Proposal of a Methodology for Prediction of Indoor PM2.5 Concentration Using Sensor-Based Residential Environments Monitoring Data and Time-Divided Multiple Linear Regression Model.

This study aims to propose an indoor air quality prediction method that can be easily utilized and reflects temporal characteristics using indoor and outdoor input data measured near the indoor target point as input to calculate indoor PM2.5 concentration through a multiple linear regression model. The atmospheric conditions and air pollution detected in one-minute intervals using sensor-based monitoring equipment (Dust Mon, Sentry Co Ltd., Seoul, Korea) inside and outside houses from May 2019 to April 2021 were used to develop the prediction model. By dividing the multiple linear regression model into one-hour increments, we attempted to overcome the limitation of not representing the multiple linear regression model's characteristics over time and limited input variables. The multiple linear regression (MLR) model classified by time unit showed an improvement in explanatory power by up to 9% compared to the existing model, and some hourly models had an explanatory power of 0.30. These results indicated that the model needs to be subdivided by time period to more accurately predict indoor PM2.5 concentrations.

Dissolving microneedles for long-term storage and transdermal delivery of extracellular vesicles.

Extracellular vesicles (EVs) have shown great potential in disease diagnosis and treatment; however, their clinical applications remain challenging due to their unsatisfactory long-term stability and the lack of effective delivery strategies. In this study, we prepared human adipose stem cell-derived EV (hASC-EV)-loaded hyaluronic acid dissolving microneedles (EV@MN) to investigate the feasibility of EVs for their clinical applications. The biological activities of the EVs in this formulation were maintained for more than six months under mild storage conditions, especially at temperatures lower than 4 °C. Moreover, the EV@MN enabled precise and convenient intradermal delivery for sustained release of EVs in the dermis layer. Therefore, EV@MN significantly improved the biological functions of hASC-EVs on dermal fibroblasts by promoting syntheses of proteins for the extracellular matrix such as collagen and elastin, enhancing fibroblast proliferation, and regulating the phenotype of fibroblast, compared with other administration methods. This research revealed a possible and feasible formulation for the clinical application of EVs.

Polymeric Hydrogels for Controlled Drug Delivery to Treat Arthritis.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are disabling musculoskeletal disorders that affect joints and cartilage and may lead to bone degeneration. Conventional delivery of anti-arthritic agents is limited due to short intra-articular half-life and toxicities. Innovations in polymer chemistry have led to advancements in hydrogel technology, offering a versatile drug delivery platform exhibiting tissue-like properties with tunable drug loading and high residence time properties This review discusses the advantages and drawbacks of polymeric materials along with their modifications as well as their applications for fabricating hydrogels loaded with therapeutic agents (small molecule drugs, immunotherapeutic agents, and cells). Emphasis is given to the biological potentialities of hydrogel hybrid systems/micro-and nanotechnology-integrated hydrogels as promising tools. Applications for facile tuning of therapeutic drug loading, maintaining long-term release, and consequently improving therapeutic outcome and patient compliance in arthritis are detailed. This review also suggests the advantages, challenges, and future perspectives of hydrogels loaded with anti-arthritic agents with high therapeutic potential that may alter the landscape of currently available arthritis treatment modalities.

Stem Cell-Derived Extracellular Vesicle-Bearing Dermal Filler Ameliorates the Dermis Microenvironment by Supporting CD301b-Expressing Macrophages.

