Biotransformation of Flavonoids by Newly Isolated and Characterized Lactobacillus pentosus NGI01 Strain from Kimchi.

Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) microorganisms. This study aimed to identify novel LAB strains that can transform flavonoids into aglycones to improve bioavailability. We isolated 34 LAB strains from kimchi. The biotransformation activity of these 34 LAB strains was investigated based on α-L-rhamnosidase and ß-D-glucosidase activities. Among them, 10 LAB strains with high activities were identified by 16S rRNA sequencing analysis. All tested LAB strains converted hesperidin to hesperetin (12.5-30.3%). Of these, only the Lactobacillus pentosus NGI01 strain produced quercetin from rutin (3.9%). The optimal biotransformation conditions for the L. pentosus NGI01 producing hesperetin and quercetin were investigated. The highest final product concentrations of hesperetin and quercetin were 207 and 78 µM, respectively. Thus, the L. pentosus NGI01 strain can be a biocatalyst for producing flavonoid aglycones in the chemical and food industries.

Genomic-, phenotypic-, and toxicity-based safety assessment and probiotic potency of Bacillus coagulans IDCC 1201 isolated from green malt.

Probiotics are beneficial microorganisms, and the evaluation of their safety for human use in the food industry has become critical. This study examines the safety of Bacillus coagulans IDCC 1201 isolated from green malt by analyzing its genomic and phenotypic characteristics and determining its toxicity. The presence of antibiotic resistance and toxigenic genes and gene transferability were investigated using whole-genome analysis. The strain's hemolytic and enzyme activities, minimum inhibitory concentrations of antibiotics, and biogenic amine and D-lactate production were also examined. Furthermore, the principal properties of B. coagulans IDCC 1201 as probiotics, such as resistance to abiotic stress and intestinal adhesion, were studied. The whole-genome analysis demonstrated that B. coagulans IDCC 1201 had no antibiotic resistance or toxigenic genes; the strain was susceptible to the nine antibiotics proposed by the European Food Safety Authority. Moreover, this strain lacked hemolytic and ß-glucuronidase activities. Additionally, it was confirmed that B. coagulans IDCC 1201 produced undesirable metabolites, including biogenic amines or D-lactate, at a safe level. Finally, the strain exhibited functional potential as a probiotic in terms of abiotic tolerance, such as bile tolerance and intestinal adhesion in in vitro experiments. In conclusion, B. coagulans IDCC 1201 can be considered as a safe probiotic with regard to human health.

Validation of an Operational Definition to Identify Distal Radius Fractures in a National Health Insurance Database.

PURPOSE: To develop and validate identification criteria for distal radius fractures (DRFs) and their treatment using a national health insurance database. METHODS: Patients who had at least 1 wrist radiograph taken in 2018 were recruited from a single academic referral hospital. After excluding patients who lacked immobilization code for wrist pathology, we collected data on the overall population. Because some patients might have undergone wrist radiography at another institution or had DRFs without an immobilization code, we additionally included patients who had a DRF diagnosis code at our institution. Reviews of medical records and wrist radiographs were considered for the diagnosis of DRF. We evaluated the sensitivity, specificity, and positive predictive value (PPV) of 3 operational definitions of fractures that were based on a single primary or secondary diagnosis code; all diagnosis codes, including primary and secondary codes; and all diagnosis and procedure codes. RESULTS: Among 768 patients included in the study, true DRFs were confirmed in 305. The sensitivity, specificity, and PPV for definition 1 were 91.5% (95% CI, 88.3%-94.6%), 97.5% (95% CI, 95.9%-99.1%), and 96.9% (95% CI, 94.9%-98.9%), respectively. Although the sensitivity of definition 2 was higher (92.1%; 95% CI, 89.1%-95.2%), its specificity and PPV were lower (96.4% [95% CI, 94.4%-98.3%] and 95.6% [95% CI, 93.2%-97.9%], respectively). The sensitivity of definition 3 was the lowest (88.2%; 95% CI, 84.6%-91.8%), but its specificity and PPV were the highest among the 3 definitions (98.6% [95% CI, 97.4%-98.8%] and 98.2% [95% CI, 96.6%-99.8%], respectively). CONCLUSIONS: Patients with DRFs can be identified from claims databases with high accuracy using an operational definition based on DRF diagnosis and procedure codes, including codes for surgical and nonoperative methods. CLINICAL RELEVANCE: Verified operational definitions will increase the consistency of results in future national health insurance database studies related to DRFs.

