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Acta Histochemica Et Cytochemica Feb 2018Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach...
Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.
PubMed: 29622846
DOI: 10.1267/ahc.17020 -
Scientific Reports Mar 2018Lesion verification and quantification is traditionally done via histological examination of sectioned brains, a time-consuming process that relies heavily on manual...
Lesion verification and quantification is traditionally done via histological examination of sectioned brains, a time-consuming process that relies heavily on manual estimation. Such methods are particularly problematic in posterior cortical regions (e.g. visual cortex), where sectioning leads to significant damage and distortion of tissue. Even more challenging is the post hoc localization of micro-electrodes, which relies on the same techniques, suffers from similar drawbacks and requires even higher precision. Here, we propose a new, simple method for quantitative lesion characterization and electrode localization that is less labor-intensive and yields more detailed results than conventional methods. We leverage staining techniques standard in electron microscopy with the use of commodity micro-CT imaging. We stain whole rat and zebra finch brains in osmium tetroxide, embed these in resin and scan entire brains in a micro-CT machine. The scans result in 3D reconstructions of the brains with section thickness dependent on sample size (12-15 and 5-6 microns for rat and zebra finch respectively) that can be segmented manually or automatically. Because the method captures the entire intact brain volume, comparisons within and across studies are more tractable, and the extent of lesions and electrodes may be studied with higher accuracy than with current methods.
Topics: Animals; Brain; Finches; Humans; Microscopy, Electron; Osmium Tetroxide; Rats; Staining and Labeling; Visual Cortex; X-Ray Microtomography
PubMed: 29581439
DOI: 10.1038/s41598-018-23247-z -
Frontiers in Neuroscience 2018Stable posture and body movement in humans is dictated by the precise functioning of the ampulla organs in the semi-circular canals. Statistical analysis of the...
Stable posture and body movement in humans is dictated by the precise functioning of the ampulla organs in the semi-circular canals. Statistical analysis of the interrelationship between bony and membranous compartments within the semi-circular canals is dependent on the visualization of soft tissue structures. Thirty-one human inner ears were prepared, post-fixed with osmium tetroxide and decalcified for soft tissue contrast enhancement. High resolution X-ray microtomography images at 15 μm voxel-size were manually segmented. This data served as templates for centerline generation and cross-sectional area extraction. Our estimates demonstrate the variability of individual specimens from averaged centerlines of both bony and membranous labyrinth. Centerline lengths and cross-sectional areas along these lines were identified from segmented data. Using centerlines weighted by the inverse squares of the cross-sectional areas, plane angles could be quantified. The fit planes indicate that the bony labyrinth resembles a Cartesian coordinate system more closely than the membranous labyrinth. A widening in the membranous labyrinth of the lateral semi-circular canal was observed in some of the specimens. Likewise, the cross-sectional areas in the perilymphatic spaces of the lateral canal differed from the other canals. For the first time we could precisely describe the geometry of the human membranous labyrinth based on a large sample size. Awareness of the variations in the canal geometry of the membranous and bony labyrinth would be a helpful reference in designing electrodes for future vestibular prosthesis and simulating fluid dynamics more precisely.
PubMed: 29535601
DOI: 10.3389/fnins.2018.00107 -
Viruses Mar 2018Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select...
Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV () in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV (). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.
Topics: Animals; Antiviral Agents; Cell Line; Chlorocebus aethiops; Cytopathogenic Effect, Viral; Disinfectants; Ebolavirus; Hemorrhagic Fever, Ebola; Humans; Microbial Sensitivity Tests; Sensitivity and Specificity; Vero Cells; Virus Inactivation
PubMed: 29533988
DOI: 10.3390/v10030126 -
RSC Advances Feb 2018This report illustrates the applications of Asymmetric Sharpless Aminohydroxylation (ASAH) in the stereoselective synthesis of vicinal amino alcohols as important... (Review)
Review
This report illustrates the applications of Asymmetric Sharpless Aminohydroxylation (ASAH) in the stereoselective synthesis of vicinal amino alcohols as important intermediates in the total synthesis of complex molecules and natural products with significant biological activities. The ASHA allows the regio- -selective synthesis of 1,2-amino alcohols reaction of alkenes with salts of -halosulfonamides, -amides and -carbamates employing osmium tetroxide (OsO) as an efficient catalyst. In this reaction, chirality is induced the addition of dihydroquinine- and dihydroquinidine as derived chiral ligands.
