Real-Time Autophagic Flux Measurements in Live Cells Using a Novel Fluorescent Marker DAPRed.

Autophagy is a conserved homeostatic mechanism involved in cellular homeostasis and many disease processes. Although it was first described in yeast cells undergoing starvation, we have learned over the years that autophagy gets activated in many stress conditions and during development and aging in mammalian cells. Understanding the fundamental mechanisms underlying autophagy effects can bring us closer to better insights into the pathogenesis of many disease conditions (e.g., cardiac muscle necrosis, Alzheimer's disease, and chronic lung injury). Due to the complex and dynamic nature of the autophagic processes, many different techniques (e.g., western blotting, fluorescent labeling, and genetic modifications of key autophagy proteins) have been developed to delineate autophagy effects. Although these methods are valid, they are not well suited for the assessment of time-dependent autophagy kinetics. Here, we describe a novel approach: the use of DAPRed for autophagic flux measurement via live cell imaging, utilizing A549 cells, that can visualize and quantify autophagic flux in real time in single live cells. This approach is relatively straightforward in comparison to other experimental procedures and should be applicable to any in vitro cell/tissue models. Key features • Allows real-time qualitative imaging of autophagic flux at single-cell level. • Primary cells and cell lines can also be utilized with this technique. • Use of confocal microscopy allows visualization of autophagy without disturbing cellular functions.

Functionally antagonistic polyelectrolyte for electro-ionic soft actuator.

Electro-active ionic soft actuators have been intensively investigated as an artificial muscle for soft robotics due to their large bending deformations at low voltages, small electric power consumption, superior energy density, high safety and biomimetic self-sensing actuation. However, their slow responses, poor durability and low bandwidth, mainly resulting from improper distribution of ionic conducting phase in polyelectrolyte membranes, hinder practical applications to real fields. We report a procedure to synthesize efficient polyelectrolyte membranes that have continuous conducting network suitable for electro-ionic artificial muscles. This functionally antagonistic solvent procedure makes amphiphilic Nafion molecules to assemble into micelles with ionic surfaces enclosing non-conducting cores. Especially, the ionic surfaces of these micelles combine together during casting process and form a continuous ionic conducting phase needed for high ionic conductivity, which boosts the performance of electro-ionic soft actuators by 10-time faster response and 36-time higher bending displacement. Furthermore, the developed muscle shows exceptional durability over 40 days under continuous actuation and broad bandwidth below 10 Hz, and is successfully applied to demonstrate an inchworm-mimetic soft robot and a kinetic tensegrity system.

Kinetics of autophagic activity in nanoparticle-exposed lung adenocarcinoma (A549) cells.

Autophagy, a homeostatic mechanism, is crucial in maintaining normal cellular function. Although dysregulation of autophagic processes is recognized in certain diseases, it is unknown how maintenance of cellular homeostasis might be affected by the kinetics of autophagic activity in response to various stimuli. In this study, we assessed those kinetics in lung adenocarcinoma (A549) cells in response to exposure to nanoparticles (NP) and/or Rapamycin. Since NP are known to induce autophagy, we wished to determine if this phenomenon could be a driver of the harmful effects seen in lung tissues exposed to air pollution. A549 cells were loaded with a fluorescent marker (DAPRed) that labels autophagosomes and autolysosomes. Autophagic activity was assessed based on the fluorescence intensity of DAPRed measured over the entire cell volume of live single cells using confocal laser scanning microscopy (CLSM). Autophagic activity over time was determined during exposure of A549 cells to single agents (50 nM Rapamycin; 80 µg/mL, 20 nm carboxylated polystyrene NP (PNP); or, 1 µg/mL ambient ultrafine particles (UFP) (<180 nm)), or double agents (Rapamycin + PNP or Rapamycin + UFP; concomitant and sequential), known to stimulate autophagy. Autophagic activity increased in all experimental modalities, including both single agent and double agent exposures, and reached a steady state in all cases ~2 times control from ~8 to 24 hrs, suggesting the presence of an upper limit to autophagic capacity. These results are consistent with the hypothesis that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic response to additional stimulation, thereby making nanoparticle-exposed cells more susceptible to secondary injury due to autophagic overload.

Submerged leaves of live indoor foliage plants adsorb H1N1 influenza virus from suspension.