Safety assessment of Streptococcus thermophilus IDCC 2201 used for product manufacturing in Korea.

Safety evaluation of probiotics has become increasingly important for human consumption in food industry. The aims of this study were to assess safety of Streptococcus thermophilus IDCC 2201 through in vitro and in vivo tests. In results, this strain was found to be negative for hemolytic and ß-glucuronidase activity. In addition, thermophilus IDCC 2201 was susceptible to nine antibiotics suggested by EFSA. In accordance with MIC tests, whole-genome analysis indicated that S. thermophilus IDCC 2201 neither harbors antibiotic resistance nor toxigenic genes. Furthermore, none of the biogenic amines including tyramine and histamine was produced and negligible amounts of D-lactate were produced by S. thermophilus IDCC 2201. Finally, it was confirmed that there was no mortality and toxicity throughout single-dose oral toxicity tests in rats. Therefore, we report that S. thermophilus IDCC 2201 is considered to be safe for human consumption as probiotics.

CPPF, A Novel Microtubule Targeting Anticancer Agent, Inhibits the Growth of a Wide Variety of Cancers.

In the past, several microtubule targeting agents (MTAs) have been developed into successful anticancer drugs. However, the usage of these drugs has been limited by the acquisition of drug resistance in many cancers. Therefore, there is a constant demand for the development of new therapeutic drugs. Here we report the discovery of 5-5 (3-cchlorophenyl)-N-(3-pyridinyl)-2-furamide (CPPF), a novel microtubule targeting anticancer agent. Using both 2D and 3D culture systems, we showed that CPPF was able to suppress the proliferation of diverse cancer cell lines. In addition, CPPF was able to inhibit the growth of multidrug-resistant cell lines that are resistant to other MTAs, such as paclitaxel and colchicine. Our results showed that CPPF inhibited growth by depolymerizing microtubules leading to mitotic arrest and apoptosis. We also confirmed CPPF anticancer effects in vivo using both a mouse xenograft and a two-step skin cancer mouse model. Using established zebrafish models, we showed that CPPF has low toxicity in vivo. Overall, our study proves that CPPF has the potential to become a successful anticancer chemotherapeutic drug.

Inhibition of osteoclasts differentiation by CDC2-induced NFATc1 phosphorylation.

Bone homeostasis is regulated by a balance of bone formation and bone resorption; dysregulation of bone homeostasis may cause bone-related diseases (e.g., osteoporosis, osteopetrosis, bone fracture). Members of the nuclear factor of activated T cells (NFAT) family of transcription factors play crucial roles in the regulation of immune system, inflammatory responses, cardiac formation, skeletal muscle development, and bone homeostasis. Of these, NFATc1 is a key transcription factor mediating osteoclast differentiation, which is regulated by phosphorylation by distinct NFAT kinases including casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), and dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs). In this study, we report that cell division control protein 2 homolog (cdc2) is a novel NFAT protein kinase that inhibits NFATc1 activation by direct phosphorylation of the NFATc1 S263 residue. Cdc2 inhibitors such as Roscovitine and BMI-1026 induce reduction of phosphorylation of NFATc1, and this process leads to the inhibition of NFATc1 translocation from the nucleus to the cytoplasm, consequently increasing the nuclear pool of NFATc1. Additionally, the inhibition of cdc2-mediated NFATc1 phosphorylation causes an elevation of osteoclast differentiation or TRAP-positive staining in zebrafish scales. Our results suggest that cdc2 is a novel NFAT protein kinase that negatively regulates osteoclast differentiation.

