-
Laboratory Investigation; a Journal of... Jan 2023Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus...
Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.
Topics: Humans; Microscopy, Electron, Scanning; Paraffin Embedding; 3,3'-Diaminobenzidine; Formaldehyde; Virion
PubMed: 36748195
DOI: 10.1016/j.labinv.2022.100020 -
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 -
Cancers Jan 2023Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of...
Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.
PubMed: 36672277
DOI: 10.3390/cancers15020328 -
Nature Methods Dec 2022We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that...
We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.
Topics: Luminescent Proteins; Microscopy; Green Fluorescent Proteins; Fluorescence Resonance Energy Transfer; Light
PubMed: 36344833
DOI: 10.1038/s41592-022-01660-7 -
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 -
ELife Oct 2022Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks...
Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high-quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here, we present an in situ time-lapsed X-ray-assisted staining procedure that opens the 'black box' of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method, we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in potassium ferrocyanide reduced osmium solution. X-ray-assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray-assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys, or humans.
Topics: Humans; Mice; Animals; Osmium Tetroxide; Osmium; X-Rays; Staining and Labeling; Microscopy, Electron
PubMed: 36263931
DOI: 10.7554/eLife.72147 -
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 -
The Journal of Organic Chemistry Nov 2022Reactions that result in the oxy-functionalization of sp C-H bonds to give phenols are relatively rare. Here we report experiments and density functional theory (DFT)...
Experimental Demonstration and Density Functional Theory Mechanistic Analysis of Arene C-H Bond Oxidation and Product Protection by Osmium Tetroxide in a Strongly Basic/Nucleophilic Solvent.
Reactions that result in the oxy-functionalization of sp C-H bonds to give phenols are relatively rare. Here we report experiments and density functional theory (DFT) calculations that demonstrate selective C-H bond hydroxylation of nitroarenes to their corresponding mono-phenoxide as the exclusive product using OsO in a highly basic solvent mixture of water, hydroxide, and pyridine. DFT calculations using a mixed explicit/continuum solvent approach indicate that there is likely a mixture of OsO-hydroxide/pyridine ground-state structures that have competitive reactivity and that the mechanism involves the nucleophilic addition of an anionic metal-oxo species to the arene followed by a hydride transfer process that is different from the standard [3 + 2] mechanism often invoked for the OsO oxidation of σ and π bonds. This work demonstrates the utility of using a strongly basic solvent for C-H bond oxidation reactions as this effectively converts any reactive phenolic product into the corresponding phenoxide, which is protected and essentially inert to further oxidation by the nucleophilic metal-oxo species.
PubMed: 36191170
DOI: 10.1021/acs.joc.2c01159 -
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 -
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