Application of an Antioxidant Response Element-Nuclear Factor Erythroid 2 Luciferase Assay for Assessing the Skin Sensitization Potential of Agrochemicals.

The skin sensitization potential of agrochemicals can be assessed using laboratory methods such as the keratinocyte activation assay so that their use in regulatory toxicology might replace experimental animal testing. Here, we evaluated the skin sensitization potential of 11 agrochemicals by using an antioxidant response element-nuclear factor erythroid 2 luciferase assay in KeratinoSens and LuSens cells and applying a skin sensitization adverse outcome pathway (AOP). The KeratinoSens and LuSens assays consistently evaluated the skin sensitization potential of 10/11 agrochemicals with reference to animal testing databases. Benomyl, pretilachlor, fluazinam, terbufos, butachlor, and carbosulfan were correctly detected as sensitizers, and glufosinate ammonium, oxiadiazon, tebuconazole, and etofenprox were correctly detected as non-sensitizers. For diazinon, the skin sensitizing potential was positive in the KeratinoSens assay but not in the LuSens assay. These results suggest that the evaluation of in vitro skin sensitization using the AOP mechanism can be applied to assess active agrochemicals.

Optimization of Solid-Phase Extraction of a Degradation Product of Novichok (A234) and Its Application to Environmental Samples.

There have been no detailed investigations regarding solid-phase-extraction (SPE) optimization and screening for the degradation products of ethyl (1-(diethylamino)ethylidene)phosphoramidofluoridate (A234) in various environmental samples. Therefore, as a first step in the selective SPE of the degradation products of A234, we optimized the SPE adsorption and extraction parameters for the A234 degradation product ethylhydrogen (1-(diethylamino)ethylidene)phosphoramidate (cpd 1). Among various SPE cartridges, the Si cartridge (500 mg, 3 mL) selectively extracted cpd 1 using an elution volume of 4 mL of 25% H2O in acetonitrile, which eliminated most interference without cpd 1 loss during loading and washing. In addition, the sorbent capacity is also critical in the adsorption of cpd 1. The Si cartridge (500 mg, 3 mL) retained cpd 1 in the concentration range 1-10 µg/mL. The linearity of detector response of cpd 1 in deionized H2O was studied in the range of 1.0-100 ng/mL and showed good linearity with γ2 ranging from 0.9979 to 0.996. The limits of detection for cpd 1 are 10 ng/mL in the product-scan mode and 100 ng/mL in the full-scan mode. Also, after we optimized the SPE method, we validated the precision and accuracy of the Si-cartridge extraction method in real soil samples with diverse concentrations. The precision ranged from 2.5% to 5.3%. This newly developed SPE is applicable to the analysis of a degradation product of Novichok A234 in various environmental matrices, such as water, soil and sand, in the Organization for the Prohibition of Chemical Weapons (OPCW) proficiency test and unknown samples collected from suspected sites.

Genome editing using preassembled CRISPR-Cas9 ribonucleoprotein complexes in Fusarium graminearum.

Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has greatly facilitated the genetic analysis of fungal pathogens. The head blight fungus, Fusarium graminearum, causes destructive losses of economically important cereal crops. The recent development of the CRISPR-Cas9 system for use with F. graminearum has enabled more efficient genome editing. In this study, we described a CRISPR-Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins (RNPs) into the protoplasts of F. graminearum. The use of RNPs significantly increased both the number of transformants and percentage of transformants in which the target gene was successfully replaced with a selectable marker. We showed that a single double-strand DNA break mediated by the Cas9 ribonucleoprotein was sufficient for gene deletion. In addition, short-homology recombination required only 50 base pair regions flanking the target gene. The high efficiency of Cas9 RNPs enables large-scale functional analysis, the identification of essential genes, and gene deletion that is difficult with conventional methods. We expect that our approach will accelerate genetic studies of F. graminearum.

How Bereaved Parents Experience Public, Self Stigma Years After a Child's Death.

