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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 -
Archives Italiennes de Biologie Dec 2022Spinal cord injury (SCI) causes various neurological consequences that disrupt the structure of axons. The C/EBP Homologous Protein (CHOP) acts in neuronal death by...
PURPOSE
Spinal cord injury (SCI) causes various neurological consequences that disrupt the structure of axons. The C/EBP Homologous Protein (CHOP) acts in neuronal death by apoptosis has been demonstrated in experimental models. Rosmarinic acid (RA) is a phenolic compound used for therapeutic purposes in many diseases. In this study, we investigated the therapeutic effect of Rosmarinic acid application on inflammation and apoptotic development after spinal cord injury.
METHODS
Male Wistar albino rats (n: 24) were assigned to three group: control, SCI and SCI+ RA. All rats were fixed on the operating table after anesthesia, the skin of the thoracic region was opened with a midline incision and the paravertebral muscles were dissected and T10-T11 laminas were exposed. A cylindrical tube of 10 cm length was fixed to the area to be laminectomy. A metal weight of 15 grams was left down the tube. Spinal damage was created, skin incisions were sutured. 50 mg/kg rosmarinic acid was given orally for 7 days after the spinal injury. Spinal tissues were fixed in formaldehyde solution and processed for paraffin wax tissue protocol and 4-5 μm sections were taken with microtome for further immunohistochemical examination. Caspase-12 and CHOP antibodies were applied to sections. Remaining tissues were carried out in glutaraldehyde for the first fixation then in osmium tetroxide for the second. Tissues were kept in pure araldite and thin sections were taken for transmission electron microscope.
RESULTS
Values of malondialdehyde (MDA), myeloperoxidase (MPO), glutathione peroxidase (GSH), neuronal degeneration, vascular dilation, inflammation, CHOP and Caspase-12 expression were increased in SCI group compared to control group. Only glutathione peroxidase content was decreased in SCI group. In SCI group, disruption of basement membrane structure in canalis ependymalis, degeneration in structures of unipolar bipolar and multipolar neurons, and apoptotic changes were seen with increased inflammation in the piamater region and positive CHOP expression in vascular endothelial cells. In SCI+RA group, reorganization of basement membrane pill in canalis ependymalis were observed with mild Caspase-12 activity in some canalis ependymal and glial cells. Also, moderate CHOP expression in multipolar and bipolar neurons and glia cells were observed.
CONCLUSIONS
The application of RA has a significant effect on preventing damage in SCI. It was thought that CHOP and Caspase-12 mediated oxidative stress could be a guide in showing the potential and therapeutic target to stop the apoptotic course after SCI injury.
Topics: Male; Rats; Animals; Rats, Wistar; Caspase 12; Endothelial Cells; Spinal Cord Injuries; Rosmarinic Acid
PubMed: 36881913
DOI: 10.12871/000398292022341 -
Methods in Molecular Biology (Clifton,... 2023Histochemical and fluorescence-based techniques enable the specific identification of myelin by bright-field or fluorescence microscopy. In this chapter, we describe...
Histochemical and fluorescence-based techniques enable the specific identification of myelin by bright-field or fluorescence microscopy. In this chapter, we describe four histological methods for the evaluation of myelin on peripheral nerve tissue sections. The first method combines the Luxol fast blue (LFB) technique with a modified Picrosirius staining contrasted with Harris hematoxylin, called MCOLL. This method simultaneously stains myelin, collagen fibers, and cell nuclei, thus giving an integrated overview of the histology, collagen network, and myelin content of the tissue in paraffin-embedded or cryosectioned samples. Secondly, we describe the osmium tetroxide method, which provides a permanent positive reaction for myelin as well as other lipids present in the tissue. The third method is the immunofluorescence-based detection of myelin proteins that allows to combine information about their expression status with other proteins of interest. Finally, the FluoroMyelin™ stains enable a fast detection of the myelin content that can be easily implemented in immunofluorescence staining panels for cryosectioned tissues. Together, this chapter provides a variety of methods to accurately identify myelin in different experimental approaches.
