Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity

Histochemical detection of tartrate-resistant acid phosphatase (TRAP) activity is a fundamental technique for visualizing osteoclastic bone resorption and assessing osteoclast activity status in tissues. This approach has mostly employed colorimetric detection, which has limited quantification of activity in situ and co-labelling with other skeletal markers. Here we report simple colorimetric and fluorescent TRAP assays in zebrafish and medaka, two important model organisms for investigating the pathogenesis of bone disorders. We show fluorescent TRAP staining, utilising the ELF97 substrate, is a rapid, robust and stable system to visualise and quantify osteoclast activity in zebrafish, and is compatible with other fluorescence stains, transgenic lines and antibody approaches. Using this approach, we show that TRAP activity is predominantly found around the base of the zebrafish pharyngeal teeth, where osteoclast activity state appears to be heterogeneous.

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Estrogen Decreases Cytoskeletal Organization by Forming an ERα/SHP2/c-Src Complex in Osteoclasts to Protect against Ovariectomy-Induced Bone Loss in Mice

Antioxidants ◽

10.3390/antiox10040619 ◽

2021 ◽

Vol 10 (4) ◽

pp. 619

Author(s):

Hyun-Jung Park ◽

Malihatosadat Gholam-Zadeh ◽

Sun-Young Yoon ◽

Jae-Hee Suh ◽

Hye-Seon Choi

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Ring Formation ◽

Tartrate Resistant Acid Phosphatase ◽

Actin Ring ◽

Collagen Crosslinks ◽

Trap Staining ◽

Src Activation

Loss of ovarian function is closely related to estrogen (E2) deficiency, which is responsible for increased osteoclast (OC) differentiation and activity. We aimed to investigate the action mechanism of E2 to decrease bone resorption in OCs to protect from ovariectomy (OVX)-induced bone loss in mice. In vivo, tartrate-resistant acid phosphatase (TRAP) staining in femur and serum carboxy-terminal collagen crosslinks-1 (CTX-1) were analyzed upon E2 injection after OVX in mice. In vitro, OCs were analyzed by TRAP staining, actin ring formation, carboxymethylation, determination of reactive oxygen species (ROS) level, and immunoprecipitation coupled with Western blot. In vivo and in vitro, E2 decreased OC size more dramatically than OC number and Methyl-piperidino-pyrazole hydrate dihydrochloride (MPPD), an estrogen receptor alpha (ERα) antagonist, augmented the OC size. ERα was found in plasma membranes and E2/ERα signaling affected receptor activator of nuclear factor κB ligand (RANKL)-induced actin ring formation by rapidly decreasing a proto-oncogene tyrosine-protein kinase, cellular sarcoma (c-Src) (Y416) phosphorylation in OCs. E2 exposure decreased physical interactions between NADPH oxidase 1 (NOX1) and the oxidized form of c-Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), leading to higher levels of reduced SHP2. ERα formed a complex with the reduced form of SHP2 and c-Src to decrease c-Src activation upon E2 exposure, which blocked a signal for actin ring formation by decreased Vav guanine nucleotide exchange factor 3 (Vav3) (p–Y) and Ras-related C3 botulinum toxin substrate 1 (Rac1) (GTP) activation in OCs. E2/ERα signals consistently inhibited bone resorption in vitro. In conclusion, our study suggests that E2-binding to ERα forms a complex with SHP2/c-Src to attenuate c-Src activation that was induced upon RANKL stimulation in a non-genomic manner, resulting in an impaired actin ring formation and reducing bone resorption.

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Abstract 426: Laminin Downregulation Facilitates Cardiomyocyte Coupling in situ to Increase Contractile Function

Circulation Research ◽

10.1161/res.119.suppl_1.426 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Ayla Sessions ◽

Gaurav Kaushik ◽

Adam Engler

Keyword(s):

Basement Membrane ◽

Contractile Function ◽

Current Model ◽

Model Systems ◽

Model Organisms ◽

Muscle Physiology ◽

Heart Tube ◽

Age Related ◽

New Evidence

Aging is associated with extensive remodeling of the heart, including basement membrane extracellular matrix (ECM) components that surround cardiomyocytes. Remodeling is thought to contribute to impaired cardiac mechanotransduction, but the contribution of specific basement membrane ECM components to age-related cardiac remodeling is unclear, owing to current model systems being complex and slow to age. To investigate the effect of basement membrane remodeling on mechanical function in genetically tractable, rapidly aging, and simple model organisms, we employed Drosophila melanogaster, which has a simple trilayered heart tube composed of only basement membrane ECM. We observed differential regulation of collagens between laboratory Drosophila strains , i.e. yellow-white ( yw ) and white-1118 ( w 1118 ), leading to changes in muscle physiology, which were linked to severity of dysfunction with age. Therefore, we sought to understand the extent to which basement membrane ECM modulates lateral cardiomyocyte coupling and contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin , Laminin A , and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression decreased levels of other genes that co-assemble in ECM, leading to overall preservation of contractile velocity and extension of median organismal lifespan by 3 weeks or 39%. These data provide new evidence of a direct link between basement membrane ECM homeostasis, contractility, and maintenance of lifespan.