This study identifies the stigma experienced by 12 bereaved parents 2 and 5 years after losing a child in the Sewol ferry disaster in South Korea. Using thematic analysis, we categorized the experience of stigma into two components based on Corrigan and Kosyluk's social cognitive model of stigma: public stigma and self-stigma, and each was analyzed into three types of stigma: stereotype, prejudice, and discrimination. We identified four additional factors related to stigma mitigation. The potential implications for characterizing the experiences of bereaved parents, particularly those facing stigma, are discussed in light of these findings in the longitudinal perspective.

Octaphyrin(1.0.1.0.1.0.1.0) as an Organic Electrode for Li and Na Rechargeable Batteries.

Organic electrode materials for rechargeable batteries have come into the spotlight due to their structural tunability and diversity. In this study, it is found that bisnickel(II) meso-mesityloctaphyrin(1.0.1.0.1.0.1.0) (Oct) is exhibiting multiple oxidation states with extended π-conjugation pathways to afford an active electrode material in Li and Na-organic batteries and secure interactions with Li+ (or Na+ ) and anions enabling efficient dual ionic charge/discharge behaviors. Cyclic voltammograms of the Oct electrode elucidate constantly reversible redox processes in both Li and Na organic batteries and pseudocapacitive behaviors at high currents. Subsequent absorption transformations in CV-UV/VIS/NIR spectroscopic analysis and TD-DFT calculations upon the different redox states of Oct conclusively indicate that six electrons are involved in redox-interconversions per unit cycle with corresponding absorption transformations, which also assessed with charge-and-discharge cell capacities. Significant contributions of the pseudocapacitive processes over the diffusion-controlled processes proceeding in Li- and Na-Oct cells induced fast charge/discharge performance and long-term cyclability.

A broad-spectrum and highly potent human monoclonal antibody cocktail for rabies prophylaxis.

Post-exposure prophylaxis (PEP) is highly effective in preventing disease progression of rabies when used in timely and appropriate manner. The key treatment for PEP is infiltration of rabies immune globulin (RIG) into lesion site after bite exposure, besides wound care and vaccination. Unfortunately, however, RIG is expensive and its supply is limited. Currently, several anti-rabies virus monoclonal antibody (mAb) products are under development as alternatives to RIG, and two recently received regulatory approval in India. In this study, fully human mAbs that recognize different rabies virus glycoprotein conformational antigenic site (II and III) were created from peripheral blood mononuclear cells of heathy vaccinated subjects. These mAbs neutralized a diverse range of lyssavirus types. As at least two anti-rabies virus mAbs are recommended for use in human PEP to ensure broad coverage against diverse lyssaviruses and to minimize possible escape variants, two most potent mAbs, NP-19-9 and 11B6, were selected to be used as cocktail treatment. These two mAbs were broadly reactive to different types of lyssaviruses isolates, and were shown to have no interference with each other. These results suggest that NP-19-9 and 11B6 are potent candidates to be used for PEP, suggesting further studies involving clinical studies in human.

Intramyocardial delivery of human cardiac stem cell spheroids with enhanced cell engraftment ability and cardiomyogenic potential for myocardial infarct repair.

