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Pathogens can affect physiological and immunological reactions in immunocompromised animals and genetically engineered mice. Specifically, murine norovirus (MNV), Helicobacter, and intestinal protozoa are prevalent in rodent laboratory facilities worldwide. In this study, microbiological test results of the soiled bedding of sentinel mice showed the prevalence of MNV (50.9%, 28/55), Helicobacter hepaticus (29.1%, 16/55), Trichomonas spp. (14.5%, 8/55), and Entamoeba spp. (32.7%, 18/55). No single infections were detected as all cases were confirmed to have complex infections with two or four pathogens. In previous studies, the success rate of the cross-fostering method was not perfect; therefore, in this study, the entire mouse strain of the SPF rodent facility was rederived using embryo transfer. For up to three years, we confirmed that the results were negative with regular health surveillance tests. Embryo transfer was, thus, determined to be an effective method for the rederivation of specific pathogen free (SPF) barrier mouse facilities. This is the report for the effectiveness of embryo transfer as an example of successful microbiological clean-up of a mouse colony with multiple infections in an entire SPF mouse facility and embryo transfer may be useful for rederiving.

The patterning of poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) hydrogels with excellent electrical property and spatial resolution is a challenge for bioelectronic applications. However, most PEDOT:PSS hydrogels are fabricated by conventional manufacturing processes such as photolithography, inkjet printing, and screen printing with complex fabrication steps or low spatial resolution. Moreover, the additives used for fabricating PEDOT:PSS hydrogels are mostly cytotoxic, thus requiring days of detoxification. Here, we developed a previously unexplored ultrafast and biocompatible digital patterning process for PEDOT:PSS hydrogel via phase separation induced by a laser. We enhanced the electrical properties and aqueous stability of PEDOT:PSS by selective laser scanning, which allowed the transformation of PEDOT:PSS into water-stable hydrogels. PEDOT:PSS hydrogels showed high electrical conductivity of 670 S/cm with 6-μm resolution in water. Furthermore, electrochemical properties were maintained even after 6 months in a physiological environment. We further demonstrated stable neural signal recording and stimulation with hydrogel electrodes fabricated by laser.

Background Recent developments in gene editing, using transcriptional activator-like effector nucleases (TALENs), have greatly helped the generation of genetically engineered animal models. The NK3 homeobox 1 (NKX3.1) protein plays important roles in prostate development and protein production, and functions as a tumor suppressor. Recently, NKX3.1 was shown to be associated with breast cancer in humans. Methods Our aim was to create a new rat model to elucidate the functions of NKX3.1. To that end, we generated Nkx3.1 knockout rats using TALENs and analyzed their phenotype. TALEN-mediated Nkx3.1 knockout was confirmed by T7 endonuclease I (T7E1) assay and DNA sequencing. Prostate weight and fertility were evaluated in the knockout rats, besides determining the proportion of epithelial cells and messenger RNA (mRNA) expression of genes associated with carcinogenesis. Breast tumors were examined by histopathology. Results Results suggested Nkx3.1 knockout rats have reduced fertility, decreased prostate weights, and increased epithelial cell layers. The mRNA expression of genes related to prostate carcinogenesis, namely Ar, Akt, and Pi3k, also increased. Moreover, the Nkx3.1 knockout rats often developed malignant breast tumors. Conclusions We, therefore, successfully created the first Nkx3.1 knockout rat model, using TALEN-mediated gene targeting, and used it to identify defects associated with Nkx3.1 deficiency, not previously observed in mice. Loss of Nkx3.1 in rats led to lower reproductive capacity, and decreased prostate weights, apart from the risk of developing breast cancer. We, thus, proposed Nkx3.1 knockout rats as reliable models for studying the role of NKX3.1 in decreased prostate weights, fertility, and breast cancer, as well as in prostate cancer.