Generation of a human induced pluripotent stem cell line (MUSIi001-A) from caesarean section scar fibroblasts using Sendai viral vectors.

We generated a human induced pluripotent stem cell (iPSC) line from caesarean section scar fibroblasts of a 33-year-old healthy woman using transgene-free Sendai viral vectors under feeder-free condition. The established iPSC line, designated as MUSIi001-A, exhibited a normal karyotype, expressed pluripotent markers, differentiated into cells of three embryonic germ layers. Further analyses showed that the Sendai viral genome was absent at passage 25. The MUSIi001-A line can serve as a control for studying developmental biology and phenotypic comparison with disease-specific iPSCs.

Streamlined System for Conducting In Vitro Studies Using Decellularized Kidney Scaffolds.

Kidney regeneration is a complex process that can only be studied in vitro at a limited capacity due to the inherent structural and functional complexity of its tissues. Thus, a suitable platform for conducting cellular response and development should be established from decellularized tissues with intact microarchitecture. In this study, a modular streamlined system was developed to allow manageable handling and setup of in vitro studies using decellularized rat kidneys. The system is composed of commercially available parts that can be reused, interchanged, and reconfigured based on the desired experimental stage and process. Decellularization was confirmed through time-lapse observation, stained tissue sections, genetic material quantification, and protein analysis. The capacity of the bioreactor design to support cell-seeded decellularized kidney constructs was tested by determining viability of seeded podocytes and endothelial cells. Based on the results, decellularized kidneys with renal proteins and intact microstructures can be achieved in relatively shorter periods compared (12 h) to established protocols (96-120 h). The minimalistic kidney bioreactor design not only maintained sterility of decellularized kidney without cells but also permitted manageable maintenance of cell-immobilized constructs for up to 1 week. Through this streamlined system, sustainable and reproducible in vitro experiments for kidney regeneration can be designed and conducted using decellularized kidney as a platform for cell growth and development.

Preparation and characterization of polycaprolactone-polyethylene glycol methyl ether and polycaprolactone-chitosan electrospun mats potential for vascular tissue engineering.

Recently, natural polymers are frequently comingled with synthetic polymers either by physical or chemical modification to prepare numerous tissue-engineered graft with promising biological function, strength, and stability. The aim of this study was to determine the efficiency for vascular tissue engineering of two distinctly different mats, one that comprised polycaprolactone-polyethylene glycol methyl ether and other that comprised polycaprolactone-chitosan. Nano/microfibrous mats prepared from electro-spinning were characterized for fiber diameter, porosity, wettability, and mechanical strength. Biological efficacy on both biodegradable mats was assessed by rat bone marrow mesenchymal stem cells, and polycaprolactone-polyethylene glycol methyl ether showed feasibility for use as an inner layer by inducing endothelial-specific gene expression and polycaprolactone-chitosan as an outer layer on dual layered without sacrificing tensile strength, small-diameter blood vessels. Therefore, scaffolds fabricated from this research could be potential sources for tissue-engineered vascular graft and could also overcome the well-known drawbacks, such as thrombogenicity and stenosis, in managing vascular disease.

Anticancer activity of a novel small molecule tubulin inhibitor STK899704.

