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Cytometry. Part a : the Journal of the... Jan 2017Mass cytometry offers the advantage of allowing the simultaneous measurement of a greater number parameters than conventional flow cytometry. However, to date, mass...
Mass cytometry offers the advantage of allowing the simultaneous measurement of a greater number parameters than conventional flow cytometry. However, to date, mass cytometry has lacked a reliable alternative to the light scatter properties that are commonly used as a cell size metric in flow cytometry (forward scatter intensity-FSC). Here, we report the development of two plasma membrane staining assays to evaluate mammalian cell size in mass cytometry experiments. One is based on wheat germ agglutinin (WGA) staining and the other on Osmium tetroxide (OsO ) staining, both of which have preferential affinity for cell membranes. We first perform imaging and flow cytometry experiments to establish a relationship between WGA staining intensity and traditional measures of cell size. We then incorporate WGA staining in mass cytometry analysis of human whole blood and show that WGA staining intensity has reproducible patterns within and across immune cell subsets that have distinct cell sizes. Lastly, we stain PBMCs or dissociated lung tissue with both WGA and OsO ; mass cytometry analysis demonstrates that the two staining intensities correlate well with one another. We conclude that both WGA and OsO may be used to acquire cell size-related parameters in mass cytometry experiments, and expect these stains to be broadly useful in expanding the range of parameters that can be measured in mass cytometry experiments. © 2016 International Society for Advancement of Cytometry.
Topics: Animals; Cell Membrane; Cell Size; Flow Cytometry; Humans; Osmium Tetroxide; Wheat Germ Agglutinins
PubMed: 27768827
DOI: 10.1002/cyto.a.23000 -
Fungal Genetics and Biology : FG & B Mar 2023Extracellular vesicles (EVs) are nanosized structures containing proteins, lipids, and nucleic acids, released by living cells to the surrounding medium. EVs participate...
Extracellular vesicles (EVs) are nanosized structures containing proteins, lipids, and nucleic acids, released by living cells to the surrounding medium. EVs participate in diverse processes, such as intercellular communication, virulence, and disease. In pathogenic fungi, EVs carry enzymes that allow them to invade the host or undergo environmental adaptation successfully. In Neurospora crassa, a non-pathogenic filamentous fungus widely used as a model organism, the vesicle-dependent secretory mechanisms that lead to polarized growth are well studied. In contrast, biosynthesis of EVs in this fungus has been practically unexplored. In the present work, we analyzed N. crassa culture's supernatant for the presence of EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM) and proteomic analysis. We identified spherical membranous structures, with a predominant subpopulation averaging a hydrodynamic diameter (d) of 68 nm and a particle diameter (d) of 38 nm. EV samples stained with osmium tetroxide vapors were better resolved than those stained with uranyl acetate. Mass spectrometry analysis identified 252 proteins, including enzymes involved in carbohydrate metabolic processes, oxidative stress response, cell wall organization/remodeling, and circadian clock-regulated proteins. Some of these proteins have been previously reported in exosomes from human cells or in EVs of other fungi. In view of the results, it is suggested a putative role for EVs in cell wall biosynthesis and vegetative development in N. crassa.
Topics: Humans; Hyphae; Neurospora crassa; Proteomics; Extracellular Vesicles; Microscopy, Electron, Transmission
PubMed: 36690295
DOI: 10.1016/j.fgb.2023.103778 -
Journal of Anatomy Jan 2022Brachial plexus injury (BPI) occurs when the brachial plexus is compressed, stretched, or avulsed. Although rodents are commonly used to study BPI, these models poorly...
Brachial plexus injury (BPI) occurs when the brachial plexus is compressed, stretched, or avulsed. Although rodents are commonly used to study BPI, these models poorly mimic human BPI due to the discrepancy in size. The objective of this study was to compare the brachial plexus between human and Wisconsin Miniature Swine (WMS ), which are approximately the weight of an average human (68-91 kg), to determine if swine would be a suitable model for studying BPI mechanisms and treatments. To analyze the gross anatomy, WMS brachial plexuses were dissected both anteriorly and posteriorly. For histological analysis, sections from various nerves of human and WMS brachial plexuses were fixed in 2.5% glutaraldehyde, and postfixed with 2% osmium tetroxide. Subsequently paraffin sections were counter-stained with Masson's Trichrome. Gross anatomy revealed that the separation into three trunks and three cords is significantly less developed in the swine than in human. In swine, it takes the form of upper, middle, and lower systems with ventral and dorsal components. Histological evaluation of selected nerves revealed differences in nerve trunk diameters and the number of myelinated axons in the two species. The WMS had significantly fewer myelinated axons than humans in median (p = 0.0049), ulnar (p = 0.0002), and musculocutaneous nerves (p = 0.0454). The higher number of myelinated axons in these nerves for humans is expected because there is a high demand of fine motor and sensory functions in the human hand. Due to the stronger shoulder girdle muscles in WMS, the WMS suprascapular and axillary nerves were larger than in human. Overall, the WMS brachial plexus is similar in size and origin to human making them a very good model to study BPI. Future studies analyzing the effects of BPI in WMS should be conducted.