Trimethyltin chloride (TMT), which is an organotin compound widely used in the agricultural and industrial fields, is a well-known neurotoxicant. In particular, excitotoxicity is suspected to be an important mechanism underlying TMT toxicity; however, the effect of TMT exposure on the retina during development and the mechanisms have not been fully elucidated to date. Therefore, in this study, we exposed postnatal mice to TMT and performed a comprehensive analysis of the retina in terms of developmental abnormalities, histopathology, apoptosis, electrophysiological function, glutamate concentration, gene expression, and fluorescence immunostaining. Exposure to 0.75 and 1.5 mg/kg of TMT up to postnatal day 14 caused a decrease in body weight and length, delayed eye opening, and induced thinning of the inner nuclear layer of the retina. In addition, apoptosis was observed in the retinal layer along with b-wave changes and a decrease in retinal ganglion cell spiking activity in the micro-electroretinogram. This change was accompanied by an increase in the concentration of glutamate in the retina, upregulation of astrocyte-related genes, and increased expression of excitatory amino acid transporter (EAAT) 1 and 2. Conversely, EAAT 3, 4, and 5, located in the retinal neurons, were decreased, and this was consistent with the immunostaining results. Our results are the first to prove that TMT induces excitotoxicity and changes in EAAT expression in the retina, and this mechanism causes functional as well as morphological retinal developmental toxicity.

Several humanized mouse models are being used to study humanspecific immune responses and diseases. However, the pivotal needs of fetal tissues for the humanized mice model have been huddled because of the demand for ethical and medical approval. Thus, we have verified the hematopoietic and immunomodulatory function of HepaRG and developed a new and easy humanized mouse model to replace the use of fetal liver tissue. HepaRG co-transplanted Hu-NSG mice significantly increased CD45+ lymphocytes and CD19+ B cells and CD3+ T cells than normal Hu-NSG, suggesting enhanced reconstitution of the human immune system. These results have improved the applicability of humanized mice by developing new models easily accessible. [BMB Reports 2020; 53(9): 466-471].

Background Glutathione peroxidase 3 (Gpx3) protects cells from oxidative stress, and its reduced expression in human prostate cancer has been reported. Objectives We hypothesized that Gpx3 might play an important role in the development of prostatic intraepithelial neoplasia (PIN), a pre-cancerous state of the prostate, and aimed to highlight the underlying molecular mechanism. Materials and methods The following double-knockout mice Nkx3.1-/-; Gpx3+/+, Nkx3.1-/-; Gpx3+/-, Nkx3.1-/-; Gpx3-/- were produced. Randomly divided animals were weighed, and their genitourinary tract (GUT) weights were determined after euthanasia at 4, 8, and 12 months. The mRNA expression of the genes involved in oxidative stress and Wnt signaling was analyzed in the prostate. Histopathology, ROS, and superoxide dismutase (SOD) activities were also measured. Results Loss of Gpx3 did not affect body weight and GUT weight in Nkx3.1 knockout mice. The mRNA expression of SOD3, iNOS, Hmox, and CISD2, which are associated with oxidative stress, was increased in Nkx3.1-/-; Gpx3-/- mice at 4 months but decreased at 8 and 12 months. There was no change in β-catenin and its targets associated with Wnt signaling. Increased ROS and decreased SOD activity were observed in Nkx3.1-/-; Gpx3-/- mice at 12 months of age. The histopathologic score and epithelium thickness were increased, and lumen area was decreased in Gpx3 knockout mice. Discussion and conclusions Gpx3 loss increased the hyperplasia of PIN in the pre-cancerous stage of the prostate. Loss of Gpx3 induced oxidative stress. Histopathologically, no invasive carcinoma was identified, and Gpx3 loss did not increase Wnt/β-catenin signaling. Further research on the role of GPX3 in the transition of PIN to invasive carcinoma is needed. We show, for the first time, that the antioxidant enzyme GPX3 plays a vital role in inhibiting hyperplasia in the PIN stage of the prostate gland in vivo.

Pathogens can affect physiological and immunological reactions in immunocompromised animals and genetically engineered mice. Specifically, murine norovirus (MNV), Helicobacter, and intestinal protozoa are prevalent in rodent laboratory facilities worldwide. In this study, microbiological test results of the soiled bedding of sentinel mice showed the prevalence of MNV (50.9%, 28/55), Helicobacter hepaticus (29.1%, 16/55), Trichomonas spp. (14.5%, 8/55), and Entamoeba spp. (32.7%, 18/55). No single infections were detected as all cases were confirmed to have complex infections with two or four pathogens. In previous studies, the success rate of the cross-fostering method was not perfect; therefore, in this study, the entire mouse strain of the SPF rodent facility was rederived using embryo transfer. For up to three years, we confirmed that the results were negative with regular health surveillance tests. Embryo transfer was, thus, determined to be an effective method for the rederivation of specific pathogen free (SPF) barrier mouse facilities. This is the report for the effectiveness of embryo transfer as an example of successful microbiological clean-up of a mouse colony with multiple infections in an entire SPF mouse facility and embryo transfer may be useful for rederiving.

