The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is responsible for hyperacute rejection (HAR) during transgenic pig-to-non-human primate xenotransplantation. To overcome HAR after xenografts, it is essential for the inactivation of α1,3Galactosyltransferase (GT) gene by the homozygotic knocked out of GT-/- and the isoglobotrihexosylceramide synthase (iGb3s-/-). This study was performed to investigate the generation and characterization of the α1,3GT-MCP/-MCP+iGb3-/- transgenic cells. Ear fibroblast cells from the GT-MCP/-MCP pig were cultured and used to positive control. For iGb3s knock out, the Cas9-GFP-iGb3s vector was transfected into the GT-MCP/-MCP cells. The Cas9-GFP-iGb3s transfected cells were sorted and sequenced for the selection of GT-MCP/-MCP+ iGb3s-/- cells. Among the three sorted cell lines, one transgenic cell line was homozygously deleted 3 bases and 10 bases in each chromosome, respectively. To characterize an expression of α-Gal epitope, a wild type and the transgenic cells were measured by FACS Aria using BS-IB4 lectin antibody. The expression of α-Gal epitope in GT-MCP/-MCP cells (<0.01 %) were significantly down-regulated to the range of wild type (99.4 %) fibroblast cells (p<0.05). To analyze the function of iGb3s, α -Gal epitope expressions were measured for the GT-MCP/-MCP, GT-MCP/-MCP+iGb3s-/+, and GT-MCP/-MCP+iGb3s-/-. The range was 95.8%, 94.2%, and 75.8%, respectively. Interestingly, there was a negative range (16.2%) of α-Gal epitope -/- section in GT-MCP/-MCP+iGb3s-/-, compared to 2.74% of GT-MCP/-MCP+iGb3s-/+ and 1.4% of WT, respectively. Our results demonstrated that iGb3s-/-combined with GT-/- had a function to inhibit α-Gal epitope expression in pig cells. Further studies are needed to evaluate the functions of double gene knock out to minimize a HAR response after xenotransplantation.

The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is responsible for hyperacute rejection (HAR) during transgenic pig-to-non-human primate xenotransplantation. There are genes related to the expression of α-Gal epitope such as α1,3Galactosyltransferase gene (GT-/-) and the isoglobotrihexosylceramide synthase (iGb3s-/-). This study was performed to investigate the expression of α-Gal epitope in the skin derived from GT-/- transgenic pig. The skin (7/1000 inches) was obtained by dermatome (Zimmer® Electric Dermatome) from one month old of wildtype (WT) and GT-/- piglets, respectively. The skins were fixed, dehydrated, cleaned, and embedded. To analyze the expression of α-Gal epitope, the paraffin section of WT and GT-/- were stained with BS-IB4 lectin and isoglobotrihexosylceramide synthase antibody. There was a strong BS-IB4 lectin signal in the skin of WT, but not detected in GT-/-. However, the iGb3s positive signals were stained in the skin of both WT and GT-/-. Taken together, it can be postulated that the knocked out of GT gene may not enough to inhibit the expression of α-Gal epitope. Further studies are needed to evaluate the functions of the double knock out of GT and iGb3s on the expression of α-Gal epitope.

It is still challenging to establish pESCs due to differences in the genetic backgrounds of mouse, human, and pig. So it is required to find pig specific pluripotency markers and cellular signaling. In this experiments, doxycycline-inducible vectors carrying OCT4, SOX2, NANOG, KLF4 and MYC known as reprogramming factors, were infected into pig stem cells for analyzing gene expression pattern. When cultured without doxycycline, pig stem cells were stably maintained in bFGF supplemented media. However, when treated with doxycycline, pig stem cells lost alkaline phosphatase activity and were differentiated within two weeks. And then, we investigated the expression of genes related to pluripotency in doxycycline-treated pig stem cells by using qRT-PCR. The qRT-PCR data revealed that expression of OCT4, CDH1 and FUT4 were significantly increased by OCT4 overexpression and OCT4 and FUT4 were also upregulated in SOX2-infected group. When infected with combination of two factors including OCT4 or SOX2, some groups could stably maintain at LIF supplemented media, having alkaline phosphatase activity. Given these data, although ectopic gene expression induced differentiation in pig stem cells, ectopic expression of OCT4 and SOX2 could upregulate pluripotent genes and overexpreession of two factors help pig stem cells adapt LIF-contained media. This study could improve understanding of pluripotent networks as well as aid in establishing bona fide pluripotent stem cells in pig.

