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Scientific Reports Jul 2022Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity...
Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity labeling for in-resin correlative light-electron microscopy of Epon-embedded cells. In this study, we established a proximity-labeled in-resin CLEM of Epon-embedded cells using miniTurbo, a biotin ligase. Biotinylation by miniTurbo was observed in cells within 10 min following the addition of biotin to the medium. Using fluorophore-conjugated streptavidin, intracellular biotinylated proteins were labeled after fixation of cells with a mixture of paraformaldehyde and glutaraldehyde. Fluorescence of these proteins was resistant to osmium tetroxide staining and was detected in 100-nm ultrathin sections of Epon-embedded cells. Ultrastructures of organelles were preserved well in the same sections. Fluorescence in sections was about 14-fold brighter than that in the sections of Epon-embedded cells expressing mCherry2 and was detectable for 14 days. When mitochondria-localized miniTurbo was expressed in the cells, mitochondria-like fluorescent signals were detected in the sections, and ultrastructures of mitochondria were observed as fluorescence-positive structures in the same sections by scanning electron microscopy. Proximity labeling using miniTurbo led to more stable and brighter fluorescent signals in the ultrathin sections of Epon-embedded cells, resulting in better performance of in-resin CLEM.
Topics: Biotin; Microscopy, Electron, Scanning; Organelles; Osmium Tetroxide; Resins, Plant; Staining and Labeling
PubMed: 35778550
DOI: 10.1038/s41598-022-15438-6 -
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 -
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 -
Scientific Reports Apr 2022We aimed to develop and validate a deep learning model for automated segmentation and histomorphometry of myelinated peripheral nerve fibers from light microscopic...
We aimed to develop and validate a deep learning model for automated segmentation and histomorphometry of myelinated peripheral nerve fibers from light microscopic images. A convolutional neural network integrated in the AxonDeepSeg framework was trained for automated axon/myelin segmentation using a dataset of light-microscopic cross-sectional images of osmium tetroxide-stained rat nerves including various axonal regeneration stages. In a second dataset, accuracy of automated segmentation was determined against manual axon/myelin labels. Automated morphometry results, including axon diameter, myelin sheath thickness and g-ratio were compared against manual straight-line measurements and morphometrics extracted from manual labels with AxonDeepSeg as a reference standard. The neural network achieved high pixel-wise accuracy for nerve fiber segmentations with a mean (± standard deviation) ground truth overlap of 0.93 (± 0.03) for axons and 0.99 (± 0.01) for myelin sheaths, respectively. Nerve fibers were identified with a sensitivity of 0.99 and a precision of 0.97. For each nerve fiber, the myelin thickness, axon diameter, g-ratio, solidity, eccentricity, orientation, and individual x -and y-coordinates were determined automatically. Compared to manual morphometry, automated histomorphometry showed superior agreement with the reference standard while reducing the analysis time to below 2.5% of the time needed for manual morphometry. This open-source convolutional neural network provides rapid and accurate morphometry of entire peripheral nerve cross-sections. Given its easy applicability, it could contribute to significant time savings in biomedical research while extracting unprecedented amounts of objective morphologic information from large image datasets.
Topics: Animals; Artificial Intelligence; Axons; Microscopy; Myelin Sheath; Nerve Fibers, Myelinated; Rats
PubMed: 35396530
DOI: 10.1038/s41598-022-10066-6 -
Accounts of Chemical Research May 2016The standard method of screening ligands for selectivity in asymmetric, transition metal-catalyzed reactions requires experimental testing of hundreds of ligands from...
