scholarly journals Innate Biomineralization

2020 ◽  
Vol 21 (14) ◽  
pp. 4820 ◽  
Author(s):  
Erming Tian ◽  
Fumiya Watanabe ◽  
Betty Martin ◽  
Maurizio Zangari
Keyword(s):  
Calcium Ion ◽  
Mammalian Cells ◽  
Mononuclear Cells ◽  
Collagen Matrix ◽  
Living Cells ◽  
Maturation Stage ◽  
Cell Type ◽  
Biomineralization Process ◽  
Concomitant Exposure ◽  
Innate Ability

In vertebrates, biomineralization is a feature considered unique to mature osteoblasts and odontoblasts by which they synthesize hydroxyapatite (HAP), which is deposited in the collagen matrix to construct endoskeleton. For many decades, the mechanisms that modulate differentiation and maturation of these specialized cells have been sought as a key to understanding bone-remodeling defects. Here, we report that biomineralization is an innate ability of all mammalian cells, irrespective of cell type or maturation stage. This innate biomineralization is triggered by the concomitant exposure of living cells to three indispensable elements: calcium ion, phosphoester salt, and alkaline phosphatase. Any given somatic cell, including undifferentiated mononuclear cells, can undergo a biomineralization process to produce calcium-phosphate agglomerates. The biologically generated minerals under such conditions are composed of genuine HAP crystallites of Ca10(PO4)6(OH)2 and 5–10 nanometer (nm) in size. This discovery will profoundly improve our understanding of bone metabolism and ectopic calcifications.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation

2001 ◽  
Vol 204 (3) ◽  
pp. 443-455
Author(s):  
C. Faucheux ◽  
S. Nesbitt ◽  
M. Horton ◽  
J. Price
Keyword(s):  
Type I Collagen ◽  
Mononuclear Cells ◽  
Osteoclast Differentiation ◽  
Cathepsin K ◽  
Collagen Matrix ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Deer Antler

Deer antlers are a rare example of mammalian epimorphic regeneration. Each year, the antlers re-grow by a modified endochondral ossification process that involves extensive remodelling of cartilage by osteoclasts. This study identified regenerating antler cartilage as a site of osteoclastogenesis in vivo. An in vitro model was then developed to study antler osteoclast differentiation. Cultured as a high-density micromass, cells from non-mineralised cartilage supported the differentiation of large numbers of osteoclast-like multinucleated cells (MNCs) in the absence of factors normally required for osteoclastogenesis. After 48 h of culture, tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (osteoclast precursors) were visible, and by day 14 a large number of TRAP-positive MNCs had formed (783+/−200 per well, mean +/− s.e.m., N=4). Reverse transcriptase/polymerase chain reaction (RT-PCR) showed that receptor activator of NF κ B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) mRNAs were expressed in micromass cultures. Antler MNCs have the phenotype of osteoclasts from mammalian bone; they expressed TRAP, vitronectin and calcitonin receptors and, when cultured on dentine, formed F-actin rings and large resorption pits. When cultured on glass, antler MNCs appeared to digest the matrix of the micromass and endocytose type I collagen. Matrix metalloproteinase-9 (MMP-9) may play a role in the resorption of this non-mineralised matrix since it is highly expressed in 100 % of MNCs. In contrast, cathepsin K, another enzyme expressed in osteoclasts from bone, is only highly expressed in resorbing MNCs cultured on dentine. This study identifies the deer antler as a valuable model that can be used to study the differentiation and function of osteoclasts in adult regenerating mineralised tissues.

scholarly journals Trends in SPR Cytometry: Advances in Label-Free Detection of Cell Parameters

Biosensors ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 102 ◽  
Author(s):  
Richard Schasfoort ◽  
Fikri Abali ◽  
Ivan Stojanovic ◽  
Gestur Vidarsson ◽  
Leon Terstappen
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Mammalian Cells ◽  
Living Cells ◽  
Label Free ◽  
Intact Cells ◽  
Cell Parameters ◽  
Spr Imaging ◽  
Label Free Detection

SPR cytometry entails the measurement of parameters from intact cells using the surface plasmon resonance (SPR) phenomenon. Specific real-time and label-free binding of living cells to sensor surfaces has been made possible through the availability of SPR imaging (SPRi) instruments and researchers have started to explore its potential in the last decade. Here we will discuss the mechanisms of detection and additionally describe the problems and issues of mammalian cells in SPR biosensing, both from our own experience and with information from the literature. Finally, we build on the knowledge and applications that has already materialized in this field to give a forecast of some exciting applications for SPRi cytometry.

scholarly journals Measuring the Cell-Induced Deformation of Collagen Matrix Detected With Digital Holographic Microscopy

2007 ◽  
Author(s):  
Kaveh Azartash ◽  
Enrico Gratton
Keyword(s):  
Mammalian Cells ◽  
Collagen Matrix ◽  
Spatial Arrangement ◽  
Digital Holographic Microscopy ◽  
Physical Aspect ◽  
Reconstruction Algorithms ◽  
Phase Images ◽  
Holographic Microscopy ◽  
Set Up ◽  
Charged Couple Device