Control of hazardous indoor particles using plants has attracted interest due to the increasing worldwide air pollution and spread of pandemic-causing viruses. However, the interaction between human pathogenic viruses (HPVs) and live plants has not been examined largely due to issues in detecting tiny amounts of infectious viruses in a carrier (such as an aerosol) and the lack of suitable examination methods. In this study, as a novel evaluation method, the effect of submerged leaves of live plants on HPVs in water was examined, using the H1N1 influenza virus as a model. Selected plant foliage of a live plant was immersed in a small bag containing HPV water suspension. In an initial screening test, the activities of 20 different plant species on the virus suspension were evaluated using a rapid virus detection kit. Ten plant species had the capability to decrease virus concentrations in the water suspension within 72 h. Among the experimental plant species, Epipremnum aureum showed the highest virus decreasing characteristics when examined using both the kit and quantitative real time polymerase chain reaction. The capacity of immersed leaf of live E. aureum to decrease viral content was enhanced when the plant-containing pot was electrically grounded to the earth (approximately 70% decrease in virus concentration). The foliage sample analysis showed that virus adsorption to the plant foliage surface could be the major reason for the decrease in the suspension. These results suggest that the proposed method can be applied to select plants to further investigate plant-HPV interactions.

Morphological and Chemical Evaluations of Leaf Surface on Particulate Matter2.5 (PM2.5) Removal in a Botanical Plant-Based Biofilter System.

Particulate matter has been increasing worldwide causing air pollution and serious health hazards. Owing to increased time spent indoors and lifestyle changes, assessing indoor air quality has become crucial. This study investigated the effect of watering and drought and illumination conditions (constant light, light/dark cycle, and constant dark) on particulate matter2.5 (PM2.5) removal and surface characterization of leaf in a botanical plant-based biofilter system. Using Ardisia japonica and Hedera helix as experimental plants in the plant-based biofilter system, PM2.5, volatile organic carbon, and CO2, as the evaluators of indoor air quality, were estimated using a sensor. Morphological and chemical changes of the leaf surface (i.e., roughness and wax) associated with PM2.5 removal were characterized via scanning electron microscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The highest PM2.5 removal efficiency, stomata closure, high leaf roughness, and wax layer were observed under drought with constant light condition. Consequently, PM2.5 removal was attributed to the combined effect of leaf roughness and wax by adsorption rather than stomatal uptake. These results suggest that operating conditions of indoor plant-based biofilter system such as watering (or drought) and illumination may be applied as a potential strategy for enhancing PM2.5 removal.

In vitro and ex vivo models in inhalation biopharmaceutical research - advances, challenges and future perspectives.

Oral inhalation results in pulmonary drug targeting and thereby reduces systemic side effects, making it the preferred means of drug delivery for the treatment of respiratory disorders such as asthma, chronic obstructive pulmonary disease or cystic fibrosis. In addition, the high alveolar surface area, relatively low enzymatic activity and rich blood supply of the distal airspaces offer a promising pathway to the systemic circulation. This is particularly advantageous when a rapid onset of pharmacological action is desired or when the drug is suffering from stability issues or poor biopharmaceutical performance following oral administration. Several cell and tissue-based in vitro and ex vivo models have been developed over the years, with the intention to realistically mimic pulmonary biological barriers. It is the aim of this review to critically discuss the available models regarding their advantages and limitations and to elaborate further which biopharmaceutical questions can and cannot be answered using the existing models.

Austalide K from the Fungus Penicillium rudallense Prevents LPS-Induced Bone Loss in Mice by Inhibiting Osteoclast Differentiation and Promoting Osteoblast Differentiation.

Osteoporosis is a chronic disease that has become a serious public health problem due to the associated reduction in quality of life and its increasing financial burden. It is known that inhibiting osteoclast differentiation and promoting osteoblast formation prevents osteoporosis. As there is no drug with this dual activity without clinical side effects, new alternatives are needed. Here, we demonstrate that austalide K, isolated from the marine fungus Penicillium rudallenes, has dual activities in bone remodeling. Austalide K inhibits the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and improves bone morphogenetic protein (BMP)-2-mediated osteoblast differentiation in vitro without cytotoxicity. The nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), and cathepsin K (CTSK) osteoclast-formation-related genes were reduced and alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN) (osteoblast activation-related genes) were simultaneously upregulated by treatment with austalide K. Furthermore, austalide K showed good efficacy in an LPS-induced bone loss in vivo model. Bone volume, trabecular separation, trabecular thickness, and bone mineral density were recovered by austalide K. On the basis of these results, austalide K may lead to new drug treatments for bone diseases such as osteoporosis.

Methodology: non-invasive monitoring system based on standing wave ratio for detecting water content variations in plants.

