Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell–derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

This study is the first report about the toxicity of pesticides to the mycophagous predator, I. koebelei, of powdery mildew of agricultural crops. Pesticides we tested are composed of synthetic and environmental-friendly products and being used conventionally for the control of insect or microbial pests on cucumber in Korea. our study was conducted to determine the relative toxicities of several pesticides used in Korea cucumber production to mycophagous natural enemy, I. koebelei and to provide a background for implementation of integrated powdery mildew management programs. Based on IOBC classification, three insecticides, bifenthrin + imidacloprid, acetamiprid + indoxacarb, acetamiprid + etopheprox are classified as having a Class 4 (harmful). Spiromesifen showed the low toxicity to the survival and the fecundity of I. koebelei when this chemical had been exposed to 3rd larva or newly emerged adult via feeding with cucumber powdery mildew. However, pyriproxyfen not only decreased the fecundity of female adult but also strongly prohibited from pupation. Many commercial biological or botanical pesticides can restrict the population of I. koebelei. However, Q pact (a.i. Ampelomyces quisqualis 94013), Top seed (a.i. Paenibacillus polymyxa AC-1), BT one (Bacillus thuringiensis) and Solbitchae (insecticidal microorganism) had no toxicity to I. koebelei when this chemical had been exposed to 3rd larva or newly emerged adult feeding with cucumber powdery mildew.

The centipede Scolopendra subspinipes mutilans has been a medically important arthropod species by using it as a traditional medicine for the treatment of various diseases. In this study, we derived a novel lactoferricin B like peptide (LBLP) from the whole bodies of adult centipedes, S. s. mutilans, and investigated the antifungal effect of LBLP. LBLP exerted an antifungal and fungicidal activity without hemolysis. To investigate the antifungal mechanism of LBLP, a membrane study with propidium iodide was first conducted against Candida albicans. The result showed that LBLP caused fungal membrane permeabilization. The assays of the three dimensional flow cytometric contour plot and membrane potential further showed cell shrinkage and membrane depolarization by the membrane damage. Finally, we confirmed the membrane-active mechanism of LBLP by synthesizing model membranes, calcein and FITC-dextran loaded large unilamellar vesicles. These results showed that the antifungal effect of LBLP on membrane was due to the formation of pores with radii between 0.74 nm and 1.4 nm. In conclusion, this study suggests that LBLP exerts a potent antifungal activity by pore formation in the membrane, eventually leading to fungal cell death.

This study has investigated the effect of isometric contractile force and muscle activity applying sperficial heat according to the time from the biceps brachii muscle. In this study, 20 university students participants without musculoskeletal and neurological disorders. By applying a hot pack 5min, 10min, 20min and 30min respectively. After that measurement are skin temperature, contractile force and muscle activity. Skin temperature of the hot 5 min applied that rapidly changing. Increasing the time it takes to apply a variance has been reduced(p<.001). Isometric contractile force was not statistically significant but highest when applying the hot pack 5 minutes and lowest when applying the hot pack 30 minutes(p<.001). Muscle activity and median frequency was highest when applying the hot pack 5 minutes. To analyze the above results, it was found that isometric contractile force and muscle activity changed according to the applying time. These result lead us to the conclusion that this study will be more evidence for changes in muscle contraction to apply hot pack on clinic.

Suspension culture is a useful tool for culturing embryonic stem (ES) cells in large-scale, but the stability of pluripotency and karyotype has to be maintained in vitro for clinical application. Therefore, we investigated whether the chromosomal abnormality of ES cells was induced in suspension culture or not. The ES cells were cultured in suspension as a form of aggregate with or without mouse embryonic fibroblasts (MEFs), and 0 or 1,000 U/ml leukemia inhibitory factor (LIF) was treated to suspended ES cells. After culturing ES cells in suspension, their karyotype, DNA content, and properties of pluripotency and differentiation were evaluated. As a result, the formation of tetraploid ES cell population was significantly increased in suspension culture in which ES cells were co-cultured with both MEFs and LIF. Tetraploid ES cell population was also generated when ES cells were cultured alone in suspension regardless of the existence of LIF. On the other hand, the formation of tetraploid ES cell population was not detected in LIF-free condition, in which MEFs were included. The origin of tetraploid ES cell population was turned out to be E14 ES cells and not MEFs by microsatellite analysis and the basic properties of them were still maintained despite ploidy-conversion to tetraploidy. Furthermore, we identified the ploidy shift from tetraploidy to near-triploidy as tetraploid ES cells were differentiated spontaneously. From these results, we demonstrated that suspension culture system could induce ploidy-conversion generating tetraploid ES cell population. Moreover, optimization of suspension culture system may make possible mass-production of ES cells.