Samples from 29 adult Gentoo (Pygoscelis papua), Chinstrap (Pygoscelis antarcticus), and Adélie Penguins (Pygoscelis adeliae) at the King Sejong Station on Nar̢ ebski Point, King George Island, Antarctica, were investigated to detect antibodies to avian influenza, Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, Mycoplasma, and Salmonella. Antibodies were identified from one Gentoo Penguin and one Chinstrap Penguin against infectious bronchitis virus; from one Gentoo Penguin against Newcastle disease virus; from one Gentoo Penguin against Mycoplasma synoviae; and from two Chinstrap Penguins against Salmonella pullorum. Thirty-three dead penguin chicks were collected from the breeding colony for necropsy, histopathological examination, and polymerase chain reaction. Pulmonary hemorrhage and congestion were the main necropsy findings.

The Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU) plays a key role in monitoring and managing the humane use of animals in scientific research. Here, as one of the pioneers of the IACUC in Korea, we reported SNU-IACUC operations and activities including committee establishment and legal formulation, protocol review, and post-approval monitoring of protocols, which the IACUC has undertaken in the last decade. In addition, legal regulations and improvements were also discussed, and encompassed the limited number of committee members and the single IACUC policy in Korea. As of December, 2020, amendments are on the table at the National Assembly. We also emphasized the independent nature of the IACUC in protecting activities, including approval and monitoring animal experiments, and its public role in narrowing the knowledge gap between society and scientists. Thus, the aim of this report is to help society and scientists understand the operations of the SNU-IACUC and its role in animal welfare.

Germinal centers (GCs) elicit protective humoral immunity through a combination of antibody-secreting cells and memory B cells, following pathogen invasion or vaccination. However, the possibility of a GC response inducing protective immunity against reinfection following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown. We found GC activity was consistent with seroconversion observed in recovered macaques and humans. Rechallenge with a different clade of virus resulted in significant reduction in replicating virus titers in respiratory tracts in macaques with high GC activity. However, diffuse alveolar damage and increased fibrotic tissue were observed in lungs of reinfected macaques. Our study highlights the importance of GCs developed during natural SARS-CoV-2 infection in managing viral loads in subsequent infections. However, their ability to alleviate lung damage remains to be determined. These results may improve understanding of SARS-CoV-2–induced immune responses, resulting in better coronavirus disease 2019 (COVID-19) diagnosis, treatment, and vaccine development., Recovery from natural SARS-CoV-2 infection may induce a protective germinal center-induced immunity against reinfection.

The gut microbiome has been known to contribute up to ~30% of the energy absorption of the host. Although various beneficial mechanisms of probiotics have been suggested for non-alcoholic fatty liver disease (NAFLD), whether and which probiotics impact the host’s intestinal energy absorption have not yet been quantitatively studied. Here, we suggest a novel mechanism of probiotics against NAFLD, in which Lactobacillus rhamnosus GG, the most common probiotic, shares intestinal fatty acids and prevents the development of diet-induced hepatic steatosis. By using quantitative methods (radioactive tracers and LC–MS) under both in vitro and in vivo conditions, we found that bacteria and hosts competed for fatty acid absorption in the intestine, resulting in decreased weight gain, body fat mass, and hepatic lipid accumulation without differences in calorie intake and excretion in mice fed the probiotic bacteria., Liver disease: Beneficial bacteria divert dietary fats Intestinal bacteria that consume common fatty acids could help protect their hosts against non-alcoholic fatty liver disease (NAFLD). The accumulation of lipids over the course of NAFLD can produce serious inflammation and ultimately lead to liver failure. The gut microbiome plays a prominent role in metabolic health and South Korean researchers led by Hui-Young Lee of Gachon University, Incheon, and Jae-Hak Park of Seoul National University, Seoul, have identified a bacterial species that could help prevent NAFLD. They found that Lactobacillus rhamnosus GG can absorb oleic acid, a fatty acid commonly found in the human diet. Transplantation of these bacteria into high-fat diet-fed mice resulted in weight loss and reduced fat accumulation in the liver. These findings providing a new mechanistic insight into the impact of certain probiotic species on NAFLD.