We have identified the small molecule STK899704 as a structurally novel tubulin inhibitor. STK899704 suppressed the proliferation of cancer cell lines from various origins with IC50 values ranging from 0.2 to 1.0 µM. STK899704 prevented the polymerization of purified tubulin in vitro and also depolymerized microtubule in cultured cells leading to mitotic arrest, associated with increased Cdc25C phosphorylation and the accumulation of both cyclin B1 and polo-like kinase 1 (Plk1), and apoptosis. Unlike many anticancer drugs such as Taxol and doxorubicin, STK899704 effectively displayed antiproliferative activity against multidrug-resistant cancer cell lines. The proposed binding mode of STK899704 is at the interface between αß-tubulin heterodimer overlapping with the colchicine-binding site. Our in vivo carcinogenesis model further showed that STK 899704 is potent in both the prevention and regression of tumors, remarkably reducing the number and volume of skin tumor by STK899704 treatment. Moreover, it was significant to note that the efficacy of STK899704 was surprisingly comparable to 5-fluorouracil, a widely used anticancer therapeutic. Thus, our results demonstrate the potential of STK899704 to be developed as an anticancer chemotherapeutic and an alternative candidate for existing therapies.

The Pepper WPP Domain Protein, CaWDP1, Acts as a Novel Negative Regulator of Drought Stress via ABA Signaling.

Plants are constantly challenged by various environmental stresses, including high salinity and drought, and they have evolved defense mechanisms to counteract the deleterious effects of these stresses. The plant hormone ABA regulates plant growth and developmental processes and mediates abiotic stress responses. Here, we report the identification and characterization of a novel CaWDP1 (Capsicum annuum) protein. The expression of CaWDP1 in pepper leaves was induced by ABA, drought and NaCl treatments, suggesting its role in the abiotic stress response. CaWDP1 proteins show conserved sequence homology with other known WDP1 proteins, and they are localized in the nucleus and cytoplasm. We generated CaWDP1-silenced peppers via virus-induced gene silencing (VIGS). We evaluated the responses of these CaWDP1-silenced pepper plants and CaWDP1-overexpressing (OX) transgenic Arabidopsis plants to ABA and drought. CaWDP1-silenced pepper plants displayed enhanced tolerance to drought stress, and this was characterized by low levels of leaf water loss in the drought-treated leaves. In contrast to CaWDP1-silenced plants, CaWDP1-OX plants exhibited an ABA-hyposensitive and drought-susceptible phenotype, which was accompanied by high levels of leaf water loss, low leaf temperatures, increased stomatal pore size and low expression levels of stress-responsive genes. Our results indicate that CaWDP1, a novel pepper negative regulator of ABA, regulates the ABA-mediated defense response to drought stress.

Pepper CaREL1, a ubiquitin E3 ligase, regulates drought tolerance via the ABA-signalling pathway.

Drought stress conditions in soil or air hinder plant growth and development. Here, we report that the hot pepper (C apsicum a nnuum) RING type E3 Ligase 1 gene (CaREL1) is essential to the drought stress response. CaREL1 encodes a cytoplasmic- and nuclear-localized protein with E3 ligase activity. CaREL1 expression was induced by abscisic acid (ABA) and drought. CaREL1 contains a C3H2C3-type RING finger motif, which functions in ubiquitination of the target protein. We used CaREL1-silenced pepper plants and CaREL1-overexpressing (OX) transgenic Arabidopsis plants to evaluate the in vivo function of CaREL1 in response to drought stress and ABA treatment. CaREL1-silenced pepper plants displayed a drought-tolerant phenotype characterized by ABA hypersensitivity. In contrast, CaREL1-OX plants exhibited ABA hyposensitivity during the germination, seedling, and adult stages. In addition, plant growth was severely impaired under drought stress conditions, via a high level of transpirational water loss and decreased stomatal closure. Quantitative RT-PCR analyses revealed that ABA-related drought stress responsive genes were more weakly expressed in CaREL1-OX plants than in wild-type plants, indicating that CaREL1 functions in the drought stress response via the ABA-signalling pathway. Taken together, our results indicate that CaREL1 functions as a negative regulator of ABA-mediated drought stress tolerance.

Ginsenoside Re Promotes Osteoblast Differentiation in Mouse Osteoblast Precursor MC3T3-E1 Cells and a Zebrafish Model.