Hyaluronic acid-based hydrogels (Hyal-Gels) have the potential to reduce wrinkles by physically volumizing the skin. However, they have limited ability to stimulate collagen generation, thus warranting repeated treatments to maintain their volumizing effect. In this study, stem cell-derived extracellular vesicle (EV)-bearing Hyal-Gels (EVHyal-Gels) were prepared as a potential dermal filler, ameliorating the dermis microenvironment. No significant differences were observed in rheological properties and injection force between Hyal-Gels and EVHyal-Gels. When locally administered to mouse skin, Hyal-Gels significantly extended the biological half-life of EVs from 1.37 d to 3.75 d. In the dermis region, EVHyal-Gels induced the overexpression of CD301b on macrophages, resulting in enhanced proliferation of fibroblasts. It was found that miRNAs, such as let-7b-5p and miR-24-3p, were significantly involved in the change of macrophages toward the CD301bhi phenotype. The area of the collagen layer in EVHyal-Gel-treated dermis was 2.4-fold higher than that in Hyal-Gel-treated dermis 4 weeks after a single treatment, and the collagen generated by EVHyal-Gels was maintained for 24 weeks in the dermis. Overall, EVHyal-Gels have the potential as an antiaging dermal filler for reprogramming the dermis microenvironment.

Vitamin A-coupled stem cell-derived extracellular vesicles regulate the fibrotic cascade by targeting activated hepatic stellate cells in vivo.

Allogeneic transplantation of mesenchymal stem cell-derived extracellular vesicles (EVs) offers great potential for treating liver fibrosis. However, owing to their intrinsic surface characteristics, bare EVs are non-specifically distributed in the liver tissue after systemic administration, leading to limited therapeutic efficacy. To target activated hepatic stellate cells (HSCs), which are responsible for hepatic fibrogenesis, vitamin A-coupled small EVs (V-EVs) were prepared by incorporating vitamin A derivative into the membrane of bare EVs. No significant differences were found in the particle size and morphology between bare and V-EVs. In addition, surface engineering of EVs did not affect the expression of surface marker proteins (e.g., CD63 and CD9), as demonstrated by flow cytometry. Owing to the surface incorporation of vitamin A, V-EVs were selectively taken up by activated HSCs via receptor-mediated endocytosis. When systemically administered to mice with liver fibrosis, V-EVs effectively targeted activated HSCs in the liver tissue, resulting in reversal of the fibrotic cascade. Consequently, even at a 10-fold lower dose, V-EVs exhibited comparable anti-fibrotic effects to those of bare EVs, substantiating their therapeutic potential for liver fibrosis.

Metabolically engineered stem cell-derived exosomes to regulate macrophage heterogeneity in rheumatoid arthritis.

Despite the remarkable advances in therapeutics for rheumatoid arthritis (RA), a large number of patients still lack effective countermeasures. Recently, the reprogramming of macrophages to an immunoregulatory phenotype has emerged as a promising therapeutic strategy for RA. Here, we report metabolically engineered exosomes that have been surface-modified for the targeted reprogramming of macrophages. Qualified exosomes were readily harvested from metabolically engineered stem cells by tangential flow filtration at a high yield while maintaining their innate immunomodulatory components. When systemically administered into mice with collagen-induced arthritis, these exosomes effectively accumulated in the inflamed joints, inducing a cascade of anti-inflammatory events via macrophage phenotype regulation. The level of therapeutic efficacy obtained with bare exosomes was achievable with the engineered exosomes of 10 times less dose. On the basis of the boosted nature to reprogram the synovial microenvironment, the engineered exosomes display considerable potential to be developed as a next-generation drug for RA.

Effects of Salt Treatment Time on the Metabolites, Microbial Composition, and Quality Characteristics of the Soy Sauce Moromi Extract.

Salt is one of the most important factors for fermented foods, but the effect of salt treatment time on the quality of fermented foods has rarely been studied. In this study, the effect of different salt treatment times (0, 48, and 96 h) after the start of fermentation on the quality of the soy sauce moromi extract (SSME) was investigated. As the salt treatment time was delayed, the population of Aspergillus oryzae, Lactobacillaceae, and Enterococcaecea in SSME increased, whereas the population of Staphylococcaceae and Bacillaceae decreased, leading to changes in the enzymatic activity and metabolite profiles. In particular, the contents of amino acids, peptides, volatile compounds, acidic compounds, sugars, and secondary metabolites were significantly affected by the salt treatment time, resulting in changes in the sensory quality and appearance of SSME. The correlation data showed that metabolites, bacterial population, and sensory parameters had strong positive or negative correlations with each other. Moreover, based on metabolomics analysis, the salt treatment-time-related SSME metabolomic pathway was proposed. Although further studies are needed to elucidate the salt treatment mechanism in fermented foods, our data can be useful to better understand the effect of salt treatment time on the quality of fermented foods.