BACKGROUND: Water content variation during plant growth is one of the most important monitoring parameters in plant studies. Conventional parameters (such as dry weight) are unreliable; thus, the development of rapid, accurate methods that will allow the monitoring of water content variation in live plants is necessary. In this study, we aimed to develop a non-invasive, radiofrequency-based monitoring system to rapidly and accurately detect water content variation in live plants. The changes in standing wave ratio (SWR) caused by the presence of stem water and magnetic particles in the stem water flow were used as the basis of plant monitoring systems. RESULTS: The SWR of a coil probe was used to develop a non-invasive monitoring system to detect water content variation in live plants. When water was added to the live experimental plants with or without illumination under drought conditions, noticeable SWR changes at various frequencies were observed. When a fixed frequency (1.611 GHz) was applied to a single experimental plant (Radermachera sinica), a more comprehensive monitoring, such as water content variation within the plant and the effect of illumination on water content, was achieved. CONCLUSIONS: Our study demonstrated that the SWR of a coil probe could be used as a real-time, non-invasive, non-destructive parameter for detecting water content variation and practical vital activity in live plants. Our non-invasive monitoring method based on SWR may also be applied to various plant studies.

Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers.

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on "equivalent" pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92-99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.

Biokinetic modeling of nanoparticle interactions with lung alveolar epithelial cells: uptake, intracellular processing, and egress.

Studies on health effects of engineered nanomaterials (ENMs) in the lung have provided information on ENM toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the vasculature, and distribute in several end organs, including the heart, liver, spleen, and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs are exocytosed. We used the data from these experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results suggest that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by proposed regulatory mechanisms.

Wavelet scattering networks for atomistic systems with extrapolation of material properties.

The dream of machine learning in materials science is for a model to learn the underlying physics of an atomic system, allowing it to move beyond the interpolation of the training set to the prediction of properties that were not present in the original training data. In addition to advances in machine learning architectures and training techniques, achieving this ambitious goal requires a method to convert a 3D atomic system into a feature representation that preserves rotational and translational symmetries, smoothness under small perturbations, and invariance under re-ordering. The atomic orbital wavelet scattering transform preserves these symmetries by construction and has achieved great success as a featurization method for machine learning energy prediction. Both in small molecules and in the bulk amorphous LiαSi system, machine learning models using wavelet scattering coefficients as features have demonstrated a comparable accuracy to density functional theory at a small fraction of the computational cost. In this work, we test the generalizability of our LiαSi energy predictor to properties that were not included in the training set, such as elastic constants and migration barriers. We demonstrate that statistical feature selection methods can reduce over-fitting and lead to remarkable accuracy in these extrapolation tasks.

Multidirectional Cylindrical Piezoelectric Force Sensor: Design and Experimental Validation.

A common design concept of the piezoelectric force sensor, which is to assemble a bump structure from a flat or fine columnar piezoelectric structure or to use a specific type of electrode, is quite limited. In this paper, we propose a new design of cylindrical piezoelectric sensors that can detect multidirectional forces. The proposed sensor consists of four row and four column sensors. The design of the sensor was investigated by the finite element method. The response of the sensor to various force directions was observed, and it was demonstrated that the direction of the force applied to the sensor could be derived from the signals of one row sensor and three column sensors. As a result, this sensor proved to be able to detect forces in the area of 225° about the central axis of the sensor. In addition, a cylindrical sensor was fabricated to verify the proposed sensor and a series of experiments were performed. The simulation and experimental results were compared, and the actual sensor response tended to be similar to the simulation.

Evaluation of IAQ Management Using an IoT-Based Indoor Garden.

This study was designed to verify the effectiveness of smart gardens by improving indoor air quality (IAQ) through the installation of an indoor garden with sensor-based Internet-of-Things (IoT) technology that identifies pollutants such as particulate matter. In addition, the study aims to introduce indoor gardens for customized indoor air cleaning using the data and IoT technology. New apartments completed in 2016 were selected and divided into four households with indoor gardens installed and four households without indoor gardens. Real-time data and data on PM2.5, CO2, temperature, and humidity were collected through an IoT-based IAQ monitoring system. In addition, in order to examine the effects on the health of occupants, the results were analyzed based on epidemiological data, prevalence data, current maintenance, and recommendation criteria, and were presented and evaluated as indices. The indices were classified into a comfort index, which reflects the temperature and humidity, an IAQ index, which reflects PM2.5 and CO2, and an IAQ composite index. The IAQ index was divided into five grades from "good" to "hazardous". Using a scale of 1 to 100 points, it was determined as follows: "good (0-20)", "moderate (21-40)", "unhealthy for sensitive group (41-60)", "bad (61-80)", "hazardous (81-100)". It showed an increase in the "good" section after installing the indoor garden, and the "bad" section decreased. Additionally, the comfort index was classified into five grades from "very comfortable" to "very uncomfortable". In the comfort index, the "uncomfortable" section decreased, and the "comfortable" section increased after the indoor garden was installed.

