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2. Cryo-EM Map Anisotropy Can Be Attenuated by Map Post-Processing and a New Method for Its Estimation.
- Author
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Sanchez-Garcia, Ruben, Gaullier, Guillaume, Cuadra-Troncoso, Jose Manuel, and Vargas, Javier
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ANISOTROPY ,ELECTRON microscopy - Abstract
One of the most important challenges in cryogenic electron microscopy (cryo-EM) is the substantial number of samples that exhibit preferred orientations, which leads to an uneven coverage of the projection sphere. As a result, the overall quality of the reconstructed maps can be severely affected, as manifested by the presence of anisotropy in the map resolution. Several methods have been proposed to measure the directional resolution of maps in tandem with experimental protocols to address the problem of preferential orientations in cryo-EM. Following these works, in this manuscript we identified one potential limitation that may affect most of the existing methods and we proposed an alternative approach to evaluate the presence of preferential orientations in cryo-EM reconstructions. In addition, we also showed that some of the most recently proposed cryo-EM map post-processing algorithms can attenuate map anisotropy, thus offering alternative visualization opportunities for cases affected by moderate levels of preferential orientations. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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3. Automated Mass Photometry of Adeno-Associated Virus Vectors from Crude Cell Extracts.
- Author
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Wagner, Christina, Fuchsberger, Felix F., Innthaler, Bernd, Pachlinger, Robert, Schrenk, Irene, Lemmerer, Martin, and Birner-Gruenberger, Ruth
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ADENO-associated virus ,PHOTOMETRY ,GENETIC vectors ,MOLAR mass ,LIFTING & carrying (Human mechanics) ,ULTRACENTRIFUGATION ,ELECTRON microscopy - Abstract
Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires minimal sample volumes between 5–10 µL, which makes it a promising candidate over orthogonal techniques such as analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (Cryo-TEM) or charge-detection mass spectrometry (CDMS). However, these methods are limited in their application to purified samples only. Here we developed a purification step based on single-domain monospecific antibody fragments immobilised on either a poly(styrene-divinylbenzene) resin or on magnetic beads prior to MP analysis that allows the quantification of empty, partially filled, full and overfull AAV vectors in crude cell extracts. This is aimed at identifying potentially promising harvest conditions that yield large numbers of filled AAV vectors during the early stages of the viral vector development platform, e.g., the type of transfection reagent used. Furthermore, we provide a direct comparison of the automated and manual handling of the mass photometer with respect to the quantities of AAV subspecies, molar mass of the capsid and payload, and highlight the differences between the "buffer-free" sample measurement and the "buffer-dilution" mode. In addition, we provide information on which candidates to use for calibration and demonstrate the limitations of the mass photometer with respect to the estimation of the capsid titer. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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4. Interaction of tRNA with Eukaryotic Ribosome.
- Author
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Graifer, Dmitri and Karpova, Galina
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TRANSFER RNA ,EUKARYOTES ,RIBOSOMES ,ELECTRON microscopy ,PROKARYOTES ,PHOTOCHEMISTRY - Abstract
This paper is a review of currently available data concerning interactions of tRNAs with the eukaryotic ribosome at various stages of translation. These data include the results obtained by means of cryo-electron microscopy and X-ray crystallography applied to various model ribosomal complexes, site-directed cross-linking with the use of tRNA derivatives bearing chemically or photochemically reactive groups in the CCA-terminal fragment and chemical probing of 28S rRNA in the region of the peptidyl transferase center. Similarities and differences in the interactions of tRNAs with prokaryotic and eukaryotic ribosomes are discussed with concomitant consideration of the extent of resemblance between molecular mechanisms of translation in eukaryotes and bacteria. [ABSTRACT FROM AUTHOR]
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- 2015
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5. Correction: Kim et al. Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy. Int. J. Mol. Sci. 2021, 22 , 4273.
- Author
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Kim, Hong-Lim, Riew, Tae-Ryong, Park, Jieun, Lee, Youngchun, and Kim, In-Beom
- Subjects
MICROSCOPY ,ELECTRON microscopy ,IMMUNOGOLD labeling ,SCANNING electron microscopy ,SCANNING electron microscopes - Abstract
33924132 2 Kusumi S., Koga D., Watanabe T., Shibata M. Combination of a cryosectioning method and section scanning electron microscopy for immuno-scanning electron microscopy. Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy. Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy. [Extracted from the article]
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- 2022
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6. The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine β-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain.
- Author
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Zamakhov, Ilya M., Anashkin, Viktor A., Moiseenko, Andrey V., Orlov, Victor N., Vorobyeva, Natalia N., Sokolova, Olga S., and Baykov, Alexander A.
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CATALYTIC domains ,CYSTATHIONINE ,INORGANIC pyrophosphatase ,ADENINE nucleotides ,CYTOSKELETAL proteins ,POLYKETIDE synthases ,ADENINE - Abstract
Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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7. Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis.
- Author
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Vénien-Bryan, Catherine and Fernandes, Carlos A. H.
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MEMBRANE proteins ,ELECTRON microscopy ,MICROSCOPY ,BANKING industry ,PROTEIN structure ,VITRIFICATION - Abstract
Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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8. Perisomatic Inhibition and Its Relation to Epilepsy and to Synchrony Generation in the Human Neocortex.
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Tóth, Estilla Zsófia, Szabó, Felicia Gyöngyvér, Kandrács, Ágnes, Molnár, Noémi Orsolya, Nagy, Gábor, Bagó, Attila G., Erőss, Loránd, Fabó, Dániel, Hajnal, Boglárka, Rácz, Bence, Wittner, Lucia, Ulbert, István, and Tóth, Kinga
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INTERNEURONS ,CANNABINOID receptors ,SYNCHRONIC order ,EPILEPSY ,PYRAMIDAL neurons ,NEOCORTEX - Abstract
Inhibitory neurons innervating the perisomatic region of cortical excitatory principal cells are known to control the emergence of several physiological and pathological synchronous events, including epileptic interictal spikes. In humans, little is known about their role in synchrony generation, although their changes in epilepsy have been thoroughly investigated. This paper demonstraits how parvalbumin (PV)- and type 1 cannabinoid receptor (CB1R)-positive perisomatic interneurons innervate pyramidal cell bodies, and their role in synchronous population events spontaneously emerging in the human epileptic and non-epileptic neocortex, in vitro. Quantitative electron microscopy showed that the overall, PV+ and CB1R+ somatic inhibitory inputs remained unchanged in focal cortical epilepsy. On the contrary, the size of PV-stained synapses increased, and their number decreased in epileptic samples, in synchrony generating regions. Pharmacology demonstrated—in conjunction with the electron microscopy—that although both perisomatic cell types participate, PV+ cells have stronger influence on the generation of population activity in epileptic samples. The somatic inhibitory input of neocortical pyramidal cells remained almost intact in epilepsy, but the larger and consequently more efficient somatic synapses might account for a higher synchrony in this neuron population. This, together with epileptic hyperexcitability, might make a cortical region predisposed to generate or participate in hypersynchronous events. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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9. The Advances and Limitations of the Determination and Applications of Water Structure in Molecular Engineering.
- Author
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Zsidó, Balázs Zoltán, Bayarsaikhan, Bayartsetseg, Börzsei, Rita, Szél, Viktor, Mohos, Violetta, and Hetényi, Csaba
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MOLECULAR structure ,STRUCTURAL engineering ,DRUG design ,DRUG interactions ,METHANE hydrates ,BINDING energy ,THERMODYNAMICS - Abstract
Water is a key actor of various processes of nature and, therefore, molecular engineering has to take the structural and energetic consequences of hydration into account. While the present review focuses on the target–ligand interactions in drug design, with a focus on biomolecules, these methods and applications can be easily adapted to other fields of the molecular engineering of molecular complexes, including solid hydrates. The review starts with the problems and solutions of the determination of water structures. The experimental approaches and theoretical calculations are summarized, including conceptual classifications. The implementations and applications of water models are featured for the calculation of the binding thermodynamics and computational ligand docking. It is concluded that theoretical approaches not only reproduce or complete experimental water structures, but also provide key information on the contribution of individual water molecules and are indispensable tools in molecular engineering. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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10. Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs.
