisture content, sugar, and cellulose showed the opposite trend. The sensory analysis results indicated that the addition of apple powder or apple powder with osmotic pre-treatment (apple OT+Lyo powder) to the ingredients of muffins positively affected the taste, smell, mastication, and appearance of the final product. Consumers rated the muffins with 30% apple OT+Lyo powder as the most acceptable. Principal component analysis, an artificial neural network, and global sensitivity analysis were utilized to differentiate among muffin samples, and to estimate the corresponding influence of the substitution of spelt flour with apple powder or apple OT powder on the observed quality and nutritional parameters of the muffins.
PubMed: 35741946
DOI: 10.3390/foods11121750