Profiling Bioactive Components of Natural Eggshell Membrane (NEM) for Cartilage Protection and Its Protective Effect on Oxidative Stress in Human Chondrocytes.

The current study aimed to investigate the physicochemical properties of the natural eggshell membrane (NEM) and its protective effects against H2O2-induced oxidative stress in human chondrocytes (SW-1353). Bioactive components from NEM related to cartilage were profiled, consisting of 1.1 ± 0.07% hyaluronic acid, 1.2 ± 0.25% total sulfated glycosaminoglycans as chondroitin sulfate, 3.1 ± 0.33% collagen, and 54.4 ± 2.40% total protein. Protein was hydrolyzed up to 43.72 ± 0.76% using in vitro gastro-intestinal digestive enzymes. Peptides eluted at 9.58, 12.46, and 14.58 min using nano-LC-ESI-MS were identified as TEW, SWVE, and VYL peptides with an M/Z value of 435.1874, 520.2402, and 394.2336, respectively. Radical scavenging activity of NEM at 10 mg/mL using the ABTS assay was revealed to be 2.1 times higher than that of the positive control. NEM treatment significantly enhanced cellular SOD expression (p < 0.05). Pre-treatment with NEM (0.1, 1, and 10 mg/mL) dose-dependently reduced H2O2-induced ROS levels in SW-1353. Cell live imaging confirmed that NEM pre-treatment led to a significant reduction in apoptosis expression compared to control. Results from the present study suggest that NEM rich in cartilage protective components including hyaluronic acid, collagen, and chondroitin antioxidative peptides could be a potential therapeutic agent for osteoarthritis (OA) by scavenging oxidative stress.

Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF-ß/SMAD2/3 pathway.

Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin-dependent kinase inhibitor p21. The activation of the TGF-ß/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF-ß/SMAD2/3 pathway.

Solution plasma synthesis of Pt-decorated Bi12O17Cl2 photocatalysts for efficient upcycling of plastics.

Photocatalytic upcycling of plastic waste is a promising approach to relieving pressure caused by solid waste, but the rational design of novel efficient photocatalysts remains a challenge. Herein, we utilize subnano-sized platinum (Pt)-based photocatalysts for plastic upcycling. A solution plasma strategy is developed to fabricate Pt-decorated Bi12O17Cl2 (SP-BOC). The Pt in an oxidant state and oxygen vacancies optimize the electronic structure for fast charge transfer. As a result, SP-BOC displays high performance for upcycling polyvinyl chloride (PVC) and polylactic acid (PLA) into acetic acid and formic acid, with yield rate and selectivity of 6.07 mg g-1cat. h-1 and 94 %, and 47.43 mg g-1cat. h-1 and 55.1 %, respectively. In addition, the dichlorination efficiency of PVC reaches 78.1 % within 10 h reaction, effectively reducing the environmental hazards associated with PVC waste disposal treatments. This research provides insight into the effective conversion of plastics into high-value chemicals, contributing to the reduction of carbon and toxic emissions in a practical and meaningful way, and offering a useful way for solving challenges of waste management and environmental sustainability.

Awareness survey on drug crime scene investigation and drug detection kits among drug-related police officers.

This questionnaire-based study aimed to investigate the drug crime scene experienced by drug-related police officers and the perceptions of drug test kits by them before initiating the development of drug test kits to detect 16 types of drugs. The subjects were 57 drug-related police officers. Most of the respondents (96.5%) had <10 years of experience in drug-related work. Respondents were questioned about the drug scene investigation and perceptions of drug test kits. The questionnaire about drug test kits included the question on 'simple/rapid drug test kit' and 'electronic portable drug analyzer' regarding the disadvantages of existing kits and expecting features when a new kit is developed. First, in the on-site survey, the drug-related crime occurred at the suspect's house (47.8%), and methamphetamine (35.0%) and γ-hydroxybutyric acid (19.5%) were mainly found. In the awareness survey on drug test kits, most respondents (67.2%) had an experience of using 'simple/rapid drug test kits', whereas 17.5% for the 'electronic portable drug analyzer'. In the case of 'simple/rapid drug test kit', the false-positive rate reached 53.8% by a misinterpretation due to ambiguous color change (47.6%). The inaccuracy of the result (33.3%) was the most concern in 'electronic portable drug analyzer'. Respondents most favored pipette type for sample collector when a new kit is developed. In addition, they preferred the smaller kit with short detection times in both kit types. This survey could be applied to the development of efficient and practical kits for police officers working in drug-related fields.

