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Novo, Sergi, Leonardo Barrios, Elena Ibáñez i Carme Nogués. "The Zona Pellucida Porosity: Three-Dimensional Reconstruction of Four Types of Mouse Oocyte Zona Pellucida Using a Dual Beam Microscope". Microscopy and Microanalysis 18, nr 6 (grudzień 2012): 1442–49. http://dx.doi.org/10.1017/s1431927612013487.
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AbstractIn the last decade, the applicability of focus ion beam–field emission scanning electron microscopy (FIB-FESEM) in the biological field has begun to get relevance. Among the possibilities offered by FIB-FESEM, high-resolution three-dimensional (3D) reconstruction of biological structures is one of the most interesting. Using this tool, the 3D porosity of four different types of mouse oocyte zona pellucida (ZP) was analyzed. A surface analysis of the mouse oocyte ZP was first performed by SEM. Next, one oocyte per ZP type was selected, and an area of its ZP was completely milled, using the cut and view mode, in the FIB-FESEM. Through a 3D reconstruction of the milled area, a map of the distribution of the pores across the ZP was established and the number and volume of pores were quantified, thus enabling for the first time the study of the inner porosity of the mouse ZP. Differences in ZP porosity observed among the four types analyzed allowed us to outline a model to explain the changes that the ZP undergoes through immature, mature, predegenerative, and degenerative stages.
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Reynaud, A. "Filtration des inclusions par les matériaux poreux et les mousses en fonderie de fonte". Revue de Métallurgie 97, nr 1 (styczeń 2000): 73–82. http://dx.doi.org/10.1051/metal/200097010073.
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Matsuoka, Shuji, Yasuyuki Ishii, Atsuhito Nakao, Hiroshi Masutani, Satoshi Takahashi, Yusuke Nakauchi i Okio Hino. "Establishment of a Therapeutic Anti-Pan HLA-Class II Monoclonal Antibody That Directly Induces Lymphoma Cell Death Via Large Pore Formation". Blood 124, nr 21 (6.12.2014): 5488. http://dx.doi.org/10.1182/blood.v124.21.5488.5488.
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Abstract To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pores formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma. Disclosures No relevant conflicts of interest to declare.
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Henry, Marianna M., i V. Ranga. "A Quantitative Study of the Development of Interalveolar Pores in the Postnatal Mouse". Experimental Lung Research 9, nr 3-4 (styczeń 1985): 277–87. http://dx.doi.org/10.3109/01902148509057528.
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Bastacky, J., i J. Goerke. "Pores of Kohn are filled in normal lungs: low-temperature scanning electron microscopy". Journal of Applied Physiology 73, nr 1 (1.07.1992): 88–95. http://dx.doi.org/10.1152/jappl.1992.73.1.88.
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Interalveolar pores of Kohn, small uniform-sized epithelium-lined openings in alveolar walls of normal lung, have historically been demonstrated with electron-microscopic techniques that remove water. We show these pores to be present but almost invariably filled with material when water and surfactant are preserved in frozen hydrated lung examined with low-temperature scanning electron microscopy. In the normal mouse, 16 open empty pores per alveolus were found in instillation-fixed dried lung vs. less than 1 per alveolus in frozen hydrated lungs (P less than 0.001). In the normal rat, 13 pores were seen per alveolus in instillation-fixed dried lung vs. less than 1 per alveolus in frozen hydrated lungs (P less than 0.001). We suggest that pores of Kohn 1) function primarily as conduits for interalveolar movement of alveolar liquid, surfactant components, and macrophages, 2) provide distributed sites for tubular myelin storage without increasing gas diffusion pathway thickness in the alveolar subphase itself, and 3) do not function as pathways for collateral ventilation during normal breathing in the absence of atelectasis or obstruction.
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Ho, Han-Chen. "Redistribution of nuclear pores during formation of the redundant nuclear envelope in mouse spermatids". Journal of Anatomy 216, nr 4 (kwiecień 2010): 525–32. http://dx.doi.org/10.1111/j.1469-7580.2009.01204.x.
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Guo, Yuxuan, i Yixian Zheng. "Lamins position the nuclear pores and centrosomes by modulating dynein". Molecular Biology of the Cell 26, nr 19 (październik 2015): 3379–89. http://dx.doi.org/10.1091/mbc.e15-07-0482.
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Lamins, the type V nuclear intermediate filament proteins, are reported to function in both interphase and mitosis. For example, lamin deletion in various cell types can lead to an uneven distribution of the nuclear pore complexes (NPCs) in the interphase nuclear envelope, whereas deletion of B-type lamins results in spindle orientation defects in mitotic neural progenitor cells. How lamins regulate these functions is unknown. Using mouse cells deleted of different combinations or all lamins, we show that lamins are required to prevent the aggregation of NPCs in the nuclear envelope near centrosomes in late G2 and prophase. This asymmetric NPC distribution in the absence of lamins is caused by dynein forces acting on NPCs via the dynein adaptor BICD2. We further show that asymmetric NPC distribution upon lamin depletion disrupts the distribution of BICD2 and p150 dynactin on the nuclear envelope at prophase, which results in inefficient dynein-driven centrosome separation during prophase. Therefore lamins regulate microtubule-based motor forces in vivo to ensure proper NPC distribution in interphase and centrosome separation in the mitotic prophase.
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Xu, Tai-Min, Dong-Mei Wu, Neng Gao, Long Zeng, Yi-Hua Xu, Xiang-Ping Fan, Yi-Fei Sun i Bao-Kai Cui. "Five New Species of Wood-Decaying Brown-Rot Fungi within Postiaceae (Polyporales, Basidiomycota) from Xinjiang, Northwest China". Journal of Fungi 10, nr 9 (17.09.2024): 655. http://dx.doi.org/10.3390/jof10090655.
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Brown-rot fungi are an important group of wood-decaying fungi, but there has been limited research on the species diversity of brown-rot fungi in Xinjiang, China. During an investigation of brown-rot fungi in Xinjiang, from July 2018 to July 2023, five new species belonging to the family Postiaceae were discovered based on morphological and molecular evidence. Amaropostia altaiensis is characterized by a conchate pileus, circular pores (5–8 per mm), and growing on Populus. Amaropostia tianshanensis is characterized by a flabelliform-to-conchate pileus, angular pores (5–6 per mm), and growing on Picfea. Cyanosporus latisporus is characterized by a hirsute and dark greyish blue pileal surface with fresh, larger pores (3–6 per mm) and broad basidiospores (4.3–5.9 × 1.4–2 µm). Cyanosporus tianshanensis is characterized by a smooth and white-to-cream pileal surface with fresh, smaller pores (6–9 per mm). Osteina altaiensis is characterized by a light mouse-grey-to-honey-yellow pileal surface, smaller pores (4–6 per mm), and slightly wide basidiospores (5–6 × 1.7–2.2 µm). Each of these five new species form independent lineages in phylogenetic analyses based on the seven gene loci (ITS + nLSU + nSSU + mtSSU + TEF1 + RPB1 + RPB2). This research enriches the diversity of brown-rot fungi species, while also demonstrating the substantial discovery potential and research value of brown-rot fungi in Xinjiang.
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Scherthan, Harry, Martin Jerratsch, Bibo Li, Susan Smith, Maj Hultén, Tycho Lock i Titia de Lange. "Mammalian Meiotic Telomeres: Protein Composition and Redistribution in Relation to Nuclear Pores". Molecular Biology of the Cell 11, nr 12 (grudzień 2000): 4189–203. http://dx.doi.org/10.1091/mbc.11.12.4189.
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Mammalian telomeres consist of TTAGGG repeats, telomeric repeat binding factor (TRF), and other proteins, resulting in a protective structure at chromosome ends. Although structure and function of the somatic telomeric complex has been elucidated in some detail, the protein composition of mammalian meiotic telomeres is undetermined. Here we show, by indirect immunofluorescence (IF), that the meiotic telomere complex is similar to its somatic counterpart and contains significant amounts of TRF1, TRF2, and hRap1, while tankyrase, a poly-(ADP-ribose)polymerase at somatic telomeres and nuclear pores, forms small signals at ends of human meiotic chromosome cores. Analysis of rodent spermatocytes reveals Trf1 at mouse, TRF2 at rat, and mammalian Rap1 at meiotic telomeres of both rodents. Moreover, we demonstrate that telomere repositioning during meiotic prophase occurs in sectors of the nuclear envelope that are distinct from nuclear pore-dense areas. The latter form during preleptotene/leptotene and are present during entire prophase I.
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Kim, Jeonghan, Rajeev Gupta, Luz P. Blanco, Shutong Yang, Anna Shteinfer-Kuzmine, Kening Wang, Jun Zhu i in. "VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease". Science 366, nr 6472 (19.12.2019): 1531–36. http://dx.doi.org/10.1126/science.aav4011.