Topics: Animals; Brachial Plexus; Hand; Humans; Shoulder; Swine; Swine, Miniature; Upper Extremity
PubMed: 34355792
DOI: 10.1111/joa.13525 -
Membranes Oct 2022Liquid membranes based on nanoparticles follow a continuous development, both from obtaining methods and characterization of techniques points of view. Lately, osmium...
Liquid membranes based on nanoparticles follow a continuous development, both from obtaining methods and characterization of techniques points of view. Lately, osmium nanoparticles have been deposited either on flat membranes, with the aim of initiating some reaction processes, or on hollow fiber membranes, with the aim of increasing the contact surface with the phases of the membrane system. This paper presents the obtainment and characterization of a liquid membrane based on osmium nanoparticles (Os-NP) dispersed in decanol (Dol) for the realization of a membrane system with a large contact surface between the phases, but without using a liquid membrane support. The dispersion of osmium nanoparticles in -decanol is carried out by the method of reducing osmium tetroxide with 1-undecenoic acid (UDA). The resulting membrane was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive spectroscopy analysis (EDAX), thermoanalysis (TG, DSC), Fourier transform infra-red (FTIR) spectroscopy and dynamic light scattering (DLS). In order to increase the mass transfer surface, a design for the membrane system was realized with the dispersion of the membrane through the receiving phase and the dispersion of the source phase through the membrane (DBLM-dispersion bulk liquid membrane). The process performance was tested for the reduction of -nitrophenol (pNP) from the source phase, using sodium tetra-borohydride (NaBH), to -aminophenol (pAP), which was transported and collected in the receiving phase. The obtained results show that membranes based on the dispersion of osmium nanoparticles in -decanol can be used with an efficiency of over 90% for the reduction of -nitrophenol and the separation of -aminophenol.
PubMed: 36295782
DOI: 10.3390/membranes12101024 -
Microscopy Research and Technique Jun 2020For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice,...
For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.
Topics: Antibodies; Desulfurococcaceae; Epoxy Compounds; Histological Techniques; Immunohistochemistry; Microalgae; Microscopy, Immunoelectron; Microtomy; Osmium Tetroxide
PubMed: 32057162
DOI: 10.1002/jemt.23459 -
Molecules (Basel, Switzerland) Mar 2023Sharpless asymmetric dihydroxylation is an important reaction in the enantioselective synthesis of chiral vicinal diols that involves the treatment of alkene with osmium... (Review)
Review
Sharpless asymmetric dihydroxylation is an important reaction in the enantioselective synthesis of chiral vicinal diols that involves the treatment of alkene with osmium tetroxide along with optically active quinine ligand. Sharpless introduced this methodology after considering the importance of enantioselectivity in the total synthesis of medicinally important compounds. Vicinal diols, produced as a result of this reaction, act as intermediates in the synthesis of different naturally occurring compounds. Hence, Sharpless asymmetric dihydroxylation plays an important role in synthetic organic chemistry due to its undeniable contribution to the synthesis of biologically active organic compounds. This review emphasizes the significance of Sharpless asymmetric dihydroxylation in the total synthesis of various natural products, published since 2020.
Topics: Hydroxylation; Biological Products; Alkenes; Stereoisomerism
PubMed: 36985698
DOI: 10.3390/molecules28062722 -
Microscopy (Oxford, England) Oct 2023Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as 'in-resin CLEM', have...
Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as 'in-resin CLEM', have been developed to improve the positional accuracy and Z-axis resolution limitations of conventional correlative light and electron microscopy (CLEM). High-pressure freezing and quick-freezing substitution result in in-resin CLEM of acrylic-based resin-embedded cells expressing green fluorescent protein, yellow fluorescent protein, mVenus and mCherry, which are sensitive to osmium tetroxide. The identification of osmium-resistant fluorescent proteins leads to the development of in-resin CLEM of Epon-embedded cells. Using subtraction-based fluorescence microscopy with a photoconvertible fluorescent protein, mEosEM-E, its green fluorescence can be observed in thin sections of Epon-embedded cells, and two-color in-resin CLEM using mEosEM-E and mScarlet-H can be performed. Green fluorescent proteins, CoGFP variant 0 and mWasabi, and far-red fluorescent proteins, mCherry2 and mKate2, are available for in-resin CLEM of Epon-embedded cells using the standard procedure for Epon-embedding with additional incubation. Proximity labeling is applied to in-resin CLEM to overcome the limitations of fluorescent proteins in epoxy resin. These approaches will contribute significantly to the future of CLEM analysis.
Topics: Humans; Epoxy Resins; Microscopy, Electron; Microscopy, Fluorescence; Green Fluorescent Proteins; HeLa Cells
PubMed: 37217182
DOI: 10.1093/jmicro/dfad028 -
Endocrine Jan 2024Accumulation of bone marrow adipose tissue (BMAT) is always seen in osteoporosis induced by estrogen deficiency. Herein, we aimed to investigate the mechanisms and...