Topics: Collagen; Coloring Agents; Hematoxylin; Lipids; Myelin Sheath; Osmium Tetroxide; Staining and Labeling
PubMed: 36152252
DOI: 10.1007/978-1-0716-2675-7_15 -
Scientific Reports Oct 2022Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of...
Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of brain lesions was performed using two techniques: (1) histological methods, such as TTC (Triphenyltetrazolium chloride), a time-consuming and inaccurate process; or (2) MRI imaging, a faster, 3D imaging method, that comes at a high cost. In the last decade, high-resolution micro-CT for 3D sample analysis turned into a simple, fast, and cheaper solution. Here, we successfully describe the application of brain contrasting agents (Osmium tetroxide and inorganic iodine) for high-resolution micro-CT imaging for fine location and quantification of ischemic lesion and edema in mouse preclinical stroke models. We used the intraluminal transient MCAO (Middle Cerebral Artery Occlusion) mouse stroke model to identify and quantify ischemic lesion and edema, and segment core and penumbra regions at different time points after ischemia, by manual and automatic methods. In the transient-ischemic-attack (TIA) mouse model, we can quantify striatal myelinated fibers degeneration. Of note, whole brain 3D reconstructions allow brain atlas co-registration, to identify the affected brain areas, and correlate them with functional impairment. This methodology proves to be a breakthrough in the field, by providing a precise and detailed assessment of stroke outcomes in preclinical animal studies.
Topics: Animals; Mice; Osmium Tetroxide; X-Ray Microtomography; Stroke; Infarction, Middle Cerebral Artery; Disease Models, Animal; Iodine
PubMed: 36261475
DOI: 10.1038/s41598-022-21494-9 -
Journal of Neural Engineering Nov 2022Vagus nerve stimulation (VNS) is Food and Drug Administration-approved for epilepsy, depression, and obesity, and stroke rehabilitation; however, the morphological...
Vagus nerve stimulation (VNS) is Food and Drug Administration-approved for epilepsy, depression, and obesity, and stroke rehabilitation; however, the morphological anatomy of the vagus nerve targeted by stimulatation is poorly understood. Here, we used microCT to quantify the fascicular structure and neuroanatomy of human cervical vagus nerves (cVNs).We collected eight mid-cVN specimens from five fixed cadavers (three left nerves, five right nerves). Analysis focused on the 'surgical window': 5 cm of length, centered around the VNS implant location. Tissue was stained with osmium tetroxide, embedded in paraffin, and imaged on a microCT scanner. We visualized and quantified the merging and splitting of fascicles, and report a morphometric analysis of fascicles: count, diameter, and area.In our sample of human cVNs, a fascicle split or merge event was observed every ∼560m (17.8 ± 6.1 events cm). Mean morphological outcomes included: fascicle count (6.6 ± 2.8 fascicles; range 1-15), fascicle diameter (514 ± 142m; range 147-1360m), and total cross-sectional fascicular area (1.32 ± 0.41 mm; range 0.58-2.27 mm).The high degree of fascicular splitting and merging, along with wide range in key fascicular morphological parameters across humans may help to explain the clinical heterogeneity in patient responses to VNS. These data will enable modeling and experimental efforts to determine the clinical effect size of such variation. These data will also enable efforts to design improved VNS electrodes.
Topics: Humans; Cross-Sectional Studies; Vagus Nerve; Vagus Nerve Stimulation; Epilepsy; Cadaver
PubMed: 36174538
DOI: 10.1088/1741-2552/ac9643 -
Nanomaterials (Basel, Switzerland) Sep 2021Composite membranes play a very important role in the separation, concentration, and purification processes, but especially in membrane reactors and membrane...