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Mitochondrial stress releases mitochondrial DNA (mtDNA) into the cytosol, thereby triggering the type Ι interferon (IFN) response. Mitochondrial outer membrane permeabilization, which is required for mtDNA release, has been extensively studied in apoptotic cells, but little is known about its role in live cells. We found that oxidatively stressed mitochondria release short mtDNA fragments via pores formed by the voltage-dependent anion channel (VDAC) oligomers in the mitochondrial outer membrane. Furthermore, the positively charged residues in the N-terminal domain of VDAC1 interact with mtDNA, promoting VDAC1 oligomerization. The VDAC oligomerization inhibitor VBIT-4 decreases mtDNA release, IFN signaling, neutrophil extracellular traps, and disease severity in a mouse model of systemic lupus erythematosus. Thus, inhibiting VDAC oligomerization is a potential therapeutic approach for diseases associated with mtDNA release.
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MATSUDA, Takeshi, Yoshifumi FUKUO, Harumichi SHINOHARA, Satoshi MORISAWA i Toshio NAKATANI. "The Postnatal Development of the Mouse Pericardium; the Time and Mechanism of Formation of Pericardial Pores". Okajimas Folia Anatomica Japonica 67, nr 2-3 (1990): 115–20. http://dx.doi.org/10.2535/ofaj1936.67.2-3_115.
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Vautier, D., P. Chesne, C. Cunha, A. Calado, J. P. Renard i M. Carmo-Fonseca. "Transcription-dependent nucleocytoplasmic distribution of hnRNP A1 protein in early mouse embryos". Journal of Cell Science 114, nr 8 (15.04.2001): 1521–31. http://dx.doi.org/10.1242/jcs.114.8.1521.
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A unique feature of certain members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins is that they shuttle continuously between nucleus and cytoplasm and their accumulation in the nucleus is transcription-dependent. An extensively characterised protein of this group is hnRNP A1. To date, most studies addressing the transcription-dependent transport of hnRNP A1 have been performed on cultured cell lines treated with transcription inhibitors. Here we have analysed the nucleocytoplasmic distribution of hnRNP A1 in early mouse embryos, where the haploid pronuclei remain transcriptionally inactive for a period of several hours. Consistent with its small molecular size (36 kDa), the hnRNP A1 protein diffuses passively through the nuclear pores and equilibrates between the nucleus and the cytoplasm of transcriptionally inactive embryos. In contrast, following transcriptional activation the A1 protein becomes accumulated in the nucleus. This accumulation of the A1 protein in the nucleus is blocked by the lectin wheat germ agglutinin (WGA), which binds to nuclear pore proteins and prevents translocation of receptor-cargo complexes through the pores. This indicates that a carrier-mediated transport pathway is required for the concentration of A1 in transcriptionally active nuclei. To further analyse how transcription is coupled to nucleocytoplasmic transport, we transplanted transcriptionally inactive pronuclei into the cytoplasm of transcriptionally active embryos. The results show that the presence of newly synthesised RNAs in the cytoplasm is not sufficient to induce the accumulation of hnRNP A1 in the nucleus. Rather, the appearance of nascent transcripts in the nucleus appears to be the crucial event. Since hnRNP A1 is a shuttling protein, an increase in its steady state nuclear concentration could be the result of either faster nuclear import or slower export to the cytoplasm. We propose that binding of A1 to nascent transcripts retards its export to the cytoplasm and therefore contributes to its concentration in the nucleus.
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D'Cruz, Akshay A., Meghan Bliss-Moreau, Maria Ericcson i Ben A. Croker. "Mlkl Pores Release Neutrophil Extracellular Traps in Necroptotic Neutrophils". Blood 126, nr 23 (3.12.2015): 2200. http://dx.doi.org/10.1182/blood.v126.23.2200.2200.
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Abstract Neutrophil extracellular traps (NETs) are networks of extracellular nuclear DNA and microbicidal proteins released from neutrophils in response to tissue damage and infection. Despite evidence of pathogenic roles for NETs in systemic lupus erythematosus, rheumatoid arthritis, diabetes, artherosclerosis and Alzheimer's disease, the major biochemical pathways controlling their formation remains poorly understood. Apoptosis does not contribute to NET formation but the role of regulated non-apoptotic cell death pathways such as necroptosis is not known. We have investigated the role of positive and negative regulators of necroptosis including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase domain-like (MLKL), receptor-interacting protein kinase-1 (RIPK1) and Caspase-8. Using immunogold electron microscopy, flow cytometry, imaging flow cytometry and fluorescence microscopy, we demonstrate that necroptosis can drive NET formation via MLKL pore formation at the cell surface. This process is caspase-independent but reactive oxygen species-dependent. Genetically-modified mouse peripheral blood and bone marrow neutrophils were used to show that Caspase-8 and RIPK1 negatively regulate NET formation driven by RIPK3 and MLKL. Mice that lack MLKL are deficient in necroptosis and NET formation, and were sensitive to methicillin-resistant Staphylococcus aureus (MRSA). Neutrophil-specific Caspase-8-deficiency also leads to increased susceptibility to MRSA due to increased rates of necroptotic neutrophil death. Killing of MRSA by necroptotic neutrophils is sensitive to DNase, and is dependent on MLKL, suggesting that necroptosis-driven NET formation contributes to the bactericidal activity of neutrophils. Human peripheral blood neutrophils also generate NETs that are sensitive to pharmacological inhibitors of necroptosis, suggesting that targeting necroptosis in general may help combat autoimmune responses to DNA. This study provides a framework to investigate the role of extracellular DNA release and cell death in the setting of infection, autoimmunity and autoinflammatory disease. Disclosures No relevant conflicts of interest to declare.
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Lin, Patricia W., Peter O. Simon, Andrew T. Gewirtz, Andrew S. Neish, Andre J. Ouellette, James L. Madara i Wayne I. Lencer. "Paneth Cell Cryptdins Actin Vitroas Apical Paracrine Regulators of the Innate Inflammatory Response". Journal of Biological Chemistry 279, nr 19 (27.02.2004): 19902–7. http://dx.doi.org/10.1074/jbc.m311821200.
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Intestinal-specific antimicrobial α-defensins, termed cryptdins, are secreted into the intestinal lumen by mouse Paneth cells in response to microbial pathogens. Cryptdins kill microbes by forming pores in their limiting membranes. The cryptdin isoforms 2 and 3 also can form anion-conductive pores in eukaryotic cell membranes, thus affecting cell physiology. Here, we find that when applied to apical membranes of the human intestinal cell line T84, cryptdin 3 (Cr3) induces secretion of the proinflammatory cytokine interleukin 8 (IL-8) in a dose-dependent manner. The induction of IL-8 secretion is specific to the cryptdins that form channels in mammalian cell membranes because cryptdin 4, which does not form pores in T84 cells, does not induce IL-8 secretion. Cr3 induces inflammatory cytokine secretion by activating NF-κB and p38 mitogen-activated protein kinase in a Ca2+-dependent manner, but influx by extra-cellular Ca2+is not involved. Unlike other known inflammatory agonists, signal transduction by Cr3 occurs slowly, suggesting a novel mechanism of action. These results show that selective cryptdins may amplify their roles in innate immunity by acting as novel paracrine agonists to coordinate an inflammatory response with the antimicrobial secretions of Paneth cells.
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Kadlečková, Markéta, Kateřina Skopalová, Barbora Ptošková, Erik Wrzecionko, Eliška Daďová, Karolína Kocourková, Aleš Mráček i in. "Hierarchically Structured Surfaces Prepared by Phase Separation: Tissue Mimicking Culture Substrate". International Journal of Molecular Sciences 23, nr 5 (25.02.2022): 2541. http://dx.doi.org/10.3390/ijms23052541.
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The pseudo 3D hierarchical structure mimicking in vivo microenvironment was prepared by phase separation on tissue culture plastic. For surface treatment, time-sequenced dosing of the solvent mixture with various concentrations of polymer component was used. The experiments showed that hierarchically structured surfaces with macro, meso and micro pores can be prepared with multi-step phase separation processes. Changes in polystyrene surface topography were characterized by atomic force microscopy, scanning electron microscopy and contact profilometry. The cell proliferation and changes in cell morphology were tested on the prepared structured surfaces. Four types of cell lines were used for the determination of impact of the 3D architecture on the cell behavior, namely the mouse embryonic fibroblast, human lung carcinoma, primary human keratinocyte and mouse embryonic stem cells. The increase of proliferation of embryonic stem cells and mouse fibroblasts was the most remarkable. Moreover, the embryonic stem cells express different morphology when cultured on the structured surface. The acquired findings expand the current state of knowledge in the field of cell behavior on structured surfaces and bring new technological procedures leading to their preparation without the use of problematic temporary templates or additives.
