A novel G3BP1-GFP reporter human lung cell system enabling real-time monitoring of stress granule dynamics for in vitro lung toxicity assessment.

Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.

Establishment of an inspection standard for decomposition and methane production rates of organic waste co-digestion facilities in Korea.

Domestic organic waste resources have increased over the past decade and treatment of this waste via co-digested biogasification facilities is increasing annually. However, inspection standards for such facilities are not well-established. Herein, we aimed to derive calculation formulas and factors related to organic matter decomposition efficiency and methane production rate in accordance with waste treatment facility inspection standards. We also aimed to determine the optimum waste mixing ratio. Sample (field) surveys of 18 treatment facilities and complete enumeration of 110 facilities were conducted. Calculation formulas and factors were derived using the survey data and biochemical methane potential (BMP) test. The calculated coefficients derived through the BMP test were 0.512 m3 CH4/kgVSin for food waste, 0.601 m3 CH4/kgVSin for livestock manure, and 0.382 m3 CH4/kgVSin for sewage sludge. The final derived calculation factors were 65.0% for food waste, 36.0% for livestock manure, and 20.0% for sewage sludge for organic matter decomposition efficiency, and 0.380 m3 CH4/kgVSin for food waste, 0.27 m3 CH4/kgVSin for livestock manure, and 0.140 m3 CH4/kgVSin for sewage sludge for methane production rates. The derived effective capacity calculation factors can be utilized in future waste treatment facility inspection methods by aiding in the establishment of appropriate inspection standards for co-digested biogasification facilities other than single food waste treatment facilities. In addition, the optimum mixing ratio can be used as design data for co-digested biogasification facilities.

UNC93B1 is essential for the plasma membrane localization and signaling of Toll-like receptor 5.

The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.

Acidic amino acid residues in the juxtamembrane region of the nucleotide-sensing TLRs are important for UNC93B1 binding and signaling.

TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane protein UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolysosomes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid-sensing TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles.

A new G3BP1-GFP reporter system for assessing skin toxicity by real-time monitoring of stress granules in vitro.

The skin, the organ with the largest surface area in the body, is the most susceptible to chemical exposure from the external environment. In this study, we aimed to establish an in vitro skin toxicity monitoring system that utilizes the mechanism of stress granule (SG) formation induced by various cellular stresses. In HaCaT cells, a keratinocyte cell line that comprises the human skin, a green fluorescent protein (GFP) was knocked in at the C-terminal genomic locus of Ras GTPase-activating protein-binding protein 1 (G3BP1), a representative component of SGs. The G3BP1-GFP knock-in HaCaT cells and wild-type (WT) HaCaT cells formed SGs containing G3BP1-GFP upon exposure to arsenite and household chemicals, such as bisphenol A (BPA) and benzalkonium chloride (BAC), in real-time. In addition, the exposure of G3BP1-GFP knock-in HaCaT cells to BPA and BAC promoted the phosphorylation of eukaryotic initiation factor 2 alpha and protein kinase R-like endoplasmic reticulum kinase, which are cell signaling factors involved in SG formation, similar to WT HaCaT cells. In conclusion, this novel G3BP1-GFP knock-in human skin cell system can monitor SG formation in real-time and be utilized to assess skin toxicity to various substances.

1,2,4-trihydroxybenzene induces stress granule formation and causes DNA damage in human keratinocytes.

Household chemical products are typically evaluated for toxicity through ingestion and inhalation, with limited information on skin absorption. Furthermore, current research focuses on the long-term toxic effects of harmful substances contained in these household chemical products, however not much is known about their acute toxic effects. In this study, the effects of 1,2,4-trihydroxybenzene (THB) in human keratinocytes by examining its effects on stress granule (SG) formation, a marker of acute stress response, and DNA double strand breaks caused by repeated exposure. THB effectively induced SG formation via endoplasmic reticulum stress-mediated eIF2α phosphorylation in keratinocytes. Furthermore, repeated exposure to THB causes apoptotic cell death due to DNA double strand breaks. Collectively, THB exposure leads to skin toxicity, suggesting precautions for the use of THB-containing household chemical products.

Nitrate vulnerability of groundwater in Jeju Volcanic Island, Korea.

Groundwater is the sole source of water for about 670,000 residents of Jeju Island, which is a volcanic Korean island. Since the 1990s, nitrate contamination of groundwater has emerged as a major environmental issue. To ensure the sustainability of water resources, this study aimed to develop a vulnerability model for nitrate contamination as a preventive measure. Based on intrinsic vulnerability determined using the DRASTIC model, the effects of anthropogenic parameters related to NO3 sources and groundwater use (land use and the hydraulic gradient, respectively) on contamination were tested using a geographic information system (GIS). The correlation between groundwater nitrate distribution and vulnerability was considerably stronger compared to the DRASTIC method, with the correlation coefficients (r) increasing from -0.048 to 0.562 and -0.069 to 0.481 in the western and eastern regions, respectively. However, in the southern and northern regions, nitrate concentrations in groundwater are low, likely due to the heavily paved land surface that resulted from urbanisation, such that groundwater vulnerability appeared negligible. To prevent further nitrate contamination in coastal groundwater, management policies for land use and groundwater exploitation should be enacted along with continuous groundwater monitoring at the regional scale.

Nitrate contamination of coastal groundwater: Sources and transport mechanisms along a volcanic aquifer.

