Plants synthesize antioxidant compounds as a defense mechanism against reactive oxygen species. Recently, plant-derived antioxidant compounds have attracted attention due to the increasing consumer awareness in the heath industry. However, traditional methods for measuring the antioxidant activity of these compounds are time-consuming and costly. Therefore, our study constructed a quantitative structure-activity relationship (QSAR) model that can predict antioxidant activity using graph convolutional networks (GCN) from plant structural data. The accuracy (Acc) of the model reached 0.6 and the loss reached 0.03. Although with lower accuracy than previously reported QSAR models, our model showed the possibility of predicting DPPH antioxidant activity in a wide range of plant compounds (phenolics, polyphenols, vitamins, etc.) based on their graph structure.

Fulvic acid, a humic substance with unique properties, has sparked interest due to its potential applications in the treatment of allergic diseases, Alzheimer's disease, and as a microplastic adsorbent. However, conventional extraction methods produce insufficient quantities for commercial use, which has prompted research to enhance fulvic acid production. In this study, we investigated the impact of Saccharomyces cerevisiae fermentation on the yield and spectral characteristics of fulvic acid extracted from white peat. Fulvic acid was extracted from both S. cerevisiae-treated and untreated white peats using acid precipitation. The yield of fulvic acid from the S. cerevisiae treated group reached its highest at 3.5 % after 72 hr of fermentation, which was significantly higher than the untreated group (1.1 %). Fourier Transform Infrared (FTIR) analysis revealed similarities in functional groups and characteristic absorption bands between the treated and untreated fulvic acid samples. These findings suggest that S. cerevisiae fermentation can increase the yield of fulvic acid extracted from white peat, providing a promising approach for enhancing the commercial viability of fulvic acid production.

Chicken embryonic stem (ES) cells have great potential and provide a powerful tool to investigate embryonic development and to manipulate genetic modification in a genome. However, very limited studies are available on the functional characterization and robust expansion of chicken ES cells compared to other species. Here, we have developed a method to generate chicken embryonic stem cell-like cells under pluripotent culture conditions. The chicken embryonic stem cell-like cells were cultivated long-term over several passages of culture without loss of pluripotency in vitro and had the specific expression of key stem cell markers. Furthermore, they showed severe changes in morphology and a significant reduction in pluripotent genes after siRNA-mediated NANOG knockdown. Collectively, these results demonstrate the efficient generation of chicken embryonic stem cell-like cells from EGK stage X blastoderm-derived singularized cells and will facilitate their potential use for various purposes, such as biobanking genetic materials and understanding stemness in the fields of animal biotechnology.

Recent progress has been made to establish intestinal organoids for an in vitro model as a potential alternative to an in vivo system in animals. We previously reported a reliable method for the isolation of intestinal crypts from the small intestine and robust three-dimensional (3D) expansion of intestinal organoids (basal-out) in adult bovines. The present study aimed to establish next-generation intestinal organoids for practical applications in disease modeling-based host-pathogen interactions and feed efficiency measurements. In this study, we developed a rapid and convenient method for the efficient generation of intestinal organoids through the modulation of the Wnt signaling pathway and continuous apical-out intestinal organoids. Remarkably, the intestinal epithelium only takes 3-4 days to undergo CHIR (1 µM) treatment as a Wnt activator, which is much shorter than that required for spontaneous differentiation (7 days). Subsequently, we successfully established an apical-out bovine intestinal organoid culture system through suspension culture without Matrigel matrix, indicating an apical-out membrane on the surface. Collectively, these results demonstrate the efficient generation and next-generation of bovine intestinal organoids and will facilitate their potential use for various purposes, such as disease modeling, in the field of animal biotechnology.

Three-dimensional (3D) organoids act as model systems because they mimic in vivo tissue morphology. Recent advancements in the field have demonstrated that organoids derived from various organs have assisted in understanding the underlying mechanisms of disease modeling and expanded our knowledge of organ development in vitro. Furthermore, these organoids have become a promising biomaterial in regenerative medicine for therapeutic purposes as well as in nutritional research for feed efficiency measurement in livestock. Intestinal organoids of livestock, including pigs, cattle, chickens, and horses, have been developed. These could be used to examine host-pathogen interactions, such as interaction between enteric viruses and epithelial cells, and are potential alternatives to in vivo systems. However, there are very limited studies regarding species-specific medium to cultivate and establish intestinal organoids of livestock. Species-specific medium is applied differently between species for the cultivation of intestinal organoids, and its modification is important for the maintenance of specific cell types or genes from the cellular diversity of the intestinal epithelium. In this study, we introduce the histological development of a 3D culture system and a species-specific medium for the cultivation of intestinal organoids in livestock. Finally, we discuss the importance and future perspectives of intestinal organoids in the fields of agriculture and biotechnology for various purposes.