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Rinde, Mia, Natalia Kupferschmidt, Muhammad Naeem Iqbal, Ghislaine Robert-Nicoud, Eric V. Johnston, Maria Lindgren i Tore Bengtsson. "Mesoporous silica with precisely controlled pores reduces food efficiency and suppresses weight gain in mice". Nanomedicine 15, nr 2 (styczeń 2020): 131–44. http://dx.doi.org/10.2217/nnm-2019-0262.
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Aim: Obesity is a risk factor for cardiovascular disease and diabetes. We aimed to elucidate the effects of distinct mesoporous silica particles (MSPs) supplemented in food on metabolic parameters in obesity. Materials & methods: MSPs with precisely controlled pore size were synthesized, characterized and compared with a control in a C57Bl/6 mouse diet-induced obesity model, studying weight, adiposity, metabolic regulation and food efficiency. Results: The most effective MSPs reduced adipose tissue formation to 6.5 ± 0.5 g compared with 9.4 ± 1.2 g, leptin levels nearly halved from 32.8 ± 7.4 to 16.9 ± 1.9 ng/ml and a 33% reduction of food efficiency. Control MSP showed no effects. Conclusion: Results demonstrate potential of distinct MSPs to improve metabolic risk factors. Further studies investigating mechanism of action and confirming human safety are needed.
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Aglietti, Robin A., Alberto Estevez, Aaron Gupta, Monica Gonzalez Ramirez, Peter S. Liu, Nobuhiko Kayagaki, Claudio Ciferri, Vishva M. Dixit i Erin C. Dueber. "GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes". Proceedings of the National Academy of Sciences 113, nr 28 (23.06.2016): 7858–63. http://dx.doi.org/10.1073/pnas.1607769113.
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Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca2+ from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.
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Hurt, E. C. "Targeting of a cytosolic protein to the nuclear periphery." Journal of Cell Biology 111, nr 6 (1.12.1990): 2829–37. http://dx.doi.org/10.1083/jcb.111.6.2829.
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The yeast nuclear envelope protein NSP1 is located at the nuclear pores and mediates its essential function via the carboxy-terminal domain. The passenger protein, cytosolic dihydrofolate reductase from mouse, was fused to the 220 residue long NSP1 carboxy-terminal domain. When expressed in yeast, this chimeric protein was tightly associated with nuclear structures and was localized at the nuclear periphery very similar to authentic NSP1. Furthermore, the DHFR-C-NSP1 fusion protein was able to complement a yeast mutant lacking a functional NSP1 gene showing that DHFR-C-NSP1 fulfils the same basic function as compared to the endogenous NSP1 protein. These data also show that the NSP1 protein is composed of separate functional moieties: a carboxy-terminal domain that is sufficient to mediate the association with the nuclear periphery and an amino-terminal and middle repetitive domain with an as yet unknown function. It is suggested that heptad repeats found in the NSP1 carboxy-terminal domain, which are similar to those found in intermediate filament proteins, are crucial for mediating the association with the nuclear pores.
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Kiradzhiyska, Denitsa, Nikolina Milcheva, Rositsa Mancheva, Tsvetelina Batsalova, Balik Dzhambazov i Nikolay Zahariev. "Preparation and Preliminary Evaluation of Silver-Modified Anodic Alumina for Biomedical Applications". Metals 12, nr 1 (27.12.2021): 51. http://dx.doi.org/10.3390/met12010051.
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The present study reports a specific method for preparation of silver-modified anodic alumina substrates intended for biomaterial applications. Al2O3 coatings were obtained by anodization of technically pure aluminum alloy in sulfuric acid electrolyte. Silver deposition into the pores of the anodic structures was carried out employing in situ thermal reduction for different time periods. The obtained coatings were characterized using scanning electron microscopy (SEM), potentiodynamic scanning after 168 h in 3.5% NaCl solution and bioassays with human fibroblast and NIH/3T3 cell lines. The modified alumina substrates demonstrated better biocompatibility compared to the control anodic Al2O3 pads indicated by increased percent cell survival following in vitro culture with human and mouse fibroblasts. The Ag-deposition time did not affect considerably the biocompatibility of the investigated anodic layers. SEM analyses indicated that mouse NIH/3T3 cells and human fibroblasts adhere to the silver-coated alumina substrates retaining normal morphology and ability to form cell monolayer. Therefore, the present studies demonstrate that silver coating of anodic alumina substrates improves their biocompatibility and their eventual biomedical application.
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Okazaki, M., Y. Tieliewuhan i I. Hirata. "Osteoblast Behavior at the Surface of CO3Ap-Collagen Sponges". Key Engineering Materials 309-311 (maj 2006): 989–92. http://dx.doi.org/10.4028/www.scientific.net/kem.309-311.989.
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Carbonate apatite (CO3Ap) was synthesized at 60+1°C and pH 7.4+0.2, to develop a new biodegradable scaffold biomaterial. The synthetic CO3Ap was mixed with a neutralized collagen gel and the CO3Ap-collagen mixtures with different kinds of CO3Ap contents and porosity were frozen and dried in lyophilized into the sponges. CO3Ap-collagen mixtures were also lyophilized into sponges in a HAp frame ring with 0.5 mm pores. To examine the degree of cell invasion, mouse MC3T3-E1 cells were grown in αMEM with 10% heat-inactivated FBS in 96-well plates containing the CO3Ap-collagen sponges at 37°C in a 5% humidified atmosphere. Under pentobarbital anesthesia, samples of UV-irradiated CO3Ap-collagen sponges with frames were surgically implanted beneath the periosteum cranii of rats. SEM observation of CO3Ap-collagen sponges showed favorable pores for cell invasion. Approximately 50~300 µm size pores seemed to continue into the deep bottom. X-ray high-resolution microtomography revealed a clear image of 3D structure of the sponges. 70 wt% CO3Ap-collagen sponge seemed to be most favorable biomaterial from the viewpoint of the natural bone properties. Then, to avoid the shrinkage of the sponges, we successfully made a hybridized CO3Ap-collagen sponge with a frame. When these sponge-frame complexes were implanted beneath the periosteum cranii of rats, newly created bone was observed toward the inner core of the complex from the surface of the periosteum cranii.
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Chaly, Nathalie, Gerald St. Aubin i David L. Brown. "Ultrastructural localization of nuclear antigens during interphase in mouse 3T3 fibroblasts". Biochemistry and Cell Biology 67, nr 9 (1.09.1989): 563–74. http://dx.doi.org/10.1139/o89-088.
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Thin sections of mouse 3T3 fibroblast nuclei labelled by immunoperoxidase with anti-nuclear antibodies I1, PI1, PI2, anti-peripherin, -lamin, and -centromere have been examined in the electron microscope. Staining was compared with the corresponding immunofluorescence labelling patterns, and was correlated with nuclear ultrastructure in conventionally fixed and uranyl-lead stained samples and in unlabelled immunoperoxidase controls. Peripherin was detected at the nuclear rim in a band broader and more irregular than the lamins/lamina. The peripheral components of PI1 and PI2 appear to be localized at nuclear pores and the nuclear envelope, respectively. The internal component of PI1 staining consisted of irregular patches and strands in the nucleoplasm, closely resembling snRNP staining as reported by others. Internal PI2 labelling consisted of spots distributed apparently at random in interchromatinic regions. The spots resembled labelling by antibody I1, but were fewer and more irregular in size. Neither the PI2 nor the I1 spots were centromere associated, nor could they be correlated with specific interchromatinic structures in conventional preparations and in unlabelled controls. The results support the hypothesis that the nucleus is segregated into function-specific domains, distinguished by morphology and (or) composition from surrounding regions of the nucleus.Key words: nuclear matrix, peripherin, immunoperoxidase, electron microscopy, nuclear antigens.
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Feng, Dian, Janice A. Nagy, John Hipp, Kathryn Pyne, Harold F. Dvorak i Ann M. Dvorak. "Reinterpretation of endothelial cell gaps induced by vasoactive mediators in guinea-fig, mouse and rat: many are transcellular pores". Journal of Physiology 504, nr 3 (listopad 1997): 747–61. http://dx.doi.org/10.1111/j.1469-7793.1997.747bd.x.
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Matsuoka, Shuji, Hiromichi Tsurui, Masaaki Abe, Kazuo Terashima, Kazuhiro Nakamura, Yoshitomo Hamano, Mareki Ohtsuji i in. "A Monoclonal Antibody to the α2 Domain of Murine Major Histocompatibility Complex Class I that Specifically Kills Activated Lymphocytes and Blocks Liver Damage in the Concanavalin A Hepatitis Model". Journal of Experimental Medicine 198, nr 3 (28.07.2003): 497–503. http://dx.doi.org/10.1084/jem.20021301.