Austalides, Osteoclast Differentiation Inhibitors from a Marine-Derived Strain of the Fungus Penicillium rudallense.

Four new meroterpenoids, austalides V-X (1-3) and a farnesylated phthalide derivative (4), were isolated from the culture of the marine fungus Penicillium rudallense, together with eight known meroterpenoids derivatives (5-12). Their structures, including absolute configurations, were determined by spectroscopic methods. All of the isolated compounds were evaluated for their inhibitory activities on the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation. Compounds 1, 2, 5-7, and 10 exhibited potent osteoclast differentiation inhibitory activity with ED50 values of 1.9-2.8 µM.

Water Extracts of Hull-less Waxy Barley (Hordeum vulgare L.) Cultivar 'Boseokchal' Inhibit RANKL-induced Osteoclastogenesis.

Osteoporosis is a disease that leads to reduced bone mineral density. The increase in patient and medical costs because of global aging is recognized as a problem. Decreased bone mass is a common symptom of bone diseases such as Paget's disease, rheumatoid arthritis, and multiple myeloma. Osteoclasts, which directly affect bone mass, show a marked increase in differentiation and activation in the aforementioned diseases. Moreover, these multinucleated cells made from monocytes/macrophages under the influence of RANKL and M-CSF, are the only cells capable of resorbing bones. In this study, we found that the water extracts of Boseokchal (BSC-W) inhibited osteoclast differentiation in vitro and investigated its inhibitory mechanism. BSC-W was obtained by extracting flour of Boseokchal using hexane and water. To osteoclast differentiation, bone marrow-derived macrophage cells (BMMs) were cultured with the vehicle (0.1% DMSO) or BSC-W in the presence of M-CSF and RANKL for 4 days. Cytotoxicity was measured by CCK-8. Gene expression of cells was confirmed by real-time PCR. Protein expression of cells was observed by western blot assay. Bone resorption activity of osteoclast evaluated by bone pit formation assay using an Osteo Assay Plate. BSC-W inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner without exerting a cytotoxic effect on BMMs. BSC-W decreased the transcriptional and translational expression of c-Fos and NFATc1, which are regulators of osteoclastogenesis and reduced the mRNA expression level of TRAP, DC-STAMP, and cathepsin K, which are osteoclast differentiation marker. Furthermore, BSC-W reduced the resorption activity of osteoclasts. Taken together, our results indicate that BSC-W is a useful candidate for health functional foods or therapeutic agents that can help treat bone diseases such as osteoporosis.

Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells.

BACKGROUND: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. METHODS: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. RESULTS: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. CONCLUSIONS: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.

Non-Einstein Viscosity Phenomenon of Acrylonitrile-Butadiene-Styrene Composites Containing Lignin-Polycaprolactone Particulates Highly Dispersed by High-Shear Stress.

Lignin powder was modified via ring-opening polymerization of caprolactone to form a lignin-polycaprolactone (LPCL) particulate. The LPCL particulates were mixed with an acrylonitrile-butadiene-styrene (ABS) matrix at an extremely high rotational speed of up to 3000 rpm, which was achieved by a closed-loop screw mixer and in-line melt extruder. Using this high-shear extruding mixer, the LPCL particulate size was controlled in the range of 3395 nm (conventional twin-screw extrusion) down to 638 nm (high-shear mixer of 3000 rpm) by altering the mixing speed and time. The resulting LPCL/ABS composites clearly showed non-Einstein viscosity phenomena, exhibiting reduced viscosity (2130 Pa·s) compared to the general extruded composite one (4270 Pa·s) at 1 s-1 and 210 °C. This is due to the conformational rearrangement and the increased free volume of ABS molecular chains in the vicinity of LPCL particulates. This was supported by the decreased glass transition temperature (T g, 83.7 °C) of the LPCL/ABS composite specimens, for example, giving a 21.8% decrement compared to that (107 °C) of the neat ABS by the incorporation of 10 wt % LPCL particulates in ABS. The LPCL particulate morphology, damping characteristics, and light transmittance of the developed composites were thoroughly investigated at various levels of applied shear rates and mixing conditions. The non-Einstein rheological phenomena stemming from the incorporation of LPCL particulates suggest an interesting plasticization methodology: to improve the processability of high-loading filler/polymer composites and ultra-high molecular weight polymers that are difficult to process because of their high viscosity.