The standard method of screening ligands for selectivity in asymmetric, transition metal-catalyzed reactions requires experimental testing of hundreds of ligands from ligand libraries. This "trial and error" process is costly in terms of time as well as resources and, in general, is scientifically and intellectually unsatisfying as it reveals little about the underlying mechanism behind the selectivity. The accurate computational prediction of stereoselectivity in enantioselective catalysis requires adequate conformational sampling of the selectivity-determining transition state but has to be fast enough to compete with experimental screening techniques to be useful for the synthetic chemist. Although electronic structure calculations are accurate and general, they are too slow to allow for sampling or fast screening of ligand libraries. The combined requirements can be fulfilled by using appropriately fitted transition state force fields (TSFFs) that represent the transition state as a minimum and allow fast conformational sampling using Monte Carlo. Quantum-guided molecular mechanics (Q2MM) is an automated force field parametrization method that generates accurate, reaction-specific TSFFs by fitting the functional form of an arbitrary force field using only electronic structure calculations by minimization of an objective function. A key feature that distinguishes the Q2MM method from many other automated parametrization procedures is the use of the Hessian matrix in addition to geometric parameters and relative energies. This alleviates the known problems of overfitting of TSFFs. After validation of the TSFF by comparison to electronic structure results for a test set and available experimental data, the stereoselectivity of a reaction can be calculated by summation over the Boltzman-averaged relative energies of the conformations leading to the different stereoisomers. The Q2MM method has been applied successfully to perform virtual ligand screens on a range of transition metal-catalyzed reactions that are important from both an industrial and an academic perspective. In this Account, we provide an overview of the continued improvement of the prediction of stereochemistry using Q2MM-derived TSFFs using four examples from different stages of development: (i) Pd-catalyzed allylation, (ii) OsO4-catalyzed asymmetric dihydroxylation (AD) of alkenes, (iii) Rh-catalyzed hydrogenation of enamides, and (iv) Ru-catalyzed hydrogenation of ketones. In the current form, correlation coefficients of 0.8-0.9 between calculated and experimental ee values are typical for a wide range of substrate-ligand combinations, and suitable ligands can be predicted for a given substrate with ∼80% accuracy. Although the generation of a TSFF requires an initial effort and will therefore be most useful for widely used reactions that require frequent screening campaigns, the method allows for a rapid virtual screen of large ligand libraries to focus experimental efforts on the most promising substrate-ligand combinations.
Topics: Alkenes; Alkylation; Amides; Catalysis; Hydrogenation; Hydroxylation; Ketones; Models, Chemical; Osmium Tetroxide; Palladium; Quantum Theory; Rhodium; Stereoisomerism
PubMed: 27064579
DOI: 10.1021/acs.accounts.6b00037 -
European Journal of Histochemistry : EJH Feb 2022The spread technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and...
The spread technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and chromatin in an isolated and distended conformation. The final step of staining the spread chromatin is of critical importance because it can strongly influence the interpretation of the results. We evaluated different staining techniques and the most part of them provided a good result. Specifically, well contrasted micrographs were obtained when staining with H3PW12O40 (PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate staining. Quite good contrast of the spread DNA could be achieved also by using Osmium Ammine; while no or few contrast of nucleic acids was observed by staining with KMnO₄ and H3PMo12O40 (PMA) respectively.
Topics: Chromatin; Coloring Agents; Microscopy, Electron; Nucleic Acids; Staining and Labeling
PubMed: 35212500
DOI: 10.4081/ejh.2022.3364 -
Molecules (Basel, Switzerland) Jun 2021The energy and structural parameters were obtained for all forms of the carbonyl complex of osmium Os(CO) with D and D symmetries using density functional theory (DFT)...
The energy and structural parameters were obtained for all forms of the carbonyl complex of osmium Os(CO) with D and D symmetries using density functional theory (DFT) methods. The calculations took into account various levels of relativistic effects, including those associated with nonconservation of spatial parity. It was shown that the ground state of Os(CO) corresponds to the D symmetry and thus may be characterized either as left-twisted (D) or right-twisted (D). The D↔D transitions occur through the D transition state with an activation barrier of ~10 kJ/mol. Parity violation energy difference (PVED) between D and D states equals to ~5 × 10 kJ/mol. An unusual three-center exchange interaction was found inside the {Os} fragment. It was found that the cooperative effects of the mutual influence of osmium atoms suppress the chirality of the electron system in the cluster.
PubMed: 34206080
DOI: 10.3390/molecules26113333 -
Scientific Reports Nov 2017Rhenium and osmium isotopes have been used for decades to date the formation of molybdenite (MoS), a common mineral in ore deposits and the world's main source of...