Strategies for stem cell-based cardiac regeneration and repair are key issues for the ischemic heart disease (IHD) patients with chronic complications related to ischemic necrosis. Cardiac stem cells (CSCs) have demonstrated high therapeutic efficacy for IHD treatment owing to their specific cardiac-lineage commitment. The therapeutic potential of CSCs could be further enhanced by designing a cellular spheroid formulation. The spheroid culture condition of CSCs was optimized to ensure regulated size and minimal core necrosis in the spheroids. The CSC spheroids revealed mRNA profiles of the factors related to cardiac regeneration, angiogenesis, anti-inflammatory, and cardiomyocyte differentiation with a higher expression level than the CSCs. Intramyocardially delivered CSC spheroids in the rat IHD model resulted in a significant increase in retention rate by 1.82-fold (day 3) and 1.98-fold (day 14) compared to CSCs. Endothelial cell differentiation and neovascularization of the engrafted CSC spheroids were noted in the infarcted myocardium. CSC spheroids significantly promoted cardiac regeneration: i.e., decreased infarction and fibrotic area (11.22% and 4.18%) and increased left ventricle thickness (0.62 mm) compared to the untreated group. Cardiac performance was also improved by 2.04-fold and 1.44-fold increase in the ejection fraction and fractional shortening, respectively. Intramyocardial administration of CSC spheroids might serve as an advanced therapeutic modality with enhanced cell engraftment and regenerative abilities for cardiac repair after myocardial infarction.

Development of surface water exposure scenarios for risk assessment of pesticides in Korea.

Surface water exposure scenarios used in the risk assessment of Korea's aquatic ecosystems, were developed to represent the 90th percentile pesticide exposure situation as a part of the country's pesticide registration procedure. The scenarios are used to estimate the pesticide concentration in the water of a rice paddy and small streams for three protection goals: (i) mudfish in rice paddies, (ii) the aquatic ecosystem of small streams located near rice paddies, and (iii) the aquatic ecosystem of small streams located near fruit orchards. The scenarios were derived taking into account major exposure routes, such as spray drift, runoff, and drainage. The scenarios were parameterized for appropriate models including the pesticide root zone model (PRZM) and the toxic substances in surface waters model (TOXSWA). A total of 17 pesticide compounds and 28 formulated products were selected to test the risk assessment using the developed scenarios. The simulated predicted environmental concentrations (PECs) fully reflected a) the exposure routes for each protection goal b) the use patterns of the products c) physicochemical properties of the pesticides, and d) meteorological conditions of Korea. However, while assessing the risks for aquatic organisms we observed that for most of the selected pesticides the calculated exposure concentrations were higher than the regulatory acceptable concentration (RAC). To implement the exposure scenarios and models for pesticide authorization in Korea, further research on the RACs is needed. We also recommend studies to develop a higher-tier model and risk-mitigation measures that can be applied to the Korean situation.

The synthesis and characterization of the octahedral CoIII complex of a pyrrolopyrrolizine derivative formed with dicyanovinylene-bis-(meso-aryl)dipyrrin.

A low spin state and diamagnetic CoIII complex 1 possessing pyrrolopyrrolizine ligands formed with dicyanovinylene-bis-(meso-aryl)dipyrrin was synthesized via the thermally activated metalation with CoCl2 and isolated via column chromatography. The nuclear magnetic resonance of complex 1 revealed diamagnetism, thereby confirming the structure of the octahedral CoIII-complex of strong-field ligands. The resulting molecular structure of 1 was elucidated by the X-ray diffraction analysis. An arrangement of two pyrrolizine-ligands for the metal chelation was found in the AB-BA order, which was distinct from the case observed during the formation of bis-NiII-expanded porphyrinoids.

Reactions of Antiaromatic Norcorrole Ni(II) Complex with Carbenes.

Norcorrole is a ring-contracted porphyrinoid, exhibiting distinct antiaromaticity. Herein, we report the reactions of meso-dimesitylnorcorrole Ni(II) complex with two types of carbenes: dichlorocarbene and an N-heterocyclic carbene (NHC). The reaction with in-situ-generated dichlorocarbene resulted in the double insertion of two chloromethine units to provide a mixture of 5,15-dichloroporphyrin and chlorinated isopyricorroles. The nucleophilic NHC attacked the 3-position of the norcorrole core and the subsequent proton transfer furnished a nonconjugated macrocycle incorporating a diazafulvene segment.

Improvement of power conversion efficiency by a stepwise band-gap structure for silicon quantum dot solar cells.