High-performance polyvinyl chloride gel artificial muscle actuator with graphene oxide and plasticizer.

A transparent and electroactive plasticized polyvinyl chloride (PVC) gel was investigated to use as a soft actuator for artificial muscle applications. PVC gels were prepared with varying plasticizer (dibutyl adipate, DBA) content. The prepared PVC gels were characterized using Fourier-transform infrared spectroscopy, thermogravimetric analysis, and dynamic mechanical analysis. The DBA content in the PVC gel was shown to have an inverse relationship with both the storage and loss modulus. The electromechanical performance of PVC gels was demonstrated for both single-layer and stacked multi-layer actuators. When voltage was applied to a single-layer actuator and then increased, the maximum displacement of PVC gels (for PVC/DBA ratios of 1:4, 1:6, and 1:8) was increased from 105.19, 123.67, and 135.55 µm (at 0.5 kV) to 140.93, 157.13, and 172.94 µm (at 1.0 kV) to 145.03, 191.34, and 212.84 µm (at 1.5 kV), respectively. The effects of graphene oxide (GO) addition in the PVC gel were also investigated. The inclusion of GO (0.1 wt.%) provided an approximate 20% enhancement of displacement and 41% increase in force production, and a 36% increase in power output for the PVC/GO gel over traditional plasticizer only PVC gel. The proposed PVC/GO gel actuator may have promising applications in artificial muscle, small mechanical devices, optics, and various opto-electro-mechanical devices due to its low-profile, transparency, and electrical response characteristics.

Idelalisib inhibits osteoclast differentiation and pre-osteoclast migration by blocking the PI3Kδ-Akt-c-Fos/NFATc1 signaling cascade.

Since increased number of osteoclasts could lead to impaired bone structure and low bone mass, which are common characteristics of bone disorders including osteoporosis, the pharmacological inhibition of osteoclast differentiation is one of therapeutic strategies for preventing and/or treating bone disorders and related facture. However, little data are available regarding the functional relevance of phosphoinositide 3-kinase (PI3K) isoforms in the osteoclast differentiation process. To elucidate the functional involvement of PI3Kδ in osteoclastogenesis, here we investigated how osteoclast differentiation was influenced by idelalisib (also called CAL-101), which is p110δ-selective inhibitor approved for the treatment of specific human B cell malignancies. Here, we found that receptor activator of nuclear factor kappa B ligand (RANKL) induced PI3Kδ protein expression, and idelalisib inhibited RANKL-induced osteoclast differentiation. Next, the inhibitory effect of idelalisib on RANKL-induced activation of the Akt-c-Fos/NFATc1 signaling cascade was confirmed by western blot analysis and real-time PCR. Finally, idelalisib inhibited pre-osteoclast migration in the last stage of osteoclast differentiation through down-regulation of the Akt-c-Fos/NFATc1 signaling cascade. It may be possible to expand the clinical use of idelalisib for controlling osteoclast differentiation. Together, the present results contribute to our understanding of the clinical value of PI3Kδ as a druggable target and the efficacy of related therapeutics including osteoclastogenesis.

Extracts of Flavoparmelia sp. Inhibit Receptor Activator of Nuclear Factor-κB Ligand-Mediated Osteoclast Differentiation.

BACKGROUND: Osteoporosis is a geriatric disease with diminished bone density. The increase in the number of patients and medical expenses due to a global aging society are recognized as problems. Bone loss is the most common symptom of bone disease, not only osteoporosis but Paget's disease, rheumatoid arthritis, multiple myeloma, and other diseases. The main cause of this symptoms is excessive increase in the number and activity of osteoclasts. Osteoclasts are multinucleated giant cells that can resorb bone. They are differentiated and activation from monocytes/macrophages in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). METHODS: The effect of extract of Flavoparmelia sp. (EFV), a genus of lichenized fungi within the Parmeliaceae, on the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts was examined by phenotype assay and the cell cytotoxicity was evaluated by cell counting kit-8. The osteoclast differentiation-related genes and proteins were investigated by real-time polymerase chain reaction and immunoblotting. The functional activity of osteoclast in response to EFV treatment was evaluated by an Osteo Assay plate. RESULTS: In this study, we found that EFV, a genus of lichenized fungi within the Parmeliaceae, inhibited osteoclast formation. And we investigated its inhibitory mechanism. EFV reduced RANKL-mediated osteoclast formation and activation by inhibiting expression of nuclear factor of activated T cells 1, a key factor of osteoclastogenesis. CONCLUSIONS: Taken together, our results show that EFV is a promising candidate for health functional foods or therapeutic agents that can help treat bone diseases such as osteoporosis.