- Author
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Surya, Wahyu, Yong, Clare Pei Yii, Tyagi, Anu, Bhushan, Shashi, and Torres, Jaume
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ESCHERICHIA coli ,MEMBRANE proteins ,OLIGOMERIZATION ,SCAFFOLD proteins ,DETERGENTS ,AQUAPORINS - Abstract
Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addition to this quaternary organization, some aquaporins can act as adhesion molecules in membrane junctions, when tetramers located in opposing membranes interact via their extracellular domains. These stacked forms have been observed in a range of aquaporins, whether using lipidic membrane environments, in electron crystallography, or using detergent micelles, in single-particle cryo-electron microscopy (cryo-EM). In the latter technique, structural studies can be performed when the aquaporin is reconstituted into nanodiscs of lipids that are surrounded by a protein scaffold. During attempts to study E. coli Aquaporin Z (AqpZ), we have found that in some conditions these nanodiscs tend to form filaments that appear to be either thicker head-to-tail or thinner side-to-side stacks of nanodiscs. Nanodisc oligomerization was observed using orthogonal analytical techniques analytical ultra-centrifugation and mass photometry, although the nature of the oligomers (head-to-tail or side-to-side) could not be determined. Using the latter technique, the AqpZ tetramer itself formed oligomers of increasing size when solubilized only in detergent, which is consistent with multiple stacking of AqpZ tetramers. We observed images consistent with both of these filaments in negative staining EM conditions, but only thicker filaments in cryo-EM conditions. We hypothesize that the apparent nanodisc side-to-side arrangement that can only be visualized in negative staining conditions is related to artifacts due to the sample preparation. Filaments of any kind were not observed in EM when nanodiscs did not contain AqpZ, or after addition of detergent into the nanodisc cryo-EM preparation, at concentrations that did not disrupt nanodisc formation. To our knowledge, these filaments have not been observed in nanodiscs preparations of other membrane proteins. AqpZ, like other aquaporins has a charge asymmetry between the cytoplasmic (more positive) and the extracellular sides, which may explain the likely head-to-tail stacking observed, both in nanodisc preparations and also in detergent micelles. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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11. Synaptic Targets of Glycinergic Neurons in Laminae I–III of the Spinal Dorsal Horn.
- Author
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Miranda, Camila Oliveira, Hegedüs, Krisztina, Kis, Gréta, and Antal, Miklós
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NEURONS ,GLYCINE receptors ,INTERNEURONS ,NEURAL circuitry ,GLUTAMATE transporters ,IN situ hybridization ,GLUTAMATE receptors ,ELECTRON microscopy - Abstract
A great deal of evidence supports the inevitable importance of spinal glycinergic inhibition in the development of chronic pain conditions. However, it remains unclear how glycinergic neurons contribute to the formation of spinal neural circuits underlying pain-related information processing. Thus, we intended to explore the synaptic targets of spinal glycinergic neurons in the pain processing region (laminae I–III) of the spinal dorsal horn by combining transgenic technology with immunocytochemistry and in situ hybridization accompanied by light and electron microscopy. First, our results suggest that, in addition to neurons in laminae I–III, glycinergic neurons with cell bodies in lamina IV may contribute substantially to spinal pain processing. On the one hand, we show that glycine transporter 2 immunostained glycinergic axon terminals target almost all types of excitatory and inhibitory interneurons identified by their neuronal markers in laminae I–III. Thus, glycinergic postsynaptic inhibition, including glycinergic inhibition of inhibitory interneurons, must be a common functional mechanism of spinal pain processing. On the other hand, our results demonstrate that glycine transporter 2 containing axon terminals target only specific subsets of axon terminals in laminae I–III, including nonpeptidergic nociceptive C fibers binding IB4 and nonnociceptive myelinated A fibers immunoreactive for type 1 vesicular glutamate transporter, indicating that glycinergic presynaptic inhibition may be important for targeting functionally specific subpopulations of primary afferent inputs. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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12. Modern Microscopic Approaches to Astrocytes.
- Author
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Morita, Mitsuhiro
- Subjects
LIGHT absorption ,ASTROCYTES ,INFORMATION technology ,OPTICAL properties ,RAMAN spectroscopy - Abstract
Microscopy started as the histological analysis based on intrinsic optical properties of tissues such as the refractive index and light absorption, and is expanding to include the visualization of organelles by chemical staining, localization of molecules by immunostaining, physiological measurements such as Ca
2+ imaging, functional manipulation by optogenetics, and comprehensive analysis of chemical composition by Raman spectra. The microscope is one of the most important tools in neuroscience, which aims to reveal the complex intercellular communications underlying brain function and pathology. Many aspects of astrocytes, including the structures of their fine processes and physiological activities in concert with neurons and blood vessels, were revealed in the course of innovations in modern microscopy. The evolution of modern microscopy is a consequence of breakthroughs in spatiotemporal resolutions and expansions in molecular and physiological targets due to the progress in optics and information technology, as well as the inventions of probes using organic chemistry and molecular biology. This review overviews the modern microscopic approach to astrocytes. [ABSTRACT FROM AUTHOR]- Published
- 2023
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13. Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes.
- Author
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Zanotti, Simona, Magri, Francesca, Salani, Sabrina, Napoli, Laura, Ripolone, Michela, Ronchi, Dario, Fortunato, Francesco, Ciscato, Patrizia, Velardo, Daniele, D'Angelo, Maria Grazia, Gualandi, Francesca, Nigro, Vincenzo, Sciacco, Monica, Corti, Stefania, Comi, Giacomo Pietro, and Piga, Daniela
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EXTRACELLULAR matrix ,GENETIC variation ,MUSCULAR dystrophy ,MUSCLE diseases ,G protein coupled receptors ,COLLAGEN - Abstract
Collagen VI is a heterotrimeric protein expressed in several tissues and involved in the maintenance of cell integrity. It localizes at the cell surface, creating a microfilamentous network that links the cytoskeleton to the extracellular matrix. The heterotrimer consists of three chains encoded by COL6A1, COL6A2 and COL6A3 genes. Recessive and dominant molecular defects cause two main disorders, the severe Ullrich congenital muscular dystrophy and the relatively mild and slowly progressive Bethlem myopathy. We analyzed the clinical aspects, pathological features and mutational spectrum of 15 COL6-mutated patients belonging to our cohort of muscular dystrophy probands. Patients presented a heterogeneous phenotype ranging from severe forms to mild adult-onset presentations. Molecular analysis by NGS detected 14 different pathogenic variants, three of them so far unreported. Two changes, localized in the triple-helical domain of COL6A1, were associated with a more severe phenotype. Histological, immunological and ultrastructural techniques were employed for the validation of the genetic variants; they documented the high variability in COL6 distribution and the extracellular matrix disorganization, highlighting the clinical heterogeneity of our cohort. The combined use of these different technologies is pivotal in the diagnosis of COL6 patients. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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14. TERT Extra-Telomeric Roles: Antioxidant Activity and Mitochondrial Protection.
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Marinaccio, Jessica, Micheli, Emanuela, Udroiu, Ion, Di Nottia, Michela, Carrozzo, Rosalba, Baranzini, Nicolò, Grimaldi, Annalisa, Leone, Stefano, Moreno, Sandra, Muzzi, Maurizio, and Sgura, Antonella
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TELOMERES ,TELOMERASE reverse transcriptase ,MITOCHONDRIA ,CELLULAR aging ,REACTIVE oxygen species ,MEMBRANE potential ,MITOCHONDRIAL membranes - Abstract
Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H
2 O2 treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H2 O2 treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality. [ABSTRACT FROM AUTHOR]- Published
- 2023
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15. Delimiting CD34+ Stromal Cells/Telocytes Are Resident Mesenchymal Cells That Participate in Neovessel Formation in Skin Kaposi Sarcoma.