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We earlier found that a rat monoclonal antibody (mAb) RE2 can induce rapid death of murine activated, but not resting, lymphocytes and lymphocyte cell lines, in a complement-independent manner, a cell death differing from typical apoptosis or necrosis. We here found that this cell death is independent of pathways involving Fas, caspase, and phosphoinositide-3 kinase. With the advantage of producing human B cell line transfectants with stable expression of human/mouse xeno-chimeric MHC class I genes, we found that RE2 epitope resides on the murine class I α2 domain. However, the α3 domain plays a key role in transducing the death signal, which mediates extensive aggregation of the MHC class I-integrin-actin filament system, giving rise to membrane blebs and pores. In mouse models with T/NKT cell activation-associated fulminant hepatitis, administration of mAb RE2 almost completely inhibited the development of liver cell injuries. Taken collectively, this form of cell death may be involved in homeostatic immune regulation, and induction of this form of cell death using the mAbs may be potentially therapeutic for subjects with immunological diseases mediated by activated lymphocytes.
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Hamada, Masakazu, Anna Haeger, Karthik B. Jeganathan, Janine H. van Ree, Liviu Malureanu, Sarah Wälde, Jomon Joseph, Ralph H. Kehlenbach i Jan M. van Deursen. "Ran-dependent docking of importin-β to RanBP2/Nup358 filaments is essential for protein import and cell viability". Journal of Cell Biology 194, nr 4 (22.08.2011): 597–612. http://dx.doi.org/10.1083/jcb.201102018.
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RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)–mediated protein import, nuclear export signal–mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)–importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP–importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.
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Okumura, Noriko, Takafumi Yoshikawa, Akitaka Nonomura i Yoshinori Takakura. "Organ Regeneration in Porous Hydoroxyapatite". Key Engineering Materials 309-311 (maj 2006): 1017–22. http://dx.doi.org/10.4028/www.scientific.net/kem.309-311.1017.
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HA has a high affinity for bone as well as various tissues. In the present study, we investigated an affinity for abdominal organs. Coralline hydroxyapatite ceramic (HA, cubic structure 4x4x4mm, Interpore 500) was used in this experiment. We made two incisions in the lower back of a 5-week-old male nude mouse, and implanted HA blocks. One was placed around the liver at the right side and another one was placed around the kidney at the left side. The organ fibrous capsule was not removed. At 6 weeks after implantation, mice were sacrificed under overanesthesia and HA blocks were retrieved and prepared for histological analysis. In the HE stain of HA blocks around liver, liver tissue is invaded into the HA pore areas. Hepatocyte proliferation in trabecular pattern was seen in contact with the surfaces of many HA pores. Within some pores, hepatic lobular pattern, Glisson sheath or central vein could be detected. In the HA around kidney, renal tissue was observed in many pores. The pore areas of HA were fullfilled with grumerulus and urinary tube tissues. In contact with the surfaces of some HA blocks, the tissue invasion of pancreas and spleen tissue were recognized. These results indicate that porous HA has a high affinity for the celiac organs, and has a stimulatory effect on celiac organ regeneration. Especially, concerning the regeneration of kidney, it has not been reported yet, so this report is very interesting. HA is also very useful as a scaffold of the organ regeneration.
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Zhang, Steven B., David Maguire, Mei Zhang, Yeping Tian, Shanmin Yang, Amy Zhang, Katherine Casey-Sawicki i in. "Mitochondrial DNA and Functional Investigations into the Radiosensitivity of Four Mouse Strains". International Journal of Cell Biology 2014 (2014): 1–8. http://dx.doi.org/10.1155/2014/850460.
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We investigated whether genetic radiosensitivity-related changes in mtDNA/nDNA ratios are significant to mitochondrial function and if a material effect on mtDNA content and function exists. BALB/c (radiosensitive), C57BL/6 (radioresistant), and F1 hybrid mouse strains were exposed to total body irradiation. Hepatic genomic DNA was extracted, and mitochondria were isolated. Mitochondrial oxygen consumption, ROS, and calcium-induced mitochondrial swelling were measured. Radiation influenced strain-specific survivalin vivo. F1 hybrid survival was influenced by maternal input. Changes in mitochondrial content corresponded to survivalin vivoamong the 4 strains. Calcium-induced mitochondrial swelling was strain dependent. Isolated mitochondria from BALB/c mice were significantly more sensitive to calcium overload than mitochondria from C57BL/6 mice. Maternal input partially influenced the recovery effect of radiation on calcium-induced mitochondrial swelling in F1 hybrids; the hybrid with a radiosensitive maternal lineage exhibited a lower rate of recovery. Hybrids had a survival rate that was biased toward maternal input. mtDNA content and mitochondrial permeability transition pores (MPTP) measured in these strains before irradiation reflected a dominant input from the parent. After irradiation, the MPTP opened sooner in radiosensitive and hybrid strains, likely triggering intrinsic apoptotic pathways. These findings have important implications for translation into predictors of radiation sensitivity/resistance.
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ZHANG, QIU-YUE, HONG-GAO LIU, GUANG-YU ZENG i YU-CHENG DAI. "Polyporus miscanthi (Polyporaceae, Basidiomycota), a new polypore on Miscanthus (Poaceae) from Guangxi, South China". Phytotaxa 655, nr 3 (26.06.2024): 249–60. http://dx.doi.org/10.11646/phytotaxa.655.3.3.
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Polyporus is a widespread genus with highly variable morphological features and intricate phylogenetic relationships. Polyporus miscanthi, collected from Guangxi Autonomous Region, South China, is presented as a new species based on both morphological and phylogenetic analyses. It is characterized by annual, solitary, eccentrically stipitate basidiomata with irregularly circular, depressed center or infundibuliform pilei, yellowish to buff pileal surface with radially aligned stripes, white to cream pores, 4–5 per mm, a mouse gray to black stipe, cylindrical basidiospores measuring 9–11 × 3.5–4.5 μm, and growth on rotten Miscanthus. We present the new species with illustrated macro- and micro morphological description and comparison with morphologically similar or phylogenetically related species. Additionally, we provide a key to the known species of Polyporus in China.
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Forysenkova, Anna A., Inna V. Fadeeva, Dina V. Deyneko, Alevtina N. Gosteva, Georgy V. Mamin, Darya V. Shurtakova, Galina A. Davydova, Viktoriya G. Yankova, Iulian V. Antoniac i Julietta V. Rau. "Polyvinylpyrrolidone—Alginate—Carbonate Hydroxyapatite Porous Composites for Dental Applications". Materials 16, nr 12 (20.06.2023): 4478. http://dx.doi.org/10.3390/ma16124478.
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An alternative approach for the currently used replacement therapy in dentistry is to apply materials that restore tooth tissue. Among them, composites, based on biopolymers with calcium phosphates, and cells can be applied. In the present work, a composite based on polyvinylpyrrolidone (PVP) and alginate (Alg) with carbonate hydroxyapatite (CHA) was prepared and characterized. The composite was investigated by X-ray diffraction, infrared spectroscopy, electron paramagnetic resonance (EPR) and scanning electron microscopy methods, and the microstructure, porosity, and swelling properties of the material were described. In vitro studies included the MTT test using mouse fibroblasts, and adhesion and survivability tests with human dental pulp stem cells (DPSC). The mineral component of the composite corresponded to CHA with an admixture of amorphous calcium phosphate. The presence of a bond between the polymer matrix and CHA particles was shown by EPR. The structure of the material was represented by micro- (30–190 μm) and nano-pores (average 8.71 ± 4.15 nm). The swelling measurements attested that CHA addition increased the polymer matrix hydrophilicity by 200%. In vitro studies demonstrated the biocompatibility of PVP-Alg-CHA (95 ± 5% cell viability), and DPSC located inside the pores. It was concluded that the PVP-Alg-CHA porous composite is promising for dentistry applications.
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Singh, Mandakini B., Jesse A. White, Eric J. McKimm, Milena M. Milosevic i Srdjan D. Antic. "Mechanisms of Spontaneous Electrical Activity in the Developing Cerebral Cortex—Mouse Subplate Zone". Cerebral Cortex 29, nr 8 (28.08.2018): 3363–79. http://dx.doi.org/10.1093/cercor/bhy205.
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Abstract Subplate (SP) neurons exhibit spontaneous plateau depolarizations mediated by connexin hemichannels. Postnatal (P1–P6) mice show identical voltage pattern and drug-sensitivity as observed in slices from human fetal cortex; indicating that the mouse is a useful model for studying the cellular physiology of the developing neocortex. In mouse SP neurons, spontaneous plateau depolarizations were insensitive to blockers of: synaptic transmission (glutamatergic, GABAergic, or glycinergic), pannexins (probenecid), or calcium channels (mibefradil, verapamil, diltiazem); while highly sensitive to blockers of gap junctions (octanol), hemichannels (La3+, lindane, Gd3+), or glial metabolism (DLFC). Application of La3+ (100 μM) does not exert its effect on electrical activity by blocking calcium channels. Intracellular application of Gd3+ determined that Gd3+-sensitive pores (putative connexin hemichannels) reside on the membrane of SP neurons. Immunostaining of cortical sections (P1–P6) detected connexins 26, and 45 in neurons, but not connexins 32 and 36. Vimentin-positive glial cells were detected in the SP zone suggesting a potential physiological interaction between SP neurons and radial glia. SP spontaneous activity was reduced by blocking glial metabolism with DFLC or by blocking purinergic receptors by PPADS. Connexin hemichannels and ATP release from vimentin-positive glial cells may underlie spontaneous plateau depolarizations in the developing mammalian cortex.