Groundwater is the sole water supply source on Jeju volcanic island in Korea and increasing levels of nitrate contamination have raised serious public concerns. This study was objected to understand the nitrate contamination mechanisms of the coastal groundwater overlain by a low-permeability layer in the western part of the island. The 2-yrs of quarterly groundwater monitoring from Sep. 2016 to Oct. 2018 revealed that NO3-N concentrations of 60.7% of sampled wells exceeded 10 mg/L, and those of 17.8% in the range of 3-10 mg/L, indicating those wells are already contaminated or under influence of potential sources. The spatio-temporal variation of groundwater chemistry and stable isotopic signatures suggested that nitrate originated mainly from chemical fertilizers due to agricultural activities and in part from liquefied manure affected by local livestock industries. Two transport processes appeared to be responsible for the nitrate in the confined coastal aquifer: 1) the direct penetration from the land surface through the wellbore leakage at incompletely grouted wells, and 2) the addition of nitrate-contaminated groundwater from upgradient areas where the confining layer is pinched out. Presently the potential nitrate sources are distributed up to 600 m above mean sea level(amsl), and the isotopic signatures implies that the provenance of coastal groundwater could be upland areas of up to 1280 m amsl. Subsequently, nitrate sources at upland areas could be picked up and carried down to the coastal groundwater under the confined condition of the basaltic aquifer that the regional groundwater recharge occurs. Consequently, groundwater contamination in the coastal aquifer should be considered in the big picture of groundwater system accommodating both regional and local flows.

Quantification of cellular uptake and in vivo tracking of transduction using real-time monitoring.

Protein transduction domains (PTDs) are short amino acid sequences that promote their own translocation across the cell plasma membrane and have been studied for possible use in drug delivery and gene therapy. However, no direct method to quantify transduction is available. Here, using a new luciferase-tagged human PTD, we show that cellular uptake levels can be determined in a reliable manner. Furthermore, we show that enhanced in vivo tracking by human PTD can be quantified in a mouse model. This is the first report on the direct quantification of PTD transduction in vitro and in vivo, which will be necessary for studying its possible therapeutic application in drug delivery and gene therapy.

Activation and expression of urokinase-type plasminogen activator are modulated by freezing/thawing process through activation of redox signal pathway in primary porcine endometrial cells.

Plasminogen activators (PAs) play a pivotal role in a variety of uterine physiologies, such as endometrial function, trophoblast invasion, and implantation process, but its alteration in expression or activity during cryopreservation of primary uterine cells has received little attention. In this study, we investigated whether PA expression and activity were modulated in first passage primary porcine uterus endometrial epithelium cells (PUEECs) treated with or without a freezing-thawing procedure. Western blotting and zymographic analysis showed that uPA expression and activity increased significantly in frozen-thawed PUEECs in a passage-dependent manner as compared to freshly prepared control cells. Moreover, intracellular reactive oxygen species (ROS) were increased by freezing-thawing and longer culturing, and were more prominent in frozen-thawed PUEECs than in control cells. However, the increase in both uPA expression and activity was greatly reduced or alleviated by treatment with either ROS scavenger N-acetylcysteine or extracellular signal-regulated kinase (ERK) inhibitor PD98059. These results suggest that ROS/ERK-mediated uPA activation may be an important factor in cryo-damage of primary uterine cells.

Characteristics of PCDDs/PCDFs in stack gas from medical waste incinerators.

Toxic polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in 45 stack gas samples were measured from 19 medical waste incinerators in South Korea using high-resolution gas chromatography equipped with a high-resolution mass spectrometer. The average concentrations of the sum of 17 toxic PCDD/PCDF congeners emitted from the medical waste incinerators ranged from 0.153 to 101.9 ng/Sm3. Based on the World Health Organization toxic equivalency factor, they ranged from 0.007 to 5.437 ng-TEQ/Sm3. The congener patterns of PCDDs/PCDFs were described using principal component analysis and presented four patterns. In the evaluation of annual average total TEQ concentrations, outlying stack gas samples were excluded. In this study, the number of chlorine substitutions was evaluated as an important factor in congener patterns. Coefficient of determination values were employed for evaluation of correlation between PCDDs and PCDFs. 1,2,3,4,6,7,8-HpCDF was measured as the most emitted congener, while 2,3,4,7,8-PeCDF was the greatest TEQ contributor.

Skin whitening and anti-corrugation activities of glycoprotein fractions from liquid extracts of boiled sea cucumber.

OBJECTIVE: To determine skin whitening and wrinkle improvement efficacy, glycoprotein fractions were extracted from liquid extracts of boiled sea cucumber and their effects on tyrosine and elastase inhibitory activities were assayed. METHODS: Fractions above and below 50 kDa (>50 kDa and <50 kDa) were extracted via a series of steps involving: boiling, filtering, desalting and freeze drying. Cytotoxicity, skin whitening and wrinkle-removing effects of boiled liquid were determined. RESULTS: Our MTT data showed that neither glycoprotein fraction of boiled liquid induces cellular cytotoxicity up to a concentration of 10 mg/mL treatment of the mouse melanoma cell line, B16F10, with 10 mg/mL >50 kDa enhanced tyrosinase and elastase inhibitory activities by 50.84% and 28.78%, respectively. Correlations of the >50 kDa concentration with tyrosinase inhibitory (R2 = 0.968) and elastase inhibitory (R2 = 0.983) efficacy were significant. CONCLUSIONS: >50 kDa glycoprotein fraction isolated from liquid extracts of boiled sea cucumber, which can serve as a functional cosmetic ingredient for whitening and wrinkle improvement of skin.