Crocin is a carotenoid that may protect cells against oxidative stress by scavenging free radicals particularly superoxide anions. It has been reported that oocyte maturation is influenced by the free radicals generated during in vitro culture (IVC) process. The objective of study was to examine the effect of crocin in in vitro maturation (IVM) medium as an antioxidant on oocyte maturation and embryonic development after parthenogenesis (PA). Cumulus-oocyte complexes (COCs) were collected from ovaries of prepubertal gilts. The basic medium for IVM was medium-199 containing 10% pig follicular fluid, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Oocytes were treated for 44 hours with crocin at 0, 25, 50, and 100 μg/ml during IVM. Oocytes reached the metaphase II stage were induced for PA and cultured for 7 days in porcine zygote medium-3. Nuclear maturation of oocytes was not influenced by various concentrations of crocin (89.0, 87.3, 84.3, and 94.1% for control, 25, 50, and 100 μg/ml crocin, respectively). IVM oocytes treated with 50 μg/ml crocin showed a higher (P<0.05) intraoocyte glutathione (GSH) contents than untreated oocytes (1.00 vs. 1.29 pixels/oocyte). Blastocyst formation of PA embryos treated with 50 (42.9%) and 100 μg/ml crocin (43.8%) was significantly higher (P<0.05) than oocytes treated with 25 μg/ml crocin (30.5%) but not different from that (35.2%) of untreated oocytes. In summary, crocin increases cytoplasmic maturation in terms of intraoocyte GSH content which may be beneficial for later embryonic development by protecting from harmful effect of reactive oxygen species. Further studies are needed to determine whether the beneficial effect of crocin treatment during IVC would be shown in embryonic development after in vitro fertilization and somatic cell nuclear transfer.

In mature oocytes, maturation promoting factor (MPF) activity is playing important roles in arrest at M-phase and its continuous phenomenon, oocyte aging. In most mammals, metaphase II oocytes show high MPF activity and have been used as ooplasts in somatic cell nuclear transfer (SCNT). Caffeine has been found to regulate MPF activity in mammalian oocytes. Caffeine inhibits p34cdc2 phosphorylation and increases MPF activity. The present study investigated the effects of caffeine treatment during last 4 hours of in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenesis (PA) and SCNT. The IVM medium was medium-199, 10% (v/v) PFF, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Immature oocytes were matured in IVM medium without or with 2.5 mM caffeine during the last 4 hours of IVM. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) bovine serum albumin. Nuclear maturation (83.6–87.2%) and intraoocyte glutathione contents (0.9–1.0 pixels/oocyte) of oocytes were not influenced by the caffeine treatment. The membrane fusion of cell-cytoplast couplets (75.5–76.5%) and cleavage (85.4–86.2%) were also not altered by the caffeine treatment. However, caffeine-treated oocytes showed higher (P<0.05) blastocyst formation after SCNT (47.5 vs. 34.3%) than untreated oocytes. Our results demonstrate that caffeine treatment during last 4 hour of IVM improves the developmental competence of SCNT embryos probably by influencing MPF activity.

Crocin is a carotenoid that may protect cells against oxidative stress by scavenging free radicals particularly superoxide anions. It has been reported that oocyte maturation is influenced by the free radicals generated during in vitro culture (IVC) process. The objective of study was to examine the effect of crocin in in vitro maturation (IVM) medium as an antioxidant on oocyte maturation and embryonic development after parthenogenesis (PA). Cumulus-oocyte complexes (COCs) were collected from ovaries of prepubertal gilts. The basic medium for IVM was medium-199 containing 10% pig follicular fluid, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Oocytes were treated for 44 hours with crocin at 0, 25, 50, and 100 μg/ml during IVM. Oocytes reached the metaphase II stage were induced for PA and cultured for 7 days in porcine zygote medium-3. Nuclear maturation of oocytes was not influenced by various concentrations of crocin (89.0, 87.3, 84.3, and 94.1% for control, 25, 50, and 100 μg/ml crocin, respectively). IVM oocytes treated with 50 μg/ml crocin showed a higher (P<0.05) intraoocyte glutathione (GSH) contents than untreated oocytes (1.00 vs. 1.29 pixels/oocyte). Blastocyst formation of PA embryos treated with 50 (42.9%) and 100 μg/ml crocin (43.8%) was significantly higher (P<0.05) than oocytes treated with 25 μg/ml crocin (30.5%) but not different from that (35.2%) of untreated oocytes. In summary, crocin increases cytoplasmic maturation in terms of intraoocyte GSH content which may be beneficial for later embryonic development by protecting from harmful effect of reactive oxygen species. Further studies are needed to determine whether the beneficial effect of crocin treatment during IVC would be shown in embryonic development after in vitro fertilization and somatic cell nuclear transfer.