Composite membranes play a very important role in the separation, concentration, and purification processes, but especially in membrane reactors and membrane bioreactors. The development of composite membranes has gained momentum especially through the involvement of various nanoparticles, polymeric, oxide, or metal, that have contributed to increasing their reactivity and selectivity. This paper presents the preparation and characterization of an active metal nanoparticle-support polymer type composite membrane, based on osmium nanoparticles obtained in situ on a polypropylene hollow fiber membrane. Osmium nanoparticles are generated from a solution of osmium tetroxide in butyl alcohol by reduction with molecular hydrogen in a contactor with a polypropylene membrane. The composite osmium-polypropylene hollow fiber obtained membranes (Os-PPM) were characterized from the morphological and structural points of view: scanning electron microscopy (SEM), high resolution SEM (HR-SEM), energy dispersive spectroscopy analysis (EDAX), X-ray diffraction analysis (XRD), Fourier transform Infrared (FTIR) spectroscopy, thermal gravimetric analysis, and differential scanning calorimetry (TGA, DSC). The process performance was tested in a redox process of nitrophenol and 10-undecylenic (10-undecenoic) acid, as a target substance of biological or biomedical interest, in solutions of lower aliphatic alcohols in a membrane contactor with a prepared composite membrane. The characteristics of osmium nanoparticles-polypropylene hollow fiber membranes open the way to biological and biotechnological applications. These membranes do not contaminate the working environment, operate at relatively low temperatures, provide a large contact area between reactants, allow successive oxidation and reduction operations in the same module, and help to recover the reaction mass by ultrafiltration. The results obtained show that the osmium-polypropylene composite membrane allows the reduction of nitrophenol or the oxidation of 10-undecylenic acid, the conversion depending on the concentration in the lower aliphatic alcohol, the nature of the lower aliphatic alcohol, and the oxidant or reducing flow through the membrane contactor.
PubMed: 34684968
DOI: 10.3390/nano11102526 -
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 -
Microscopy (Oxford, England) Nov 2023Biological nanoparticles, such as bacterial outer membrane vesicles (OMVs), are routinely characterized through transmission electron microscopy (TEM). In this study, we...
Biological nanoparticles, such as bacterial outer membrane vesicles (OMVs), are routinely characterized through transmission electron microscopy (TEM). In this study, we report a novel method to prepare OMVs for TEM imaging. To preserve vesicular shape and structure, we developed a dual fixation protocol involving osmium tetroxide incubation prior to negative staining with uranyl acetate. Combining osmium tetroxide with uranyl acetate resulted in preservation of sub-50 nm vesicles and improved morphological stability, enhancing characterization of lipid-based nanoparticles by TEM.
Topics: Microscopy, Electron; Coloring Agents; Osmium Tetroxide; Bacterial Outer Membrane; Microscopy, Electron, Transmission; Staining and Labeling; Osmium
PubMed: 37148329
DOI: 10.1093/jmicro/dfad027 -
Biomedical Research (Tokyo, Japan) 2020The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the...
The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the conventional osmium maceration method, tissues are immersed in a diluted osmium tetroxide solution for several days at 20°C to remove soluble cytosolic proteins from the freeze-cracked surface of cells, and the optimal duration of this process is dependent on the cell type. To improve the efficiency of the osmium maceration procedure, we have examined systematically the relationship between the reaction temperature and time of the osmium maceration procedure. Treatment at temperatures higher than 20°C drastically shortened the time required to remove cytosolic proteins from the freeze-cracked surface of specimens with optimal durations for the osmium maceration of hepatocytes at 30, 40, 50 and 60°C being 30, 15, 5 and 1 h, respectively. Considering the stability and reproducibility of the macerated specimens, we concluded that the most appropriate temperature was 30 to 40°C. This rapid osmium maceration procedure was used successfully to observe the 3D ultrastructure of Purkinje cells in the cerebellum and proximal convoluted tubule cells in the kidney. This simple and reproducible rapid osmium maceration protocol should find wide appeal for the 3D analysis of cellular organelles in various cell types.
Topics: Animals; Cryopreservation; Cryoprotective Agents; Dimethyl Sulfoxide; Formaldehyde; Glutaral; Hepatocytes; Liver; Male; Microscopy, Electron, Scanning; Osmium Tetroxide; Polymers; Rats; Rats, Wistar; Temperature; Time Factors; Tissue Fixation
PubMed: 32801265
DOI: 10.2220/biomedres.41.161