Phloretin is a flavonoid with known anticancer activities. However, we do not fully understand how phloretin mitigates prostate cancer on the molecular level. In the present study, we examined changes in proliferation, colony formation, and migration after phloretin treatment in human prostate cancer cells PC3 and DU145. We measured reactive oxygen species (ROS) and gene expression. Phloretin increased ROS and suppressed cell proliferation, migration, and colony formation in both cell lines. Additionally, phloretin treatment increased oxidative stress, as demonstrated through lower antioxidant enzymes (catalase, SOD2, Gpx1, Gpx3). In addition, their regulator CISD2 decreased in expression. We also found that increased ROS significantly downregulated multiple components of the Wnt/β-catenin signaling pathway (β-catenin, TCF4, FoxA2, c-Myc) and Twist1. Thus, anticancer activity of phloretin against human prostate cancer cells occurs through generating ROS to influence Wnt/β-catenin signaling. The results of this study suggest that phloretin has a therapeutic effect on prostate cancer in vitro, inhibiting the proliferation and migration of cancer cell lines PC3 and DU145. The mechanism of phloretin appears to be increasing ROS production. We thus recommend phloretin as a promising anticancer therapeutic agent.

Trimethyltin chloride (TMT), one of the most widely used organotin compounds in industrial and agricultural fields, is widespread in soil, aquatic systems, foodstuffs and household items. TMT reportedly has toxic effects on the nervous system; however, there is limited information about its effects on eye development and no clear associated mechanisms have been identified. Therefore, in the present study, we investigated eye morphology, vison-related behavior, reactive oxygen species (ROS) production, apoptosis, histopathology, and gene expression to evaluate the toxicity of TMT during ocular development in zebrafish embryos. Exposure to TMT decreased the axial length and surface area of the eye and impaired the ability of zebrafish to recognize light. 2′,7′-dichlorofluorescein diacetate and acridine orange assays revealed dose-dependent increases in ROS formation and apoptosis in the eye. Furthermore, pyknosis of retinal cells was confirmed through histopathological analysis. Antioxidative enzyme-related genes were downregulated and apoptosis-inducing genes were upregulated in TMT-treated zebrafish compared to expression in controls. Retinal cell-specific gene expression was suppressed mainly in retinal ganglion cells, bipolar cells, and photoreceptor cells, whereas amacrine cell-, horizontal cell-, and Muller cell-specific gene expression was enhanced. Our results demonstrate for the first time the toxicity of TMT during eye development, which occurs through the induction of ROS-mediated apoptosis in retinal cells during ocular formation.

Environmental pollution (EP) is a well-known threat to wild animals, but its toxicological impact is poorly understood. In vitro toxicity evaluation using cells of lower predators could be a promising way to assess and monitor the effects of EPs on whole wildlife populations that are related in the food web. Here, we describe EPs' toxic effect and mechanism in the primary fibroblast derived from the embryo of the striped field mouse, Apodemus agrarius. Characterization of the primary fibroblast was via morphology, genetics, immunocytochemistry, and stable culture conditions for optimal toxicity screening. Cell viability assays-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH)-were performed to observe cytotoxicity, and quantitative PCR was conducted to confirm gene alteration by EP exposure. MTT and LDH assays confirmed the cytotoxicity of transfluthrin (TF), benzyl butyl phthalate (BBP), and 17β-estradiol (E2) with IC50 values of 10.56 μM, 10.82 μM, and 24.08 μM, respectively, following 48-h exposures. mRNA expression of androgen-binding protein, growth hormone receptor, cytochrome C oxidase, and cytochrome P450-1A1 was induced after exposure to TF, BBP, and E2. We unveiled new EP mechanisms at the mammalian cellular level and discovered potential biomarker genes for monitoring of EPs. Based on our findings, we propose the primary fibroblast of A. agrarius as a valuable model to assess the toxicological effects of EP on wildlife.

Using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we exposed macaques to high titers of SARS-CoV-2 via combined transmission routes. We observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. All macaques had a significant loss of total lymphocytes during infection, which were restored over time. These data show that SARS-CoV-2 causes a coronavirus disease 2019 (COVID-19)-like disease in macaques. This new model could investigate the interaction between SARS-CoV-2 and the immune system to test therapeutic strategies.