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Autoamplification and competition drive symmetry breaking: Initiation of centriole duplication by the PLK4-STIL network

10.1101/430264 ◽

2018 ◽

Author(s):

Marcin Leda ◽

Andrew J. Holland ◽

Andrew B. Goryachev

Keyword(s):

Symmetry Breaking ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Break Symmetry ◽

Biophysical Model ◽

Model Organisms ◽

Centriole Duplication ◽

Mother Centriole ◽

Autocatalytic Activation

SummarySymmetry breaking, a central principle of physics, has been hailed as the driver of self-organization in biological systems in general and biogenesis of cellular organelles in particular, but the molecular mechanisms of symmetry breaking only begin to become understood. Centrioles, the structural cores of centrosomes and cilia, must duplicate every cell cycle to ensure their faithful inheritance through cellular divisions. Work in model organisms identified conserved proteins required for centriole duplication and found that altering their abundance affects centriole number. However, the biophysical principles that ensure that, under physiological conditions, only a single procentriole is produced on each mother centriole remain enigmatic. Here we propose a mechanistic biophysical model for the initiation of procentriole formation in mammalian cells. We posit that interactions between the master regulatory kinase PLK4 and its activator-substrate STIL form the basis of the procentriole initiation network. The model faithfully recapitulates the experimentally observed transition from PLK4 uniformly distributed around the mother centriole, the “ring”, to a unique PLK4 focus, the “spot”, that triggers the assembly of a new procentriole. This symmetry breaking requires a dual positive feedback based on autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4-STIL complexes by phosphorylated STIL. We find that, contrary to previous proposals,in situdegradation of active PLK4 is insufficient to break symmetry. Instead, the model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes explains both the instability of the PLK4 ring and formation of the unique PLK4 spot. In the model, strong competition at physiologically normal parameters robustly produces a single procentriole, while increasing overexpression of PLK4 and STIL weakens the competition and causes progressive addition of procentrioles in agreement with experimental observations.

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Collecting eco-evolutionary data in the dark: Impediments to subterranean research and how to overcome them

10.32942/osf.io/u5z3e ◽

2020 ◽

Author(s):

Stefano Mammola ◽

Enrico Lunghi ◽

Helena Bilandžija ◽

Pedro Cardoso ◽

Volker Grimm ◽

...

Keyword(s):

Model Systems ◽

Ex Situ ◽

Model Organisms ◽

Physiological Tolerance ◽

Advantages And Disadvantages ◽

General Scientific ◽

Experimental Model Systems ◽

Scientific Questions ◽

Subterranean Species

(1) Caves and other subterranean habitats fulfill the requirements of experimental model systems to address general questions in ecology and evolution. Yet, the harsh working conditions of these environments and the uniqueness of the subterranean organisms have challenged most attempts to pursuit standardized research(2) Two main obstacles have synergistically hampered previous attempts. First, there is a habitat impediment related to the objective difficulties of exploring subterranean habitats and our inability to access the network of fissures that represent the elective habitat for the so-called “cave species.” Second, there is a biological impediment illustrated by the rarity of most subterranean species and their low physiological tolerance, often limiting sample size and complicating lab experiments.(3) We explore the advantages and disadvantages of four general experimental setups (in-situ, quasi in-situ, ex-situ, and in-silico) in the light of habitat and biological impediments. We also discuss the potential of indirect approaches to research. Furthermore, using bibliometric data, we provide a quantitative overview of the model organisms that scientists have exploited in the study of subterranean life.(4) Our over-arching goal is to promote caves as model systems where one can perform standardised scientific research. This is important not only to achieve an in-depth understanding of the functioning of subterranean ecosystems but also to fully exploit their long-discussed potential in addressing general scientific questions with implications beyond the boundaries of this discipline.