Kap1 Regulates the Stability of Lin28A in Embryonic Stem Cells.

Lin28A is an RNA-binding protein that controls mammalian development and maintenance of the pluripotency of embryonic stem cells (ESCs) via regulating the processing of the microRNA let-7. Lin28A is highly expressed in ESCs, and ectopic expression of this protein facilitates reprogramming of somatic cells to induced pluripotent stem cells. However, the mechanisms underlying the post-translational regulation of Lin28A protein stability in ESCs remain unclear. In the present study, we identified Kap1 (KRAB-associated protein 1) as a novel Lin28A-binding protein using affinity purification and mass spectrometry. Kap1 specifically interacted with the N-terminal region of Lin28A through its coiled-coil domain. Kap1 overexpression significantly attenuated Lin28A ubiquitination and increased its stability. However, small interfering RNA-mediated knockdown of Kap1 promoted the ubiquitination of Lin28A, leading to its proteasomal degradation. Trim71, an E3 ubiquitin ligase, induced Lin28A degradation and Kap1 knockdown accelerated the Trim71-dependent degradation of Lin28A. Mutation of the lysine 177 residue of Lin28A to arginine abrogated the ubiquitination and degradation of Lin28A which were accelerated by Kap1 silencing. Moreover, Kap1 overexpression led to the accumulation of Lin28A in the cytoplasm, but not in the nucleus, and reduced the levels of let-7 subtypes. These results suggest that Kap1 plays a key role in regulation of the stability of Lin28A by modulating the Trim71-mediated ubiquitination and subsequent degradation of Lin28A, thus playing a pivotal role in the regulation of ESC self-renewal and pluripotency.

Effect of Aged and Fermented Garlic Extracts as Natural Antioxidants on Lipid Oxidation in Pork Patties.

The aim of this study was to investigate the natural antioxidant activity of raw garlic (RG), aged black garlic (AG), and garlic fermented with Bacillus subtilis (FG) extracts on pork patty lipid oxidation throughout refrigerated storage. The total polyphenol, total flavonoid content, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity of three different types of garlic extracts were measured. The total phenolic and flavonoid content of AG was significantly higher than that of FG and RG; FG also showed a significantly higher total phenolic content than that of RG (p<0.05). The DPPH and ABTS radical scavenging activity of AG and FG was significantly higher than that of RG and that of AG was significantly higher than that of FG (p<0.05). To investigate the effect of processed garlic extracts on pork patty lipid oxidation, freeze-dried extracts of RG, FG, and AG were added to the patties at levels of 0.5% (w/w). Patties containing 0.01% (w/w) ascorbic acid (AA) and patties without treatment (CON) were compared with patties containing garlic extracts. The pH value, 2-thiobarbituric acid reactive substances value, and volatile basic nitrogen value of pork patties containing AG and FG extracts were significantly decreased compared to the other groups (CON, AA, and RG; p<0.05). Taken together, these results suggest that AG and FG extracts possess strong antioxidative activity and can serve as natural antioxidative additives to prevent pork patty lipid oxidation.

Biological lipid membranes for on-demand, wireless drug delivery from thin, bioresorbable electronic implants.