- Author
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Díaz-Flores, Lucio, Gutiérrez, Ricardo, González-Gómez, Miriam, García, Maria del Pino, Palmas, Marta, Carrasco, Jose Luis, Madrid, Juan Francisco, and Díaz-Flores Jr., Lucio
- Subjects
STROMAL cells ,KAPOSI'S sarcoma ,CD34 antigen ,ENDOTHELIAL cells ,BLOOD vessels ,ELECTRON microscopy - Abstract
Kaposi sarcoma (KS) is an angioproliferative lesion in which two main KS cell sources are currently sustained: endothelial cells (ECs) and mesenchymal/stromal cells. Our objective is to establish the tissue location, characteristics and transdifferentiation steps to the KS cells of the latter. For this purpose, we studied specimens of 49 cases of cutaneous KS using immunochemistry and confocal and electron microscopy. The results showed that delimiting CD34+ stromal cells/Telocytes (CD34+SCs/TCs) in the external layer of the pre-existing blood vessels and around skin appendages form small convergent lumens, express markers for ECs of blood and lymphatic vessels, share ultrastructural characteristics with ECs and participate in the origin of two main types of neovessels, the evolution of which gives rise to lymphangiomatous or spindle-cell patterns—the substrate of the main KS histopathological variants. Intraluminal folds and pillars (papillae) are formed in the neovessels, which suggests they increase by vessel splitting (intussusceptive angiogenesis and intussusceptive lymphangiogenesis). In conclusion, delimiting CD34+SCs/TCs are mesenchymal/stromal cells that can transdifferentiate into KS ECs, participating in the formation of two types of neovessels. The subsequent growth of the latter involves intussusceptive mechanisms, originating several KS variants. These findings are of histogenic, clinical and therapeutic interest. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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16. Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a.
- Author
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Leyder, Tanguy, Mignon, Julien, Mottet, Denis, and Michaux, Catherine
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ZINC-finger proteins ,TERTIARY structure ,ELECTRON microscopy ,NEURODEGENERATION ,PROTEINS ,DENATURATION of proteins - Abstract
Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically disordered proteins. DPF3 isoforms differ from their C-terminal region (C-TERb and C-TERa), containing zinc fingers and disordered domains. Herein, we investigated the disorder aggregation properties of C-TER isoforms. In agreement with the predictions, spectroscopy highlighted a lack of a highly ordered structure, especially for C-TERa. Over a few days, both C-TERs were shown to spontaneously assemble into similar antiparallel and parallel β-sheet-rich fibrils. Altered metal homeostasis being a neurodegeneration hallmark, we also assessed the influence of divalent metal cations, namely Cu
2+ , Mg2+ , Ni2+ , and Zn2+ , on the C-TER aggregation pathway. Circular dichroism revealed that metal binding does not impair the formation of β-sheets, though metal-specific tertiary structure modifications were observed. Through intrinsic and extrinsic fluorescence, we found that metal cations differently affect C-TERb and C-TERa. Cu2+ and Ni2+ have a strong inhibitory effect on the aggregation of both isoforms, whereas Mg2+ impedes C-TERb fibrillation and, on the contrary, enhances that of C-TERa. Upon Zn2+ binding, C-TERb aggregation is also hindered, and the amyloid autofluorescence of C-TERa is remarkably red-shifted. Using electron microscopy, we confirmed that the metal-induced spectral changes are related to the morphological diversity of the aggregates. While metal-treated C-TERb formed breakable and fragmented filaments, C-TERa fibrils retained their flexibility and packing properties in the presence of Mg2+ and Zn2+ cations. [ABSTRACT FROM AUTHOR]- Published
- 2022
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17. Low-Intensity Pulsed Ultrasound Counteracts Advanced Glycation End Products-Induced Corpus Cavernosal Endothelial Cell Dysfunction via Activating Mitophagy.
- Author
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Chen, Yuzhuo, Xiao, Ming, Zhao, Liangyu, Huang, Yongquan, Lin, Yuhong, Xie, Ting, Tian, Jiali, Wang, Qi, Tang, Yuxin, and Su, Zhongzhen
- Subjects
ADVANCED glycation end-products ,ENDOTHELIUM diseases ,RECEPTOR for advanced glycation end products (RAGE) ,ULTRASONIC imaging ,HIGH-intensity focused ultrasound ,REACTIVE oxygen species ,ENDOTHELIAL cells ,CORPORA ,ELECTRON microscopy - Abstract
Injury to corpus cavernosal endothelial cells (CCECs) is an important pathological basis of diabetes mellitus-induced erectile dysfunction (DMED), while low-intensity pulsed ultrasound (LIPUS) has been shown to improve erectile function in DMED. To further understand its therapeutic mechanism of action, in this study, we first demonstrated increased apoptosis and shedding in the CCECs of DMED patients, accompanied by significant mitochondrial injury by immunohistochemistry and electron microscopy of corpus cavernosum tissue. Next, we used advanced glycation end products (AGEs) to simulate the diabetic environment in vitro and found that AGES damaged mitochondria and inhibited angiogenesis in CCECs in a dose-dependent manner, while LIPUS treatment significantly reversed its effects. Mechanistic studies based on transcriptome sequencing showed that LIPUS significantly up-regulated LC3 and PARKIN protein levels in mitochondria, promoted mitophagy, and affected mitochondrial dynamics and reactive oxygen species (ROS) production. In addition, the protective effects of LIPUS were abrogated when mitophagy was inhibited by 3-methyladenine. In summary, LIPUS exerted potent inhibitory effects on AGES-induced CCEC failure via mitophagy, providing a theoretical basis for DMED treatment that encompasses the protection of endothelial structure and function. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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18. Ligand-Based Discovery of a Small Molecule as Inhibitor of α-Synuclein Amyloid Formation.
- Author
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De Luca, Laura, Vittorio, Serena, Peña-Díaz, Samuel, Pitasi, Giovanna, Fornt-Suñé, Marc, Bucolo, Federica, Ventura, Salvador, and Gitto, Rosaria
- Subjects
SMALL molecules ,ALPHA-synuclein ,AMYLOID beta-protein ,PARKINSON'S disease ,AMYLOID ,ELECTRON microscopy ,MEDICAL screening - Abstract
α-Synuclein (α-Syn) aggregates are implicated in Parkinson's disease (PD), so inhibitors of α-Syn aggregation have been intensively explored. It has been demonstrated that small molecules might be able to reduce α-Syn aggregation in fibrils, thus exerting neuroprotective effects in models of PD. To expand our knowledge about the structural requirements for blocking the recognition process into the oligomeric assembly of α-Syn aggregates, we performed a ligand-based virtual screening procedure using two well-known α-Syn aggregation inhibitors, SynuClean-D and ZPD-2, as query compounds. A collection of thirty-four compounds bearing distinct chemical functionalities and mutual chemical features were studied in a Th-T fluorescence test, thus identifying 5-(2,6-dinitro-4-(trifluoromethyl)benzyl)-1-methyl-1H-tetrazole (named MeSC-04) as a potent α-Syn amyloid formation inhibitor that demonstrated similar behavior when compared to SynuClean-D in the thioflavin-T-monitored kinetic assays, with both molecules reducing the number and size of amyloid fibrils, as evidenced by electron microscopy. Molecular modeling studies suggested the binding mode of MeSC-04 through the identification of putative druggable pockets on α-syn fibrils and a subsequent consensus docking methodology. Overall, this work could furnish new insights in the development of α-Syn amyloid inhibitors from synthetic sources. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
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19. Estimation of SPIO Nanoparticles Uptakes by Macrophages Using Transmission Electron Microscopy.
- Author
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Aleid, Adham, Alhussaini, Khalid, Almijalli, Mohammed, and Saad, Ali S.
- Subjects
TRANSMISSION electron microscopy ,NANOMEDICINE ,MACROPHAGES ,TARGETED drug delivery ,HIGH resolution imaging ,IRON oxide nanoparticles ,MONOCYTES ,RED - Abstract
Due to their interesting size-dependent magnetic characteristics and relative biocompatibility, magnetic superparamagnetic iron oxide (SPIO) nanoparticles have been widely exploited as probes for cell and subcellular structure identification, as well as medication and gene delivery. A thorough understanding of the mechanics of the interaction between nanoparticles and macrophages is vital in managing dynamic processes in nanomedicine. In this study, the interaction behavior and uptake of SPIO nanoparticles by M1- and M2-type macrophages were investigated. Mice monocytes were differentiated into M1 and M2 macrophages, and the uptake of SPIO nanoparticles was studied using a TEM microscope. A high resolution image of 1 nm resolution, an image processing technique, was developed to extract the SPIO-NPs from tomographic TEM microscopic images. Lysosomes appear to be the zones of high concentrations of SPIO inside macrophages. Lysosomes were first selected in each image, and then segmentation by the Otsu thresholding method was used to extract the SPIO-NPs. The Otsu threshold method is a global thresholding technique used to automatically differentiate SPIOs from the background. The SPIO-NPs appear in red colors, and the other pixels in the image are considered background. Then, an estimation of the SPIO-NP uptakes by lysosomes is produced. Higher uptake of all-sized nanoparticles was observed in M1- and M2-type macrophages. An accurate estimation of the number of SPIO-NPs was obtained. This result will help in controlling targeted drug delivery and assessing the safety impact of the use of SPIO-NPs in nanomedicine for humans. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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20. The High Plasticity of Nonpathogenic Mycobacterium brumae Induces Rapid Changes in Its Lipid Profile during Pellicle Maturation: The Potential of This Bacterium as a Versatile Cell Factory for Lipid Compounds of Therapeutic Interest.
