The purpose of our study was to determine the effect of Maitland orthopedic manual therapy, Silver Spike Point, dietary fiber and gymnastic exercise on the improvement of constipation. Forty patients with constipation participated in the study (Maitland Orthopedic Manual Therapy Group(n=10), Silver Spike Point Therapy Group(n=10), Dietary Fiber Group(n=10) and Gymnastics Exercise Group(n=10)). The assessment scale and weekly bowel frequency were measured before and after the experiment. Assessment scale was significantly increased in Silver Spike Point Therapy, Maitland orthopedic therapy, gymnastic exercise compare to dietary fiber. Weekly bowel frequency was significantly increased in gymnastic exercise compared to dietary fiber. The results of this study suggest that Silver Spike Point Therapy, Maitland orthopedic therapy, gymnastic exercise improve the symptom in patients with constipation.

Human interferon alpha 2b (hIFNα-2b) is an important immune regulator widely used in clinic, for the treatment of chronic hepatitis, hairy cell leukemia, chronic myelogenous leukemia and multiple myeloma, etc. The clinically used hIFNα-2b is generally produced by E. Coli, which lacks the post-translational O-glycosylation of naturally synthesized protein, and has a short serum half-life. In this study, we report the successful generation of transgenic chickens that produce hIFNα-2b in the egg white using a feline immunodeficiency virus (FIV)-based lentiviral vector. In preliminary in vitro study, the hIFNα-2b gene under the control of CMV promoter expressed as much as 2,650 ng/㎖ in CEF-LNC-hIFNα-2bW cell. A FIV vector packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected underneath the blastoderm of freshly laid chicken eggs (stage X) to produce a hIFNα -2b transgenic chicken. Out of 187 injected eggs, 55 chicks were hatched after 21 days of incubation, and 27 of the G0 hatched chicks expressed the vector-encoded hIFNα-2b gene. The expression of recombinant hIFNα-2b in transgenic chickens constitutes an important step towards low-cost and full biological activity production of this protein drug in bioreactor. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.

In the present study, using a MoMLV-based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of hEPO. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). One rooster was mated to wild-type hens to produce 748 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were seven G1 transgenic offspring, corresponding to a 0.9% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from three G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood samples taken from G1 transgenic chickens resulted in 4,150 ～ 10,823 IU/㎖ (34.6 ～ 90.2 ㎍/㎖) of hEPO in the blood. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. Red blood cell numbers were more than three-fold higher in the transgenic chickens compared to the non-transgenic chickens. Successful germline transmission of the transgene was also confirmed in G2 transgenic chicks produced from crossing G1 transgenic roosters with non-transgenic hens. We confirmed that 13 transgenic chicks of 45 G2 progeny, corresponding to a 28.9% germline transmission rate. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.

CRISPRs(clustered regularly interspaced short palindromic repeats) / CRISPR - associated(CAS) system has been used genome editing technology. Genome stage modification using CRISPR/CAS9 system can be used to wide research for the gene functional study and therapeutics. However, improving of CRISPR/CAS9 system in efficiency is essential for application in various fields. Here, we treated various chemicals during the procine early embryo development to increase the mutation of target site by NHEJ(non-homologous end joining). Firstly, we confirmed the chemical toxicity after parthenogenetic activation and then check embryo puality using by counting of total cell number and TUNEL Assay in blastocyst satge. To check any improvement on mutation rate by NHEJ pathway. AZT(3′-Azido-3′-deoxythymidine, antiretroviral drug – 0.1 μM) was treated after injection of cas9 and sgRNA target to OCT4 exon 5 during the zygote stage, followed by PCR sequencing. As a result, AZT treated group shows a significantly increased in knock-out efficiency as a consequence of NHEJ. Nocodazole(anti-neoplastic agent – 200ng/ml), RO-3306 (specific inhibitor of CDK1 - 10 μM) and NU-7026(PKC signalling inhibitor - 50 μM) was treated after injection of cas9 and sgRNA with eGFP vector during the zygote stage(hpa8~hpa20) and checked a efficiency of knock-in by PCR sequencing. Interestingly, nocodazole treatment groups increased of insertion of eGFP sequence in blastocyst stage compared with non-treat group(control : 8.33%, nocodazole treatment : 16.67%). However, RO-3306 and NU-7026 made a no impact. In summary, CRISPR/CAS9 system with treatment of chemicals during porcine embryogenesis can be improving of site-specific mutation and enhancement of CRISPR genome editing.

Unlike somatic cells mitosis, germ cell meiosis consists of two consecutive rounds of divisions that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric divisions. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it was localized to meiotic spindles, and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration; and symmetric division or large polar body emission. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.