On-demand, localized release of drugs in precisely controlled, patient-specific time sequences represents an ideal scenario for pharmacological treatment of various forms of hormone imbalances, malignant cancers, osteoporosis, diabetic conditions and others. We present a wirelessly operated, implantable drug delivery system that offers such capabilities in a form that undergoes complete bioresorption after an engineered functional period, thereby obviating the need for surgical extraction. The device architecture combines thermally actuated lipid membranes embedded with multiple types of drugs, configured in spatial arrays and co-located with individually addressable, wireless elements for Joule heating. The result provides the ability for externally triggered, precision dosage of drugs with high levels of control and negligible unwanted leakage, all without the need for surgical removal. In vitro and in vivo investigations reveal all of the underlying operational and materials aspects, as well as the basic efficacy and biocompatibility of these systems.

Vibrio vulnificus IlpA induces MAPK-mediated cytokine production via TLR1/2 activation in THP-1 cells, a human monocytic cell line.

Vibrio vulnificus is a pathogenic bacterium causing primary septicemia, which is followed by a classical septic shock pathway including an overwhelming inflammatory cytokine response. V. vulnificus IlpA is a potent immunogenic lipoprotein that triggers cytokine production in human monocytes by activating the toll-like receptor 2 (TLR2). In this study, we further defined the IlpA signaling pathways involved in cytokine production in the human monocytic cell line, THP-1. TLR2 was involved in cytokine production by complexing with TLR1, but not with TLR6. MyD88 was necessary for IlpA-induced cytokine expression through TLR1/TLR2. Three mitogen activated protein kinases (MAPK), p38, ERK1/2, and JNK, were activated in THP-1 cells stimulated with recombinant IlpA (rIlpA). Selective inhibition of each MAPK resulted in significant decrease of rIlpA-induced cytokine production. Especially, functional TLR2 was necessary for IlpA-induced activation of p38 and JNK. IlpA augmented the DNA-binding activity of nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) transcriptional factors to their recognition sites in THP-1 cells. These results suggest that serial activation of TLR1/TLR2, MyD88, the three MAPKs, and NF-κB/AP-1 comprises the signaling pathway responsible for proinflammatory cytokine production by V. vulnificus IlpA.

Cell death mediated by Vibrio parahaemolyticus type III secretion system 1 is dependent on ERK1/2 MAPK, but independent of caspases.

Vibrio parahaemolyticus, which causes gastroenteritis, wound infection, and septicemia, has two sets of type III secretion systems (TTSS), TTSS1 and TTSS2. A TTSS1- deficient vcrD1 mutant of V. parahaemolyticus showed an attenuated cytotoxicity against HEp-2 cells, and a significant reduction in mouse lethality, which were both restored by complementation with the intact vcrD1 gene. V. parahaemolyticus also triggered phosphorylation of mitogenactivated protein kinases (MAPKs) including p38 and ERK1/2 in HEp-2 cells. The ability to activate p38 and ERK1/2 was significantly affected in a TTSS1-deficient vcrD1 mutant. Experiments using MAPK inhibitors showed that p38 and ERK1/2 MAPKs are involved in V. parahaemolyticus-induced death of HEp-2 cells. In addition, caspase-3 and caspase-9 were processed into active forms in V. parahaemolyticus-exposed HEp-2 cells, but activation of caspases was not essential for V. parahaemolyticusinduced death of HEp-2 cells, as shown by both annexin V staining and lactate dehydrogenase release assays. We conclude that secreted protein(s) of TTSS1 play an important role in activation of p38 and ERK1/2 in HEp-2 cells that eventually leads to cell death via a caspaseindependent mechanism.

Functional characterization of EpsC, a component of the type II secretion system, in the pathogenicity of Vibrio vulnificus.

EpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a human-pathogenic bacterium, Vibrio vulnificus, to evaluate its role in eliciting virulence. An espC-deleted mutant of V. vulnificus displayed a reduced cytotoxicity to the human cell line HEp-2 and an attenuated virulence in a mouse model. This mutant exhibited dramatic defects in the secretion of diverse extracellular proteins, such as outer membrane proteins, transporters, and the known secreted factors, notably, a hemolysin (VvhA) and an elastase (VvpE). A defect in its secretion of proteins was restored by in trans complementation of the intact epsC gene. Analyses of cellular fractions revealed that VvhA and VvpE of the ΔepsC mutant were not excreted outside the cell but were present mainly in the periplasmic space. Examination of a V. vulnificus mutant deficient in TolC, a component of the T1SS, showed that it is not involved in the secretion of VvhA and VvpE but that it is necessary for the secretion of another major toxin of V. vulnificus, RtxA. Therefore, the T2SS is required for V. vulnificus pathogenicity, which is mediated by at least two secreted factors, VvhA and VvpE, via facilitating the secretion and exposure of these factors to host cells.

A mammalian insulysin homolog is regulated by enzyme IIA(Glc) of the glucose transport system in Vibrio vulnificus.

Vibrio vulnificus is an opportunistic human pathogen that causes severe infections in susceptible individuals. While the components of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) have been shown to regulate numerous targets, little such information is available for the V. vulnificus PTS. Here we show that enzyme IIA(Glc) of the PTS regulates the peptidase activity of a mammalian insulysin homolog in V. vulnificus. While interaction of IIA(Glc) with the insulysin homolog is independent of the phosphorylation state of IIA(Glc), only unphosphorylated IIA(Glc) activates the insulysin homolog. Taken together, our results suggest that the V. vulnificus insulysin-IIA(Glc) complex plays a role in survival in the host by sensing glucose.

Autotrophic and heterotrophic respiration in needle fir and Quercus-dominated stands in a cool-temperate forest, central Korea.

To investigate annual variation in soil respiration (R (S)) and its components [autotrophic (R (A)) and heterotrophic (R (H))] in relation to seasonal changes in soil temperature (ST) and soil water content (SWC) in an Abies holophylla stand (stand A) and a Quercus-dominated stand (stand Q), we set up trenched plots and measured R (S), ST and SWC for 2 years. The mean annual rate of R (S) was 436 mg CO(2) m(-2) h(-1), ranging from 76 to 1,170 mg CO(2) m(-2) h(-1), in stand A and 376 mg CO(2) m(-2) h(-1), ranging from 82 to 1,133 mg CO(2) m(-2) h(-1), in stand Q. A significant relationship between R (S) and its components and ST was observed over the 2 years in both stands, whereas a significant correlation between R (A) and SWC was detected only in stand Q. On average over the 2 years, R (A) accounted for approximately 34% (range 17-67%) and 31% (15-82%) of the variation in R (S) in stands A and Q, respectively. Our results suggested that vegetation type did not significantly affect the annual mean contributions of R (A) or R (H), but did affect the pattern of seasonal change in the contribution of R (A) to R (S).

Functional characterization of the IlpA protein of Vibrio vulnificus as an adhesin and its role in bacterial pathogenesis.

Vibrio vulnificus is a Gram-negative bacterium that causes a fatal septicemia. One of its virulence factors is a membrane-bound lipoprotein, IlpA, which can induce cytokine production in human immune cells. In the present study, the role of IlpA as an adhesion molecule was investigated. An ilpA-deleted V. vulnificus mutant showed significantly decreased adherence to INT-407 human intestinal epithelial cells, which in turn resulted in reduced cytotoxicity. The DeltailpA mutant recovered the adherence ability of the wild type by complementation in trans with the intact ilpA gene. In addition, pretreatment of V. vulnificus with anti-IlpA polyclonal antibodies resulted in a significant reduction of bacterial adherence. To localize the domain of IlpA required for cytoadherence, three truncated recombinant IlpA polypeptides were constructed and tested for the ability to adhere to human cells by a ligand-binding immunoblot assay and fluorescence microscopy. The polypeptide containing the carboxy (C)-terminal hydrophilic domain exhibited direct binding to INT-407 cells. Therefore, the C-terminal domain of IlpA allows this protein to be an adhesion molecule of V. vulnificus.