Rhenium and osmium isotopes have been used for decades to date the formation of molybdenite (MoS), a common mineral in ore deposits and the world's main source of molybdenum and rhenium. Understanding the distribution of parent Re and radiogenic daughter Os isotopes in molybdenite is critical in interpreting isotopic measurements because it can compromise the accurate determination and interpretation of mineralization ages. In order to resolve the controls on the distribution of these elements, chemical and isotope mapping of MoS grains from representative porphyry copper-molybdenum deposits were performed using electron microprobe and nano-scale secondary ion mass spectrometry. Our results show a heterogeneous distribution of Re and Os isotopes in MoS, and that both Re and Os isotopes are not decoupled as previously thought. We conclude that Re and Os are structurally bound or present as nanoparticles in or next to molybdenite grains, recording a complex formation history and hindering the use of microbeam techniques for Re-Os molybdenite dating. Our study opens new avenues to explore the effects of isotope nuggeting in geochronometers.
PubMed: 29167505
DOI: 10.1038/s41598-017-16380-8 -
Microscopy (Oxford, England) Oct 2022The Golgi apparatus, which plays a role in various biosynthetic pathways, is usually identified in electron microscopy by the morphological criteria of lamellae. A...
The Golgi apparatus, which plays a role in various biosynthetic pathways, is usually identified in electron microscopy by the morphological criteria of lamellae. A 3-dimensional analyses with serial block-face scanning electron microscope (SBF-SEM), a volume-SEM proficient in obtaining large volumes of data at the whole-cell level, could be a promising technique for understanding the precise distribution and complex ultrastructure of Golgi apparatus, although optimal methods for such analyses remain unclear since the observation can be hampered with sample charging and low image contrast, and manual segmentation often requires significant manpower. The present study attempted the whole-cell observation and semi-automatic classification and segmentation of the Golgi apparatus in rat hepatocytes for the first time by SBF-SEM via ZIO staining, a classical osmium impregnation. The staining electron-densely visualized individual Golgi lamellae, and their ultrastructure could stably be observed without any noticeable charging. The simple thresholding of the serial images enabled the efficient reconstruction of the labeled Golgi apparatus, which revealed plural Golgi apparatus in one hepatocyte. The combination of the heavy metal-based histochemistry of zinc, iodine and osmium (ZIO) staining and SBF-SEM was useful in the 3-dimensional observation of the Golgi apparatus at the whole-cell level because of two technical advantages: (i) visualization of the Golgi apparatus without any heavy metal staining and efficient acquisition of the block-face images without additional conductive staining or any devices for eliminating charging; (ii) easy identification of the staining and hassle-free, semi-automatic classification and segmentation by simple thresholding of the images. This novel approach could elucidate the topographic characteristics of the Golgi apparatus in hepatocytes.
Topics: Animals; Golgi Apparatus; Hepatocytes; Iodine; Microscopy, Electron, Scanning; Osmium; Rats; Zinc
PubMed: 35535544
DOI: 10.1093/jmicro/dfac024 -
Metallomics : Integrated Biometal... Feb 2021The organo-osmium complex [OsII(ɳ6-p-cym)(PhAzPy-NMe2)I]+ (FY26) exhibits promising in vitro antitumour activity against mouse hepatocarcinoma Hepa1-6 and other mouse...
The organo-osmium complex [OsII(ɳ6-p-cym)(PhAzPy-NMe2)I]+ (FY26) exhibits promising in vitro antitumour activity against mouse hepatocarcinoma Hepa1-6 and other mouse or human cancer cell lines. Here, we drastically enhance water solubility of FY26 through the replacement of the PF6- counter-anion with chloride using a novel synthesis method. FY26⋅PF6 and FY26⋅Cl displayed similar in vitro cytotoxicity in two cancer cell models. We then show the moderate and late anticancer efficacy of FY26⋅PF6 and FY26⋅Cl in a subcutaneous murine hepatocarcinoma mouse model. Both efficacy and tolerability varied according to FY26 circadian dosing time in hepatocarcinoma tumour-bearing mice. Tumour and liver uptake of the drug were determined over 48 h following FY26⋅Cl administration at Zeitgeber time 6 (ZT6), when the drug is least toxic (in the middle of the light span when mice are resting). Our studies suggest the need to administer protracted low doses of FY26 at ZT6 in order to optimize its delivery schedule, for example through the use of chrono-releasing nanoparticles.
Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Coordination Complexes; Dose-Response Relationship, Drug; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Tissue Distribution
PubMed: 33595653
DOI: 10.1093/mtomcs/mfaa003