Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution

High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

C(sp3)–H bond activation by carboxylate-adduct of osmium tetroxide (OsO4)

The reaction of osmium tetroxide (OsO4) and carboxylate anions (acetate: X = AcO– and benzoate: X = BzO–) gave 1 : 1 adducts, [OsO4(X)]– (1X), the structures of which were...

Osmium Recovery as Membrane Nanomaterials through 10–Undecenoic Acid Reduction Method

The recovery of osmium from residual osmium tetroxide (OsO4) is a necessity imposed by its high toxicity, but also by the technical-economic value of metallic osmium. An elegant and extremely useful method is the recovery of osmium as a membrane catalytic material, in the form of nanoparticles obtained on a polymeric support. The subject of the present study is the realization of a composite membrane in which the polymeric matrix is the polypropylene hollow fiber, and the active component consists of the osmium nanoparticles obtained by reducing an alcoholic solution of osmium tetroxides directly on the polymeric support. The method of reducing osmium tetroxide on the polymeric support is based on the use of 10-undecenoic acid (10–undecylenic acid) (UDA) as a reducing agent. The osmium tetroxide was solubilized in t–butanol and the reducing agent, 10–undecenoic acid (UDA), in i–propanol, t–butanol or n–decanol solution. The membranes containing osmium nanoparticles (Os–NP) were characterized morphologically by the following: scanning electron microscopy (SEM), high-resolution SEM (HR–SEM), structurally: energy-dispersive spectroscopy analysis (EDAX), Fourier transform infrared (FTIR) spectroscopy. In terms of process performance, thermal gravimetric analysis was performed by differential scanning calorimetry (TGA, DSC) and in a redox reaction of an organic marker, p–nitrophenol (PNP) to p–aminophenol (PAP). The catalytic reduction reaction with sodium tetraborate solution of PNP to PAP yielded a constant catalytic rate between 2.04 × 10−4 mmol s–1 and 8.05 × 10−4 mmol s−1.

Comparative evaluation of methods for studying the degree of assimilation of feed by animals

Relevance. The activity of the digestive organs is largely determined by the feeding regime, in particular, the frequency, timeliness, and the order of feeding the feed. The digestibility of nutrients is influenced by the preparation of feed for feeding, which facilitates mechanical processing in the digestive tract and the action of digestive enzymes. Against the background of a decrease in the natural resistance of the animal body, an important aspect is the use of mineral supplements and the enrichment of diets with biologically active substances that increase the nutritional value and the degree of assimilation of feed by animals. The aim of the work was a comparative assessment of methods for studying the degree of assimilation of feed by animals.Methods. To determine the degree of food assimilation, 2 drops of isotonic (0.9%) sodium chloride solution and a small amount of intestinal contents were applied to a glass slide, a thin smear was prepared, dried in air for 30 min, and large particles were removed. The preparations were fixed for 15 min with 96% ethyl alcohol, then 3–5 drops of a 1.0% aqueous solution of methylene blue, Lugol, Sudan III were applied. Along with the indicated conventional methods, we tested a method for preparing preparations using fixation in vapors of a 25.0% solution of glutaraldehyde for 30–40 minutes, in vapors of a 1.0% aqueous solution of osmium tetroxide.Results. The kinetics of the formation of intestinal microbiocenoses is characterized by a decrease in the number of lactobacilli and an increase in the number of pathogenic and opportunistic bacteria. In a comparative assessment of the preparation methods, the advantage of microscopic research methods is the possibility of a detailed study of the contents, the degree of assimilation of feed components in the gastrointestinal tract, and a visual assessment of the state of the intestinal microflora. The advantage of the method for preparing preparations with the use of vapor fixation of a 25.0% solution of glutaraldehyde and osmium tetroxide is the possibility of visualizing the formation of monospecific and polyspecies biofilms by optical and scanning electron microscopy. The preparations were sufficiently contrasting, there were no artifacts, bacteria and fungi stained brown, which made it possible to count even pinpoint colonies using a magnifying glass or an optical microscope. The easily digestible feed results in a low fecal yield and a stable consistency. The use of functional food “Pristine Gold Vet” in the nutrition of dogs and fur-bearing animals made it possible to increase the absorption of nutrients by 18,1% and 15,3%.