Biometric-based estimation of net ecosystem production in a mature Japanese cedar (Cryptomeria japonica) plantation beneath a flux tower.

Quantification of carbon budgets and cycling in Japanese cedar (Cryptomeria japonica D. Don) plantations is essential for understanding forest functions in Japan because these plantations occupy about 20% of the total forested area. We conducted a biometric estimate of net ecosystem production (NEP) in a mature Japanese cedar plantation beneath a flux tower over a 4-year period. Net primary production (NPP) was 7.9 Mg C ha(-1) year(-1) and consisted mainly of tree biomass increment and aboveground litter production. Respiration was calculated as 6.8 (soil) and 3.3 (root) Mg C ha(-1) year(-1). Thus, NEP in the plantation was 4.3 Mg C ha(-1) year(-1). In agreement with the tower-based flux findings, this result suggests that the Japanese cedar plantation was a strong carbon sink. The biometric-based NEP was higher among most other types of Japanese forests studied. Carbon sequestration in the mature plantation was characterized by a larger increment in tree biomass and lower mortality than in natural forests. Land-use change from natural forest to Japanese cedar plantation might, therefore, stimulate carbon sequestration and change the carbon allocation of NPP from an increment in coarse woody debris to an increase in tree biomass.

Carbon dioxide exchange in a cool-temperate evergreen coniferous forest over complex topography in Japan during two years with contrasting climates.

We investigated carbon dioxide (CO(2)) exchange and its environmental response during two years with contrasting climate (2006 and 2007) in a cool-temperate mixed evergreen coniferous forest dominated by Japanese cedar (Cryptomeria japonica) and Japanese cypress (Chamaecyparis obtusa). The study, which was conducted in a mountainous region of central Japan, used the eddy-covariance technique. Our results (crosschecked using the common u (*) approach and van Gorsel's alternative approach) showed that annual gross primary production (GPP) and ecosystem respiration (RE) were at least 6% higher in the dry year than in the wet year, whereas net ecosystem exchange (NEE) was similar in both years. Without soil water stress, strong light stress or seasonality of plant area index during most of the study period, the forest had high metabolic activity. GPP and RE differed greatly between the two years, especially in spring (April-May) and summer (July-September), respectively. The spring GPP difference (>20%) was influenced by different winter air temperatures and snow melt timing, which controlled photosynthetic capacity in spring, and by different spring light intensities. The annual NEE differed depending on the evaluation method used, but the mean 2-year NEE estimated by the u (*) threshold approach [-3.39 +/- 0.11 (SD) MgC ha(-1) year(-1)] appears more reasonable in comparison with results from other forests.

The prolyl isomerase Pin1 interacts with a ribosomal protein S6 kinase to enhance insulin-induced AP-1 activity and cellular transformation.

Phosphorylation of proteins on serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis-trans isomerase Pin1 and is a central signaling mechanism in cell proliferation and transformation. Although Pin1 is frequently overexpressed in hepatocellular carcinoma (HCC), the molecular mechanism of Pin1 in HCC has not been completely elucidated. Here, we show that Pin1 interacts with p70S6K in vitro and ex vivo. Overexpression of Pin1 resulted in enhanced p70S6K phosphorylation induced by insulin in SK-HEP-1 cells. In contrast, Pin1(-/-) mouse embryonic fibroblasts (MEFs) exhibited significantly decreased insulin-induced p70S6K phosphorylation compared with Pin1(+/+) MEFs. Furthermore, Pin1 enhanced the insulin-induced extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation through its interaction with p70S6K, whereas the inhibition of p70S6K activity by rapamycin suppressed insulin-induced ERK1/2 phosphorylation in SK-HEP-1 cells. Hence, Pin1 affected activator protein-1 activity through p70S6K-ERK1/2 signaling in SK-HEP-1 cells. Most importantly, Pin1-overexpressing JB6 Cl41 cells enhanced neoplastic cell transformation promoted by insulin much more than green fluorescent protein-overexpressing JB6 Cl41 control cells. These results imply that Pin1 amplifies insulin signaling in hepatocarcinoma cells through its interaction with p70S6K, suggesting that Pin1 plays an important role in insulin-induced tumorigenesis and is a potential therapeutic target in hepatocarcinoma.