- Author
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Campo-Pérez, Víctor, Guallar-Garrido, Sandra, Luquin, Marina, Sánchez-Chardi, Alejandro, and Julián, Esther
- Subjects
MYCOBACTERIUM ,LIPID synthesis ,MOLECULAR weights ,CELL culture ,MYCOBACTERIA - Abstract
The immunomodulatory potential of mycobacteria to be used for therapeutic purposes varies by species and culture conditions and is closely related to mycobacterial lipid composition. Although the lipids present in the mycobacterial cell wall are relevant, lipids are mainly stored in intracellular lipid inclusions (ILIs), which have emerged as a crucial structure in understanding mycobacteria-host interaction. Little is known about ILI ultrastructure, production, and composition in nonpathogenic species. In this study, we compared the lipid profiles of the nonpathogenic immunomodulatory agent Mycobacterium brumae during pellicle maturation under different culture conditions with qualitative and quantitative approaches by using high-resolution imaging and biochemical and composition analyses to understand ILI dynamics. The results showed wax esters, mainly in early stages of development, and acylglycerols in mature ILI composition, revealing changes in dynamics, amount, and morphometry, depending on pellicle maturation and the culture media used. Low-glycerol cultures induced ILIs with lower molecular weights which were smaller in size in comparison with the ILIs produced in glycerol-enriched media. The data also indicate the simple metabolic plasticity of lipid synthesis in M. brumae, as well as its high versatility in generating different lipid profiles. These findings provide an interesting way to enhance the production of key lipid structures via the simple modulation of cell culture conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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21. Alteration in the Synaptic and Extrasynaptic Organization of AMPA Receptors in the Hippocampus of P301S Tau Transgenic Mice.
- Author
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Alfaro-Ruiz, Rocio, Aguado, Carolina, Martín-Belmonte, Alejandro, Moreno-Martínez, Ana Esther, Merchán-Rubira, Jesús, Hernández, Félix, Ávila, Jesús, Fukazawa, Yugo, and Luján, Rafael
- Subjects
AMPA receptors ,TRANSGENIC mice ,INTERNEURONS ,DENDRITIC spines ,PYRAMIDAL neurons ,NEUROFIBRILLARY tangles ,TAU proteins ,ALZHEIMER'S disease - Abstract
Tau pathology is a hallmark of Alzheimer's disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
22. Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from Sinapis alba.
- Author
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Ruedas, Rémi, Muthukumar, Soumiya Sankari, Kieffer-Jaquinod, Sylvie, Gillet, François-Xavier, Fenel, Daphna, Effantin, Grégory, Pfannschmidt, Thomas, Couté, Yohann, Blanvillain, Robert, and Cobessi, David
- Subjects
RNA polymerases ,SURFACE interactions ,MASS spectrometry ,CHLOROPLAST formation ,ELECTRON microscopy ,SEQUENCE alignment ,CHLOROPLAST DNA - Abstract
RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of the plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
23. Ibuprofen Favors Binding of Amyloid-β Peptide to Its Depot, Serum Albumin.
- Author
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Litus, Ekaterina A., Kazakov, Alexey S., Deryusheva, Evgenia I., Nemashkalova, Ekaterina L., Shevelyova, Marina P., Machulin, Andrey V., Nazipova, Aliya A., Permyakova, Maria E., Uversky, Vladimir N., and Permyakov, Sergei E.
- Abstract
The deposition of amyloid-β peptide (Aβ) in the brain is a critical event in the progression of Alzheimer's disease (AD). This Aβ deposition could be prevented by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aβ. We show here that an exogenous HSA ligand, ibuprofen (IBU), exerts the analogous effect. Plasmon resonance spectroscopy data evidence that a therapeutic IBU level increases HSA affinity to monomeric Aβ
40 /Aβ42 by a factor of 3–5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aβ40 fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aβ40 -binding sites of HSA. The revealed enhancement of the HSA–Aβ interaction by IBU and the strengthened inhibition of Aβ fibrillation by HSA in the presence of IBU could contribute to the neuroprotective effects of the latter, previously observed in mouse and human studies of AD. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
24. A Reliable Approach for Revealing Molecular Targets in Secondary Ion Mass Spectrometry.
- Author
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Li, Fengxia, Fornasiero, Eugenio F., Dankovich, Tal M., Kluever, Verena, and Rizzoli, Silvio O.
- Subjects
SECONDARY ion mass spectrometry ,DRUG target ,IMAGING systems in chemistry ,LIFE sciences ,ELECTRON microscopy ,FLUORESCENCE microscopy - Abstract
Nano secondary ion mass spectrometry (nanoSIMS) imaging is a rapidly growing field in biological sciences, which enables investigators to describe the chemical composition of cells and tissues with high resolution. One of the major challenges of nanoSIMS is to identify specific molecules or organelles, as these are not immediately recognizable in nanoSIMS and need to be revealed by SIMS-compatible probes. Few laboratories have generated such probes, and none are commercially available. To address this, we performed a systematic study of probes initially developed for electron microscopy. Relying on nanoscale SIMS, we found that antibodies coupled to 6 nm gold particles are surprisingly efficient in terms of labeling specificity while offering a reliable detection threshold. These tools enabled accurate visualization and sample analysis and were easily employed in correlating SIMS with other imaging approaches, such as fluorescence microscopy. We conclude that antibodies conjugated to moderately sized gold particles are promising tools for SIMS imaging. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
25. Elongation and Contraction of Scallop Sarcoplasmic Reticulum (SR): ATP Stabilizes Ca 2+ -ATPase Crystalline Array Elongation of SR Vesicles.
- Author
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Nakamura, Jun, Maruyama, Yuusuke, Tajima, Genichi, Suwa, Makiko, and Sato, Chikara
- Subjects
SARCOPLASMIC reticulum ,SCALLOPS ,ADENOSINE triphosphatase ,ELECTRON microscopy ,MONOMERS - Abstract
The Ca
2+ -ATPase is an integral transmembrane Ca2+ pump of the sarcoplasmic reticulum (SR). Crystallization of the cytoplasmic surface ATPase molecules of isolated scallop SR vesicles was studied at various calcium concentrations by negative stain electron microscopy. In the absence of ATP, round SR vesicles displaying an assembly of small crystalline patches of ATPase molecules were observed at 18 µM [Ca2+ ]. These partly transformed into tightly elongated vesicles containing ATPase crystalline arrays at low [Ca2+ ] (≤1.3 µM). The arrays were classified as ''tetramer", "two-rail" (like a railroad) and ''monomer". Their crystallinity was low, and they were unstable. In the presence of ATP (5 mM) at a low [Ca2+ ] of ~0.002 µM, "two-rail" arrays of high crystallinity appeared more frequently in the tightly elongated vesicles and the distinct tetramer arrays disappeared. During prolonged (~2.5 h) incubation, ATP was consumed and tetramer arrays reappeared. A specific ATPase inhibitor, thapsigargin, prevented both crystal formation and vesicle elongation in the presence of ATP. Together with the second part of this study, these data suggest that the ATPase forms tetramer units and longer tetramer crystalline arrays to elongate SR vesicles, and that the arrays transform into more stable "two-rail" forms in the presence of ATP at low [Ca2+ ]. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