Insecticidal toxicities of the isolated constituent of Eucalyptus dives oil and its analogues were bioassayed. 3-Carvomenthenone was isolated by chromatographic techniques and determined by EI-MS, 13C-NMR,1H-NMR, 1H-1H COSY, and HMQC. In the fumigant bioassay against P. interpunctella, cyclohexenone exhibited the strongest insecticidal toxicity (LD50 against larvae and adults, 2.45 and 3.63 μg/cm3), followed by methylcyclohexenone, seudenone, and 3-carvomenthenone. In the structure-activity relationships between 3-carvomenthenone analogues and insecticidal toxicity, the mode of the insecticidal action of 3-carvomenthenone, cyclohexenone, methylcyclohexenone, and seudenone was through the dermal organs of T. castaneum and P. interpunctella. This study indicates that 3-carvomenthenone, cyclohexenone, methylcyclohexenone, and seudenone have potential capacity for the development as safety natural agents to control the stored grain insects.

Cotton aphid, Aphis gossypii Gloever is one of the major pests on a wide range of economically important crops in the world. The sustained use of chemical insecticides to control the aphid has led to the emergence of resistant strains to numerous used insecticides. As an alternative strategy entomopathogenic fungi have been used as part of integrated pest management program to control aphid, especially insecticide-resistance population. In particular, Beauveria bassiana-based commercial bio-insecticide has been used to reduce the pest population under greenhouse conditions in various countries. In this study, we investigated the control efficacy of a prototype of commercial mycopesticide using an B. bassiana (wettable powder) against cotton aphid on potted cucumber plant in greenhouse conditions.

The sweetpotato whitefly Bemisia tabaci (Gennadius) is one of the most important sap sucking pests causing economic losses in a variety of vegetables in Bangladesh and as well as around the world. In the present study, the mtCOI sequence of B. tabaci was analysed using samples collected from different host plants (Potato, Brinjal, Tomato, Sweet potato, Bean) from district Gazipur, Patuakhali, Rajshahi and Nilphamari of Bangladesh. Phylogenetic analysis of our samples and relative sequences of B. tabaci in NCBI database was shown three independent clusters. Samples in Bangladesh were most similar with those of Thailand, Vietnam, Pakistan, China and India but did not show any B and Q aggressive biotypes.

Recent research has suggested that the dietary protein:carbohydrate (P:C) balance is a critical determinant of fitness in insects. In this study, we examined the effects of dietary P:C balance on life-time reproductive success in the mealworm beetle, Tenebrio molitor. Both males and females lived the longest when fed on P:C 1:1 diet. Throughout their adult lives, females fed on P:C 1:1 diet laid significantly more eggs than those on nutritionally imbalanced diets (P:C 1:5 or 5:1). When given a choice, beetles regulated their intake of protein and carbohydrate to a ratio close to 1:1. Taken together, our results indicate the balanced intake of protein and carbohydrate maximizes life-time reproductive success in this species.

This studies was conducted to compare functional diversity of terrestrial arthropods in commercial apple orchards under conventional and organic practices. We collected terrestiral arthropods using pitfall traps in four conventional and seven organic apple orchards from April to October, 2012-2014. Sampled arthropods were identified at the species level and then classified three functional groups (detritivores, herbivores and beneficial arthropods included pollinators, parasitoids and predators). Biodiversity was analyzed with species richness, abundance and shannon index for each group and compared between conventional and organic orchards. In results, species richness of detritivores and benefical arthropods were higher in organic orchards than in conventional orchards (detritivores: t=-2.68, df=9, P=0.03; beneficial arthropods: t=-3.98, df=9, P=0.003). Organic orchards showed significant difference at abundance of beneficial arthropods (t=-3.33, df=9, P=0.008) and higher shannon index at detritivores (t=-2.36, df=9, P=0.04) than conventional orchards. However, all biodiversity indices of herbivores were not significantly different between conventional and organic orchards at 95% confidence level.

The jumping plant-lice of Laos are reviewed based on material collected during recent expeditions by the Seoul National University (2012−2015) and the Naturhistorisches Museum Basel (2003−2012). To date, only three psyllid species, viz. Diaphorina citri, Heteropsylla cubana and Pseudophacopteron tuberculatum, are recorded from Laos. In the material at hand, 22 species (of 6 families and 16 genera) are represented. This collection includes the three previously reported species and eight species reported for the first time from Laos. Eleven species are identified only to genus due to insufficient material.

The essential oils of three Coriandrum sativum seeds cultivated from India, Russia and America were evaluated for acaricidal toxicities against Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae. The oils of three C. sativum seeds were analyzed by gas chromatography. The oil of C. sativum seeds cultivated from India included substantially linalool (66.8%) and camphor (6.46%). In the fumigant bioassay, based on the LD50 values against D. farinae, D. pteronyssinus, and T. putrescentiae, the oil of C. sativum seeds cultivated from America (LD50, 2.62, 2.74, and 2.88 μg/cm3) was about 3.75, 3.32, and 4.17 times more active higher than benzyl benzoate (9.85, 9.10 and, 12.01 μg/cm3). The acaricidal activity of the oil of C. sativum seeds cultivated from India was 2.27, 2.03, and 2.64 times higher than that of the benzyl benzoate, followed by Russia oil. These results suggest that the oils of three C. sativum seeds might be used as suitable acaricides.

