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Acta Parasitologica Jun 2018Balantioides coli is a ciliated protozoon that inhabits the intestine of pigs, non-human primates and humans. Light microscopy studies have described over 50 species of...
Balantioides coli is a ciliated protozoon that inhabits the intestine of pigs, non-human primates and humans. Light microscopy studies have described over 50 species of the genus Balantioides but their validity is in doubt. Due to the limited information about this genus, this study is aimed to identify morphological characteristics of Balantioides coli isolated using fluorescence microscopy and both scanning (SEM) and transmission electron microscopy (TEM). Trophozoites isolated from the feces of pig and macaque were washed and subjected to centrifugation. These cells were fixed with paraformaldehyde for immunofluorescence. Other aliquots of these trophozoites were fixed with glutaraldehyde, post fixed with osmium tetroxide and processed for SEM and TEM. Immunofluorescence studies revealed microtubules with a longitudinal distribution to the main axis of the parasite and in the constitution of cilia. SEM demonstrated a high concentration of cilia covering the oral apparatus and a poor presence of such structures in cytopyge. TEM revealed in the plasma membrane, several associated structures were observed to delineate the cellular cortex and mucocysts. The cytoskeleton of the oral region was observed in detail and had an organization pattern consisting of microtubules, which formed files and nematodesmal networks. Organelles such as hydrogenosomes like and peroxisomes were observed close to the cortex. Macronuclei were observed, but structures that were consistent with micronuclei were not identified. Ultrastructural morphological analysis of isolates confirms its similarity to Balantioides coli. In this study were identified structures that had not yet been described, such as hydrogenosomes like and cytoskeletal structures.
Topics: Animals; Cell Membrane; Feces; Humans; Intestines; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Microtubules; Organelles; Parasites; Peroxisomes; Primates; Protozoan Infections, Animal; Swine; Trophozoites
PubMed: 29654686
DOI: 10.1515/ap-2018-0033 -
Journal of Biomolecular Structure &... Feb 2023The concentrations of specific macromolecular species can be quantified using diagnostic tools that rely on molecular recognition by nucleic acid aptamers. One such...
The concentrations of specific macromolecular species can be quantified using diagnostic tools that rely on molecular recognition by nucleic acid aptamers. One such approach involves the formation of osmium tetroxide 2,2'-bipyridine protein adducts, followed by electrochemical detection of analytes that bind specifically to electrode-tethered aptamers. In conjunction with a 27-mer DNA aptamer that binds specifically to exosite II on human alpha thrombin, this technique permits, in theory, a highly sensitive diagnostic tool for the quantification of serum thrombin levels. However, thrombin's aptamer binding site is lined by two tryptophan residues and the conjugation of bulky osmium groups to these residues weakens aptamer binding by an estimated 4 to 12 kcal/mol, undermining detection sensitivity. Therefore, we have rationally modified this DNA aptamer to strengthen its thrombin binding in the presence of conjugated osmium. Specifically, aptamers carrying long hydrophobic thymine derivatives in place of guanine 21 have binding affinities for osmium-conjugated thrombin that are enhanced by 10 to 15 kcal/mol, suggesting that these modified aptamers may be effective in a highly sensitive electrochemical sensor for the quantification of low concentrations of thrombin. Our approach of using molecular simulation to subtly re-engineer a DNA aptamer may be generally applicable for the optimization of other macromolecular binding interfaces.Communicated by Ramaswamy H. Sarma.
Topics: Humans; Aptamers, Nucleotide; Thrombin; Osmium; Computer Simulation; Binding Sites
PubMed: 34895068
DOI: 10.1080/07391102.2021.2011414 -
Anatomical Record (Hoboken, N.J. : 2007) Aug 2023The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve...
The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve dissection. Little data have been reported on the normality of these nerves. The vestibular nerve is composed of the superior vestibular, inferior vestibular, and branch nerves. This study aimed to study the microscopic anatomy of the superior vestibular nerve (SVN) of guinea pigs using light microscopy and to search for normality patterns for use in experimental models in basic otologic research. We used eight male albino guinea pigs (Cavia porcellus, English strain), weighing between 400 and 500 g. After anesthetizing, the animals were perfused with a fixative solution of 2.5% glutaraldehyde. Dissection was performed by the access method to the temporal bone, coming to the rock and exposing the cochlea and vestibular nerve. The NVS fragments were removed, postfixed in osmium tetroxide, and embedded in the epoxy plastic resin Poly/Bed 812® (Polysciences Inc., Warrington, PA). Semi-thin transverse serial sections (0.5 μm) were made using a microtome MT6000-XL, RMC, Inc. and stained with toluidine blue. Morphology and morphometry were described and evaluated using the KS 400 application (Kontron 2.0, EchingBei, Munich, Germany) by macro, a computer program specially designed and developed for the study of the VIII nerve. The SVN was found to be devoid of epineurium, with only a thin conjunctive tissue layer. The myelin sheath of guinea pigs is relatively thin compared to the sensory and motor nerves found in mammals. The average fascicular area SVN was 0.19 ± 0.05 mm , with the largest area found to be 0.24 mm and the lowest was 0.12 mm . The average number of fibers was 5,753.00 ± 538 fibers. The density of myelinated fibers reached 32,316.08 ± 11,375.29 fibers/mm . Its diameter ranged from 1.0 to 9 μm and its peak was 3 μm. The measured results confirm the results of another study, indicating that the methodology is appropriate and reproducible. These findings are important for the evaluation of injured nerves in experimental models of peripheral neuropathy and basic ear disease.