The gastrointestinal tract is the most common exposure route of xenobiotics, and intestinal toxicity can result in systemic toxicity in most cases. It is important to develop intestinal toxicity assays mimicking the human system; thus, stem cells are rapidly being developed as new paradigms of toxicity assessment. In this study, we established human embryonic stem cell (hESC)-derived enterocyte-like cells (ELCs) and compared them to existing in vivo and in vitro models. We found that hESC-ELCs and the in vivo model showed transcriptomically similar expression patterns of a total of 10,020 genes than the commercialized cell lines. Besides, we treated the hESC-ELCs, in vivo rats, Caco-2 cells, and Hutu-80 cells with quarter log units of lethal dose 50 or lethal concentration 50 of eight drugs—chloramphenicol, cycloheximide, cytarabine, diclofenac, fluorouracil, indomethacin, methotrexate, and oxytetracycline—and then subsequently analyzed the biomolecular markers and morphological changes. While the four models showed similar tendencies in general toxicological reaction, hESC-ELCs showed a stronger correlation with the in vivo model than the immortalized cell lines. These results indicate that hESC-ELCs can serve as a next-generation intestinal toxicity model.

Benzimidazole derivatives are used for their antihelmintic properties, but have also been reported to exert anticancer effects. In the present study, the anticancer effects of albendazole on prostate cancer cells were assessed using proliferation, clonogenic and migration assays. To investigate the anticancer mechanisms of albendazole, reactive oxygen species (ROS) levels were measured, and the expression of genes associated with oxidative stress and Wnt/β-catenin signaling was confirmed by reverse transcription-quantitative PCR and western blotting. Albendazole selectively inhibited the proliferation of the PC3, DU145, LNCaP and AT2 prostate cancer cell lines at concentrations that did not affect the proliferation of a normal prostate cell line (RWPE-1). Albendazole also inhibited the colony formation and migration of PC3 and DU145 cells, as well as inducing ROS production. Diphenyleneiodonium chloride, an inhibitor of NADPH oxidase (NOX), one of the sources of ROS, decreased basal ROS levels in the PC3 and DU145 cells, but did not reduce albendazole-associated ROS production, suggesting that ROS production following albendazole treatment was NOX-independent. The anticancer effect was decreased when albendazole-induced ROS was reduced by treatment with antioxidants (glutathione and N-acetylcysteine). Furthermore, albendazole decreased the mRNA expression of CDGSH iron sulfur domain 2, which regulates antioxidant activity against ROS, as well as the antioxidant enzymes catalase, and glutathione peroxidase 1 and 3. Albendazole also decreased the mRNA expression of catenin β1 and transcription factor 4, which regulate Wnt/β-catenin signaling and its associated targets, Twist family BHLH transcription factor 1 and BCL2. The albendazole-related decrease in the expression levels of oxidative stress-related genes and Wnt/β-catenin signaling proteins was thought to be associated with ROS production. These results suggest that the antihelmintic drug, albendazole, has inhibitory effects against prostate cancer cells in vitro. Therefore, albendazole may potentially be used as a novel anticancer agent for prostate cancer.

In accordance with requirements of the ICH S7B safety pharmacology guidelines, numerous next-generation cardiotoxicity studies using human stem cell-derived cardiomyocytes (CMs) are being conducted globally. Although several stem cell-derived CMs are being developed for commercialization, there is insufficient research to verify if these CMs can replace animal experiments. In this study, in vitro high-efficiency CMs derived from human embryonic stem cells (hESC-CMs) were compared with Sprague-Dawley rats as in vivo experimental animals, and primary cultured in vitro rat-CMs for cardiotoxicity tests. In vivo rats were administrated with two consecutive injections of 100 mg/kg isoproterenol, 15 mg/kg doxorubicin, or 100 mg/kg nifedipine, while in vitro rat-CMs and hESC-CMs were treated with 5 μM isoproterenol, 5 μM doxorubicin, and 50 μM nifedipine. We have verified the equivalence of hESC-CMs assessments over various molecular biological markers, morphological analysis. Also, we have identified the advantages of hESC-CMs, which can distinguish between species variability, over electrophysiological analysis of ion channels against cardiac damage. Our findings demonstrate the possibility and advantage of high-efficiency hESC-CMs as next-generation cardiotoxicity assessment. [BMB Reports 2020; 53(8): 437-441].