Bone homeostasis is tightly regulated to balance bone formation and bone resorption. Many anabolic drugs are used as bone-targeted therapeutic agents for the promotion of osteoblast-mediated bone formation or inhibition of osteoclast-mediated bone resorption. Previous studies showed that ginsenoside Re has the effect of the suppression of osteoclast differentiation in mouse bone-marrow derived macrophages and zebrafish. Herein, we investigated whether ginsenoside Re affects osteoblast differentiation and mineralization in in vitro and in vivo models. Mouse osteoblast precursor MC3T3-E1 cells were used to investigate cell viability, alkaline phosphatase (ALP) activity, and mineralization. In addition, we examined osteoblastic signaling pathways. Ginsenoside Re affected ALP activity without cytotoxicity, and we also observed the stimulation of osteoblast differentiation through the activation of osteoblast markers including runt-related transcription factor 2, type 1 collagen, ALP, and osteocalcin in MC3T3-E1 cells. Moreover, Alizarin red S staining indicated that ginsenoside Re increased osteoblast mineralization in MC3T3-E1 cells and zebrafish scales compared to controls. These results suggest that ginsenoside Re promotes osteoblast differentiation as well as inhibits osteoclast differentiation, and it could be a potential therapeutic agent for bone diseases.

Ginsenoside Re Inhibits Osteoclast Differentiation in Mouse Bone Marrow-Derived Macrophages and Zebrafish Scale Model.

Ginsenosides, which are the active materials of ginseng, have biological functions that include anti-osteoporotic effects. Aqueous ginseng extract inhibits osteoclast differentiation induced by receptor activator of NF-κB ligand (RANKL). Aqueous ginseng extract produces chromatography peaks characteristic of ginsenosides. Among these peaks, ginsenoside Re is a major component. However, the preventive effects of ginsenoside Re against osteoclast differentiation are not known. We studied the effect of ginsenoside Re on osteoclast differentiation, RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity, and formation of multinucleated osteoclasts in vitro. Ginsenoside Re hampered osteoclast differentiation in a dose-dependent manner. In an in vivo zebrafish model, aqueous ginseng extract and ginsenoside Re had anti-osteoclastogenesis effects. These findings suggest that both aqueous ginseng extract and ginsenoside Re prevent bone resorption by inhibiting osteoclast differentiation. Ginsenoside Re could be important for promoting bone health.

The Pepper RING-Type E3 Ligase, CaAIP1, Functions as a Positive Regulator of Drought and High Salinity Stress Responses.

Plant adaptive responses to osmotic stress are co-ordinated by restriction of growth and developmental processes and by molecular and physiological activities. The phytohormone ABA is the primary regulator that induces and responds to osmotic stress, and its sensitivity markedly influences osmotic stress tolerance levels. Several E3 ubiquitin ligases act as positive or negative regulators of ABA, thereby mediating sensitivity to osmotic stress in higher plants. Here, we report that the C3H2C3-type RING finger E3 ligase, CaAIP1, regulates osmotic stress responses via ABA-mediated signaling. CaAIP1 contains a RING finger motif, which functions during attachment of ubiquitins to the target proteins. Expression of CaAIP1 was induced by ABA, drought and NaCl treatments, suggesting its role in the osmotic stress response. CaAIP1-silenced pepper plants displayed a drought-sensitive phenotype characterized by a high level of transpirational water loss in the drought-treated leaves. CaAIP1-overexpressing (OX) plants exhibited increased sensitivity to ABA, but an NaCl- and mannitol-tolerant phenotype during seed germination and seedling growth. CaAIP1-OX plants further displayed enhanced tolerance to drought stress, characterized by low levels of transpirational water loss via increased stomatal closure and leaf temperature. Our data indicate that CaAIP1 is a positive regulator of the osmotic stress tolerance mechanism.

The Pepper CaOSR1 Protein Regulates the Osmotic Stress Response via Abscisic Acid Signaling.

