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Frontiers in Neurology 2024Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the...
BACKGROUND
Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the invasion and spread of pathogens into the inner ear. Here, we combined high-resolution light microscopy, super-resolution immunohistochemistry (SR-SIM) and synchrotron phase contrast imaging (SR-PCI) to identify the protection and barrier systems in the various parts of the human inner ear, focusing on the lateral wall, spiral ganglion, and endolymphatic sac.
MATERIALS AND METHODS
Light microscopy was conducted on mid-modiolar, semi-thin sections, after direct glutaraldehyde/osmium tetroxide fixation. The tonotopic locations were estimated using SR-PCI and 3D reconstruction in cadaveric specimens. The sections were analyzed for leucocyte and macrophage activity, and the results were correlated with immunohistochemistry using confocal microscopy and SR-SIM.
RESULTS
Light microscopy revealed unprecedented preservation of cell anatomy and several macrophage-like cells that were localized in the cochlea. Immunohistochemistry demonstrated IBA1 cells frequently co-expressing MHC II in the spiral ganglion, nerve fibers, lateral wall, spiral limbus, and tympanic covering layer at all cochlear turns as well as in the endolymphatic sac. RNAscope assays revealed extensive expression of fractalkine gene transcripts in type I spiral ganglion cells. CD4 and CD8 cells occasionally surrounded blood vessels in the modiolus and lateral wall. TMEM119 and P2Y12 were not expressed, indicating that the cells labeled with IBA1 were not microglia. The round window niche, compact basilar membrane, and secondary spiral lamina may form protective shields in the cochlear base.
DISCUSSION
The results suggest that the human cochlea is surveilled by dwelling and circulating immune cells. Resident and blood-borne macrophages may initiate protective immune responses via chemokine signaling in the lateral wall, spiral lamina, and spiral ganglion at different frequency locations. Synchrotron imaging revealed intriguing protective barriers in the base of the cochlea. The role of the endolymphatic sac in human inner ear innate and adaptive immunity is discussed.
PubMed: 38817543
DOI: 10.3389/fneur.2024.1355785 -
Journal of Lipid Research May 2024Contrast-enhanced computed tomography (CECT) offers a non-destructive approach to studying adipose tissue in 3D. Several contrast-enhancing staining agents (CESAs) have...
Contrast-enhanced computed tomography (CECT) offers a non-destructive approach to studying adipose tissue in 3D. Several contrast-enhancing staining agents (CESAs) have been explored, whereof osmium tetroxide (OsO) is the most popular nowadays. However, due to the toxicity and volatility of the conventional OsO, alternative CESAs with similar staining properties were desired. Hf-WD 1:2 POM and Hexabrix have proven effective for structural analysis of adipocytes using CECT, but fail to provide chemical information. This study introduces isotonic Lugol's iodine (IL) as an alternative CESA for adipose tissue analysis, comparing its staining potential with Hf-WD 1:2 POM and Hexabrix in murine caudal vertebrae (MCV) and bovine muscle tissue (BMT) strips. Single and sequential staining protocols were compared to assess the maximization of information extraction from each sample. The study investigated interactions, distribution, and reactivity of iodine species towards biomolecules using simplified model systems and assesses the potential of the CESA to provide chemical information. (Bio)chemical analyses on whole tissues revealed that differences in adipocyte grey values post-IL staining were associated with chemical distinctions between BMT and MCV. More specific, a difference in degree of unsaturation of fatty acids was identified as a likely contributor, though not the sole determinant of grey value differences. This research sheds light on the potential of IL as a CESA, offering both structural and chemical insights into adipose tissue composition.
PubMed: 38823780
DOI: 10.1016/j.jlr.2024.100572 -
BioRxiv : the Preprint Server For... Sep 2023Mapping the complete synaptic connectivity of a mammalian brain would be transformative, revealing the pathways underlying perception, behavior, and memory. Serial...
Mapping the complete synaptic connectivity of a mammalian brain would be transformative, revealing the pathways underlying perception, behavior, and memory. Serial section electron microscopy, via membrane staining using osmium tetroxide, is ideal for visualizing cells and synaptic connections but, in whole brain samples, faces significant challenges related to chemical treatment and volume changes. These issues can adversely affect both the ultrastructural quality and macroscopic tissue integrity. By leveraging time-lapse X-ray imaging and brain proxies, we have developed a 12-step protocol, ODeCO, that effectively infiltrates osmium throughout an entire mouse brain while preserving ultrastructure without any cracks or fragmentation, a necessary prerequisite for constructing the first comprehensive mouse brain connectome.
PubMed: 37808722
DOI: 10.1101/2023.09.26.558265 -
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 -
Ecology and Evolution Apr 2024Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly...
Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly mapping the microscale anatomies of related species poses a major impediment to using comparative anatomy approaches for identifying evolutionary adaptations. We describe a method using synchrotron source micro-x-ray computed tomography (syn-μXCT) combined with machine learning algorithms for high-throughput imaging of Lepidoptera (i.e., butterfly and moth) eyes. Our pipeline allows for imaging at rates of ~15 min/mm at 600 nm resolution. Image contrast is generated using standard electron microscopy labeling approaches (e.g., osmium tetroxide) that unbiasedly labels all cellular membranes in a species-independent manner thus removing any barrier to imaging any species of interest. To demonstrate the power of the method, we analyzed the 3D morphologies of butterfly crystalline cones, a part of the visual system associated with acuity and sensitivity and found significant variation within six butterfly individuals. Despite this variation, a classic measure of optimization, the ratio of interommatidial angle to resolving power of ommatidia, largely agrees with early work on eye geometry across species. We show that this method can successfully be used to determine compound eye organization and crystalline cone morphology. Our novel pipeline provides for fast, scalable visualization and analysis of eye anatomies that can be applied to any arthropod species, enabling new questions about evolutionary adaptations of compound eyes and beyond.
PubMed: 38571794
DOI: 10.1002/ece3.11137 -
Molecules (Basel, Switzerland) May 2024-Tetrahexylporphyrin was converted to its corresponding 7,8-dihydroxychlorin using an osmium tetroxide-mediated dihydroxylation strategy. Its diol moiety was shown to be...
-Tetrahexylporphyrin was converted to its corresponding 7,8-dihydroxychlorin using an osmium tetroxide-mediated dihydroxylation strategy. Its diol moiety was shown to be able to undergo a number of subsequent oxidation reactions to form a chlorin dione and porpholactone, the first -alkylporphyrin-based porphyrinoid containing a non-pyrrolic building block. Further, the diol chlorin was shown to be susceptible to dehydration, forming the porphyrin enol that is in equilibrium with its keto-chlorin form. The -hexylchlorin dione could be reduced and it underwent mono- and bis-methylation reactions using methyl-Grignard reagents, and trifluoromethylation using the Ruppert-Prakash reagent. The optical and spectroscopic properties of the products are discussed and contrasted to their corresponding -aryl derivatives (where known). This contribution establishes -tetrahexyl-7,8-dihydroxychlorins as a new and versatile class of chlorins that is susceptible to a broad range of conversions to generate functionalized chlorins and a pyrrole-modified chlorin analogue.
PubMed: 38731635
DOI: 10.3390/molecules29092144 -
ACS Omega Feb 2024To study transcriptome dynamics without harming cells, it is essential to convert chemical bases. 4-Thiouridine (4sU) is a biocompatible uridine analogue that can be...
To study transcriptome dynamics without harming cells, it is essential to convert chemical bases. 4-Thiouridine (4sU) is a biocompatible uridine analogue that can be converted into a cytidine analogue. Although several reactions can convert 4sU into a cytidine analogue, few studies have compared the features of these reactions. In this study, we performed three reported base conversion reactions, including osmium tetroxide, iodoacetamide, and sodium periodate treatment, as well as a new reaction using 2,4-dinitrofluorobenzene. We compared the reaction time, conversion efficacy, and effects on reverse transcription. These reactions successfully converted 4sU into a cytidine analogue quantitatively using trinucleotides. However, the conversion efficacy and effect on reverse transcription vary depending on the reaction with the RNA transcript. OsO treatment followed by NHCl treatment showed the best base-conversion efficiency. Nevertheless, each reaction has its own advantages and disadvantages as a tool for studying the transcriptome. Therefore, it is crucial to select the appropriate reaction for the target of interest.
PubMed: 38434802
DOI: 10.1021/acsomega.3c08516 -
Heliyon Sep 2023Cochlear implants (CIs) allow individuals with profound hearing loss to understand speech and perceive sounds. However, not all patients obtain the full benefits that...
Cochlear implants (CIs) allow individuals with profound hearing loss to understand speech and perceive sounds. However, not all patients obtain the full benefits that CIs can provide and the cause of this disparity is not fully understood. One possible factor for the variability in outcomes after cochlear implantation, is the development of fibrotic scar tissue around the implanted electrode. It has been hypothesised that limiting the extent of fibrosis after implantation may improve overall CI function, and longevity of the device. Currently, histology is often used to quantify the extent of intracochlear tissue growth after implantation however this method is labour intensive, time-consuming, often involves significant user bias, and causes physical distortion of the fibrosis. Therefore, this study aimed to evaluate x-ray micro computed tomography (μCT) as a method to measure the amount and distribution of fibrosis in a guinea pig model of cochlear implantation. Adult guinea pigs were implanted with an inactive electrode, and cochleae harvested eight weeks later (n = 7) and analysed using μCT, to quantify the extent of tissue reaction, followed by histological analysis to confirm that the tissue was indeed fibrotic. Cochleae harvested from an additional six animals following implantation were analysed by μCT, before and after contrast staining with osmium tetroxide (OsO), to enhance the visualisation of soft tissues within the cochlea, including the tissue reaction. Independent analysis by two observers showed that the quantification method was robust and provided additional information on the distribution of the response within the cochlea. Histological analysis revealed that μCT visualised dense collagenous material and new bone formation but did not capture loose, areolar fibrotic tissue. Treatment with OsO significantly enhanced the visible tissue reaction detected using μCT. Overall, μCT is an alternative and reliable method that can be used to quantify the extent of the CI-induced intracochlear tissue response and will be a useful tool for the assessment of novel anti-fibrotic treatments.