The United States Food and Drug Administration-approved antipsychotic drug, pimozide, has anticancer activities. However, the role of reactive oxygen species (ROS) in its effect on prostate cancer is not well-known. We examined cell proliferation, colony formation, migration, ROS production, and the expression of antioxidant-related genes after treatment of human prostate cancer PC3 and DU145 cells with pimozide. In addition, histopathology, ROS production, and superoxide dismutase (SOD) activity were analyzed after administering pimozide to TRAMP, a transgenic mouse with prostate cancer. Pimozide increased the generation of ROS in both cell lines and inhibited cell proliferation, migration, and colony formation. Oxidative stress induced by pimozide caused changes in the expression of antioxidant enzymes (SOD1, peroxiredoxin 6, and glutathione peroxidase 2) and CISD2. Co-treatment with glutathione, an antioxidant, reduced pimozide-induced ROS levels, and counteracted the inhibition of cell proliferation. Administration of pimozide to TRAMP mice reduced the progression of prostate cancer with increased ROS generation and decreased SOD activity. These results suggest that the antipsychotic drug, pimozide, has beneficial effects in prostate cancer in vivo and in vitro. The mechanism of pimozide may be related to augmenting ROS generation. We recommend pimozide as a promising anticancer agent.

Additional file 1: Supplementary Fig. 1. Cellular characteristics after re-warming compared with vitrification and slow-freezing method using various cell lines. (A) Morphology and (B) viability of MSCs after warming either vitrified or non-vitrified groups using trypan blue staining. (C) The DNA fragmentation of each cell line by TUNEL assay (blue: cell, red: DNA strand breaks) and (D) the measurement of intracellular reactive oxygen species levels (green: unfrozen control). Supplementary Fig. 2. Survivability and various gene expression of rewarmed spheroids using hepaRG cell line. (A) Viability of the largest size of spheroids after rewarming via live-dead staining. (B) Quantitative real-time polymerase chain reaction (RT-PCR) analysis for apoptosis, oxidative stress and heat shock damage after rewarming. (n ≥ 3). *p

Our previous study showed that silymarin, a mixture of flavonolignans extracted from seeds of milk thistle, modulated hepatic transsulfuration reactions, leading to an increase in glutathione (GSH) synthesis. To investigate its pharmacological significance, we determined the effect of silymarin on acetaminophen (APAP)-induced liver injury. Silymarin administration to mice prevented the induction of APAP-hepatotoxicity as measured by changes in plasma enzyme activities, hepatic lipid peroxidation and formation of nitrotyrosine protein-adducts. Depletion of hepatic GSH was inhibited significantly. Plasma APAP, APAP-glucuronide or APAP-sulfate level was not changed, but APAP-GSH, APAP-cysteine and APAP-N-acetylcysteine levels were elevated. Silymarin treatment did not induce proteins or activities of Cyp2e1, Cyp1a2, and Cyp3a11, the major cytochrome P450 subtypes responsible for the metabolic activation of APAP to a reactive metabolite, N-acetyl-p-benzoquinoneimine. The results suggest that the hepatoprotective effect of silymarin is associated with an increase in the metabolic disposition of N-acetyl-p-benzoquinoneimine via up-regulation of the GSH conjugation capacity.

Human embryonic stem cells (hESCs) are pluripotent cells widely used in conventional and regenerative medicine due to their ability to self-renew, proliferate and differentiate. Recently, genetic modification of stem cells using genome editing is the most advanced technique for treating hereditary diseases. Nevertheless, the low transfection efficiency of hESCs using enzymatic methods is still limited in in vitro preclinical research. To overcome these limitations, we have developed transfection methods using non-enzymatic treatments on hESCs. In this study, hESCs were transfected following enzymatic (TrypLE and trypsin) and non-enzymatic treatment ethylenediaminetetraacetic acid (EDTA) to increase transfection efficiency. Flow cytometric analysis using an enhanced green fluorescent protein vector showed a significantly increased transfection efficiency of EDTA method compared to standard enzyme method. In addition, the EDTA approach maintained stable cell viability and recovery rate of hESCs after transfection. Also, metabolic activity by using Extracellular Flux Analyzer revealed that EDTA method maintained as similar levels of cell functionality as normal group comparing with enzymatic groups. These results suggest that transfection using EDTA is a more efficient and safe substitute for transfection than the use of standard enzymatic methods.