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Keel-bone fractures are associated with bone quality differences in laying hens

Animal Welfare ◽

10.7120/09627286.30.1.071 ◽

2021 ◽

Vol 30 (1) ◽

pp. 71-80

Author(s):

HD Wei ◽

YJ Chen ◽

XY Zeng ◽

YJ Bi ◽

YN Wang ◽

...

Keyword(s):

Bone Quality ◽

Laying Hens ◽

Bone Fractures ◽

Quality Parameters ◽

Bone Volume ◽

Tartrate Resistant Acid Phosphatase ◽

Bone Surface ◽

Mineral Density ◽

Trabecular Thickness ◽

Trap Activity

This study aimed to investigate the relationship between bone quality in terms of metabolism, homeostasis of elements, bone mineral density (BMD), and microstructure and keel-bone fractures in laying hens (Gallusgallusdomesticus). One hundred and twenty 17 week old Lohmann White laying hens with normal keel bones were individually housed in furnished cages for 25 weeks. Birds were then euthanased and dissected to assess keel-bone status at 42 weeks. Serum and keel-bone samples from normal keel (NK) and fractured keel (FK) hens were collected to determine the previously mentioned bone quality parameters. The results showed FK hens to have higher levels of the components of osteocalcin, greater alkaline phosphatase activity in serum and keel bones, and greater tartrate-resistant acid phosphatase (TRAP) activity in keel bones, compared to NK hens. Additionally, FK hens also had higher concentrations of Li, B, K, Cu, As, Se, Sn, Hg, and Pb, but lower concentrations of Na, P, and Ca. Moreover, FK hens showed decreased bone microstructural parameters including bone volume/tissue volume, trabecular number, degree of anisotropy, connectivity density, and BMD, but increased trabecular separation. Meanwhile, no differences were detected in serum TRAP activity, trabecular thickness, bone surface, or bone surface/bone volume. Results showed laying hens with keel-bone fractures to have differences in bone metabolism, elements of homeostasis, bone microstructure parameters, and BMD. These results suggest that keel-bone fractures may be associated with bone quality.

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Similarities between IgG-bearing lymphocytes and hairy cells: cytologic and cytochemical studies

Blood ◽

10.1182/blood.v64.1.166.166 ◽

1984 ◽

Vol 64 (1) ◽

pp. 166-172 ◽

Cited By ~ 10

Author(s):

T Machii ◽

T Kitani

Keyword(s):

Peripheral Blood ◽

Phase Contrast ◽

Peripheral Blood Lymphocytes ◽

Tartrate Resistant Acid Phosphatase ◽

Normal Peripheral Blood ◽

Surface Immunoglobulin ◽

Contrast Microscope ◽

Trap Staining ◽

Hairy Cells ◽

Cytoplasmic Processes

Abstract Six patients with hairy cell leukemia (HCL) were studied for surface immunoglobulin ( sIg ). In all five sIg -positive cases, the heavy chain isotype was IgG. We performed cytologic and cytochemical studies of sIgG + lymphocytes in normal peripheral blood and compared them with hairy cells. Normal sIgM + lymphocytes were also examined. sIgG + and sIgM + lymphocytes made up 0.9% and 6.1% of normal peripheral blood lymphocytes, respectively. Under a phase-contrast microscope, 76% of sIgG + lymphocytes showed cytoplasmic processes similar to those found on hairy cells, whereas most sIgM + lymphocytes had smooth surfaces. Tartrate-resistant acid phosphatase (TRAP) staining revealed that TRAP- positive cells accounted for 65% of sIgG + lymphocytes and 19% of sIgM + lymphocytes. Some (8.3%) of the sIgM + lymphocytes expressed sIgG concomitantly. When sIgM +, sIgM +, sIgG + lymphocytes were excluded, the percentages of cells with surface processes and of TRAP-positive cells in the remaining sIgM +, sIgG - lymphocytes were 10% and 12%, respectively. A very small proportion (0.2%) of sIgM -, sIgG - lymphocytes had cytoplasmic processes. These results indicate that normal sIgG + lymphocytes are cytologically and cytochemically different from most sIgM + lymphocytes and that the phase-contrast microscopic appearances and TRAP activity of sIgG + lymphocytes are similar to those of HCL tumor cells.

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Osteoblast and Osteoclast Activity Affect Bone Remodeling Upon Regulation by Mechanical Loading-Induced Leukemia Inhibitory Factor Expression in Osteocytes

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.585056 ◽

2020 ◽

Vol 7 ◽

Author(s):

Jingke Du ◽

Jiancheng Yang ◽

Zihao He ◽

Junqi Cui ◽

Yiqi Yang ◽

...