In the present study, total methanol extracts prepared from Alpinia katsumadai showed significant protective effects against the oxidative stress induced by hydrogen peroxide, UV-C or γ-ray irradiation. These protective effects were substantially increased by treatment with 20~100 μg/ml of the extract. The A. katsumadai total methanol preparation was further fractionated into n-hexane, dichloromethane, ethylacetate, n-butanol and water fractions. Among these five fractions, the ethylacetate and butanol fractions of A. katsumadai showed the strongest protective effects against oxidative stress induced by UV-C and γ-ray irradiation. These fractions also showed high DPPH radical scavenging and lipid peroxidation inhibitory activities. In addition, both fractions displayed cell proliferation activation effects, as evidenced by significant increases in colony formation. Our current data thus suggest that the mechanisms underlying the protective effects of A. katsumadai against oxidative damage may include radical scavenging, protection against cell membrane damage and stimulation of cell proliferation.

The tight regulators of fruit set initiation, gibberellin (GA) and auxin, have been applied for decades to induce parthenocarpy, fruit set without fertilization. The integration of GA and auxin signaling mediated by either GA or auxin application during parthenocarpy has been actively reported in tomato, and recently we reported that GA application at pre-bloom also activating auxin signaling and down-regulated negative regulators of fruit set initiation in grapevines. However, the activation of auxin signaling upon GA application without up-regulation of auxin biosynthesis is still unclear. In this study, expression patterns of three auxin efflux transporter genes, VvPIN1a, VvPIN2 and VvPIN4, were monitored during inflorescence development in ‘Tamnara’ grapevines with or without GA application. Without GA application, transcription levels of VvPIN1a and VvPIN4 gradually increased from 14 days before full bloom (DBF) to 2 and 5 days after full bloom (DAF), respectively, except down-regulation of VvPIN1a during 5 DBF to full bloom. However, VvPIN2 expression declined steadily after peaking at 10 DBF. With GA application, VvPIN1a did not show significantly different expression patterns when compared to no GA application, with the exception of 4-fold up-regulation at full bloom, but transcription of VvPIN4 was reduced between 5 and 2 DBF. In addition, VvPIN2 was down-regulated between 12 and 10 DBF by more than 50% compared to levels in the absence of GA application. These reductions of both VvPIN2 and VvPIN4 with GA application prior to pollination suggest that GA application might regulate auxin distribution, instead of auxin biosynthesis, to activating auxin signaling during parthenocarpic fruit initiation.

Gibberellic acid (GA) is a well-characterized plant hormone, which plays a critical role in various plant growth and development. including stem elongation, floral indcution and seed development. GA is known to cause enlargement of ripening fruits and, especially in grapevines, GA shows a unique function: the induction of seedlessness in seeded grape varieties. However, despite extensive previous studies about GA, there has been no clear verification of the mechanism that induces seedlessness in grapes. To understand how GA treatment results in artificial parthenocarpy of seeded grapes at molecular levels, we analyzed transcriptional changes in seeded grapes with and without GA application in various inflorescence developmental stages using RNA-seq. At 14 days before flowering (DBF), seeded grapes were treated with 100 ppm GA and clusters were collected at three developmental stages: 7 DBF, full bloom, and 5 days after flowering (DAF). Of a total of 28,974 genes that were mapped to grape genome reference sequences, 7,013 and 9,064 genes were up- and down-regulated, respectively, in the GA-treated grape as compared to the non-GA-treated control at 7 DBF, full bloom, and 5 DAF. Clustering analysis revealed that these genes could be grouped into 9 clusters with different expression patterns. We also carried out functional annotation based on gene ontology categories. There were significant differences in the expression of the GA and auxin-related gene families. These findings expand our understanding of the complex molecular and cellular mechanisms of GA-induced parthenocarpy of grapes and provide a foundation for future studies on seed development in grapevines.