FOXO activity adaptation safeguards the hematopoietic stem cell compartment in hyperglycemia.

Hematopoietic stem cell (HSC) activity is tightly controlled to ensure the integrity of the hematopoietic system during the organism's lifetime. How the HSC compartment maintains its long-term fitness in conditions of chronic stresses associated with systemic metabolic disorders is poorly understood. In this study, we show that obesity functionally affects the long-term function of the most immature engrafting HSC subpopulation. We link this altered regenerative activity to the oxidative stress and the aberrant constitutive activation of the AKT signaling pathway that characterized the obese environment. In contrast, we found minor disruptions of the HSC function in obese mice at steady state, suggesting that active mechanisms could protect the HSC compartment from its disturbed environment. Consistent with this idea, we found that FOXO proteins in HSCs isolated from obese mice become insensitive to their normal upstream regulators such as AKT, even during intense oxidative stress. We established that hyperglycemia, a key condition associated with obesity, is directly responsible for the alteration of the AKT-FOXO axis in HSCs and their abnormal oxidative stress response. As a consequence, we observed that HSCs isolated from a hyperglycemic environment display enhanced resistance to oxidative stress and DNA damage. Altogether, these results indicate that chronic metabolic stresses associated with obesity and/or hyperglycemia affect the wiring of the HSCs and modify their oxidative stress response. These data suggest that the uncoupling of FOXO from its environmental regulators could be a key adaptive strategy that promotes the survival of the HSC compartment in obesity.

Inkjet Bioprinting on Parchment Paper for Hit Identification from Small Molecule Libraries.

In this study, an inkjet bioprinting-based high-throughput screening (HTS) system was designed and applied for the first time to a catecholpyrimidine-based small molecule library to find hit compounds that inhibit c-Jun NH2-terminal kinase1 (JNK1). JNK1 kinase, inactivated MAPKAPK2, and specific fluorescent peptides along with bioink were printed on parchment paper under optimized printing conditions that did not allow rapid evaporation of printed media based on Triton-X and glycerol. Subsequently, different small compounds were printed and tested against JNK1 kinase to evaluate their degree of phosphorylation inhibition. After printing and incubation, fluorescence intensities from the phosphorylated/nonphosphorylated peptide were acquired for the % phosphorylation analysis. The IM50 (inhibitory mole 50) value was determined as 1.55 × 10-15 mol for the hit compound, 22. Thus, this work demonstrated that inkjet bioprinting-based HTS can potentially be adopted for the drug discovery process using small molecule libraries, and cost-effective HTS can be expected to be established based on its low nano- to picoliter printing volume.

A FRET assay for the quantitation of inhibitors of exonuclease EcoRV by using parchment paper inkjet-printed with graphene oxide and FAM-labelled DNA.

A graphene oxide (GO)-based cost-effective, automatted strip test has developed for screening of inhibitors of endonuclease EcoRV. The method involves the use of GO and a DNA substrate for EcoRV that contains both an ssDNA region for binding of GO and a fluorescein amidite (FAM)-labelled dsDNA. All the components were inkjet printed on a piece of parchment paper. The ssDNA region binds to the surface of GO and anchors so that the fluorescence of FAM is quenched. The parchment paper strip is then incubated with a sample containing EcoRV which causes enzymatic hydrolysis, and dsDNA was separated from the GO. As a result, green fluorescence is generated at the reaction spot. Enzyme activity can be measured in the presence and absence of aurintricarboxy acid acting as an EcoRV inhibitor. This method excels by its need for 2-3 orders less reagents compared to the standard well plate assay. Thus, it is an efficient platform for GO-based screening of EcoRV enzyme inhibitors. Graphical abstract A graphene oxide (GO)-based endonuclease EcoRV inhibition FRET assay using inkjet printing was developed. Printing of GO along with assay reagents has a beneficial effect on the enzymatic reaction on paper. This method was successfully applied to evaluate EcoRV inhibitor activity.