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Zubova, Ganna, Hanna Melnyk, Iryna Zaets, Tetyana Sergeyeva, Olesia Havryliuk, Sergiy Rogalsky, Lyudmila Khirunenko i in. "Halochromic Bacterial Cellulose/Anthocyanins Hybrid Polymer Film with Wound-Healing Potential". Polymers 16, nr 16 (16.08.2024): 2327. http://dx.doi.org/10.3390/polym16162327.
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Polymer-based dressings deriving from natural biomaterials have advantages such as nontoxicity, biocompatibility, and mechanical stability, which are essential for efficient wound healing and microbial infection diagnostics. Here, we designed a prototype of an intelligent hydrogel dressing on the base of bacterial cellulose (BC) for monitoring wound microbial infection due to the uploaded natural pH dye-sensor, anthocyanins (ANC) of elderberry fruit (Sambucus nigra L.). The highest sensor responses to bacterial metabolites for ANC immobilized to BC were observed at pH 5.0 and 6.0. The detection limit of the sensor signals was 3.45 A.U., as it was evaluated with a smartphone-installed application. The FTIR spectral analysis of the hybrid BC/ANC hydrogel films has proved the presence of anthocyanins within the BC matrix. Hybrid films differed from the control ones by thicker microfibrils and larger pores, as detected with scanning electron microscopy. Halochromic BC/ANC films exhibited antimicrobial activities mainly against gram-positive bacteria and yeast. They showed no cytotoxicity for the in vitro human cell lines and mouse fibroblasts within a selected range of anthocyanin concentrations released from the BC/ANC film/dressing prototype. Compared to the control, the in vitro healing test showed overgrowth of primary mouse fibroblasts after applying 0.024–2.4 µg/mL ANC.
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Popken, J., M. Dahlhoff, T. Guengoer, V. J. Schmid, A. Strauss, T. Cremer, V. Zakhartchenko i E. Wolf. "92 NUCLEAR INVAGINATIONS ADAPT TO RABBIT EARLY EMBRYONIC DEVELOPMENT". Reproduction, Fertility and Development 27, nr 1 (2015): 139. http://dx.doi.org/10.1071/rdv27n1ab92.
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Nuclear invaginations carrying nuclear pores may facilitate increased mRNA export and protein import to areas inside the nucleus remote from the nuclear border. In this study on rabbit embryos, we investigated whether large early embryonic nuclei and the increased import/export demands around major embryonic genome activation (MGA) at the 8-cell stage affected the quantity of nuclear invaginations carrying nuclear pores. To achieve this objective, we used super-resolution 3-dimensional structured illumination microscopy on 10 pronuclei or nuclei per stage of 23 in vivo-fertilized and in vitro-cultured embryos stained with antibodies for the nucleoporin NUP153 and lamin B and stained with 4′,6-diamidino-2-phenylindole (DAPI) for chromatin. Statistical comparisons between stages were performed using the Wilcoxon rank-sum test. At the zygote stage, the female pronucleus displayed on average 16.5 and the male pronucleus featured on average 15.25 wide and narrow nuclear envelope invaginations, carrying large or tiny amounts of cytoplasm. Subsequent stages showed predominantly wide invaginations targeting nucleoli. The contact areas between nucleoli and invaginations were free of nuclear pores. In contrast, narrow invaginations, which are the almost exclusive type of invaginations during cattle and mouse pre-implantation development, were not in contact with nucleoli. At the 2-cell stage, the number of invaginations increased to an average of 27.3 invaginations per nucleus (P < 0.05) and increased again to peak at the 4-cell stage with 51.2 invaginations per nucleus (P < 0.01). At the 8-cell stage (MGA), the amount of nuclear invaginations was reduced to 25.4 invaginations per nucleus (P < 0.01). The reduced number of nuclear invaginations at the 8-cell stage could be associated with a significant decrease in average nuclear volume from 2593 µm3 at the 4-cell stage to 960 µm3 at the 8-cell stage (P < 0.001) and a subsequent reduced average distance from areas inside the nucleus to the nuclear border. Nuclear invagination numbers continued their decline at the 21-cell stage with 5.2 invaginations per nucleus (P < 0.001), whereas nuclear volumes decreased to 618 µm3 (P < 0.001). The morula stage, with 6.9 invaginations per nucleus (P = 0.9), and the blastocyst stage, with 4.5 invaginations per nucleus (P = 0.5), showed no more significant changes. Large NUP153 cytoplasmic clusters present before MGA may represent a maternally provided NUP153 deposit. MGA may mark the switch from the use of a NUP153 deposit to on-demand production. Additionally, over- and under-representation analyses on mitotic blastomeres revealed that NUP153 association with chromatin is initiated during metaphase before the initiation of the regeneration of the lamina (P < 0.001; chi-squared goodness-of-fit test). In conclusion, rabbit embryonic development is accompanied by stage-dependent changes of the amount of nuclear invaginations carrying nuclear pores. Although cattle and mouse embryos almost exclusively feature narrow invaginations restricted to the nuclear periphery and not in contact with nucleoli, rabbit embryos feature additional wide invaginations that can reach across the nucleus and target nucleoli.
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Fan, Hui, Junfeng Hui, Zhiguang Duan, Daidi Fan, Yu Mi, Jianjun Deng i Hui Li. "Novel Scaffolds Fabricated Using Oleuropein for Bone Tissue Engineering". BioMed Research International 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/652432.
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We investigated the feasibility of oleuropein as a cross-linking agent for fabricating three-dimensional (3D) porous composite scaffolds for bone tissue engineering. Human-like collagen (HLC) and nanohydroxyapatite (n-HAp) were used to fabricate the composite scaffold by way of cross-linking. The mechanical tests revealed superior properties for the cross-linked scaffolds compared to the uncross-linked scaffolds. The as-obtained composite scaffold had a 3D porous structure with pores ranging from 120 to 300 μm and a porosity of73.6±2.3%. The cross-linked scaffolds were seeded with MC3T3-E1 Subclone 14 mouse osteoblasts. Fluorescence staining, the Cell Counting Kit-8 (CCK-8) assay, and scanning electron microscopy (SEM) indicated that the scaffolds enhanced cell adhesion and proliferation. Our results indicate the potential of these scaffolds for bone tissue engineering.
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Ninivaggi, Marisa, Hilde Kelchtermans, Marijke J. Kuijpers, Bianca Hemmeryckx, Johan W. M. Heemskerk, Theo Lindhout, Marc F. Hoylaerts i Bas de Laat. "Whole blood thrombin generation in Bmal1-deficient mice". Thrombosis and Haemostasis 112, nr 08 (2014): 271–75. http://dx.doi.org/10.1160/th13-11-0910.
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SummaryThe Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20– to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437 ± 7 nM.min; mean ± SD, n=7) when compared with WT mice (ETP=220 ± 45 nM.min; mean ± SD, n=5). The peak heights also differed significantly (p=0.027). By applying SEM we found that Bmal1 deficient mice display a denser fibrin network with smaller pores compared to WT mice. In conclusion, the whole blood TG assay in mice revealed to be reproducible. As a proof-of-principle we have shown that the whole blood TG assay is capable of detecting a prothrombotic phenotype in Bmal1-KO mice.
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Grigoriev, A. M., Yu B. Basok, A. D. Belova, N. P. Shmerko, A. M. Subbot, V. K. Kulakova, V. I. Lozinsky i V. I. Sevastianov. "Biological properties of macroporous cryostructurate based on extracellular matrix components". Russian Journal of Transplantology and Artificial Organs 25, nr 4 (16.10.2023): 109–20. http://dx.doi.org/10.15825/1995-1191-2023-4-109-120.