Francisella tularensis (FT), a highly infectious pathogen, is considered to be a potential biological weapon owing to the current lack of a human vaccine against it. Tul4 and FopA, both outer membrane proteins of FT, play an important role in the bacterium's immunogenicity. In the present study, we evaluated the immune response of mice-humanized with human CD34+ cells (hu-mice)-to a cocktail of recombinant Tul4 and FopA (rTul4 and rFopA), which were codon-optimized and expressed in Escherichia coli. Not only did the cocktail-immunized hu-mice produce a significant human immunoglobulin response, they also exhibited prolonged survival against an attenuated live vaccine strain as well as human T cells in the spleen. These results suggest that the cocktail of rTul4 and rFopA had successfully induced an immune response in the hu-mice, demonstrating the potential of this mouse model for use in the evaluation of FT vaccine candidates.

We describe the first case of biliary cirrhosis in Japanese macaque. Clinical signs had not been detected. The liver was nodular. Histopathologically, portal-to-portal pattern of fibrosis might have indicated chronic cholestasis. Fibrotic septa were infiltrated with inflammatory cells. Therefore, this case could be diagnosed as active incomplete biliary cirrhosis.

Ethical issues in animal toxicity testing have led to the search for alternative methods to determine the skin sensitization potential of cosmetic products. The emergence of ethical testing issues has led to the development of many alternative methods that can reliably estimate skin sensitization potentials. However, a single alternative method may not be able to achieve high predictivity due to the complexity of the skin sensitization mechanism. Therefore, several prediction assays, including both in chemico and in vitro test methods, were investigated and integrated based on the skin sensitization adverse outcome pathway. In this study, we evaluated three different integrated approaches to predict a human skin sensitization hazard using data from in vitro assays (KeratinoSens™ and human cell line activation test [h-CLAT]), and a newly developed in chemico assay (spectrophotometric direct peptide reactivity assay [Spectro-DPRA]). When the results of the in chemico and in vitro assays were combined, the predictivity of human data increased compared with that of a single assay. The highest predictivity was obtained for the approach in which sensitization potential was determined by Spectro-DPRA followed by final determination using the result of KeratinoSens™ and h-CLAT assays (96.3% sensitivity, 87.1% specificity, 86.7% positive predictive value, 96.4% negative predictive value and 91.4% accuracy compared with human data). While further optimization is needed, we believe this integrated approach may provide useful predictive data when determining the human skin sensitization potential of chemicals.

Some cosmetic ingredients can act as a chemical hapten to induce an immune response; therefore, evaluating the sensitizing potential of cosmetic ingredients is essential. We previously developed a novel in chemico direct peptide reactivity assay involving a spectrophotometric evaluation (Spectro-DPRA) for animal skin sensitization tests (local lymph node assay; LLNA). Based on previous research, we expanded the test materials to confirm the effectiveness of the Spectro-DPRA method for predicting the animal skin sensitization potential, and further determined the feasibility of the method for estimating the human skin sensitization potential. Spectro-DPRA showed 83.1% or 89.1% accuracy compared to a conventional LLNA or prediction based on human data, respectively, with a combination model using both a cysteine peptide and lysine peptide cut-off. To identify the effect of the lipophilicity of a chemical on predicting the skin sensitization potential, we applied our prediction model to chemicals with a Log Pow range of -1 to 4. Overall predictability was increased, and the accuracy compared to the LLNA and human data was 91.5% and 94.9%, respectively, in the combination cut-off prediction model. In conclusion, Spectro-DPRA serves as an easy, rapid, and high-throughput in chemico screening method with high accuracy to predict the human skin sensitization potential of chemicals.

Trimethyltin chloride (TMT) is a neurotoxicant widely present in the aquatic environment. Chronic exposure of embryos to TMT for 4 days post-fertilization (dpf) elicited a concentration-related decrease in head & eye size and increase in axial malformation. In addition, Rohon-Beard sensory neurons and motor neurons showed decreased patterns of protein expression. These data coincide with previous research about the neurotoxicity of TMT on mRNA expression (Kim et al., 2016 [1]). These data demonstrates that TMT inhibits specific neurodevelopmental stages in zebrafish embryos and suggests a possible mechanism for the toxicity of TMT in vertebrate neurodevelopment. This paper contains data related to research concurrently published in Kim et al. (2016) [1]. Keywords: Trimethyltin chloride, Neurotoxicity, Zebrafish