Inhibitory effects of fucoidan on activation of epidermal growth factor receptor and cell transformation in JB6 Cl41 cells.

Algal fucoidan is a marine sulfated polysaccharide with a wide variety of biological activities including anti-thrombotic, anti-inflammatory, and anti-tumor activities. In this study, we tested the hypothesis that fucoidan may suppress neoplastic cell transformation by inhibiting the phosphorylation of epidermal growth factor receptor (EGFR) in mouse epidermal JB6 Cl41 cells. Our results provided the first evidence that fucoidan from Laminaria guryanovae exerted a potent inhibitory effect on EGF-induced phosphorylation of EGFR. Consistent with its inhibitory action on phosphorylation of EGFR, fucoidan clearly suppressed the phosphorylation of extracellular signal-regulated kinase or c-jun N-terminal kinases induced by EGF. Moreover, EGF-induced the c-fos and c-jun transcriptional activities were inhibited by fucoidan, resulting to suppressing of activator protein-1 (AP-1) activity and cell transformation induced by EGF. Taken together, these results indicate that fucoidan might exert chemopreventive effects through the inhibition of phosphorylation of the EGFR.

Isolation of tick-borne encephalitis viruses from wild rodents, South Korea.

To determine whether the tick-borne encephalitis virus (TBEV) is present in vector ticks and mammalian hosts in Korea, we examined two tick species, Haemaphysalis longicornis (n = 548) and Ixodes nipponensis (n = 87), and the lungs or spleens of rodents Apodemus agrarius (n = 24) and wild boars (n = 16). Tick-borne encephalitis virus was detected in samples by reverse transcriptase (RT)-nested polymerase chain reaction (PCR), after which TBEV-positive samples were inoculated into BHK-21 cells and suckling mice. Tick-borne encephalitis virus genes were detected in 4 of 38 tick pools and 5 of 24 wild rodents. Suckling mice inoculated intracerebrally with TBEV-positive rodent samples showed signs of encephalitis at six days post-inoculation. The isolation of TBEV was confirmed by inoculating samples obtained from the brains of sick mice in cell culture. Phylogenetic analysis showed that the E genes of the TBEV isolates were clustered with the Western subtype (98% identity). This study suggests the possible occurrence of tick-borne encephalitis in Korea.

Fucoidan from Laminaria cichorioides inhibits AP-1 transactivation and cell transformation in the mouse epidermal JB6 cells.

Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, has anticoagulant and antithrombotic activities. Unlike heparine, fucoidan is known to exhibit anticarcinogenic activities. However, the underlying molecular mechanisms of the chemopreventive activities of fucoidan are not understood. Here we report that fucoidan from Laminaria cichorioides inhibited the epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic cell transformation, but had less cytotoxic effects on JB6 mouse epidermal cells. The EGF-induced phosphorylation of extracellular signal-regulated kinases 1/2 and c-Jun N-terminal kinases, and c-Jun was inhibited by fucoidan, resulting from the inhibition of phosphorylation of epidermal growth factor receptor (EGFR). Fucoidan dose-dependently attenuated the c-fos or c-jun transcriptional activity, and thereby inhibited the associated activator protein-1 (AP-1) transactivation activity. In vitro binding assay revealed that fucoidan directly interacted with EGF, suggested that antitumor promoting effect of fucoidan might be due to preventing the binding of EGF to its cell surface receptor (EGFR). These findings are the first to reveal a molecular basis for the anticarcinogenic action of fucoidan and may partially account for the reported chemopreventive effects of brown seaweeds.