PubMed: 35559209
DOI: 10.1039/c7ra12625e -
Neural Regeneration Research Dec 2017Peripheral nerve injury is a serious disease and its repair is challenging. A cable-style autologous graft is the gold standard for repairing long peripheral nerve...
Peripheral nerve injury is a serious disease and its repair is challenging. A cable-style autologous graft is the gold standard for repairing long peripheral nerve defects; however, ensuring that the minimum number of transplanted nerve attains maximum therapeutic effect remains poorly understood. In this study, a rat model of common peroneal nerve defect was established by resecting a 10-mm long right common peroneal nerve. Rats receiving transplantation of the common peroneal nerve in situ were designated as the in situ graft group. Ipsilateral sural nerves (10-30 mm long) were resected to establish the one sural nerve graft group, two sural nerves cable-style nerve graft group and three sural nerves cable-style nerve graft group. Each bundle of the peroneal nerve was 10 mm long. To reduce the barrier effect due to invasion by surrounding tissue and connective-tissue overgrowth between neural stumps, small gap sleeve suture was used in both proximal and distal terminals to allow repair of the injured common peroneal nerve. At three months postoperatively, recovery of nerve function and morphology was observed using osmium tetroxide staining and functional detection. The results showed that the number of regenerated nerve fibers, common peroneal nerve function index, motor nerve conduction velocity, recovery of myodynamia, and wet weight ratios of tibialis anterior muscle were not significantly different among the one sural nerve graft group, two sural nerves cable-style nerve graft group, and three sural nerves cable-style nerve graft group. These data suggest that the repair effect achieved using one sural nerve graft with a lower number of nerve fibers is the same as that achieved using the two sural nerves cable-style nerve graft and three sural nerves cable-style nerve graft. This indicates that according to the 'multiple amplification' phenomenon, one small nerve graft can provide a good therapeutic effect for a large peripheral nerve defect.
PubMed: 29323049
DOI: 10.4103/1673-5374.221167 -
The Science of the Total Environment May 2018Diet shifts are common in mammals and birds, but little is known about how such shifts along the food web affect contaminant exposure. Voles are staple food for many...
Diet shifts are common in mammals and birds, but little is known about how such shifts along the food web affect contaminant exposure. Voles are staple food for many mammalian and avian predators. There is therefore a risk of transfer of contaminants accumulated in voles within the food chain. Osmium is one of the rarest earth elements with osmium tetroxide (OsO) as the most toxic vapor-phase airborne contaminant. Anthropogenic OsO accumulates in fruticose lichens that are important winter food of bank voles (Myodes glareolus). Here, we test if a) anthropogenic osmium accumulates in bank voles in winter, and b) accumulation rates and concentrations are lower in autumn when the species is mainly herbivorous. Our study, performed in a boreal forest impacted by anthropogenic osmium, supported the hypotheses for all studied tissues (kidney, liver, lung, muscle and spleen) in 50 studied bank voles. In autumn, osmium concentrations in bank voles were even partly similar to those in the graminivorous field vole (Microtus agrestis; n=14). In autumn but not in late winter/early spring, osmium concentrations were generally negatively correlated with body weight and root length of the first mandible molar, i.e. proxies of bank vole age. Identified negative correlations between organ-to-body weight ratios and osmium concentrations in late winter/early spring indicate intoxication. Our results suggest unequal accumulation risk for predators feeding on different cohorts of bank voles.
Topics: Air Pollutants; Animals; Arvicolinae; Diet; Food Chain; Nutritional Status; Osmium; Seasons; Sweden
PubMed: 29079088
DOI: 10.1016/j.scitotenv.2017.10.056 -
Endocrinology Oct 2017Obesity during maturation can affect the growing skeleton directly and indirectly, although these effects and the mechanisms behind them are not fully understood. Our...