Ultrastructural pathology of bioprosthetic heart valves with infectious endocarditis

Aim. To study the ultrastructure of mitral bioprosthetic heart valves (BHVs) which failed due to infective endocarditis.Materials and Methods. Here we examined 7 ethylene glycol diglycidyl ether-treated xenopericardial BHVs excised during repeated BHV replacement because of prosthetic endocarditis. After being fixed in formalin and postfixed in osmium tetroxide, BHVs were dehydrated and stained in uranyl acetate with the subsequent embedding into epoxy resin, grinding, polishing, and lead citrate counterstaining. Upon the sputter coating with carbon, we visualised the BHV microanatomy by means of backscattered scanning electron microscopy at 15 kV voltage.Results. The extracellular matrix underwent degradation and disintegration resulting in loosening, fragmentation, and reduction in the electron density of collagen and elastin fibers. We observed a number of recipient cells (macrophages, multinucleated giant cells, neutrophils, endothelial cells and smooth muscle cells) within the BHVs. The highest number of cells was localized on the valve surfaces. The localization of the recipient cells on the ventricular and atrial surfaces was different. The central part of the valves was abundantly populated by macrophages.Conclusion. Prosthetic endocarditis is accompanied by the migration of recipient cells into the BHV structure, which is the consequence of surface and extracellular matrix disintegration.

Elemental analysis of valvular and atherosclerotic calcification

Aim. To analyze the topographic patterns of valvular and atherosclerotic calcification growth.Methods. Dysfunctional aortic valves (n = 18) and atherosclerotic plaques (n = 20) were fixed in formalin, postfixed in 1% osmium tetroxide, consecutively stained by 2% osmium tetroxide and 2% uranyl acetate, and embedded into epoxy resin (Epon) with the further grinding and polishing ofthe samples. Upon the counterstaining by lead citrate and sputter coating with carbon, samples were visualized by backscattered scanning electron microscopy. Elemental analysis was conducted via energy-dispersive X-ray spectroscopy. Measurement of Ca/P ratio within the mineral deposits was carried out employing a pool table principle (i.e., in the center of the deposit, in the near and far circumferences (clockwise), and in control regions around the mineral deposit). Topographic patterns of calcifications were modeled using the correlation analysis. Results. Significant correlation was revealed between the Ca/P ratio in the deposit center and in the near and far circumferences of deposit in both in valvular (r = 0,35-0,78 - near circumference; r = 0,63-0,69 - far circumference) and atherosclerotic mineral deposits (r = 0,37-0,56 - near circumference; r = 0,48-0,63 - far circumference), suggesting the hierarchical growth of cardiovascular calcification around the initial nucleation sites.Conclusion. Valvular and atherosclerotic calcifications development is concentric.

Catalytic Effectiveness of Synchrotron and Synchrocyclotron Radiations on Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) Nano Capsules Delivery in DNA/RNA of Cancer Cells

In the current research, catalytic effectiveness of synchrotron and synchrocyclotron radiations on Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) nano capsules delivery in DNA/RNA of cancer cells is investigated. The calculation of thickness and optical constants of Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) catalytic effectiveness of synchrotron and synchrocyclotron radiations on Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) nano capsules delivery in DNA/RNA of cancer cells produced using sol-gel method over glassy medium through a single reflection spectrum is presented. To obtain an appropriate fit for reflection spectrum, the classic Drudge-Lorentz model for parametric di–electric function is used. The best fitting parameters are determined to simulate the reflection spectrum using Levenberg-Marquardt optimization method. The simulated reflectivity from the derived optical constants and thickness are in good agreement with experimental results. Keywords: Catalytic Effectiveness; Synchrotron and Synchrocyclotron Radiations; Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) Nano Capsules; Delivery; DNA/RNA; Cancer Cells

Regularities of plaque stabilization in various scenarios of neointimal calcification and vascularization

Aim. To study the relationships between phenotypes of extracranial arteries' plaques (stable/unstable), their calcification and its causes, in particular, vascularization.Material and methods. The study included 88 patients: patients (n=44) with ischemic stroke and those (n=44) with chronic brain ischemia. In all subjects, the parameters of systemic mineral homeostasis were assessed (total and ionized calcium, phosphate, total protein, albumin, and calcification propensity). Atherosclerotic plaques have been obtained during carotid endarterectomy, fixed in formalin, postfixed in 1% osmium tetroxide, stained in 2% osmium tetroxide, dehydrated in ascending ethanol series and acetone, stained with 2% alcoholic uranyl acetate and embedded into epoxy resin with its further polymerization. Epoxy resin blocks were grinded, polished, counterstained with Reynolds' lead citrate and sputter coated with carbon. Sample visualization was performed employing backscattered scanning electron microscopy. Number and area of calcium deposits and neointimal vessels were quantified using ImageJ. Statistical analysis was carried out using Mann-Whitney U-test and Spearman's rank correlation coefficient.Results. It was found that area of neointimal calcification, but not number of calcium deposits, was associated with the stable plaque phenotype. The stabilizing effect of calcification was manifested in retarding stenosis associated with plaque rupture and stroke. Calcification extent directly correlated with total and local plaque vascularization, which have been associated with unstable and stable plaque phenotype, respectively. In addition, plaque calcification negatively correlated with total protein and albumin, thereby reflecting the impaired systemic mineral homeostasis.Conclusion. Atherosclerotic plaque calcification and active local vascularization reduce stenosis extent and stabilize plaque. In contrast, total plaque calcification contributes to the atherosclerosis progression and promotes major acute cardiovascular events.