A modified Mach-Zender set-up in reflection is applied to record and reconstruct holographic amplitude and phase images. A charged couple device (CCD) is used to record a hologram and numerical reconstruction algorithms are then applied to rebuild the hologram for obtaining both phase and amplitude information. One could also focus on multiple focal planes from a single hologram, similar to the focusing control of a conventional microscope. The morphology and behavior of mammalian cells is determined by an interaction between signals from the intracellular matrix and the cellular responses. It is important to note that the physical aspect of the extracellular matrix is as significant as the chemical nature of it. Specifically the stresses, mechanical forces, and the profile of the external environment have major effects on cell behavior. The mechanical and physical characteristics of a tissue are greatly dependent on a hierarchical spatial arrangement of its extra-cellular matrix components. A key player in the ECM is collagen which exhibits significant tensile strength on the cellular scale. Digital holographic microscopy (DHM) is applied to study the deformation of collage matrix in response to cell migration.

scholarly journals Glass beads load macromolecules into living cells

1987 ◽  
Vol 88 (5) ◽  
pp. 669-678
Author(s):  
P.L. McNeil ◽  
E. Warder
Keyword(s):  
Culture Medium ◽  
Small Volume ◽  
Cultured Cells ◽  
Cell Monolayer ◽  
Cell Loss ◽  
Living Cells ◽  
Glass Beads ◽  
Cell Type ◽  
Large Numbers ◽  
Micron Diameter

We describe and characterize an exceptionally rapid and simple new technique for loading large numbers of cultured cells with large macromolecules. The culture medium of the cell monolayer is replaced by a small volume of the macromolecule to be loaded. Glass beads (75–500 micron diameter) are then sprinkled onto the cells, the cells are washed free of beads and exogenous macromolecules, and ‘bead-loading’ is completed. The conditions for bead-loading can readily be modified to accommodate cell type and loading objectives: for example, the amount of loading per cell increases if bead size is increased or if beads are agitated after sprinkling onto the monolayer, but at the expense of increased cell loss. As many as 97% of a population of bovine aortic endothelial (BAE) cells were loaded with a 10,000 Mr dextran; and 79% with a 150,000 Mr dextran using bead-loading. Various cell lines have been loaded using glass beads. Moreover, bead-loading has the advantage of producing loaded cells that remain adherent and well-spread, thus minimizing recovery time and permitting immediate microscopic examination.

HIV-1revAntisense Phosphorothioate Oligonucleotide Binding to Human Mononuclear Cells Is Cell Type Specific and Inducible

1994 ◽  
Vol 4 (4) ◽  
pp. 285-289 ◽  
Author(s):  
SAMUEL J. PIRRUCCELLO ◽  
GREG A. PERRY ◽  
PAUL J. BOCK ◽  
MOLLY S. LANG ◽  
SCOTT M. NOEL ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Cell Type ◽  
Phosphorothioate Oligonucleotide ◽  
Human Mononuclear Cells ◽  
Cell Type Specific ◽  
Oligonucleotide Binding

scholarly journals Fabricating polyacrylamide microbeads by inverse emulsification to mimic the size and elasticity of living cells

2017 ◽  
Vol 5 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Nicholas R. Labriola ◽  
Edith Mathiowitz ◽  
Eric M. Darling
Keyword(s):  
Elastic Properties ◽  
Mammalian Cells ◽  
Living Cells

Inverse emulsification was used to fabricate polyacrylamide (PAAm) microbeads with size and elastic properties similar to typical, mammalian cells.

Associated Anisotropy of intrinsic NAD(P)H for monitoring changes in the metabolic activities of breast cancer cells in three-dimensional collagen matrix

2021 ◽  
Author(s):  
Anh Cong ◽  
Rafaela M. L. Pimenta ◽  
Jon Holy ◽  
Ahmed A Heikal
Keyword(s):  
Breast Cancer ◽  
Cell Culture ◽  
Breast Cancer Cells ◽  
Monolayer Culture ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Living Cells ◽  
Two Dimensional ◽  
Metabolic Activities

The majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture. Recent studies, however, suggest that 2D cell culture promotes specific types of...

scholarly journals Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Biology ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 430
Author(s):  
Amreen ◽  
Hayssam M. Ali ◽  
Mohammad Ahmad ◽  
Mohamed Z. M. Salem ◽  
Altaf Ahmad
Keyword(s):  
Hydrogen Peroxide ◽  
Real Time ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Resonance Energy ◽  
Living Cells ◽  
Stress Conditions ◽  
Live Cells ◽  
Cell Physiology ◽  
Broad Dynamic Range

Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.

scholarly journals Cell-type-specific differences in KDEL receptor clustering in mammalian cells

PLoS ONE ◽  
2020 ◽  
Vol 15 (7) ◽  
pp. e0235864
Author(s):  
Achim Bauer ◽  
Ludger Santen ◽  
Manfred J. Schmitt ◽  
M. Reza Shaebani ◽  
Björn Becker
Keyword(s):  
Mammalian Cells ◽  
Cell Type ◽  
Receptor Clustering ◽  
Cell Type Specific ◽  
Kdel Receptor
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