26. Molecular Imaging in Nanomedical Research 2.0.
- Author
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Malatesta, Manuela
- Subjects
APTAMERS ,IRON oxide nanoparticles ,NUCLEAR magnetic resonance spectroscopy ,MAGNETIC resonance imaging ,MICROSCOPY ,MAGNETIC particle imaging ,ELECTRON microscopy - Abstract
Over the last two decades, imaging techniques have become irreplaceable tools in nanotechnology: electron microscopy techniques are routinely used to observe the structural features of newly manufactured nanoconstructs, while light and electron microscopy, magnetic resonance imaging, optical imaging, positron emission tomography, and ultrasound imaging allow dynamic monitoring of the biodistribution, targeting and clearance of nanoparticulates in living systems, either for the whole organism or at the level of single cells, tissues and organs [[1]]. After describing the concepts and characteristics of general optical coherence tomography systems, Wang and co-workers [[9]] describe the recent progress in the application of contrast agents, focusing on the combination of optical coherence tomography and nanoparticle contrast agents in the nanomedical field. Su and co-workers [[4]] aimed to develop an efficient near-infrared imaging method to monitor the kinetics of single-domain antibodies from camelids (VHH) and VHH-conjugated iron oxide nanoparticles in mice. [Extracted from the article]
- Published
- 2022
- Full Text
- View/download PDF
27. Perisomatic Inhibition and Its Relation to Epilepsy and to Synchrony Generation in the Human Neocortex
- Author
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Estilla Zsófia Tóth, Felicia Gyöngyvér Szabó, Ágnes Kandrács, Noémi Orsolya Molnár, Gábor Nagy, Attila G. Bagó, Loránd Erőss, Dániel Fabó, Boglárka Hajnal, Bence Rácz, Lucia Wittner, István Ulbert, and Kinga Tóth
- Subjects
perisomatic inhibition ,basket cell ,human ,epilepsy ,synchrony ,electron microscopy ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Inhibitory neurons innervating the perisomatic region of cortical excitatory principal cells are known to control the emergence of several physiological and pathological synchronous events, including epileptic interictal spikes. In humans, little is known about their role in synchrony generation, although their changes in epilepsy have been thoroughly investigated. This paper demonstraits how parvalbumin (PV)- and type 1 cannabinoid receptor (CB1R)-positive perisomatic interneurons innervate pyramidal cell bodies, and their role in synchronous population events spontaneously emerging in the human epileptic and non-epileptic neocortex, in vitro. Quantitative electron microscopy showed that the overall, PV+ and CB1R+ somatic inhibitory inputs remained unchanged in focal cortical epilepsy. On the contrary, the size of PV-stained synapses increased, and their number decreased in epileptic samples, in synchrony generating regions. Pharmacology demonstrated—in conjunction with the electron microscopy—that although both perisomatic cell types participate, PV+ cells have stronger influence on the generation of population activity in epileptic samples. The somatic inhibitory input of neocortical pyramidal cells remained almost intact in epilepsy, but the larger and consequently more efficient somatic synapses might account for a higher synchrony in this neuron population. This, together with epileptic hyperexcitability, might make a cortical region predisposed to generate or participate in hypersynchronous events.
- Published
- 2021
- Full Text
- View/download PDF
28. Perisomatic Inhibition and Its Relation to Epilepsy and to Synchrony Generation in the Human Neocortex
- Author
-
Estilla Zsófia Tóth, Felicia Gyöngyvér Szabó, Ágnes Kandrács, Noémi Orsolya Molnár, Gábor Nagy, Attila G. Bagó, Loránd Erőss, Dániel Fabó, Boglárka Hajnal, Bence Rácz, Lucia Wittner, István Ulbert, and Kinga Tóth
- Subjects
Adult ,Male ,QH301-705.5 ,Action Potentials ,Neocortex ,Article ,Catalysis ,Inorganic Chemistry ,Interneurons ,Humans ,perisomatic inhibition ,basket cell ,human ,epilepsy ,synchrony ,electron microscopy ,Cortical Synchronization ,Physical and Theoretical Chemistry ,Biology (General) ,Receptors, Cannabinoid ,Molecular Biology ,QD1-999 ,Spectroscopy ,Aged ,Aged, 80 and over ,Organic Chemistry ,Neural Inhibition ,General Medicine ,Middle Aged ,Computer Science Applications ,Chemistry ,Parvalbumins ,Synapses ,Female - Abstract
Inhibitory neurons innervating the perisomatic region of cortical excitatory principal cells are known to control the emergence of several physiological and pathological synchronous events, including epileptic interictal spikes. In humans, little is known about their role in synchrony generation, although their changes in epilepsy have been thoroughly investigated. This paper demonstraits how parvalbumin (PV)- and type 1 cannabinoid receptor (CB1R)-positive perisomatic interneurons innervate pyramidal cell bodies, and their role in synchronous population events spontaneously emerging in the human epileptic and non-epileptic neocortex, in vitro. Quantitative electron microscopy showed that the overall, PV+ and CB1R+ somatic inhibitory inputs remained unchanged in focal cortical epilepsy. On the contrary, the size of PV-stained synapses increased, and their number decreased in epileptic samples, in synchrony generating regions. Pharmacology demonstrated—in conjunction with the electron microscopy—that although both perisomatic cell types participate, PV+ cells have stronger influence on the generation of population activity in epileptic samples. The somatic inhibitory input of neocortical pyramidal cells remained almost intact in epilepsy, but the larger and consequently more efficient somatic synapses might account for a higher synchrony in this neuron population. This, together with epileptic hyperexcitability, might make a cortical region predisposed to generate or participate in hypersynchronous events.
- Published
- 2022
29. Inhaled Cadmium Oxide Nanoparticles: Their in Vivo Fate and Effect on Target Organs.
- Author
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Dumkova, Jana, Vrlikova, Lucie, Vecera, Zbynek, Putnova, Barbora, Docekal, Bohumil, Mikuska, Pavel, Fictum, Petr, Hampl, Ales, and Buchtova, Marcela
- Subjects
CADMIUM oxide ,NANOPARTICLES ,HEAVY metals ,POLLUTION ,MITOCHONDRIA - Abstract
The increasing amount of heavy metals used in manufacturing equivalently increases hazards of environmental pollution by industrial products such as cadmium oxide (CdO) nanoparticles. Here, we aimed to unravel the CdO nanoparticle destiny upon their entry into lungs by inhalations, with the main focus on the ultrastructural changes that the nanoparticles may cause to tissues of the primary and secondary target organs. We indeed found the CdO nanoparticles to be transported from the lungs into secondary target organs by blood. In lungs, inhaled CdO nanoparticles caused significant alterations in parenchyma tissue including hyperemia, enlarged pulmonary septa, congested capillaries, alveolar emphysema and small areas of atelectasis. Nanoparticles were observed in the cytoplasm of cells lining bronchioles, in the alveolar spaces as well as inside the membranous pneumocytes and in phagosomes of lung macrophages. Nanoparticles even penetrated through the membrane into some organelles including mitochondria and they also accumulated in the cytoplasmic vesicles. In livers, inhalation caused periportal inflammation and local hepatic necrosis. Only minor changes such as diffusely thickened filtration membrane with intramembranous electron dense deposits were observed in kidney. Taken together, inhaled CdO nanoparticles not only accumulated in lungs but they were also transported to other organs causing serious damage at tissue as well as cellular level. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
30. Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line.
- Author
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Ren, Binhai, Chang Tao, Swan, Margaret Anne, Joachim, Nichole, Martiniello-Wilks, Rosetta, Nassif, Najah T., O'Brien, Bronwyn A., and Simpson, Ann M.
- Subjects
PANCREATIC hormones ,ANIMAL models of diabetes ,GENE therapy ,TREATMENT of diabetes ,BLOOD sugar measurement ,ELECTRON microscopy - Abstract
Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone), H4IIE/ND (NeuroD1 gene alone), and H4IIEins/ND (insulin and NeuroD1 genes). The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ±l 171.8 pmol/insulin/ 5 × 106 cells, respectively. Additionally, several β cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0-20 mmol/L) was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
31. Weekly Treatment of 2-Hydroxypropyl-β-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice.
- Author
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Walenbergh, Sofie M. A., Houben, Tom, Hendrikx, Tim, Jeurissen, Mike L. J., van Gorp, Patrick J., Vaes, Nathalie, Olde Damink, Steven W. M., Verheyen, Fons, Koek, Ger H., Lütjohann, Dieter, Grebe, Alena, Latz, Eicke, and Shiri-Sverdlov, Ronit
- Subjects
CYCLODEXTRINS ,CHOLESTEROL metabolism ,FAMILIAL hypercholesterolemia ,MICE as carriers of disease ,METABOLIC syndrome treatment ,ELECTRON microscopy ,LYSOSOMES ,THERAPEUTICS - Abstract
Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Two-hydroxypropyl-β-cyclodextrin (HP-B-CD) is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs). Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr
-/- ) mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE) storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7α-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD. [ABSTRACT FROM AUTHOR]- Published
- 2015
- Full Text
- View/download PDF
32. Development of a New Monomer for the Synthesis of Intrinsic Antimicrobial Polymers with Enhanced Material Properties.