As a promising next-generation solar cell, the power conversion efficiency of a silicon quantum dot (Si-QD) solar cell is still low. In this work, the band-gap structure of a Si-QD layer was modified to improve the power conversion efficiency of a Si-QD solar cell. A stepwise band-gap Si-QD (SB Si-QD) layer with a high bandgap top layer (about 2.22 eV) and a low band-gap bottom layer (about 1.98 eV) was grown on a Si (100) substrate. The open circuit voltage and short circuit current were improved by band-gap engineering of the Si-QD absorption layer. As a result, the power conversion efficiency of the SB Si-QD solar cell increased from 16.50% to 17.50%, compared to that of a Si-QD solar cell with a uniform band gap. This results will provide a guide to design advanced Si-QD solar cells by considering the band-gap structure in the Si-QD absorption layer.

Exploration of Li-Organic Batteries Using Hexaphyrin as an Active Cathode Material.

Lithium-collaborating organic batteries (Li-[28]hexs) were investigated with [28]hexaphyrin(1.1.1.1.1.1) as an active electrode material. Each hexaphyrin of [28]Hex cathode ideally involved four electrons per unit cycle and performed a typical charge/discharge processes of Li-organic battery. Li-[28]Hex batteries set with fast charging rates showed reasonably stable charge and discharge performances over 200 cycles even though it caused incomplete (2~3 electrons) charge/discharge cycles due to failing the complete charging process. UV absorption changes of [28]hexaphyrin in CH2Cl2 were supplementary for the electrochemical oxidation, which performed a conversion from [28]hexaphyrin to [26]hexaphyrin.

Intra-articular delivery of synovium-resident mesenchymal stem cells via BMP-7-loaded fibrous PLGA scaffolds for cartilage repair.

Delivery of synovium-resident mesenchymal stem cells (synMSCs) to cartilage defect site might provide a novel therapeutic modality for treatment of articular cartilage diseases. However, low isolation efficiency of synMSCs limits their therapeutic application. Niche-preserving non-enzymatic isolation of synMSCs was firstly attempted by employing micro-organ culture system based on recapitulating tissue-specific homeostasis ex vivo. The isolated synMSCs retained superior long-term growth competency, proliferation and chondrogenic potential to bone marrow-derived MSCs (BMSCs). It was noted that synMSCs demonstrated 9-fold increase in cartilaginous micro-tissue formation and 13-fold increase in sulfated proteoglycans deposition compared to BMSCs. For delivery of synMSCs, fibrous PLGA scaffolds were specifically designed for full-thickness osteochondral defects in rabbits. The scaffolds provided effective micro-environment for growth and host-integration of synMSCs. Combined delivery of synMSCs with bone morphogenetic proteins-7 (BMP-7) was designed to achieve synergistic therapeutic efficacy. BMP-7-loaded PLGA nanoparticles electrosprayed onto the scaffolds released BMP-7 over 2 weeks to conform with its aimed role in stimulating early stage endochondral ossification. Scaffold-supported combined administration of synMSCs with BMP-7 resulted in high proteoglycan and collagen type II induction and thick hyaline cartilage formation. Intra-articular co-delivery of synMSCs with BMP-7 via fibrous PLGA scaffolds may be a promising therapeutic modality for articular cartilage repair.

In-Use Stability of the Rituximab Biosimilar CT-P10 (Truxima®) Following Preparation for Intravenous Infusion and Storage.