In our previous studies, the cardiac xenotransplantation from an alpha-1,3-galactosyltransferase knockout pig (GT-MCP-MCP) to cynomolgus monkeys showed a mean survival of 38 days. The objective of this study is to genetically upgrade the GT-MCP-MCP pig, to further enhance membrane cofactor protein (MCP) expression and to express an endothelial specific thrombomodulin (TBM). MCP is a complement regulatory protein and TBM is a coagulation inhibitor. As the dicistronic cassette for wild-type-based MCP and TBM concurrent expressions does not show any increase of MCP, we optimized the MCP codon usage (mMCP) and substituted mMCP for MCP. When the mMCP-TBM cassette was transfected to HeLa cells, we were able to find an increased expression of MCP and endothelial cell-specific TBM expression. The cassette was then transfected into ear-skin fibroblasts isolated from one-month-old #23-4 of a GT-MCP-MCP pig, and the cell populations expressing MCP were obtained by MACS cell sorting. We performed a single cell culture of the selected cells, and obtained clones over expressing 90% MCP. The cells of a clone were used as a donor for nuclear transfer and generated GT-MCP/-MCP/mMCP/TBM pig. The transgenic pig was confirmed to be carrying the cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Thus, we believe that the GT-MCP/-MCP/mMCP/TBM transgenic pig would be potential for the prolongation of xenograft survival in the recipients.

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig (GalT-MCP/-MCP). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain GalT-MCP/-MCP/CD39/CD73 pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from GalT-MCP/-MCP pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate GalT-MCP/-MCP/CD39/CD73 pig expressing CD39 and CD73 at endothelial cells.

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus (GalT-MCP/-MCP) as well as expressing MCP at GalT gene loci with CD73 expression (GalT-MCP/+/CD73). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.

It is very difficult to get the information about semen quality analysis in transgenic pigs because of limited numbers and research facilities. Therefore, in the present study, we analyzed the semen quality of transgenic boars generated for xenotransplantation research. Briefly, the semen samples were collected from 5 homozygous α1,3-Galactosyltransferase knock-out (GalT-/-) transgenic boars and immediately transported to the laboratory. These semen samples were decupled with DPBS and conducted to analyze semen parameters by a computer-assisted semen analysis (CASA) system. The boar semen were examined all 12 parameters such as total motility (TM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and hyperactivated (HYP), etc. In results, among the 5 GalT-/- boars, three boars (#134, 144, and 170) showed normal range of semen parameters, but #199 and 171 boars showed abnormal ranges of semen parameters according to standard ranges of semen parameters. Unfortunately, #171 boar showed azoospermia symptom with rare sperm counts in the original semen. Conclusively, assessment of semen parameters by CASA system is useful to pre-screening of reproductively healthy boar prior to natural mating and artificial insemination for multiplication and breeding.

Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at 38.5 ˚C under 5% CO2 in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at 118.3 ± 2.5 days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods.

하구둑이 형성되지 않은 자연 하천을 유지하고 있는 섬진강 하구의 어류상을 조사하고 주요 어종의 개체군 생태를 분석하였다. 조사 결과 섬진강 하구에서 출현한 어류는 13목 26과 68종이었으며 전체 우점종은 큰줄납자루 (37.4%), 아우점종은 황어 (10.5%)였고 피라미 (8.4%), 주둥치(5.1%)순으로 개체수가 많았다. 회유성 어종은 뱀장어, 황어, 은어, 연어, 숭어, 점농어, 민물검정망둑 7종이 출현하였고 한국고유종은 임실납자루와 칼납자루 등 20종이었다. 상류 담수역에서는 8과 35종이 채집되었고 우점종은 큰줄납자루 (38.2%), 아우점종은 참중고기 (11.5%)였으며, 하류 담수역은 13과 37종이 출현하였고 우점종은 큰줄납자루 (48.5%), 아우점종은 피라미(13.0%)였다. 하구의 기수역은 19과 34종이 확인되었고 우점종은 황어 (42.6%), 아우점종은 주둥치 (13.2%)로 나타났다. 누치 등 주요 종에 대한 개체군 건강도 지수는 하구둑이 설치된 낙동강 집단에 비해 높게 나타나 하구둑 조성이 없는 섬진강 어류 집단이 안정적인 성장을 하고 있다고 본다.