Topics: Animals; Guinea Pigs; Vestibular Nerve; Male; Myelin Sheath; Cochlea
PubMed: 37461264
DOI: 10.1002/ar.25053 -
Basic & Clinical Pharmacology &... Apr 2021
Topics: Osmium; Osmium Tetroxide; Skin
PubMed: 33459507
DOI: 10.1111/bcpt.13562 -
Wiadomosci Lekarskie (Warsaw, Poland :... 2022The aim: The objective of our study was to evaluate the features of ultramorphometric characteristics of exocrine parenchyma and microvasculature of the pancreas in the...
OBJECTIVE
The aim: The objective of our study was to evaluate the features of ultramorphometric characteristics of exocrine parenchyma and microvasculature of the pancreas in the presence of moderate dehydration by means of an experiment in laboratory rats.
PATIENTS AND METHODS
Materials and methods: The experiment involved 20 mature white male rats divided into 2 groups: control and experimental (10 rats each). In the experimental group, moderate dehydration was simulated, i.e. the animals were deprived of water for 7 days, while the control rats were provided with a normal water supply during the study. Pancreatic parenchyma samples were fixed in phosphate-buffered glutaraldehyde solution and post-fixed in osmium tetroxide solution, dehydrated and embedded in a mixture of epoxy resins. Ultrastructural analysis was performed using JEOL JEM-1230 transmission electron microscope (Japan).
RESULTS
Results: Pancreatic electron microscopy in the presence of moderate dehydration demonstrated statistically significant changes in exocrinocytes area and exocrinocyte nucleus area which increased by 8.02% (p = 0.028) and 40.28% (p < 0.001), respectively. Among the vessels of microcirculation, the largest changes occurred in the capillaries: their lumen narrowed by 22.34% (p = 0.002) as compared with the control group. The cytoplasm of endothelial cells contained a large number of vacuoles and micropinocytotic vesicles.
CONCLUSION
Conclusions: Among the organelles of exocrinocytes, mitochondria appeared the most vulnerable to the effects of dehydration. They demonstrated polymorphic changes: a part of the mitochondria was hypotrophic and had partially reduced cristae, and another part was hypertrophic.
Topics: Animals; Dehydration; Endothelial Cells; Humans; Male; Microscopy, Electron; Microvessels; Pancreas; Rats
PubMed: 35633332
DOI: 10.36740/WLek202204214 -
Oral Diseases Jan 2019A preliminary study to determine collagen fibril diameter (CF-ED) distribution on medial and lateral sides of cleft lip (CL).
OBJECTIVE
A preliminary study to determine collagen fibril diameter (CF-ED) distribution on medial and lateral sides of cleft lip (CL).
MATERIAL AND METHODS
Tissue samples from medial and lateral sides of CL were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and embedded in Araldite CY212 resin for transmission electron microscopy. The analysis of CF-ED was performed using the ImageJ program. To characterize the packaging of collagen fibrils (CFs) in the two tissues, we estimated the collagen number density (CF-ND) and fibril-area-fraction (FAF). Differences in measurements across the two sides were calculated using Wilcoxon signed-rank test.
RESULTS
The CF-ED was statistically significantly (p < 0.001) smaller on the medial side (45.69 ± 7.89 nm) than on the lateral side (54.18 ± 7.62 nm). The medial side had a higher CF-ND and a higher percentage of FAF than the lateral side.
CONCLUSION
Our finding of a smaller CF-ED and higher CF-ND and FAF for the medial side suggests possible differences in size and distribution of CFs between medial and lateral sides of CL. This finding provides knowledge toward underlying tissue biomechanics that may help reconstruction of perioral tissue scaffolds, ultimately resulting in better treatment of patients with oral clefts.
Topics: Cleft Lip; Collagen; Extracellular Matrix; Humans; Microscopy, Electron, Transmission
PubMed: 30144227
DOI: 10.1111/odi.12962 -
MethodsX 2022Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the...
Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the accumulation of lipids as a response. Considering that the distribution of lipids in an organ is directly related to the induction of inflammatory processes by aquatic contaminants, this study proposes to carry out an integrative investigation of the behavior and the distribution of lipids in the liver tissue. Techniques of light and electron microscopy were performed in order to propose a new way of assessing and quantifying the distribution of lipid droplets, also presenting methodological alternatives that can be chosen by the reader according to the interests and resources available. Thus, it is assumed that the method begins with the fixation of the liver with Glutaraldehyde 2,5% in PBS 0,1 M and continues with post fixation with osmium tretoxide 1%, which marks lipids. For this proposition, two inclusion methodologies were performed to histological analyses in Historesin and ultrastructural analyses in EMBeed 812. For light microscopy (LM) analyses, cuts were obtained with 2,5 micrometers thickness, which were stained with (1) Mayers hematoxylin and (2) toluidine blue. The images obtained were processed in software Image J Fiji to evidence the lipid distribution in liver.•Cytological reactions with osmium tetroxide constitute low complexity methods that allow the optimization of the localization, identification and quantification of lipid droplets in the liver tissue when analyzed under the conventional light microscope.•Samples included in EMBeed 812 resin commonly used in Transmission Electron Microscopy can be analyzed by SEM-BEC, as complementary analyses for the detection of lipids.•Using SEM-BEC and conventional light microscopy, it is possible to quantify the area occupied by lipid droplets using Image J Fiji software, as these are contrasted due to the reaction with osmium tetroxide.
PubMed: 35818446
DOI: 10.1016/j.mex.2022.101769 -
Developmental Dynamics : An Official... Nov 2017Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may...
BACKGROUND
Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may impair rapid and efficient transmission of action potentials and alter nerve function. Identifying ultrastructural changes resulting from defects to these cellular components via transmission electron microscopy (TEM) can provide valuable insight into nerve function and disease. However, efficiently locating spinal motor nerves in adult zebrafish for TEM is challenging and time-consuming. Because of this, we developed a protocol that allows us to quickly and precisely locate spinal motor nerve roots in adult zebrafish for TEM processing.
RESULTS
Following fixation, a transverse slab of adult zebrafish dissected from the trunk region was mounted in embedding media, sectioned, and secondary fixation with osmium tetroxide performed. Transverse sections containing motor nerves were selected for TEM ultrathin sectioning and imaging.
CONCLUSIONS
We developed an efficient protocol for locating spinal motor nerves in adult zebrafish to allow for ultrastructural characterization. Although our work focuses on spinal motor nerves, this protocol may be useful for efficiently identifying other discrete, repeated structures within the developing and mature nervous system that are difficult to find via traditional, whole organism TEM processing. Developmental Dynamics 246:956-962, 2017. © 2017 Wiley Periodicals, Inc.
Topics: Animals; Histological Techniques; Microscopy, Electron, Transmission; Spinal Nerve Roots; Zebrafish
PubMed: 28598521
DOI: 10.1002/dvdy.24529 -
Nature Methods Jan 2020Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the...
Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO) treatment for improved SR-CLEM.
Topics: Animals; CHO Cells; Cricetulus; Epoxy Resins; Fluorescence; Fluorescent Antibody Technique; Humans; Luminescent Proteins; Microscopy, Electron; Microscopy, Fluorescence; Molecular Imaging; Organelles; Osmium Tetroxide; Specimen Handling
PubMed: 31611693
DOI: 10.1038/s41592-019-0613-6 -
Langmuir : the ACS Journal of Surfaces... Mar 2022We demonstrate a facile method to fabricate a recyclable cell-alignment scaffold using nanogrooves based on sublimable liquid crystal (LC) material. Randomly and...
We demonstrate a facile method to fabricate a recyclable cell-alignment scaffold using nanogrooves based on sublimable liquid crystal (LC) material. Randomly and uniaxially arranged smectic LC structures are obtained, followed by sublimation and recondensation processes, which directly produce periodic nanogrooves with dimensions of a couple of hundreds of nanometers. After treatment with osmium tetroxide (OsO), the nanogroove can serve as a scaffold to efficiently induce directed cell growth without causing cytotoxicity, and it can be used repeatedly. Together, various cell types are applied to the nanogroove, proving the scaffold's broad applicability. Depending on the nanotopography of the LC structures, cells exhibit different morphologies and gene expression patterns, compared to cells on standard glass substrates, according to microscopic observation and qPCR. Furthermore, cell sheets can be formed, which consist of oriented cells that can be repeatedly formed and transferred to other substrates, while maintaining its organization. We believe that our cell-aligning scaffold may pave the way for the soft material field to bioengineering, which can involve fundamentals in cell behavior and function, as well as applications for regenerative medicine.
Topics: Liquid Crystals; Nanostructures; Osmium Tetroxide
PubMed: 35302783
DOI: 10.1021/acs.langmuir.1c03359