- Author
-
Brodkorb, Florian, Fischer, Björn, Kalbfleisch, Katrin, Robers, Oliver, Braun, Carina, Dohlen, Sophia, Kreyenschmidt, Judith, Lorenz, Reinhard, and Kreyenschmidt, Martin
- Subjects
ANTI-infective agents ,DEPOLYMERIZATION ,ANTIMICROBIAL polymers ,SECONDARY amines ,ELECTRON microscopy ,CELL membranes ,NEONATAL Escherichia coli infections ,STAPHYLOCOCCUS aureus - Abstract
The use of biocidal compounds in polymers is steadily increasing because it is one solution to the need for safety and hygiene. It is possible to incorporate an antimicrobial moiety to a polymer. These polymers are referred to as intrinsic antimicrobial. The biocidal action results from contact of the polymer to the microorganisms, with no release of active molecules. This is particularly important in critical fields like food technology, medicine and ventilation technology, where migration or leaching is crucial and undesirable. The isomers N-(1,1-dimethylethyl)-4-ethenyl-benzenamine and N-(1,1-dimethyl-ethyl)-3- ethenyl-benzenamine (TBAMS) are novel (Co-)Monomers for intrinsic anti-microbial polymers. The secondary amines were prepared and polymerized to the corresponding water insoluble polymer. The antimicrobial activity was analyzed by the test method JIS Z 2801:2000. Investigations revealed a high antimicrobial activity against Staphylococcus aureus and Escherichia coli with a reduction level of >4.5 log
10 units. Furthermore, scanning electron microscopy (SEM) of E. coli. in contact with the polymer indicates a bactericidal action which is caused by disruption of the bacteria cell membranes, leading to lysis of the cells. [ABSTRACT FROM AUTHOR]- Published
- 2015
- Full Text
- View/download PDF
33. Gal3 Plays a Deleterious Role in a Mouse Model of Endotoxemia.
- Author
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Fernández-Martín, Juan Carlos, Espinosa-Oliva, Ana María, García-Domínguez, Irene, Rosado-Sánchez, Isaac, Pacheco, Yolanda M., Moyano, Rosario, Monterde, José G., Venero, José Luis, and de Pablos, Rocío M.
- Subjects
ENDOTOXEMIA ,LABORATORY mice ,ANIMAL disease models ,LIPOPOLYSACCHARIDES ,SEPSIS ,INTRAPERITONEAL injections ,KNOCKOUT mice ,ELECTRON microscopy - Abstract
Lipopolysaccharide (LPS)-induced endotoxemia induces an acute systemic inflammatory response that mimics some important features of sepsis, the disease with the highest mortality rate worldwide. In this work, we have analyzed a murine model of endotoxemia based on a single intraperitoneal injection of 5 mg/kg of LPS. We took advantage of galectin-3 (Gal3) knockout mice and found that the absence of Gal3 decreased the mortality rate oflethal endotoxemia in the first 80 h after the administration of LPS, along with a reduction in the tissular damage in several organs measured by electron microscopy. Using flow cytometry, we demonstrated that, in control conditions, peripheral immune cells, especially monocytes, exhibited high levels of Gal3, which were early depleted in response to LPS injection, thus suggesting Gal3 release under endotoxemia conditions. However, serum levels of Gal3 early decreased in response to LPS challenge (1 h), an indication that Gal3 may be extravasated to peripheral organs. Indeed, analysis of Gal3 in peripheral organs revealed a robust up-regulation of Gal3 36 h after LPS injection. Taken together, these results demonstrate the important role that Gal3 could play in the development of systemic inflammation, a well-established feature of sepsis, thus opening new and promising therapeutic options for these harmful conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
34. A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules.
- Author
-
Herasimenka, Yury, Kotasinska, Marta, Walter, Stefan, and Schrempf, Hildgund
- Subjects
CHITIN ,POLYSACCHARIDES ,MICROBIOLOGICAL assay ,BIOSYNTHESIS ,ORGANIC synthesis ,BIOCHEMICAL engineering - Abstract
A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis) has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein), has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
35. Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy.
- Author
-
Božič, Darja, Hočevar, Matej, Kisovec, Matic, Pajnič, Manca, Pađen, Ljubiša, Jeran, Marko, Bedina Zavec, Apolonija, Podobnik, Marjetka, Kogej, Ksenija, Iglič, Aleš, and Kralj-Iglič, Veronika
- Subjects
LIGHT scattering ,ELECTRON scattering ,ELECTRON microscopy ,EXTRACELLULAR vesicles ,TRITON X-100 - Abstract
Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4–10), osmolarity (50–1000 mOsm/L), temperature (15–60 °C), and surfactant Triton X-100 (10–500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius (R
h ), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS–citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50–1000 mOsm/L, pH 4–10) had no significant effect on the Rh (=100–130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material. [ABSTRACT FROM AUTHOR]- Published
- 2021
- Full Text
- View/download PDF
36. Over Fifty Years of Life, Death, and Cannibalism: A Historical Recollection of Apoptosis and Autophagy.
- Author
-
Izadi, Mahmoud, Ali, Tayyiba Akbar, and Pourkarimi, Ehsan
- Subjects
AUTOPHAGY ,RECOLLECTION (Psychology) ,MEDICAL sciences ,CANNIBALISM ,CLINICAL medicine - Abstract
Research in biomedical sciences has changed dramatically over the past fifty years. There is no doubt that the discovery of apoptosis and autophagy as two highly synchronized and regulated mechanisms in cellular homeostasis are among the most important discoveries in these decades. Along with the advancement in molecular biology, identifying the genetic players in apoptosis and autophagy has shed light on our understanding of their function in physiological and pathological conditions. In this review, we first describe the history of key discoveries in apoptosis with a molecular insight and continue with apoptosis pathways and their regulation. We touch upon the role of apoptosis in human health and its malfunction in several diseases. We discuss the path to the morphological and molecular discovery of autophagy. Moreover, we dive deep into the precise regulation of autophagy and recent findings from basic research to clinical applications of autophagy modulation in human health and illnesses and the available therapies for many diseases caused by impaired autophagy. We conclude with the exciting crosstalk between apoptosis and autophagy, from the early discoveries to recent findings. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
37. Fractal-Percolation Structure Architectonics in Sol-Gel Synthesis.
- Author
-
Kononova, Irina, Kononov, Pavel, Moshnikov, Vyacheslav, and Ignat'ev, Sergey
- Subjects
THERMAL desorption ,POROUS materials ,ATOMIC force microscopy ,ELECTRON microscopy ,SOL-gel materials ,SOL-gel processes - Abstract
It was developed a new technique to assess micro- and mesopores with sizes below a few nanometers. The porous materials with hierarchical fractal-percolation structure were obtained with the sol-gel method. The tetraethoxysilane hydrolysis and polycondensation reactions were performed in the presence of salts as the sources of metal oxides. The porous materials were obtained under spinodal decomposition conditions during application of the polymer sol to the substrate surface and thermal treatment of the structures. The model is based on an enhanced Kepler net of the 4612 type with hexagonal cells filled with a quasi-two-dimensional projection of the Jullien fractal after the 2nd iteration. The materials obtained with the sol-gel method were studied using the atomic force microscopy, electron microscopy, thermal desorption, as well as an AutoCAD 2022 computer simulation of the percolation transition in a two-component system using the proposed multimodal model. Based on the results obtained, a new method was suggested to assess micro- and mesopores with sizes below a few nanometrs, which cannot be analyzed using the atomic force microscopy and electron microscopy. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
38. The Association between α-Synuclein and α-Tubulin in Brain Synapses.
- Author
-
Amadeo, Alida, Pizzi, Sara, Comincini, Alessandro, Modena, Debora, Calogero, Alessandra Maria, Madaschi, Laura, Faustini, Gaia, Rolando, Chiara, Bellucci, Arianna, Pezzoli, Gianni, Mazzetti, Samanta, and Cappelletti, Graziella
- Subjects
SYNAPSES ,NEURAL transmission ,CORPUS striatum ,TUBULINS ,SUBSTANTIA nigra ,NERVE tissue ,ELECTRON spin ,CENTRAL nervous system - Abstract
α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
39. Internalization of Neutrophil-Derived Microvesicles Modulates TNFα-Stimulated Proinflammatory Cytokine Production in Human Fibroblast-Like Synoviocytes.