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-humanprimate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

국내에서 토양 내 중금속 농도를 정량하기 위하여 다양한 분석장비 및 전처리관련 공정시험법이 존재한다. 대표적으로 환경부 토양환경공정시험법에서는 토양의 전분해(왕수추출)후 AAS 또는 ICP로 분석을 제시하고 있으며, 해양환경공정시험법에서는 퇴적물의 완전분해 후 분석이 제시되어 있다. 또한, 상기 화학적 전처리공정을 거치지 않는 XRD분석이나 입자를 레이저빔으로 승화시켜 측정하는 레이저 유도 붕괴 분광법(LIBS; Laser-Induced Breakdown Spectroscopy)도 상용화 되고 있다. 이러한 각종 전처리 및 분석 방법은 특히 전처리 정도에 따라 동일시료라 하더라도 경우에 따라 측정된 농도 값에 차이가 발생되나, 신기술이나 검증 등에 고려하지 않는 경우가 많기 때문에 혼란이 야기되는 실정이다. 따라서, 토양시료를 대상으로 전처리에 따른 분석결과차이를 증명하고, 이를 상용화 중인 LIBS분석방법과 비교하고자 한다. 중금속 오염토양 10가지를 대상으로 하였으며, 각 토양을 왕수분해(전분해)하고 충분히 반응시키고 원심분리하여 상등액을 분취하여 분석시료로 하였다. 이 왕수추출 후 잔류입자 내 잔류 중금속 농도를 정량하기 위하여 불산 등을 투입(완전분해)하여 잔류입자를 완전히 용해시켜 이를 분석시료로 하였다. 전분해와 완전분해는 각각 토양오염공정시험법과 해양환경공정시험법(-퇴적물)을 바탕으로 실험하였다. 중금속 농도 정량에는 ICP-OES(Perkin Elmer DV2100)을 사용하였으며, 총 5종(납, 아연, 구리, 크롬, 카드뮴)을 분석하였다. 추가로 동일 토양시료를 레이저유도 붕괴 분광법(LIBS)으로도 분석하여 이를 비교해 보았다. 토양시료 10개에서 납, 아연, 구리, 크롬의 분석농도는 완전분해(불산 등)대비 왕수분해(전분해)가 65~100%, 62~94%, 71~91%, 30~52%로 나타났으며, 이때 카드뮴은 검출되지 않았다. 토양 왕수추출 후에도 잔류입자(주로 실리카)에 중금속이 일부 존재하고 있는 것으로 나타났으며, 이는 전처리 후 입자유무에 따라 토양 에서 잔류중금속 농도 분석 값이 다를 수 있음을 나타낸다. 완전분해 시 납, 구리, 크롬, 아연 농도는 LIBS측정치 대비 평균회수율이 각각 87.8, 96.8%, 106.2%, 90.1%로 나타나 상대적으로 LIBS방법과 유사한 측정결과가 나타났다. 이는 완전분해 측정법과 LIBS측정법이 입자까지 모두 용해 또는 승화시켜 측정하는 방법이라는 점에서 유사하기 때문으로 판단된다.

Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced pluripotent stem cells (piPSCs) is very low. The present study was performed to investigate the effect of cell cycle inhibitors on the cell cycle synchronization of piPSCs. piPSCs were generated using combination of six human transcriptional factors under stem cell culture condition. To examine the efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel coated plate with stem cell media and they were treated with staurosporine (STA, 20 nM), daidzein (DAI, 100 μM), roscovitine (ROSC, 10 μM), or olomoucine (OLO, 200 μM) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were in G1 (37.5±0.2%), S (34.0±0.6%) and G2/M (28.5±0.4%). The proportion of cells at G1 in DAI group was significantly higher than that in control, while STA, ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of viability and apoptosis were affected by STA and ROSC treatment, but there were no significantly differences between control and DAI groups. Real-Time qPCR and FACs results revealed that DAI treatment did not affect the expression of pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and apoptosis, but Oct4 expression was significantly decreased. Our results suggest that DAI could be used for synchronizing piPSCs at G1 stage and has any deleterious effect on survival and pluripotency sustaining of piPSCs.

This study was to verity that the uptake inhibition and accumulation of nitrogen in different potassium levels. Lettuce was used as model plant in this study and grown in pot of 10cm's in diameter and depth with mixture media of vermiculite and perlite under supply of different culture solution for three weeks. Nitrogen absorption at root was inhibited by increased potassium concentration in nutrient solution, and nitrate accumulation of plant was depended on absorption of nitrogen because nitrate content of 0 K level was 4-5 times higher than that of 2 K level, Concentration of ascorbic acid was decreased by increasing the nitrogen absorption, since ascorbic acid (AsA) content of 2K level was higher than those of OK level in both of old leaf and flesh leaf