Inkjet printing-based ß-secretase fluorescence resonance energy transfer (FRET) assay for screening of potential ß-secretase inhibitors of Alzheimer's disease.

Amyloid-ß (Aß) is generated by proteolytic processing of amyloid precursor protein (APP) by beta-secretase (BACE-1) and gamma-secretase. Amyloid-ß is responsible for the formation of senile plaques in Alzheimer's disease (AD). Consequently, inhibition of ß-secretase (BACE-1), a rate-limiting enzyme in the production of Aß, constitutes an attractive therapeutic approach to the treatment of AD. This paper reports an inkjet printing-based fluorescence assay for high throughput screening of ß-secretase inhibitors achieved by employing a BACE-1 FRET substrate (Rh-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-Quencher). This peptide substrate is known to be a readily available and suitable substrate for proteolytic activity, and it has high affinity to BACE-1. The BACE-1 peptide substrate printed on parchment paper was effectively cleaved by BACE-1, which was printed on the same spot. The amount of enzyme and substrate required for this inkjet printing-based BACE-1 assay can be less than 1.4ⅹ103, permitting the evaluation of inhibitor activity with femtomolar potency. The inkjet-printing-based BACE-1 inhibitory assay revealed inhibitory effects of inhibitor IV and STA on BACE-1 with an IM50 of 1.00 × 10-15 mol and 1.01 × 10-14 mol, respectively. These data confirm that both BACE-1 inhibitors (inhibitor IV and STA) actively inhibited the BACE-1 proteolysis of BACE-1 substrate on parchment paper. It important to note that the number of mole of BACE-1-substrate and enzyme utilized in the printing-based enzymatic assay are 1.4ⅹ103 smaller than the amount used in the conventional well-plate assay. The inkjet printing-based inhibitory assay constitutes a versatile high throughput technique and the IM50 values of the inhibitors were obtained with satisfactory reproducibility, suggesting that this inkjet-printing BACE-1 inhibitory assay could be quite suitable for the screening of new potential BACE-1 inhibitors for AD.

Inkjet printing-based photo-induced electron transfer reaction on parchment paper using riboflavin as a photosensitizer.

In this study, we report the photo-induced electron transfer (PET) on parchment paper using riboflavin as a photo inducer and ultraviolet lamp (362 nm) as the light source. To this end, a conventional inkjet printer equipped with 4 cartridges was used. Parchment paper was found to be a favorable substrate due to its insignificant self-absorption while assisting efficient sample interaction. Upon UV-irradiation, riboflavin generated superoxide anion radical (O2-·) and it was available to interact with superoxide dismutase present on the same spot. A decrease in NBT formazan in the reaction spot indicates increased O2-· scavenging activity of molecule. It was estimated that the well-plate based-colorimetric method used 12.5 µM (1.25 × 10-9 mole) of riboflavin and 0.25 mM (2.50 × 10-8 mole) of NBT to react with different superoxide dismutase or drug concentrations, while the printing technique consumed 3.19 × 10-13 mole of NBT and 2.98 × 10-13 mole of riboflavin to react with gradient superoxide dismutase or drug concentration. In contrast to the conventional well plate method, inkjet printing-based molecular assay provides automatic delivery in the nanoliter range with precise time, which ensures four-to five-order lower reagent consumption. The inkjet printing-based quantitative measurement specifies the amount of a particular drug/enzyme printed on a surface. Therefore, applicability of inkjet printing technique conjoined radical scavenging assay will be more competent to determine the radical scavenging potential of natural plant products. In addition, this inkjet printing approach offers easy, fast, cost-effective, and less time-consuming method to determine PET reaction on paper.