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Objective: to study the biological properties of macroporous cryostructurate from multicomponent concentrated collagen-containing solution (MCCS) as a promising matrix for the formation of cell- and tissue-engineered constructs.Materials and methods. A macroporous spongy carrier was obtained by cryostructuring of collagencontaining extract, prepared by acetic acid hydrolysis of chicken connective tissue (BIOMIR Service, Russian Federation). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (Sigma-Aldrich, USA) was used to make the cryostructurate water insoluble. The micromorphology of the sponge surface was studied using scanning electron microscopy. The cytotoxicity of the carrier was evaluated by reaction of the mouse NIH 3T3 fibroblast cell culture using automated microscope IncuCyte ZOOM (EssenBioscience, USA). Biocompatibility of the macroporous carrier was studied on cultures of human adipose tissue-derived mesenchymal stromal cells (AD-MSC), human hepatocellular carcinoma cell line HepG2 and human umbilical vein endothelial cell line EA.hy926. The metabolic activity of cells was determined using PrestoBlue™ reagents (Invitrogen™, USA). Cell population development during long-term cultivation of the cell-engineered construct (CEC) was assessed by fluorescencelifetime imaging microscopy over the entire surface of the sample using a Leica Dmi8 inverted microscope with Leica Thunder software (Leica Microsystems, Germany).Results. Optical microscopy and scanning electron microscopy (SEM) showed the presence of pores of different sizes in the resulting biopolymer material: large pores with 237 ± 32 μm diameter, medium-sized pores with 169 ± 23 μm diameter, and small-sized pores with 70 ± 20 μm diameter; large and medium-sized pores were predominant. The studied media did not exhibit cytotoxicity. Cell adhesion and proliferation on the surface of the material and their penetration into the underlying layers during long-term cultivation were observed. The highest metabolic activity of the cells was observed for human AD-MSC on day 14, which corresponds to the normal dynamics of development of a population of cells of this type. The functional activity of HepG2 cells – albumin and urea production – was shown in the liver CEC model.Conclusion. The good adhesion and active proliferation that were shown for the three cell types indicate that the resulting biopolymer carrier is biocompatible, and that the spread of the cells into the inner volume of the sponge and active population of the sponge under prolonged culturing indicates that this material can be used to create cell- and tissue-engineered constructs.
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Agapova, O. I., A. E. Efimov, M. M. Moisenovich, V. G. Bogush i I. I. Agapov. "COMPARATIVE ANALYSIS OF THREE-DIMENSIONAL NANOSTRUCTURE OF POROUS BIOCOMPATIBLE SCAFFOLDS MADE OF RECOMBINANT SPIDROIN AND SILK FIBROIN FOR REGENERATIVE MEDICINE". Russian Journal of Transplantology and Artificial Organs 17, nr 2 (26.05.2015): 37–44. http://dx.doi.org/10.15825/1995-1191-2015-2-37-44.
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Aim.To perform a comparison of three-dimensional nanostructure of porous biocompatible scaffolds made of fibroinBombix moriand recombinant spidroin rS1/9.Materials and methods.Three-dimensional porous scaffolds were produced by salt leaching technique. The comparison of biological characteristics of the scaffolds shows that adhesion and proliferation of mouse fibroblastsin vitroon these two types of scaffolds do not differ significantly. Comparative experimentsin vivoshow that regeneration of bone tissue of rats is faster with implantation of recombinant spidroin scaffolds. Three-dimensional nanostructure of scaffolds and interconnectivity of nanopores were studied with scanning probe nanotomography (SPNT) to explain higher regenerative activity of spidroin-based scaffolds.Results.Significant differences were detected in the integral density and volume of pores: the integral density of nanopores detected on 2D AFM images is 46 μm–2 and calculated volume porosity is 24% in rS1/9-based scaffolds; in fibroin-based three-dimensional structures density of nanopores and calculated volume porosity were 2.4 μm–2 and 0.5%, respectively. Three-dimensional reconstruction system of nanopores and clusters of interconnected nanopores in rS1/9-based scaffolds showed that volume fraction of pores interconnected in percolation clusters is 35.3% of the total pore volume or 8.4% of the total scaffold volume.Conclusion.Scanning probe nanotomography method allows obtaining unique information about topology of micro – and nanopore systems of artificial biostructures. High regenerative activity of rS1/9-based scaffolds can be explained by higher nanoporosity of the scaffolds.
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Hodaei, Mohammad, i Andreas Mandelis. "Quantitative osteoporosis diagnosis of porous cancellous bone using poroelastodynamic modal analysis". Journal of the Acoustical Society of America 154, nr 5 (1.11.2023): 3101–24. http://dx.doi.org/10.1121/10.0022351.
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Osteoporosis is a skeletal disease characterized by reduced bone mass and microarchitectural deterioration, leading to increased fragility. This study presents a novel three-dimensional poroelastodynamic model for analyzing cancellous bone free vibration responses. The model incorporates the Navier-Stokes equations of linear elasticity and the Biot theory of porous media, allowing the investigation of osteoporosis-related changes. The analysis considers parameters like porosity, density, elasticity, Poisson ratio, and viscosity of bone marrow within the porous medium. Our findings indicate that natural frequencies of cancellous bone play a crucial role in osteoporosis prediction. By incorporating experimental data from 12 mouse femurs, we unveil insights into osteoporosis prediction. Increased porosity reduces bone stiffness, lowering natural frequencies. However, it also increases bone mass loss relative to stiffness, leading to higher frequencies. Therefore, the natural frequencies of osteoporotic bone are always higher than the natural frequencies of normal bone. Additionally, an increase in bone marrow within the pores, while increasing damping effects, also increases natural frequencies, which is another indication of osteoporosis growth in bone. The presence of bone marrow within the pores further influences natural frequencies, providing additional insights into osteoporosis growth. Thinner and smaller bones are found to be more susceptible to osteoporosis compared to larger and bigger bones due to their higher natural frequencies at equivalent porosity levels.
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TAN, Jing-he, Zhong-Hua LIU, Wei REN, Hua NI, Xing-shen SUN i Gui-xin HE. "The Role of Extracellular Ca2+ and Formation and Duration of Pores on the Oolemma in the Electrical Activation of Mouse Oocytes." Journal of Reproduction and Development 43, nr 4 (1997): 289–93. http://dx.doi.org/10.1262/jrd.43.289.
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Solel, E., E. Brudvik, S. Afroz, R. Choudhury, R. Bjerkvig, J. Hossain i H. Miletic. "P06.02.B GSDME-MEDIATED PYROPTOSIS IN GLIOBLASTOMA". Neuro-Oncology 25, Supplement_2 (1.09.2023): ii44—ii45. http://dx.doi.org/10.1093/neuonc/noad137.142.
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Abstract BACKGROUND Immunotherapeutic strategies have largely failed for glioblastoma , the most frequent primary brain tumor. Beyond apoptosis and autophagy, little is known about cell death mechanisms in glioblastoma, despite their potential impact on the immune microenvironment. Pyroptosis, unlike apoptosis, is an immunogenic and lytic type of programmed cell death, where gasdermin proteins form pores into the plasma membrane, releasing cell content to the tumor microenvironment. Gasdermin E (GSDME), often inactivated in cancers by mutations/low expression, is proteolytically cleaved by caspase-3 upon apoptotic stimuli leading to a shift from apoptosis to pyroptosis. Here, we investigated the relevance of GSDME-mediated pyroptosis for glioblastoma. According to The Cancer Genome Atlas, GSDME is highly expressed in glioblastomas compared to many other cancers and no mutations are found. MATERIAL AND METHODS Using the drug raptinal to induce apoptosis, we assessed expression of full length and cleaved GSDME as well as cleaved caspase-3 in syngeneic mouse and human patient-derived glioblastoma stem-like cell lines by western blotting. Lytic cell death and viability were measured by lactate dehydrogenase (LDH) and WST-1 assays respectively. PI uptake assay was performed to capture pyroptotic morphology by Incucyte®️ S3. BAPTA-AM, a fast Ca2+ chelator, was used to block plasma membrane repair in human glioblastoma cell lines and analyze its effect on pyroptosis. GSDME was knocked out by CRISPR/Cas9. RESULTS GSDME and caspase-3 cleavage were confirmed after raptinal treatment in mouse and human glioblastoma cell lines, indicating that the molecular pyroptotic machinery is intact. A significant drop in viability with raptinal was shown indicating successful execution of cell death. A significant LDH release was detected in mouse glioblastoma cell lines, which could be blocked by GSDME knockout. In contrast, human glioblastoma cells, although executing GSDME cleavage, showed less or no LDH release. Pyroptotic morphology was detected after raptinal treatment in mouse glioblastoma cell lines, while it was delayed/inhibited in human glioblastoma cells. Application of BAPTA-AM in human glioblastoma cell lines enhanced pyroptotic morphology, indicating a role for plasma membrane repair in inhibiting pyroptosis. CONCLUSION The molecular machinery to execute pyroptosis is functioning in mouse and human glioblastoma cells. However, while mouse glioblastoma cells show clear execution of pyroptosis upon apoptosis induction, human glioblastoma cells indicate less efficient pyroptotic ability which partly can be reverted by blocking plasma membrane repair. While pyroptosis can induce anti-tumor immunity in experimental cancer models, future in vivo experiments must delineate if pyroptosis is a viable strategy for immunotherapy of glioblastoma.