BACKGROUND: CT-P10 is the first biosimilar of the anti-CD20 monoclonal antibody, rituximab. CT-P10 is currently available in over 51 countries worldwide, where it is approved in the same indications as its reference product rituximab. In-use stability studies are conducted for biologics to determine how conditions (e.g., temperature, light, humidity, length of time stored) affect drug quality following dilution and storage in infusion bags. OBJECTIVE: We evaluated the in-use stability of CT-P10 for intravenous infusion stored diluted in infusion bags over longer periods than currently recommended by manufacturer guidelines. METHODS: CT-P10, within the final month of its 36-month shelf life, was diluted to 1.0 or 4.0 mg/mL and stored at 2-8 °C in polyethylene or polyvinylchloride infusion bags for 2, 4, and 6 weeks. CT-P10 infusion bags were incubated at room temperature for 24 h before analysis. Analyses included detection of sub-visible particles, formation of impurities and determination of charge variants, and heavy- and light-chain content. Cell-based CD20 binding affinity and complement-dependent cytotoxicity were also assessed. RESULTS: Diluted CT-P10 solutions remained clear, colorless, and free of visible particles irrespective of type of infusion bag, target concentration, or timepoint. Protein concentrations, sub-visible particles, pH, osmolality, and molecular weight and charge variants were stable across all timepoints and variables. The binding affinity and potency of CT-P10 remained unchanged, indicating that the efficacy of the antibody was maintained following in-use preparation. CONCLUSIONS: We demonstrated that CT-P10 was stable after refrigerated storage for up to 6 weeks followed by incubation at room temperature.

Probing Changes in Lung Physiology in COPD Using CT, Perfusion MRI, and Hyperpolarized Xenon-129 MRI.

RATIONALE AND OBJECTIVES: Chronic obstructive pulmonary disease (COPD) is highly heterogeneous and not well understood. Hyperpolarized xenon-129 (Xe129) magnetic resonance imaging (MRI) provides a unique way to assess important lung functions such as gas uptake. In this pilot study, we exploited multiple imaging modalities, including computed tomography (CT), gadolinium-enhanced perfusion MRI, and Xe129 MRI, to perform a detailed investigation of changes in lung morphology and functions in COPD. Utility and strengths of Xe129 MRI in assessing COPD were also evaluated against the other imaging modalities. MATERIALS AND METHODS: Four COPD patients and four age-matched normal subjects participated in this study. Lung tissue density measured by CT, perfusion measures from gadolinium-enhanced MRI, and ventilation and gas uptake measures from Xe129 MRI were calculated for individual lung lobes to assess regional changes in lung morphology and function, and to investigate correlations among the different imaging modalities. RESULTS: No significant differences were found for all measures among the five lobes in either the COPD or age-matched normal group. Strong correlations (R > 0.5 or < -0.5, p < 0.001) were found between ventilation and perfusion measures. Also gas uptake by blood as measured by Xe129 MRI showed strong correlations with CT tissue density and ventilation measures (R > 0.5 or < -0.5, p < 0.001) and moderate to strong correlations with perfusion measures (R > 0.4 or < -0.5, p < 0.01). Four distinctive patterns of functional abnormalities were found in patients with COPD. CONCLUSION: Xe129 MRI has high potential to uniquely identify multiple changes in lung physiology in COPD using a single breath-hold acquisition.

Synthesis of pyrrolo[3',2':4,5][1,3]diazepino[2,1,7-cd]pyrrolizine derivatives from dicyanovinylene-bis(meso-aryl)dipyrrin.

Pyrrolo[3',2':4,5][1,3]diazepino[2,1,7-cd]pyrrolizine derivatives 2 and 3 were synthesized from dicyanovinylene-bis(meso-aryl)dipyrrin in the presence of either BF3·OEt2 or InBr3, where 2 was readily oxidized in aerobic conditions to be 3. It was understood that the fully elongated π-conjugation of 3 is achieved via the conformation of 2. Crystal structures of 2 and 3 were elucidated by X-ray diffraction analysis. Furthermore, two redox states, 3ox and 3red were observed in the chemical redox processes.

Structural and biochemical analyses reveal ubiquitin C-terminal hydrolase-L1 as a specific client of the peroxiredoxin II chaperone.