Paper-based inkjet bioprinting to detect fluorescence resonance energy transfer for the assessment of anti-inflammatory activity.

For the first time, a paper-based fluorescence resonance energy transfer (FRET) determination with cyclic AMP (cAMP)-specific phosphodiesterase 4B (PDE4B) inhibitory assay using an inkjet-printing technique is proposed. Non-fabricated parchment paper is found to constitute a unique substrate to measure fluorescent energy transfer, due to its insignificant self-absorption, and enables efficient sample interaction. Here, we report the responsive FRET signals generated on paper, upon sequentially printing reaction components on parchment paper using a conventional inkjet printer equipped with four cartridges. After printing, the energy emitted by Eu chelate was transferred by FRET to ULight molecule on paper, detected at 665 nm. In the absence of free cAMP, a maximum FRET signal was achieved on paper, while a decrease in FRET signals was recorded when free cAMP produced by PDE4B inhibitors compete with Eu-cAMP, binding with ULight-mAb. The IM50 value was determined as 2.46 × 10-13 mole for roliparm and 1.86 × 10-13 mole for roflumilast, to effectively inhibit PDE4B activity. Inkjet printing-based FRET signal determination utilizes components that are less than the femtomole range, which was four-orders less than the standard assay method. The methodology reported here constitutes an innovative approach towards the determination of FRET signals generated on paper.

Obesity alters the long-term fitness of the hematopoietic stem cell compartment through modulation of Gfi1 expression.

Obesity is a chronic organismal stress that disrupts multiple systemic and tissue-specific functions. In this study, we describe the impact of obesity on the activity of the hematopoietic stem cell (HSC) compartment. We show that obesity alters the composition of the HSC compartment and its activity in response to hematopoietic stress. The impact of obesity on HSC function is progressively acquired but persists after weight loss or transplantation into a normal environment. Mechanistically, we establish that the oxidative stress induced by obesity dysregulates the expression of the transcription factor Gfi1 and that increased Gfi1 expression is required for the abnormal HSC function induced by obesity. These results demonstrate that obesity produces durable changes in HSC function and phenotype and that elevation of Gfi1 expression in response to the oxidative environment is a key driver of the altered HSC properties observed in obesity. Altogether, these data provide phenotypic and mechanistic insight into durable hematopoietic dysregulations resulting from obesity.

Superior Efficacy and Selectivity of Novel Small-Molecule Kinase Inhibitors of T790M-Mutant EGFR in Preclinical Models of Lung Cancer.

The clinical utility of approved EGFR small-molecule kinase inhibitors is plagued both by toxicity against wild-type EGFR and by metastatic progression in the central nervous system, a disease sanctuary site. Here, we report the discovery and preclinical efficacy of GNS-1486 and GNS-1481, two novel small-molecule EGFR kinase inhibitors that are selective for T790M-mutant isoforms of EGFR. Both agents were effective in multiple mouse xenograft models of human lung adenocarcinoma (T790M-positive or -negative), exhibiting less activity against wild-type EGFR than existing approved EGFR kinase inhibitors (including osimertinib). In addition, GNS-1486 showed superior potency against intracranial metastasis of EGFR-mutant lung adenocarcinoma. Our results offer a preclinical proof of concept for new EGFR kinase inhibitors with the potential to improve therapeutic index and efficacy against brain metastases in patients. Cancer Res; 77(5); 1200-11. ©2017 AACR.

Impact of Environmental Pollutant Cadmium on the Establishment of a Cancer Stem Cell Population in Breast and Hepatic Cancer.