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Seki, Shinsuke, Keisuke Edashige, Sakiko Wada i Peter Mazur. "Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation". REPRODUCTION 142, nr 4 (październik 2011): 505–15. http://dx.doi.org/10.1530/rep-10-0538.
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The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to −7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to −70 °C. IIF that occurred during the 20 °C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were −34, −40, −35, and −25 °C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10–15 °C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-strandedAqp3RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.
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Wardenburg, Juliane Bubeck, i Olaf Schneewind. "Vaccine protection against Staphylococcus aureus pneumonia". Journal of Experimental Medicine 205, nr 2 (11.02.2008): 287–94. http://dx.doi.org/10.1084/jem.20072208.
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Staphylococcus aureus pneumonia causes significant mortality in hospitalized or healthy individuals, and recent increases in morbidity are attributed to the rapid spread of methicillin-resistant S. aureus (MRSA) strains, which are often not susceptible to antibiotic therapy. α-Hemolysin (Hla), a secreted pore-forming toxin, is an essential virulence factor of MRSA in a mouse model of S. aureus pneumonia. We show that the level of Hla expression by independent S. aureus strains directly correlates with their virulence. Active immunization with a mutant form of Hla (HlaH35L), which cannot form pores, generates antigen-specific immunoglobulin G responses and affords protection against staphylococcal pneumonia. Moreover, transfer of Hla-specific antibodies protects naive animals against S. aureus challenge and prevents the injury of human lung epithelial cells during infection. Thus, Hla vaccination or immunotherapy may prevent S. aureus pneumonia in humans.
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OH, Seunguk, Rick Odland, Scott R. Wilson, Kurt M. Kroeger, Chunyan Liu, Pedro R. Lowenstein, Maria G. Castro, Walter A. Hall i John R. Ohlfest. "Improved distribution of small molecules and viral vectors in the murine brain using a hollow fiber catheter". Journal of Neurosurgery 107, nr 3 (wrzesień 2007): 568–77. http://dx.doi.org/10.3171/jns-07/09/0568.
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Object A hollow fiber catheter was developed to improve the distribution of drugs administered via direct infusion into the central nervous system (CNS). It is a porous catheter that significantly increases the surface area of brain tissue into which a drug is infused. Methods Dye was infused into the mouse brain through convection-enhanced delivery (CED) using a 28-gauge needle compared with a 3-mm-long hollow fiber catheter. To determine whether a hollow fiber catheter could increase the distribution of gene therapy vectors, a recombinant adenovirus expressing the firefly luciferase reporter was injected into the mouse striatum. Gene expression was monitored using in vivo bioluminescent imaging. To assess the distribution of gene transfer, an adenovirus expressing green fluorescent protein was injected into the striatum using a hollow fiber catheter or a needle. Results Hollow fiber catheter–mediated infusion increased the volume of brain tissue labeled with dye by 2.7 times relative to needle-mediated infusion. In vivo imaging revealed that catheter-mediated infusion of adenovirus resulted in gene expression that was 10 times greater than that mediated by a needle. The catheter appreciably increased the area of brain transduced with adenovirus relative to a needle, affecting a significant portion of the injected hemisphere. Conclusions The miniature hollow fiber catheter used in this study significantly increased the distribution of dye and adenoviral-mediated gene transfer in the mouse brain compared with the levels reached using a 28-gauge needle. Compared with standard single-port clinical catheters, the hollow fiber catheter has the advantage of millions of nanoscale pores to increase surface area and bulk flow in the CNS. Extending the scale of the hollow fiber catheter for the large mammalian brain shows promise in increasing the distribution and efficacy of gene therapy and drug therapy using CED.
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Liu, C. C., P. M. Persechini i J. D. Young. "Expression and characterization of functionally active recombinant perforin produced in insect cells." Journal of Immunology 156, nr 9 (1.05.1996): 3292–300. http://dx.doi.org/10.4049/jimmunol.156.9.3292.
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Abstract A key cytolytic mediator used by killer lymphocytes, perforin (also known as pore-forming protein or cytolysin), has been shown to be capable of undergoing polymerization to form pores in cell membranes and cause osmotic lysis of target cells. Although perforin has been purified from killer lymphocytes and the coding gene has been cloned and sequenced, information concerning the domain structure of the perforin molecule has remained scarce. To overcome the difficulty in obtaining sufficient amounts of perforin and to further assess the functional relevance of the N-terminal portion of the perforin molecule in its lytic activity, we have attempted in the present study to produce recombinant perforins. Three forms of recombinant mouse perforin, a full-length form and two N-terminal truncated forms, have been expressed in insect (Spodoptera frugiperda (Sf9)) cells using recombinant baculovirus. Biochemical and functional characterization showed the purified full-length recombinant perforin to be capable of lysing target cells, inducing Ca2+ influx into target cells, and forming structural pores in target membranes. Significant lytic activities were also detected for the two truncated recombinant perforins lacking, respectively, the N-terminal 21 amino acid residues and 121 amino acid residues. Time course study showed that the latter acted less efficiently than the former. These results suggest the N-terminal portion of the perforin molecule to be an important, but not the only, domain responsible for the lytic function of the perforin molecule.
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Arzumanyan, V. G., T. A. Artemeva i A. M. Iksanova. "ANTIFUNGAL ACTIVITY OF HUMAN AND SOME MAMMALS SERA". Journal of microbiology epidemiology immunobiology 1, nr 1 (23.08.2019): 17–22. http://dx.doi.org/10.36233/0372-9311-2019-1-17-22.
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Aim. Estimation of activity of native human serum and its antimicrobial peptides fraction against clinically important yeasts and comparison with the activity of some mammals sera. Materials and methods. Pooled samples of human, bovine, rabbit and mouse sera and collection strains of yeasts Candida albicans, Rhodotorula mucilaginosa, Malassezia furfur, Cryptococcus neoformans, Geotrichum candidum, Trichosporon cutaneum, Saccharomyces cerevisiae were used in the study. Antimicrobial peptides fractions (AMP) were obtained by filtration through molecular filters with 100 kDa pores. Activity of sera and their AMP-fractions were estimated by spectrophotometric method. Results. Activity of native mammal sera varied in diapason 73÷89% independently from yeast genus, although AMP-fractions activity varied more significantly. The minimal sensitivity to AMP-fractions of sera demonstrated M. furfur (activity values were equal 0÷13,5%) and G. candidum (0÷6,5%), but the maximal — R. mucilaginosa (12,3÷56,4%), C. albicans (22,0÷32,9%), and C. neoformans (17,1÷29,9%). Activity values of AMP-fractions of human serum were correlated meaningfully with no of the values of other mammals (Pirson coefficient r=0,459÷0,527). Considerable correlation of the indexes took place between rabbit and bovine sera (r=0,827), as well as between rabbit and mouse sera (r = 0,753). Conclusion. The differences between AMP-fractions activity towards studied yeast genera/specia indicate the occurrence of its specificity probably related with structural organization of cytoplasmic membrane of yeast cells as well as with variations in AMP composition in different mammals.
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Qiu, Yujia, Elma Sakinatus Sajidah, Sota Kondo, Shinnosuke Narimatsu, Muhammad Isman Sandira, Yoshiki Higashiguchi, Goro Nishide i in. "An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy". Cells 13, nr 3 (2.02.2024): 279. http://dx.doi.org/10.3390/cells13030279.
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Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.
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Leung, Jennifer, Michael Chang, Richard E. Moore, Jargalsaikhan Dagvadorj, Fayyaz S. Sutterwala i Suzanne L. Cassel. "Gasdermin D and Gasdermin E Are Dispensable for Silica-Mediated IL-1β Secretion from Mouse Macrophages". ImmunoHorizons 8, nr 9 (1.09.2024): 679–87. http://dx.doi.org/10.4049/immunohorizons.2400019.
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Abstract Silica crystals activate the NLRP3 inflammasome in macrophages, resulting in the caspase-1–dependent secretion of the proinflammatory cytokine IL-1β. Caspase-1–mediated cleavage of gasdermin D (GSDMD) triggers the formation of GSDMD pores, which drive pyroptotic cell death and facilitate the rapid release of IL-1β. However, the role of GSDMD in silica-induced lung injury is unclear. In this study, we show that although silica-induced lung injury is dependent on the inflammasome adaptor ASC and IL-1R1 signaling, GSDMD is dispensable for acute lung injury. Although the early rapid secretion of IL-1β in response to ATP and nigericin was GSDMD dependent, GSDMD was not required for IL-1β release at later time points. Similarly, secretion of IL-1β from macrophages in response to silica and alum proceeded in a GSDMD-independent manner. We further found that gasdermin E did not contribute to macrophage IL-1β secretion in the absence of GSDMD in vitro and was also not necessary for silica-induced acute lung injury in vivo. These findings demonstrate that GSDMD and gasdermin E are dispensable for IL-1β secretion in response to silica in vitro and in silica-induced acute lung injury in vivo.