Background: Lipid accumulation in hepatocytes constitutes a major component in the pathogenesis of chronic liver injury. However, the impact of lipid deposition in liver on drug metabolizing capacity remains unclear. Purpose of the study: The present study was undertaken to evaluate the changes in hepatic cytochrome P-450 (CYP) enzymes in acute hepatosteatosis. Methods: Rats were fasted for 48 h, and then provided with a high-carbohydrate/fat-free diet (FH) or a normal diet (FN) for 48 h. Results: Hepatic lipid accumulation was significant in the FH group. In the FN group there was a small increase in microsomal p-nitrophenol hydroxylase, p-nitroanisole O-demethylase, and erythromycin N-demethylase activities, but aminopyrine N-demethylase activity was decreased markedly. However, the microsomal enzyme activities were all inhibited by FH intake. Immunoblotting analysis showed that CYP2E1, CYP1A, CYP3A, and CYP2B1/2 proteins were decreased in the FH group. Expression of corresponding CYP mRNAs was also down-regulated. A dose of CCl4, a CYP2E1 substrate,was administered to the rats fed the different diets. The liver injury was significantly lower in the FH group as determined by elevation of serum enzyme activities and histopathological examination. Conclusions: The results show that acute hepatosteatosis may result in a significant alteration in hepatic CYP-mediated metabolizing capacity, which can precipitate erratic responses of liver to various endogenous and exogenous substances.

In order to examine the prevalence of Cryptosporidium infection in wild rodents and insectivores of South Korea and to assess their potential role as a source of human cryptosporidiosis, a total of 199 wild rodents and insectivore specimens were collected from 10 regions of South Korea and screened for Cryptosporidium infection over a period of 2 years (2012-2013). A nested-PCR amplification of Cryptosporidium oocyst wall protein (COWP) gene fragment revealed an overall prevalence of 34.2% (68/199). The sequence analysis of 18S rRNA gene locus of Cryptosporidium was performed from the fecal and cecum samples that tested positive by COWP amplification PCR. As a result, we identified 4 species/genotypes; chipmunk genotype I, cervine genotype I, C. muris, and a new genotype which is closely related to the bear genotype. The new genotype isolated from 12 Apodemus agrarius and 2 Apodemus chejuensis was not previously identified as known species or genotype, and therefore, it is supposed to be a novel genotype. In addition, the host spectrum of Cryptosporidium was extended to A. agrarius and Crosidura lasiura, which had not been reported before. In this study, we found that the Korean wild rodents and insectivores were infected with various Cryptosporidium spp. with large intra-genotypic variationa, indicating that they may function as potential reservoirs transmitting zoonotic Cryptosporidium to livestock and humans.

West Nile virus (WNV) is a mosquito-borne zoonotic pathogen that has spread throughout Europe and the United States. Recently, WNV spread to East and Southeast Asia, and great efforts have been made in South Korea to prevent the spread of WNV from neighboring countries. In this study, we diagnosed the first case of WNV in pigeons (Columba livia domestica) residing in cities using a competitive enzyme-linked immunosorbent assay and confirmed it with nested reverse transcription polymerase chain reaction analysis and sequencing. This is the first report to provide convincing evidence that WNV is present within South Korea.

Troglitazone (TGZ) is a synthetic peroxisome proliferator-activated receptor γ (PPARγ) ligand that exhibits potential antitumor effects on a number of cancer subtypes, including prostate cancer. However, little is known about the effect of TGZ on metastasis in prostate cancer. The aim of the present study was to determine the inhibitory effect and mechanism underlying TGZ on cell growth, migration and invasion using the prostate cancer PC-3 cell line. Cellular migration and invasion were evaluated by performing a wound healing assay and Matrigel assay, respectively. The expression levels of mRNA and protein were determined by reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that TGZ dose-dependently inhibited cell migration and invasion of PC-3 cells. The present study also revealed that TGZ increased the mRNA and protein levels of E-cadherin and glutathione peroxidase 3 (GPx3) in human prostate cancer PC-3 cells. In addition, GW9662, a PPARγ antagonist, attenuated the increased mRNA and protein levels of E-cadherin and GPx3, suggesting that the PPARγ-dependent signaling pathway was involved. Taken together, these results suggested that the anti-migration and anti-invasion effect of TGZ on PC-3 prostate cancer cells is, at least in part, mediated via upregulation of E-cadherin and GPx3. The present study also concluded that PPARγ may be used as a potential remedial target for the prevention and treatment of prostate cancer cell invasion and metastasis.