Peroxiredoxins (Prxs) play dual roles as both thiol-peroxidases and molecular chaperones. Peroxidase activity enables various intracellular functions, however, the physiological roles of Prxs as chaperones are not well established. To study the chaperoning function of Prx, we previously sought to identify heat-induced Prx-binding proteins as the clients of a Prx chaperone. By using His-tagged Prx I as a bait, we separated ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a heat-induced Prx I binding protein from rat brain crude extracts. Protein complex immunoprecipitation with HeLa cell lysates revealed that both Prx I and Prx II interact with UCH-L1. However, Prx II interacted considerably more favorably with UCH-L1 than Prx I. Prx II exhibited more effective molecular chaperone activity than Prx I when UCH-L1 was the client. Prx II interacted with UCH-L1 through its C-terminal region to protect UCH-L1 from thermal or oxidative inactivation. We found that chaperoning via interaction through C-terminal region (specific-client chaperoning) is more efficient than that involving oligomeric structural change (general-client chaperoning). Prx II binds either thermally or oxidatively unfolding early intermediates of specific clients and thereby shifted the equilibrium towards their native state. We conclude that this chaperoning mechanism provides a very effective and selective chaperoning activity.

Genome-wide functional characterization of putative peroxidases in the head blight fungus Fusarium graminearum.

Reactive oxygen species (ROS) are associated with various developmental processes and host-pathogen interactions in pathogenic fungi. Peroxidases are a group of ROS-detoxifying enzymes that are involved in the oxidative stress response and in a variety of physiological processes. In this study, we performed a genome-wide functional characterization of putative peroxidase genes in Fusarium graminearum, a head blight pathogen of cereal crops. We identified 31 putative peroxidase genes and generated deletion mutants for these genes. Twenty-six of the deletion mutants showed developmental phenotypes indistinguishable from that of the wild-type, and five deletion mutants exhibited phenotypic changes in at least one phenotypic category. Four deletion mutants, fca6, fca7, fpx1 and fpx15, showed increased sensitivity to extracellular H2 O2 . Deletion mutants of FCA7 also exhibited reduced virulence and increased trichothecene production compared with those of the wild-type strain, suggesting that Fca7 may play an important role in the host-pathogen interaction in F. graminearum. To identify the transcription factors (TFs) regulating FCA6, FCA7, FPX1 and FPX15 in response to oxidative stress, we screened an F. graminearum TF mutant library for growth in the presence of H2 O2 and found that multiple TFs co-regulated the expression of FCA7 under oxidative stress conditions. These results demonstrate that a complex network of transcriptional regulators of antioxidant genes is involved in oxidative stress responses in this fungus. Moreover, our study provides insights into the roles of peroxidases in developmental processes and host-pathogen interactions in plant-pathogenic fungi.

Anti-obesity effect of robusta fermented with Leuconostoc mesenteroides in high-fat diet-induced obese mice.

Robusta beans cultivated with Monascus ruber (RMR) were successively fermented with Leuconostoc mesenteroides (LM) and the antiobesity effects were examined. To produce an obese mouse model to investigate the hypolipidemic effects, ICR mice were fed the same high-fat diet for 6 weeks. Treatment groups were given 10 or 20% RMR-LM. Body weight changes in the 20% RMR-LM group were lower compared with those in the control group. Visceral adipose tissue weight and adipose size were significantly lower in the 20% RMR-LM group compared with those in the control group. Significant improvement in glucose tolerance was observed in the 10 and 20% RMR-LM groups compared with the control group. The 20% RMR-LM group exhibited a significant reduction in serum glucose concentration. Hepatic mRNA levels of sterol regulatory element-binding protein 1, fas cell surface death receptor, and peroxisome proliferator-activated receptor γ, which are associated with lipid, and fatty acid metabolism, in the 20% RMR-LM group were significantly lower compared with those in the control group. The results of the present study demonstrated that 20% RMR-LM may be used to prevent obesity, and ameliorate diabetes and lipid metabolism imbalances.

Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG) have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC). Two kinds of chimeric human antibodies (chimeric #7 and #17) were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.