Differential gene regulation is crucial for development. Indianmeal moth is a global pest of stored and processed food products. Here, we determined the whole developmental expression patterns of 13 genes (shsp, hsp70, grp78, and hsp90), ecdysone receptor (EcR), ultraspiracle (USP), hemolin, β-1,3-glucan recognition protein (βgrp), prophenoloxidase (ProPO), superoxide dismutase (SOD) and thioredoxin peroxidase (Tpx), and two hexamerin storage protein genes (SP1 and SP2) related to growth, stress, metabolism, and host defense. We also studies effects of Bracon hebetor envenomation on these gene expressions to explore their role in host regulation. We found unexpected transcriptional peaks especially hsps which were high in egg and adult stages. This study provides comprehensive understanding about development and parasitoid regulation at molecular level.

Genus Phytocoris Fallén, 1814 is the largest genus among Heteroptera, around 700 species are described worldwide. In this presentation, the genus Phytocoris from Korean Peninsula is reviewed for the first time with eight species, includes one new record, P. minakatai and one new species, P. goryeonus sp. nov. For further comprehension of this genus, figures of dorsal habitus, genitalia and diagnosis of each Phytocoris species are provided.

A novel nanocomposite LDPE film with UV protective properties was developed for active packaging applications. Initially, undoped and Mn-doped TiO2 nanoparticles (NPs) were synthesized by the sol-gel method and the resulting particles were characterized. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed an agglomerated nature and spherical morphology. X-ray diffraction (XRD) studies indicated that all products were crystalline and in the form of rutile. The reflectance spectrum of undoped TiO2 NPs demonstrated a characteristic sharp edge at 410 nm. Subsequently, nanocomposite (NC) LDPE samples were prepared with the NPs by solvent precipitation followed by film casting. The optical and thermal properties of the NC samples were investigated. Incremental increases in Mn concentration from 0.25 mol % to 1.00 mol % were associated with progressive decreases in light transmission in the UV region. The melting and maximum decomposition temperatures of all NCs were 107 and 442-449 °C, respectively. The UV protective LDPE-based NC films exhibited superior photostability. Absorption in the FTIR spectra at 1716 and 1734 cm-1 changed after 4-wk exposure to UV for all film samples as a consequence of photodegradation. Finally, the photooxidation of perilla oil was assessed as an example of a UV protective packaging application. After 12 days, protection with 1.00 mol% Mn-doped TiO2-LDPE was associated with a gradual increase in PV, while protection with TiO2-LDPE was associated with a significant increase and protection with the control treatment was associated with a dramatic increase in PV. Hence, a 1.00 mol% Mn-doped TiO2-LDPE NC showed promise for UV shielding packaging applications.

Time-temperature indicators or integrators (TTIs) indicate food quality changes based on time-temperature history. Whilst many types of TTIs have been developed and commercialized, educated consumers often refuse to purchase food products with attached TTI labels showing even a slight color change. In this study, a novel on-off diffusion-based TTI coupled with polydiacetylene/silica nanocomposites has been proposed. The prototype TTI tag has a multilayer structure comprised of a self-adhesive base layer, a middle microporous sheet, and an upper opaque white layer coupled with a square reservoir of Tween 20 attached to an activation stripe. At the end of the diffusion path, polydiacetylene/silica nanocomposites were injected into a loading site as a fine blue stripe. After activation, Tween 20 diffused and reached the loading site, where it rapidly changed from blue-to-red via solvatochromism. This alternative and innovative TTI continuously showed a blue color until reaching the end point, at which stage a red color rapidly appeared, indicating product rejection. Thus, this novel TTI it is of great benefit to the brand owner. The developed prototype was characterized and evaluated for its ability to monitor microbial quality based on published, isothermal, microbial growth data of modified-atmosphere packaged minced beef, Mediterranean fish, and ground pork. The diffusion of Tween 20 in the TTI system was measured under various isothermal conditions and a kinetic model, based on the association between diffusion and time-temperature, was investigated. The Gaussian-estimated activation energy value was 51.082kJ mol-1. Tween 20 diffusion of 6.10, 5.15 and 6.15mm along the TTI systems were considered to be end points and the 95% confidence interval between the times taken for TTI to display OFF and for the foods to reach their deterioration thresholds were 23.30-23.70, 23.00-23.50 and 23.44-24.05h for total aerobic bacteria, Shewanella putrefaciens, and Pseudomonas spp. respectively. The TTI performance test for reproducibility and accuracy revealed a normal frequency distribution with 35004.90, 1200.254.82 and 549.811.09min at 0, 11 and 25C, respectively in accordance with the investigation of diffusion in the TTI.