Keyword(s):

Bone Remodeling ◽

Mechanical Loading ◽

Leukemia Inhibitory Factor ◽

Fluid Shear Stress ◽

Bone Structure ◽

Inhibitory Factor ◽

Tartrate Resistant Acid Phosphatase ◽

Osteoclast Activity ◽

Microgravity Simulation

PurposeBone remodeling is affected by mechanical stimulation. Osteocytes are the primary mechanical load-sensing cells in the bone, and can regulate osteoblast and osteoclast activity, thus playing a key role in bone remodeling. Further, bone mass during exercise is also regulated by Leukemia inhibitory factor (LIF). This study aimed to investigate the role of LIF in the mechanical response of the bone, in vivo and in vitro, and to elucidate the mechanism by which osteocytes secrete LIF to regulate osteoblasts and osteoclasts.MethodsA tail-suspension (TS) mouse model was used in this study to mimic muscular disuse. ELISA and immunohistochemistry were performed to detect bone and serum LIF levels. Micro-computed tomography (CT) of the mouse femurs was performed to measure three-dimensional bone structure parameters. Fluid shear stress (FSS) and microgravity simulation experiments were performed to study mechanical stress-induced LIF secretion and its resultant effects. Bone marrow macrophages (BMMs) and bone mesenchymal stem cells (BMSCs) were cultured to induce in vitro osteoclastogenesis and osteogenesis, respectively.ResultsMicro-CT results showed that TS mice exhibited deteriorated bone microstructure and lower serum LIF expression. LIF secretion by osteocytes was promoted by FSS and was repressed in a microgravity environment. Further experiments showed that LIF could elevate the tartrate-resistant acid phosphatase activity in BMM-derived osteoclasts through the STAT3 signaling pathway. LIF also enhanced alkaline phosphatase staining and osteogenesis-related gene expression during the osteogenic differentiation of BMSCs.ConclusionMechanical loading affected LIF expression levels in osteocytes, thereby altering the balance between osteoclastogenesis and osteogenesis.

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Increased Osteoclast Activity in the Presence of Increased Homocysteine Concentrations

Clinical Chemistry ◽

10.1373/clinchem.2005.053363 ◽

2005 ◽

Vol 51 (12) ◽

pp. 2348-2353 ◽

Cited By ~ 94

Author(s):

Markus Herrmann ◽

Thomas Widmann ◽

Graziana Colaianni ◽

Silvia Colucci ◽

Alberta Zallone ◽

...

Keyword(s):

Mononuclear Cells ◽

Osteoporotic Fractures ◽

Nuclear Factor Κb ◽

Tartrate Resistant Acid Phosphatase ◽

Peripheral Blood Mononuclear ◽

Therapeutic Implications ◽

Trap Activity ◽

K Activity

Abstract Background: Increased plasma homocysteine (HCY) may be an independent risk factor for osteoporotic fractures and therefore may also adversely affect bone metabolism. We analyzed the effect of HCY on human osteoclast (OC) activity. Methods: We cultured peripheral blood mononuclear cells from 17 healthy male donors [median (SD) age, 30 (5) years] for 20 days with 25 μg/L macrophage-colony-stimulating factor (days 0–11), 20 μg/L receptor-activator of nuclear factor-κB ligand (days 6–20), and 4 different concentrations of HCY (0, 10, 50, and 100 μmol/L; days 0–20). For control purposes, cysteine and glutathione were tested in equimolar concentrations. OCs were identified as large, multinucleated cells with tartrate-resistant acid phosphatase (TRAP) activity and surface vitronectin receptors. We quantified OC activity by measuring TRAP activity. We analyzed cathepsin K (CP-K) activity in 9 donor samples and estimated the dentine-resorbing activity on standard dentine slices in 3 samples. Results: After 20 days of culture, most cells were fully differentiated OCs. TRAP activity increased with increasing HCY concentrations (P <0.001). HCY concentrations of 10, 50, and 100 μmol/L stimulated TRAP activity by 20%, 15%, and 42%. Additionally, HCY stimulated CP-K activity (P = 0.005): in the presence of 100 μmol/L HCY, CP-K activity was ∼38% higher than in controls (P = 0.002). Bone-resorbing activity was significantly increased in cultures with 50 and 100 μmol/L HCY. Cysteine and glutathione significantly decreased TRAP and CP-K activity. Conclusions: Increased HCY concentrations specifically stimulate OC activity in vitro, suggesting a mechanistic role of HCY for bone resorption. Future studies clarifying the mechanistic role of increased HCY concentrations in osteoporosis could have interesting therapeutic implications.

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