- Author
-
Zhan, Dong, Cross, Andrew, Wright, Helen L., Moots, Robert J., Edwards, Steven W., and Honsawek, Sittisak
- Subjects
VESICLES (Cytology) ,CYTOKINES ,TUMOR necrosis factors ,KNEE ,CELL migration inhibition ,NANOMEDICINE ,CELL survival ,ELECTRON microscopy - Abstract
Neutrophil-derived microvesicles (NDMVs) have the potential to exert anti-inflammatory effects. Our study aimed to explore the effects of NDMVs on proinflammatory cytokines expressed by tumor necrosis factor α (TNFα)-stimulated fibroblast-like synoviocytes (FLS). FLS were isolated from the synovium of knee osteoarthritis (OA) patients undergoing surgery. NDMVs, isolated from TNFα-stimulated healthy neutrophils, were characterized by electron microscopy and nanoparticle tracking analysis. MTT and scratch wound healing assays were used to measure FLS viability and migration after treatment with NDMVs, while internalization of fluorescently labeled NDMVs was appraised by flow cytometry and confocal microscopy. Levels of proinflammatory cytokines in supernatants were quantified by the Bio-Plex system. Incubation of FLS with NDMVs at a vesicle/cell ratio of 100 resulted in a time-dependent uptake, with 35% of synoviocytes containing microvesicles over a 6–24 h time period, with no significant change in cell viability. TNFα stimulated the cytokine expression in FLS, and NDMVs down-regulated TNFα-induced expression of IL-5, IL-6, IL-8, MCP-1, IFNγ and MIP-1β. However, this down-regulation was selective, as NDMVs had no significant effects on TNFα-stimulated expression of IL-2 or IL-4. NDMVs were internalized by FLS to inhibit TNFα-stimulated broad-spectrum proinflammatory cytokine secretion. NDMVs, therefore, may exhibit an anti-inflammatory role in the regulation of the FLS function. [ABSTRACT FROM AUTHOR]
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- 2021
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40. Simulated Microgravity Effects on Human Adenocarcinoma Alveolar Epithelial Cells: Characterization of Morphological, Functional, and Epigenetic Parameters.
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Degan, Paolo, Cortese, Katia, Pulliero, Alessandra, Bruno, Silvia, Gagliani, Maria Cristina, Congiu, Matteo, and Izzotti, Alberto
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REDUCED gravity environments ,EPITHELIAL cells ,CELL cycle regulation ,CELL morphology ,BIOLOGICAL systems ,LUNGS ,PLANT mitochondria - Abstract
Background: In space, the reduction or loss of the gravity vector greatly affects the interaction between cells. Since the beginning of the space age, microgravity has been identified as an informative tool in biomedicine, including cancer research. The A549 cell line is a hypotriploid human alveolar basal epithelial cell line widely used as a model for lung adenocarcinoma. Microgravity has been reported to interfere with mitochondrial activity, energy metabolism, cell vitality and proliferation, chemosensitivity, invasion and morphology of cells and organelles in various biological systems. Concerning lung cancer, several studies have reported the ability of microgravity to modulate the carcinogenic and metastatic process. To investigate these processes, A549 cells were exposed to simulated microgravity (µG) for different time points. Methods: We performed cell cycle and proliferation assays, ultrastructural analysis of mitochondria architecture, as well as a global analysis of miRNA modulated under µG conditions. Results: The exposure of A549 cells to microgravity is accompanied by the generation of polynucleated cells, cell cycle imbalance, growth inhibition, and gross morphological abnormalities, the most evident are highly damaged mitochondria. Global miRNA analysis defined a pool of miRNAs associated with µG solicitation mainly involved in cell cycle regulation, apoptosis, and stress response. To our knowledge, this is the first global miRNA analysis of A549 exposed to microgravity reported. Despite these results, it is not possible to draw any conclusion concerning the ability of µG to interfere with the cancerogenic or the metastatic processes in A549 cells. Conclusions: Our results provide evidence that mitochondria are strongly sensitive to µG. We suggest that mitochondria damage might in turn trigger miRNA modulation related to cell cycle imbalance. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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41. The Density of Group I mGlu 5 Receptors Is Reduced along the Neuronal Surface of Hippocampal Cells in a Mouse Model of Alzheimer's Disease.
- Author
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Martín-Belmonte, Alejandro, Aguado, Carolina, Alfaro-Ruiz, Rocío, Albasanz, José Luis, Martín, Mairena, Moreno-Martínez, Ana Esther, Fukazawa, Yugo, and Luján, Rafael
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LABORATORY mice ,ALZHEIMER'S disease ,GRANULE cells ,ANIMAL disease models ,NEURAL transmission ,GLUTAMATE receptors ,PYRAMIDAL neurons - Abstract
Metabotropic glutamate receptor subtype 5 (mGlu
5 ) is implicated in the pathophysiology of Alzheimer's disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the expression and localisation patterns of mGlu5 in the APP/PS1 model of AD at 12 months of age, combining immunoblots, histoblots and high-resolution immunoelectron microscopic approaches. Immunoblots revealed that the total amount of mGlu5 protein in the hippocampus, in addition to downstream molecules, i.e., Gq/11 and PLCβ1 , was similar in both APP/PS1 mice and age-matched wild type mice. Histoblots revealed that mGlu5 expression in the brain and its laminar expression in the hippocampus was also unaltered. However, the ultrastructural techniques of SDS-FRL and pre-embedding immunogold demonstrated that the subcellular localisation of mGlu5 was significantly reduced along the neuronal surface of hippocampal principal cells, including CA1 pyramidal cells and DG granule cells, in APP/PS1 mice at 12 months of age. The decrease in the surface localisation of mGlu5 was accompanied by an increase in its frequency at intracellular sites in the two neuronal populations. Together, these data demonstrate, for the first time, a loss of mGlu5 at the plasma membrane and accumulation at intracellular sites in different principal cells of the hippocampus in APP/PS1 mice, suggesting an alteration of the excitability and synaptic transmission that could contribute to the cognitive dysfunctions in this AD animal model. Further studies are required to elucidate the specificity of mGlu5 -associated molecules and downstream signalling pathways in the progression of the pathology. [ABSTRACT FROM AUTHOR]- Published
- 2021
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42. CD34+ Stromal Cells/Telocytes as a Source of Cancer-Associated Fibroblasts (CAFs) in Invasive Lobular Carcinoma of the Breast.
- Author
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Díaz-Flores, Lucio, Gutiérrez, Ricardo, González-Gómez, Miriam, García, Maria Pino, Díaz-Flores Jr., Lucio, Carrasco, José Luís, Martín-Vasallo, Pablo, and Manetti, Mirko
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STROMAL cells ,LOBULAR carcinoma ,FIBROBLASTS ,ELECTRON microscopy ,ENDOMETRIUM - Abstract
Several origins have been proposed for cancer-associated fibroblasts (CAFs), including resident CD34+ stromal cells/telocytes (CD34+SCs/TCs). The characteristics and arrangement of mammary CD34+SCs/TCs are well known and invasive lobular carcinoma of the breast (ILC) is one of the few malignant epithelial tumours with stromal cells that can express CD34 or αSMA, which could facilitate tracking these cells. Our objective is to assess whether tissue-resident CD34+SCs/TCs participate in the origin of CAFs in ILCs. For this purpose, using conventional and immunohistochemical procedures, we studied stromal cells in ILCs (n:42) and in normal breasts (n:6, also using electron microscopy). The results showed (a) the presence of anti-CD34+ or anti-αSMA+ stromal cells in varying proportion (from very rare in one of the markers to balanced) around nests/strands of neoplastic cells, (b) a similar arrangement and location of stromal cells in ILC to CD34+SCs/TCs in the normal breast, (c) both types of stromal cells coinciding around the same nest of neoplastic cells and (d) the coexpression of CD34 and αSMA in stromal cells in ILC. In conclusion, our findings support the hypothesis that resident CD34+SCs/TCs participate as an important source of CAFs in ILC. Further studies are required in this regard in other tumours. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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43. Huntingtin: A Protein with a Peculiar Solvent Accessible Surface.