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Predescu, D., i G. E. Palade. "Plasmalemmal vesicles represent the large pore system of continuous microvascular endothelium". American Journal of Physiology-Heart and Circulatory Physiology 265, nr 2 (1.08.1993): H725—H733. http://dx.doi.org/10.1152/ajpheart.1993.265.2.h725.
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In the capillary physiology literature, molecules and particles larger than 10 nm are assumed to leave the plasma mostly through large pores located at the level of intercellular junctions in microvessels lined with a continuous endothelium. In morphological studies of similar microvessels, outgoing particles > 10 nm were detected in endothelial plasmalemmal vesicles not in intercellular junctions. Because the probes may not be found in transit through the junctions because they may be swept away by strong currents generated by Starling forces, we have examined a large number of junctions in arteriolar, capillary, and venular segments of bipolar vascular fields of mouse diaphragms collected after perfusion with large pore probes. The results presented in this study indicate that 1) the perfused probes accumulate in the luminal introits of the junctions as filtration residues that decrease in size and frequency from arterioles to venules, and 2) large pore probes move across the endothelium exclusively through plasmalemmal vesicles.
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Matsuura, T., M. Kawada, S. Hasumura, S. Nagamori, T. Osata, M. Yamaguchp, Y. Hataba i in. "High Density Culture of Immortalized Liver Endothelial Cells in the Radial-flow Bioreactor in the Development of an Artificial Liver". International Journal of Artificial Organs 21, nr 4 (kwiecień 1998): 229–34. http://dx.doi.org/10.1177/039139889802100410.
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Liver endothelial cells are important components of the tissue along the hepatic sinusoid. They are responsible for microcirculation in the liver and scavenger functions. It would therefore be important to include these cells in any hybrid type of artificial liver in addition to hepatocytes. However, it is difficult to culture these cells in vitro. The development of a liver endothelial cell line, which maintains the characteristics of the primary culture, would thus be of great benefit in the development of an artificial liver. In the present study we established immortalized liver endothelial cells from the liver of an H-2Kb-tsA58 transgenic mouse, which harbors the SV40 TAg gene. Hepatic sinusoidal cells isolated from H-2Kd-tsA58 mouse proliferated In the presence of γ-interferon at 33°C. Four clones were established, out of which clone M1 had the highest amounts of PGI2 production, as well as plasminogen activator activity and internalized acetylated low density lipoprotein. On culture dishes the M1 cells grew individually and spread. Sieve plates on the cell surface were not readily visible, but small pores were detected under electron microscopic observation. These results suggest that M1 clone cells originated from liver endothelial cells. Moreover it was possible to culture the immortalized liver endothelial cells in a radial-flow bioreactor for 5 days, with a maximum 6-keto prostaglandin F1α production of 25 μg per day. This suggests that immortalized liver endothelial cells and a radial-flow bioreactor can prove useful tools in the development an artificial liver.
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Dean, Kristin T., Bockgie Jung i Buka Samten. "Identification of N-formylated peptides as the neutrophilic chemotactic factors of Mycobacterium tuberculosis". Journal of Immunology 204, nr 1_Supplement (1.05.2020): 148.19. http://dx.doi.org/10.4049/jimmunol.204.supp.148.19.
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Abstract Since tuberculosis (TB) remains a major cause of human death, it is urgent to understand the pathology of TB. Lung neutrophil infiltration initiates granuloma formation and is associated with disease severity. Though several cytokines and chemokines are associated with neutrophil infiltration in chronic inflammation, neutrophil chemotactic factors of Mycobacterium tuberculosis (Mtb) remain unexplored. Thus, we performed neutrophil chemotactic assays by culturing neutrophils of healthy human blood samples and mouse bone marrow in the upper chamber of a transwell plate with 5–8 μm pores with the culture filtrates of Mtb H37Rv (CF) in the lower chamber using N-formyl-met-leu-phe (fMLF), a bacterial neutrophil chemotactic tripeptide, as a positive control. The cell numbers in the lower chamber were determined by flow cytometry as a chemotactic indicator. Our results show that CF induced chemotaxis of both human and mouse neutrophils in an Mtb culture period dependent manner. Testing CF of 11 clinical isolates of Mtb also showed neutrophil chemotactic activity. Sulfasalazine, a formylated peptide receptor inhibitor, blocked chemotaxis of neutrophils, indicating the involvement of fPRs in CF induces chemotaxis of neutrophils. Mass spectrometry analysis of CF identified three candidate N-formylated heptapeptides. Authentication of the chemotactic activities of the identified peptides with synthetic mimetics confirmed their neutrophil chemotactic activities and indicated the requirement of N-formylation for their chemotactic activities. Thus, we have identified novel formylated peptides of Mtb with neutrophil chemotactic activities, and further studies are in progress to determine their significance in TB infection.
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Shin, Jin Hak, Seon Sook Kim i Su Ryeon Seo. "Pyrrolidine Dithiocarbamate Suppresses Cutibacterium acnes-Induced Skin Inflammation". International Journal of Molecular Sciences 24, nr 5 (23.02.2023): 4444. http://dx.doi.org/10.3390/ijms24054444.
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Cutibacterium acnes (C. acnes), a Gram-positive anaerobic bacterium, proliferates in hair follicles and pores and causes inflammation in the skin of young people. The rapid growth of C. acnes triggers macrophages to secrete proinflammatory cytokines. Pyrrolidine dithiocarbamate (PDTC) is a thiol compound that exerts antioxidant and anti-inflammatory effects. Although the anti-inflammatory function of PDTC in several inflammatory disorders has been reported, the effect of PDTC on C. acnes-induced skin inflammation remains unexplored. In the present study, we examined the effect of PDTC on C. acnes-induced inflammatory responses and determined the mechanism by using in vitro and in vivo experimental models. We found that PDTC significantly inhibited the expression of C. acnes-induced proinflammatory mediators, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and NOD-like receptor (NLR) pyrin domain-containing 3 (NLRP3), in mouse-bone-marrow-derived macrophage (BMDM) cells. PDTC suppressed C. acnes-induced activation of nuclear factor-kappa B (NF-κB), which is the major transcription factor for proinflammatory cytokine expression. In addition, we found that PDTC inhibited caspase-1 activation and IL-1β secretion through suppressing NLRP3 and activated the melanoma 2 (AIM2) inflammasome but not the NLR CARD-containing 4 (NLRC4) inflammasome. Moreover, we found that PDTC improved C. acnes-induced inflammation by attenuating C. acnes-induced IL-1β secretion in a mouse acne model. Therefore, our results suggest that PDTC has potential therapeutic value for the amelioration of C. acnes-induced skin inflammation.
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Jacobelli, Jordan, Scott B. Thompson, Adam M. Sandor, Victor Lui, Jeffrey W. Chung, Monique M. Waldman i Rachel S. Friedman. "Formin-like-1 mediates effector T cell extravasation through restrictive endothelial barriers enabling T cell entry into peripheral tissues and autoimmunity induction". Journal of Immunology 204, nr 1_Supplement (1.05.2020): 220.12. http://dx.doi.org/10.4049/jimmunol.204.supp.220.12.
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Abstract To perform their functions, T cells migrate from the blood vasculature into tissues by traversing the vascular wall through a process known as transendothelial migration (TEM). While many of the extracellular cues that guide this process have been identified, the role of downstream cytoskeletal effectors that mediate force generation and morphological changes required for TEM remains an area of active research. Formin-like-1 (FMNL1) is a cytoskeletal effector that mediates cytoskeletal remodeling. However, the role of FMNL1 in T cell functions, and migration in particular, is mostly unknown. The goal of our study was to examine the role of FMNL1 in T cell extravasation and trafficking. We generated a new FMNL1 knock-out (KO) mouse and identified a novel role for FMNL1 in promoting T cell migration through restrictive endothelial barriers during TEM. In vitro, FMNL1-deficient T cells had reduced actin polymerization in response to chemokine signaling. Furthermore, FMNL1 KO T cells had a selective impairment in chemotaxis through restrictive 3μm pores vs permissive 5μm pores. Using time-lapse microscopy, we determined that FMNL1-deficient T cells are severely impaired in their capacity to complete TEM through endothelial barriers. In vivo, we found that FMNL1 was dispensable for T cell extravasation through permissive high endothelial venules and homeostatic trafficking to lymphoid organs. Instead, FMNL1 regulated the ability of activated T cells to extravasate into inflamed peripheral tissues and enabled self-reactive T cells to induce autoimmune disease. Overall, our data identify FMNL1 as a key regulator of lymphocyte migration through restrictive barriers and as a potential target for modulating effector T cell trafficking.