PubMed: 37662829
DOI: 10.1016/j.heliyon.2023.e19343 -
Carbohydrate Research Dec 2023Bacterial natural products containing heptosides such as septacidin represent interesting scaffolds for the development of drugs to combat antimicrobial resistance....
Bacterial natural products containing heptosides such as septacidin represent interesting scaffolds for the development of drugs to combat antimicrobial resistance. However, very few synthetic strategies have been reported to grant access to these derivatives. Here, we have devised a synthetic pathway to l-glycero-l-glucoheptoside, a key building block en route to septacidin, directly from l-glucose. Importantly, we show that carbon homologation at C6, encompassing oxidation of the C6-OH followed by methylenation, is significantly influenced by the nature of the C4-moiety. In order to observe the effect of various patterns, namely azide (N), p-methoxybenzyloxy (OPMB), and benzyloxy (OBn), a thorough analysis was conducted on the corresponding l-glucosides. The results unveiled a distinct trend where the efficiency of methylenation followed the trend OBn > OPMB > N. Finally, the C6-alkene was dihydroxylated in the presence of osmium tetroxide to yield the expected l/d-glycero-l-glucoheptosides. The lead building block, which features a C-4 azide, was delivered as a phenyl thioglycoside. Added to the suitable masking of the 6,7-diol, this combination enables further functionalization to achieve versatile compounds of biological interest. The study insights into the interplay between substitution at C-4 and carbon homologation at C-6 provide valuable guidance for future endeavors in the synthesis of these carbohydrate molecules.
Topics: Glucose; Azides; Heptoses; Carbon
PubMed: 38016254
DOI: 10.1016/j.carres.2023.108985 -
Veterinary World Mar 2024The pathogenesis of staphylococcal infections is mediated by virulence factors, such as enzymes, toxins, and biofilms, which increase the resistance of microorganisms to...
BACKGROUND AND AIM
The pathogenesis of staphylococcal infections is mediated by virulence factors, such as enzymes, toxins, and biofilms, which increase the resistance of microorganisms to host immune system evasion. Testing and searching for standardized multi-level algorithms for the indication and differentiation of biofilms at the early stages of diagnosis will contribute to the development of preventive measures to control the critical points of technology and manage dangerous risk factors for the spread of infectious diseases. This research aimed to study the main stages of s biofilm formation in experiments and to analyze the dynamics of respiratory syndrome development in chickens infected with these bacteria.
MATERIALS AND METHODS
Experimental reproduction of the infectious process was performed using laboratory models: 10-day-old White Leghorn chickens (n = 20). Before the experiments, the birds were divided into two groups according to the principle of analogs: Group I (control, n = 10): the birds were intranasally inoculated with 0.5 cm of 0.9% NaCl solution; Group II (experiment, n = 10): the birds were intranasally inoculated with a suspension of bacteria, 0.5 cm, concentration 1 billion/cm.
RESULTS
Colonization of individual areas of the substrate under study occurred gradually from the sedimentation and adhesion of single motile planktonic cells to the attachment stage of microcolony development. Staining preparations with gentian violet due to the "metachromosia" property of this dye are a quick and fairly simple way to differentiate cells and the intercellular matrix of biofilms. Fixation with vapors of glutaraldehyde and osmium tetroxide preserves the natural architecture of biofilms under optical and scanning electron microscopy. Pure cultures of microorganisms were isolated from the blood, lungs, small intestine, liver, kidneys, and spleen after 5-10 days during experimental infection of chickens. Clinical signs of respiratory syndrome developed within 5-6 days after infection. Acute and subacute serous-fibrinous airsacculitis, characterized by edema and thickening of the membranes of the air sacs and the presence of turbid, watery, foamy contents in the cavity, was the most characteristic pathomorphological sign. The signs of acute congestive hyperemia and one-sided serous-fibrinous pneumonia developed with significant thickening of fibrinous deposits. In Garder's gland, there was an increase in the number of secretory sections, indicating hypersecretion of the glands. In the lymphoid follicles of Meckel's diverticulum, leukocytes, usually lymphocytes, and pseudoeosinophils were detected.
CONCLUSIONS
Hydration and heteromorphism of the internal environment of biofilms determine the localization of differentiated cells in a three-dimensional matrix for protection against adverse factors. The most characteristic pathomorphological sign was the development of acute and subacute serous-fibrinous airsacculitis when reproducing the infectious process in susceptible models. There was a significant thickening of fibrinous deposits and signs of acute congestive hyperemia and one or two serous-fibrinous pneumonia developed.
PubMed: 38680142
DOI: 10.14202/vetworld.2024.612-619