In the present study, we examined potential roles of glucose and pyruvate in nuclear and cytoplasmic maturation of porcine oocytes. In the presence and absence of 10% porcine follicular fluid (PFF), either 5.6 mM glucose or 2mM pyruvate effect on meiotic maturation and followed development ability. However, DOs doesn't take full advantage of the glucose in medium, only pyruvate can increase MII rate and follow early embryo development ability significance. COCs were matured with 200 uM pentose phosphate pathway (PPP) inhibitor (dehydroepiandrosterone, DHEA) or 2 μM glycolysis inhibitor (iodoacetate, IA), significantly lower levels of GHS in the DHEA an IA treated oocytes and the levels of ROS were higher significantly in the DHEA treated oocytes, treatment with DHEA significantly reduced the intra-oocyte ATP and NADPH level. Blastocysts from DHEA or IA treated group also presented higher apoptosis levels, meanwhile, the percentage of proliferating cells was dramatically lower than the non-treated group. In conclusion, our results suggest that 10% PFF promoted oocytes make full use of energy, glucose metabolism during in vitro maturation inseparable from the cumulus cells, PPP and glycolysis promoted porcine oocytes cytoplasmic maturation by supplying energy and reducing oxidative stress.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) have a important role in influence of pre-messenger RNA (pre-mRNA) processing and mRNA metabolism and transportation in cells. Recently, hnRNP A2/B1 can recognize m6A modifications on pre-mRNA or pre-miRNA and affect alternative splicing and miRNA processing in HeLa Cells. However, roles of hnRNP A2/B1 in various cells and tissues, especially in elary embryo development, are unclear. Here, we investigated the temporal and spatial expression patterns of hnRNPA2B13 during mammalian early embryo development. In mouse, hnRNPA2B1 was localized at the nucleus after 1-cell stage, however, hnRNPA2B1 was expressed after 2-cell stage in pig. Then, knockdown of hnRNP A2/B1 induced by RNA interference (RNAi) was used to analyze the effect of hnRNP A2/B1 in preimplantation develop in pigs. Knockdown of hnRNP A2/B1 delayed embryo development. Interestingly, ICM marker OCT4 and Sox2 was significantly decreased in blastocyst stage. mRNA expression show that transcription factors which is Pou5f1, Sox2, Nanog, Cdx2 and AP2γwas decreased the transcription levels without the changing of junction protein, ZO-1, occludin, and CXADR. Outgrowth results indicated that knock-down of hnRNPA2B1 embryos cannot format the colony. Knock-down of Methyltransferase like 3(METTL3) embryos mislocalized the hnRNPA2/B1 at the nucleus. In summary, the expression patterns of hnRNPA2/B1 differ between mouse and porcine embryos, and these differences may reflect species-specific functions during preimplantation embryo development. Our results suggested that hnRNPA2/B1 is necessary for newly synthesis of mRNA related with transcription factor, and early embryo development by the RNA epigenetic modification.

CDK2 inhibition plays a central role in DNA damage–induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or MII arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage as demonstrated by the formation of distinct γH2AX foci in nuclei of day 3- and day 5-embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activating of the ATM-P53-P21 pathway. However, the mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for double strand break (DSB) repair reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in day 7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

Mitotic spindle formation is regulated by centrosomes, composed of a centriole pair surrounded by pericentriolar materials(PCM) proteins. However, mammalian oocytes rely on acentriolar MTOCs for the function of meiotic spindle. The composition of acentriolar MTOCs and the molecular precesses that regulate the localization and accumulation in mammalian oocyte are not well understood. In this study, we analyzed the mechanisms of spindle microtubule nucleation and stability from MTOCs in mouse oocyte, and indentified Centrosomal protein192(CEP192) as a key regulator for acentriolar MTOC formation. CEP192 specifically colocalized with pericentrin (PCNT) during the oocyte maturaion. CEP192 proteins are localized throughout cytoplasm and around nucleus at GV stage, and then after BD stage, CEP192 proteins were further fragmented into smaller MTOCs around chromosomes. At metaphase, CEP192 proteins were concentrated in spindle pole. Knockdown of CEP192 using siRNAs resulted in metaphase I arrest. The arrested oocytes were characterized by reduced microtubule intensity and misalignment chromosome. Also at BD and ProMI stage, the oocytes reduced microtubule density and PCNT intensity. To confirm the mechanism of CEP192 regulation, we confirmed that PLK1 and AuroraA kinase were involved in CEP192 activation. The investigations for detailed molecular mechanisms of CEP192 and RanGTP for microtubule nucleation in oocytes are underway using various techniques including siRNA, mRNA, and positive or negative dominant injection and inhibitors.

Maturation-promoting factor (MPF) is well-known as cell cycle regulator during oocyte maturation and fertilization. MPF activity maintains high levels and arrest the cell cycle progression until fertilization. After fertilization, Anaphase-promoting complex/cyclosome (APC/C) mediated degradation of cyclin B causes decrease of MPF activity. One of the cytostatic factor (CSF), Emi2 inhibits APC/C activity by binding to APC/C-cdc20, therefore blocks the proteolysis of cyclin B. Degradation of Emi2 requires phosphorylation by Polo-like kinase 1 (Plk1). Thus recognition and phosphorylation of Emi2 by Plk1 are essential step for meiotic cell cycle resumption. In our previous research, we found that two phosphorylated threonine regions at amino acid position 152 and 176 in Emi2 are respectively contributed for recognition by polo-box domain of Plk1. Peptidomimetics 103-8 can block the interaction between Plk1-PBD and Emi2, and therefore meiotic maturation and meiosis resumption via parthenogenetic activation were impaired. However, major drawback of 103-8 was the limitation of penetration through the cell membrane. We synthesized the new peptidomimetics and checked bioavailability in mammalian oocyte by injection and media treatment. Medium treatment with peptidomimetics C-4, meiotic maturation has significantly decreased and meiotic resumption via parthenogenetic activation has perfectly impaired. For the next experiment, we are preparing X-ray crystallography to identify the binding modes between Plk1-PBD and peptidomimetics C-4.

Pine wilt disease (PWD) is one of the most important forest tree diseases, especially in the East Asian countries of Japan, China, and Korea. The Japanese pine sawyer, Monochamus alternatus Hope, is the PWN vector for Japanese red pine and Japanese black pine while Monochamus saltuarius Gebler is the vector for Korean white pine. Various control methods, such as aerial pesticide applications, biological control using parasitoids and fumigant such as methyl bromide are used. But the PWD still has spread. Therefore, we were selected effective aerial insecticides. Susceptibility of M. saltuarius and M. alternatus adults were investigated using 9 insecticides which are available in the market in Korea. And then, we tested them to the Apis mellifera to vertify the environmental impact.