Cadmium, a heavy metal pollutant, causes cancer. The existence of cancer stem cells (CSCs) in tumors is widely considered to be the reason for the recurrence and treatment failure of cancer. Increasing evidence has confirmed that under certain conditions non-CSCs could be converted into CSCs. The impact of cadmium on the development of CSC lineage in the bulk tumor cell population is not yet studied. The aim of this study was to evaluate the effect of cadmium on the conversion of non-CSCs to CSCs and the identification of CSCs based on the concurrent monitoring of multiple CSC markers. High-content monitoring of molecular markers was performed using quantum dot (QD) nanoprobes and an acousto-optical tunable filter (AOTF)-based imaging device. Cadmium treatment significantly increased the CSC population in MCF-7 and HepG2 cell lines. The cadmium-induced CSCs were identified by a concurrent analysis of stem-cell markers, namely, CD44, CD24, CD133, and ALDH1. Moreover, increased m-RNA expression of CD44, ALDH1, and CD133 and protein expression of p-Ras, p-Raf-1, p-MEK-1, and p-ERK-1 were observed in the cadmium-treated MCF-7 and HepG2 cells. This study demonstrates that cadmium induces the gene expression of CSC markers in the breast and liver cancer cell lineage and promotes the conversion of non-CSCs to CSCs.

Inkjet-Printing Enzyme Inhibitory Assay Based on Determination of Ejection Volume.

An accurate, rapid, and cost-effective methodology for enzyme inhibitor assays is highly needed for large-scale screening to evaluate the efficacy of drugs at the molecular level. For the first time, we have developed an inkjet printing-based enzyme inhibition assay for the assessment of drug activity using a conventional inkjet printer composed of four cartridges. The methodology is based on the determination of the number of moles of the drug on the printed surface. The number of moles was quantified through the volume of substance ejected onto the printed surface. The volume ejected on the reaction spot was determined from the density of reagent ink solution and its weight loss after printing. A xanthine oxidase (XOD) inhibition assay was executed to quantitatively evaluate antioxidant activities of the drug based on the determination of the number of moles of the drug ejected by inkjet printing. The assay components of xanthine, nitro blue tetrazolium (NBT), superoxide dismutase (SOD)/drug, and XOD were printed systematically on A4 paper. A gradient range of the number of moles of SOD/drug printed on A4 paper could be successfully obtained. Because of the effect of enzyme activity inhibition, incrementally reduced NBT formazan colors appeared on the paper in a number-of-moles-dependent manner. The observed inhibitory mole (IM50) values of tested compounds exhibited a similar tendency in their activity order, compared to the IC50 values observed through absorption assay in well plates. Inkjet printing-based IM50 assessment consumed a significantly smaller reaction volume (by 2-3 orders of magnitude) and more rapid reaction time, compared to the well-plate-based absorption assay.

Urinary benzophenone concentrations and their association with demographic factors in a South Korean population.

Benzophenone (BP) and its derivatives are widely used in various cosmetics, personal care products, and food packaging ink. The use of BP has raised concerns about the potential health risks associated with its endocrine-disrupting effects. This study evaluated urinary concentrations of BP derivatives in a national sample of the South Koreans population aged 6-89 years. From July to September in each 2010 and 2011, 1576 urine samples were collected. Urinary concentrations of benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), benzophenone-4 (BP-4), benzophenone-8 (BP-8), and 4-hydroxybenzophenone (4-OH-BP) were analyzed using liquid chromatography-mass spectrometry. The detection rate for BP-1 and 4-OH-BP were 56% [limit of detection (LOD) 0.59ng/mL] and 88% (LOD 0.04ng/mL), respectively, whereas those for BP-2, BP-3, BP-4, and BP-8 were all below 25%. The geometric means of urinary BP-1 and 4-OH-BP concentrations were 1.24ng/mL and 0.45ng/mL, respectively. Multiple linear regression analysis indicated that concentrations of BP-1 in and of 4-OH-BP in adults were associated with sex and age. The BP-1 and 4-OH-BP concentration of children and adolescents was associated with sex, age, income, and current area of residence. The correlation was observed between urinary concentrations of BP derivatives, which is an important indication of exposure biomarkers and the metabolic pathways from BP-3. This is the first national study to evaluate the presence of BP derivatives in urine samples from the South Korean population, stratified by demographic factors.