BACKGROUND
Accumulation of bone marrow adipose tissue (BMAT) is always seen in osteoporosis induced by estrogen deficiency. Herein, we aimed to investigate the mechanisms and consequences of this phenomenon by establishing a mouse model of osteoporosis caused by ovariectomy (OVX)-mimicked estrogen deficiency.
METHODS
Micro-CT, osmium tetroxide staining, and histological analyses were performed to examine the changes in bone microstructure, BMAT and white adipose tissue (WAT) in OVX mice compared to sham mice. The osteogenesis and adipogenesis of primary bone marrow stromal cells (BMSCs) isolated from sham and OVX mice were compared in vitro. The molecular phenotypes of BMAT and WAT were determined and compared by quantitative PCR (qPCR). Bone marrow adipocyte-conditioned medium (BMA CM) was prepared from sham or OVX mice for coculture assays, and BMSCs or bone marrow monocytes/macrophages (BMMs) were isolated and subjected to osteoblast and osteoclast differentiation, respectively. Cell staining and qPCR were used to assess the effects of BMAT on bone metabolism.
RESULTS
OVX-induced estrogen deficiency induced reductions in both cortical and trabecular bone mass along with an expansion of BMAT volume. At the cellular level, loss of estrogen inhibited BMSC osteogenesis and promoted BMSC adipogenesis, whereas addition of estradiol exerted the opposite effects. In response to estrogen deficiency, despite the common proinflammatory molecular phenotype observed in both fat depots, BMAT, unlike WAT, unexpectedly exhibited an increase in adipocyte differentiation and lipolytic activity as well as the maintenance of insulin sensitivity. Importantly, BMAT, but not WAT, presented increased mRNA levels of both BMP receptor inhibitors (Grem1, Chrdl1) and Rankl following OVX. In addition, treatment with BMA CM, especially from OVX mice, suppressed the osteoblast differentiation of BMSCs while favoring the osteoclast differentiation of BMMs.
CONCLUSION
Our study illustrates that OVX-induced estrogen deficiency results in bone loss and BMAT expansion by triggering imbalance between the osteogenesis and adipogenesis of BMSCs. Furthermore, expanded BMAT, unlike typical WAT, may negatively regulate bone homeostasis through paracrine inhibition of osteoblast-mediated bone formation and promotion of osteoclast-mediated bone resorption.
Topics: Female; Mice; Animals; Humans; Bone Marrow; Adipose Tissue; Osteoporosis; Osteogenesis; Cell Differentiation; Estrogens; Ovariectomy; Eye Proteins; Nerve Tissue Proteins
PubMed: 37682419
DOI: 10.1007/s12020-023-03504-6 -
Dalton Transactions (Cambridge, England... Jan 2022The reaction of osmium tetroxide (OsO) and carboxylate anions (acetate: X = AcO and benzoate: X = BzO) gave 1 : 1 adducts, [OsO(X)] (1X), the structures of which...
The reaction of osmium tetroxide (OsO) and carboxylate anions (acetate: X = AcO and benzoate: X = BzO) gave 1 : 1 adducts, [OsO(X)] (1X), the structures of which were determined by X-ray crystallographic analysis. In both cases, the carboxylate anion X coordinates to the osmium centre to generate a distorted trigonal bipyramidal osmium(VIII) complex. The carboxylate adducts show a negative shift of the redox potentials () and a red shift of the stretches as compared to those of tetrahedral OsO itself. Despite the negative shift of , the reactivity of these adduct complexes 1X was enhanced compared to that of OsO in benzylic C(sp)-H bond oxidation. The reaction obeyed the first-order kinetics on both 1X and the substrates, giving the second-order rate constant (), which exhibits a linear correlation with the C-H bond dissociation energy (BDE) of the substrates (xanthene, 9,10-dihydroanthracene, fluorene and 1,2,3,4-tetrahydronaphthalene) and a kinetic deuterium isotope effect (KIE) of 9.7 ((xanthene-)/(xanthene-)). On the basis of these kinetic data together with the DFT calculation results, we propose a stepwise reaction mechanism involving rate-limiting benzylic hydrogen atom abstraction and subsequent rebound of the generated organic radical intermediate to a remaining oxido group on the osmium centre.
PubMed: 34951431
DOI: 10.1039/d1dt03819b -
Frontiers in Endocrinology 2020The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis,... (Review)
Review
Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.
The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) BMA imaging, (3) BMA imaging, (4) cell isolation, culture, differentiation and modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced μCT for quantification, specific MRI sequences (WFI and H-MRS) for studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., , Kit) and development of specific BMA deletion models would be highly desirable for this purpose.
Topics: Adipogenesis; Adiposity; Animals; Bone Marrow; Guidelines as Topic; Humans; International Agencies; Obesity; Research Design; Research Report; Societies, Scientific
PubMed: 32180758
DOI: 10.3389/fendo.2020.00065