Plants are sessile organisms, and their growth and development is detrimentally affected by environmental stresses such as drought and high salinity. Defense mechanisms are tightly regulated and complex processes, which respond to changing environmental conditions; however, the precise mechanisms that function under adverse conditions remain unclear. Here, we report the identification and functional characterization of the CaOSR1 gene, which functions in the adaptive response to abiotic stress. We found that CaOSR1 gene expression in pepper leaves was up-regulated after exposure to abscisic acid (ABA), drought, and high salinity. In addition, we demonstrated that the fusion protein of CaOSR1 with green fluorescent protein (GFP) is localized in the nucleus. We used CaOSR1-silenced pepper plants and CaOSR1-OX-overexpressing (OX) transgenic Arabidopsis plants to show that the CaOSR1 protein regulates the osmotic stress response. CaOSR1-silenced pepper plants showed increased drought susceptibility, and this was accompanied by a high transpiration rate. CaOSR1-OX plants displayed phenotypes that were hypersensitive to ABA and hyposensitive to osmotic stress, during the seed germination and seedling growth stages; furthermore, these plants exhibited enhanced drought tolerance at the adult stage, and this was characterized by higher leaf temperatures and smaller stomatal apertures because of ABA hypersensitivity. Taken together, our data indicate that CaOSR1 positively regulates osmotic stress tolerance via ABA-mediated cell signaling. These findings suggest an involvement of a novel protein in ABA and osmotic stress signalings in plants.

Effects of delisting nonprescription combination drugs on health insurance expenditures for pharmaceuticals in Korea.

In November 2006, 727 combination drugs in 24 therapeutic classes were delisted in the national formulary in Korea to reduce the country's pharmaceutical expenditure, making these 727 drugs non-reimbursable. This study examines the effects of this delisting on national health insurance expenditures for pharmaceuticals. An interrupted time series analysis was conducted for the period from January 2005 to August 2007 by using administrative claims from Korea's National Health Insurance. The main outcome variable was the total pharmaceutical expenditure measured as a whole and based on the level of health care institutions and therapeutic classes. Comparison between exposure group (delisted drugs) and non-exposure group (listed drugs) were done. No changes in the level of or trend in the total pharmaceutical expenditure were detected. However, the delisting reduced pharmaceutical expenditures in clinics. Delisting effects were also observed according to therapeutic classes of drugs. The results indicate that any delisting should take into account the type of drug.

Trends of Incidence, Mortality, and Future Projection of Spinal Fractures in Korea Using Nationwide Claims Data.

Spinal fractures have been recognized as a major health concern. Our purposes were to evaluate the trends in the incidence and mortality of spinal fractures between 2008 and 2012 and predict the number of spinal fractures that will occur in Korea up to 2025, using nationwide data from the National Health Insurance Service (NHIS). A nationwide data set was evaluated to identify all new visits to medical institutes for spinal fractures in men and women aged 50 years or older between 2008 and 2012. The incidence, mortality rates and estimates of the number of spinal fractures were calculated using Poisson regression. The number of spinal fractures increased over the time span studied. Men and women experienced 14,808 and 55,164 vertebral fractures in 2008 and 22,739 and 79,903 in 2012, respectively. This reflects an increase in the incidence of spinal fractures for both genders (men, 245.3/100,000 in 2008 and 312.5/100,000 in 2012; women, 780.6/100,000 in 2008 and 953.4/100,000 in 2012). The cumulative mortality rate in the first year after spinal fractures decreased from 8.51% (5,955/69,972) in 2008 to 7.0% (7,187/102,642) in 2012. The overall standardized mortality ratio (SMR) of spinal fractures at 1 year post-fracture was higher in men (7.76, 95% CI: 7.63-7.89) than in women (4.70, 95% CI: 4.63-4.76). The total number of spinal fractures is expected to reach 157,706 in 2025. The incidence of spinal fractures increased in Korea in the last 5 years, and the socioeconomic burden of spinal fractures will continue to increase in the near future.