Obesity during maturation can affect the growing skeleton directly and indirectly, although these effects and the mechanisms behind them are not fully understood. Our objective was to determine how a high-fat diet with or without metformin treatment affects skeletal development. We also sought to characterize changes that occur in white adipose tissue, circulating metabolites, lipids, and gut microbiota. A diet-induced obesity C57BL/6J mouse model was used to test the effects of obesity and metformin on bone using bone histomorphometry and microcomputed tomography. Bone marrow adipose tissue was quantified with osmium tetroxide microcomputed tomography and histology. Dual-energy x-ray absorptiometry was used to analyze body composition. Hematoxylin and eosin staining was used to assess changes in white adipose depots, mass spectrometry was used for circulating lipids and protein metabolite analysis, and ribosomal RNA sequencing was used for gut microbiome analysis. Mice fed a high fat-diet since wean displayed increased medullary areas and decreased osteoblast numbers in the long bones; this phenotype was partially normalized by metformin. Marrow and inguinal adipose expansion was also noted in obese mice, and this was partially normalized by metformin. A drug-by-diet interaction was noted for circulating lipid molecules, protein metabolites, and gut microbiome taxonomical units. Obesity was not detrimental to trabecular bone in growing mice, but bone marrow medullary expansion was observed, likely resulting from inhibition of osteoblastogenesis, and this was partially reversed by metformin treatment.
Topics: Absorptiometry, Photon; Adipose Tissue, White; Adiposity; Animals; Body Composition; Bone Marrow; Cell Count; Chromatography, Liquid; Cortical Bone; Diet, High-Fat; Gastrointestinal Microbiome; Immunohistochemistry; Lipid Metabolism; Male; Mass Spectrometry; Metabolomics; Metformin; Mice; Mice, Inbred C57BL; Obesity; Organ Size; Osteoblasts; Phenotype; RNA, Bacterial; RNA, Ribosomal, 16S; Tandem Mass Spectrometry; X-Ray Microtomography
PubMed: 28977604
DOI: 10.1210/en.2017-00299 -
Chemical & Pharmaceutical Bulletin 2017Total synthesis of sphingofungin E and 4,5-di-epi-sphingofungin E was achieved from an intermediate same as that of myriocin and mycestericin D via antipodal...
Total synthesis of sphingofungin E and 4,5-di-epi-sphingofungin E was achieved from an intermediate same as that of myriocin and mycestericin D via antipodal stereoselective dihydroxylations.
Topics: Amino Acids; Fatty Acids, Unsaturated; Magnetic Resonance Spectroscopy; Spectrometry, Mass, Fast Atom Bombardment; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet
PubMed: 28674344
DOI: 10.1248/cpb.c17-00322 -
Developmental Dynamics : An Official... Nov 2017Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may...
BACKGROUND
Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may impair rapid and efficient transmission of action potentials and alter nerve function. Identifying ultrastructural changes resulting from defects to these cellular components via transmission electron microscopy (TEM) can provide valuable insight into nerve function and disease. However, efficiently locating spinal motor nerves in adult zebrafish for TEM is challenging and time-consuming. Because of this, we developed a protocol that allows us to quickly and precisely locate spinal motor nerve roots in adult zebrafish for TEM processing.
RESULTS
Following fixation, a transverse slab of adult zebrafish dissected from the trunk region was mounted in embedding media, sectioned, and secondary fixation with osmium tetroxide performed. Transverse sections containing motor nerves were selected for TEM ultrathin sectioning and imaging.
CONCLUSIONS
We developed an efficient protocol for locating spinal motor nerves in adult zebrafish to allow for ultrastructural characterization. Although our work focuses on spinal motor nerves, this protocol may be useful for efficiently identifying other discrete, repeated structures within the developing and mature nervous system that are difficult to find via traditional, whole organism TEM processing. Developmental Dynamics 246:956-962, 2017. © 2017 Wiley Periodicals, Inc.
Topics: Animals; Histological Techniques; Microscopy, Electron, Transmission; Spinal Nerve Roots; Zebrafish
PubMed: 28598521
DOI: 10.1002/dvdy.24529