Risk assessment based on urinary bisphenol A levels in the general Korean population.

Bisphenol A (BPA) is a high-volume industrial chemical used in the global production of polycarbonate plastics and epoxy resins, which are used in food and drink containers, such as tableware (plates and mugs). Due to its broad applications, BPA has been detected in human blood, urine and breast milk as well as environmental substances, including water, indoor and outdoor air, and dust. Indeed, exposure to high concentrations of BPA can result in a variety of harmful effects, including reproductive toxicity, through a mechanism of endocrine disruption. Our comparison of reported BPA urinary concentrations among different countries revealed that exposures in Korea may be higher than those in other Asian countries and North America, but lower than or similar to those in European countries. The current study included a total of 2044 eligible subjects of all ages. The subjects were evenly divided between males and females (48.58% and 51.42%, respectively). The geometric mean (GM) of pre-adjusted (adjusted) urinary BPA concentrations was 1.83µg/L (2.01µg/g creatinine) for subjects of all ages, and there was no statistically difference in BPA concentrations between males (1.90µg/L, 1.87µg/g creatinine) and females (1.76µg/L, 2.16µg/g creatinine). Multiple regression analysis revealed only one positive association between creatinine pre-adjusted urinary BPA concentration and age (ß=-0.0868, p<0.001). The 95th percentile levels of 24-hour recall (HR), food frequency questionnaires (FFQ) and estimated daily intake (EDI) through urinary BPA concentrations were 0.14, 0.13, and 0.22µg/kg bw/day, respectively. According to the Ministry of Food and Drug Safety (MFDS), a tolerable daily intake (tDI) of 20µg/kg bw/day was established for BPA from the available toxicological data. Recently, the European Food Safety Authority (EFSA) established a temporary TDI of 4µg/kg bw/day based on current toxicological data. By comparing these TDIs with subjects' exposure, we conclude that there are no health concerns for any age group as a result of current levels of dietary exposure to BPA.

Tgfbi deficiency leads to a reduction in skeletal size and degradation of the bone matrix.

Transforming growth factor-ß-induced gene product-h3 (TGFBI/BIGH3) is an extracellular matrix protein expressed in a wide variety of tissues. TGFBI binds to type I, II, and IV collagens, as well as to biglycan and decorin and plays important roles in cell-to-cell, cell-to-collagen, and cell-to-matrix interactions. Furthermore, TGFBI is involved in cell growth and migration, tumorigenesis, wound healing, and apoptosis. To investigate whether TGFBI is involved in the maintenance of skeletal tissues, Tgfbi knockout mice were generated by crossing male and female Tgfbi heterozygous mice. Skeletal preparation showed that the skeletal size in Tgfbi knockout mice was smaller than in wild-type and heterozygous mice. However, chondrocytic cell alignment in the growth plates, bone mineral density, and bone forming rates were similar in Tgfbi knockout, wild-type, and heterozygous mice. Alterations in skeletal tissue arrangements in Tgfbi knockout mice were estimated from safranin O staining, trichrome staining, and immunohistochemistry for type II and X collagen, and matrix metalloproteinase 13 (MMP13). Cartilage matrix degradation was observed in the articular cartilage of Tgfbi knockout mice. Although the detection of type II collagen in the articular cartilage was lower in Tgfbi knockout mice than wild-type mice, the detection of MMP13 was markedly higher, indicating that Tgfbi deficiency is associated with the degradation of cartilage matrix. These results suggest that TGFBI plays an important role in maintaining skeletal tissues and the cartilage matrix in mice.