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Babbi, Giulia, Savojardo, Castrense, Martelli, Pier Luigi, Casadio, Rita, and Morea, Veronica
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HUNTINGTIN protein ,SOLVENTS ,MEMBRANE proteins ,ELECTRON microscopy ,PROTEIN-protein interactions - Abstract
Taking advantage of the last cryogenic electron microscopy structure of human huntingtin, we explored with computational methods its physicochemical properties, focusing on the solvent accessible surface of the protein and highlighting a quite interesting mix of hydrophobic and hydrophilic patterns, with the prevalence of the latter ones. We then evaluated the probability of exposed residues to be in contact with other proteins, discovering that they tend to cluster in specific regions of the protein. We then found that the remaining portions of the protein surface can contain calcium-binding sites that we propose here as putative mediators for the protein to interact with membranes. Our findings are justified in relation to the present knowledge of huntingtin functional annotation. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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44. Supra-Molecular Assemblies of ORAI1 at Rest Precede Local Accumulation into Puncta after Activation.
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Peckys, Diana B., Gaa, Daniel, Alansary, Dalia, Niemeyer, Barbara A., and Jonge, Niels de
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ELECTRON microscopy ,ENDOPLASMIC reticulum ,SINGLE molecules ,CALCIUM channels ,MEMBRANE proteins ,SUPRAMOLECULES - Abstract
The Ca
2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM), the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand ORAI1 positions revealed a number of ORAI1 channels had formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, a fraction of ORAI1 assembled in micron-sized two-dimensional structures, such as the known puncta at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating dense ORAI1 accumulations upon SOCE-activation. [ABSTRACT FROM AUTHOR]- Published
- 2021
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45. Novel Approach to Tooth Chemistry: Quantification of Human Enamel Apatite in Context for New Biomaterials and Nanomaterials Development.
- Author
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Kuczumow, Andrzej, Chałas, Renata, Nowak, Jakub, Smułek, Wojciech, and Jarzębski, Maciej
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DENTAL enamel ,ELECTRON probe microanalysis ,APATITE ,MOLARS ,TEETH - Abstract
A series of linear profiles of the elements of the enamel in human molar teeth were made with the use of an electron microprobe and a Raman microscope. It is postulated that the enamel can be treated as the superposition of variable "overbuilt" enamel on the stable "core" enamel at the macro-, micro- and nanoscale level. The excessive values characterize the "overbuilt enamel". All the profiles of excessive parameters along the enamel thickness from the enamel surface to the dentin enamel junction (DEJ) can be approximated very precisely with the use of exponential functions, where Ca, P, Cl and F spatial profiles are decaying while Mg, Na, K and CO
3 2− ones are growing distributions. The "overbuilt" apatite formed on the boundary with DEJ, enriched in Na, Mg, OH and carbonates, reacts continuously with Ca, Cl and F, passing into an acid-resistant form of the "overbuilt" enamel. The apparent phases arriving in boundary regions of the "overbuilt enamel" were proposed. Microdiffraction measurements reveal relative variation of energy levels during enamel transformations. Our investigations are the milestones for a further new class of biomaterial and nanomaterial development for biomedical applications. [ABSTRACT FROM AUTHOR]- Published
- 2021
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46. New Model for Stacking Monomers in Filamentous Actin from Skeletal Muscles of Oryctolagus cuniculus.
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Glyakina, Anna V., Surin, Alexey K., Grishin, Sergei Yu., Selivanova, Olga M., Suvorina, Mariya Yu., Bobyleva, Liya G., Vikhlyantsev, Ivan M., and Galzitskaya, Oxana V.
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EUROPEAN rabbit ,ACTIN ,MONOMERS ,MASS analysis (Spectrometry) ,F-actin ,MICROFILAMENT proteins - Abstract
To date, some scientific evidence (limited proteolysis, mass spectrometry analysis, electron microscopy (EM)) has accumulated, which indicates that the generally accepted model of double-stranded of filamentous actin (F-actin) organization in eukaryotic cells is not the only one. This entails an ambiguous understanding of many of the key cellular processes in which F-actin is involved. For a detailed understanding of the mechanism of F-actin assembly and actin interaction with its partners, it is necessary to take into account the polymorphism of the structural organization of F-actin at the molecular level. Using electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling we demonstrated that F-actin presented in the EM images has no double-stranded organization, the regions of protease resistance are accessible for action of proteases in F-actin models. Based on all data, a new spatial model of filamentous actin is proposed, and the F-actin polymorphism is discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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47. Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses.
- Author
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Kleindienst, David, Montanaro, Jacqueline, Bhandari, Pradeep, Case, Matthew J., Fukazawa, Yugo, and Shigemoto, Ryuichi
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CALCIUM channels ,GLUTAMATE receptors ,SYNAPSES ,AMPA receptors ,METHYL aspartate receptors ,NEUROPLASTICITY - Abstract
The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
48. Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.
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Grishin, Sergei Y., Deryusheva, Evgeniya I., Machulin, Andrey V., Selivanova, Olga M., Glyakina, Anna V., Gorbunova, Elena Y., Mustaeva, Leila G., Azev, Vyacheslav N., Rekstina, Valentina V., Kalebina, Tatyana S., Surin, Alexey K., and Galzitskaya, Oxana V.
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ELECTRON microscopy ,RIBOSOMAL DNA ,BIOINFORMATICS ,PEPTIDES ,ESCHERICHIA coli ,RIBOSOMAL proteins ,THERMUS thermophilus - Abstract
Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
49. Structure of Nanotubes Self-Assembled from a Monoamide Organogelator.
- Author
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Zapién-Castillo, Samuel, Díaz-Zavala, Nancy P., Melo-Banda, José A., Schwaller, Duncan, Lamps, Jean-Philippe, Schmutz, Marc, Combet, Jérôme, and Mésini, Philippe J.
- Subjects
NANOTUBES ,ELECTRON density ,BENZOIC acid ,ALKANES ,SMALL-angle X-ray scattering ,ELECTRON microscopy ,ORGANIC compounds ,X-ray scattering - Abstract
Some organic compounds are known to self-assemble into nanotubes in solutions, but the packing of the molecules into the walls of the tubes is known only in a very few cases. Herein, we study two compounds forming nanotubes in alkanes. They bear a secondary alkanamide chain linked to a benzoic acid propyl ester (HUB-3) or to a butyl ester (HUB-4). They gel alkanes for concentrations above 0.2 wt.%. The structures of these gels, studied by freeze fracture electron microscopy, exhibit nanotubes: for HUB-3 their external diameters are polydisperse with a mean value of 33.3 nm; for HUB-4, they are less disperse with a mean value of 25.6 nm. The structure of the gel was investigated by small- and wide-angle X-ray scattering. The evolution of the intensities show that the tubes are metastable and transit slowly toward crystals. The intensities of the tubes of HUB-4 feature up to six oscillations. The shape of the intensities proves the tubular structure of the aggregates, and gives a measurement of 20.6 nm for the outer diameters and 11.0 nm for the inner diameters. It also shows that the electron density in the wall of the tubes is heterogeneous and is well described by a model with three layers. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
50. Force-Generating Mechanism of Axonemal Dynein in Solo and Ensemble.
- Author
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Ishibashi, Kenta, Sakakibara, Hitoshi, and Oiwa, Kazuhiro
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DYNEIN ,GENERATING functions ,X-ray crystallography ,ELECTRON microscopy ,MOLECULAR motor proteins ,CILIA & ciliary motion - Abstract
In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowledge and perspectives of the force-generating mechanism of axonemal dyneins in solo and in ensemble. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
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