The mesenchymal stem cells (MSCs) that reside in dental tissues hold a great potential for future applications in regenerative dentistry. In this study, we used human dental pulp cells, isolated from the molars (DPCs), in order to establish the organoid culture. DPCs were established after growing pulp cells in an MSC expansion media (MSC-EM). DPCs were subjected to organoid growth media (OGM) in comparison with human dental pulp stem cells (DPSCs). Inside the extracellular matrix in the OGM, the DPCs and DPSCs readily formed vessel-like structures, which were not observed in the MSC-EM. Immunocytochemistry analysis and flow cytometry analysis showed the elevated expression of CD31 in the DPCs and DPSCs cultured in the OGM. These results suggest endothelial cell-prone differentiation of the DPCs and DPSCs in organoid culture condition.

Recently, we published a microinjection method for generating transgenic cattle using the DNA transposon system and their analysis by next-generation sequencing (Yum et al. Sci Rep. 2016 Jun 21;6:27185). In that study, we generated transgenic cattle using two different types of DNA transposon system, sleeping beauty (SB) and piggybac (PB), carrying Yellow fluorescent protein with SB (SB-YFP, female) and green fluorescent protein with PB (PB-GFP, male) under the control of the ubiquitous CAG promoter, respectively. The female and male founder cattle have been grown up to date (the female age: 40 months old, the male age: 33 months old) without any health issues. In genomic instability and blood analysis, there was no significant differences between wild type and founder cattle. In the present study, we confirmed germ-line transmission of the transposon-mediated transgene integrations and ubiquitous and persistent expression of transgene in second generation of offspring (F1). The F1 was born without any assistance and expressed GFP in the eyes without UV light. The ubiquitous expression of GFP was detected in skin fibroblast from the ear tissue and confirmed by genomic DNA PCR, which suggest that the transgene from the PB-GFP was successfully transmitted. Unfortunately, no transgene from SB-YFP were identified. To confirm the transgene integration site, the genomic DNA from blood was extracted and performed next-generation sequencing (NGS). The GFP gene was integrated in chromosome 4 (two copies), and 6. As results, a total of two copies of paternal transgene transmitted into the F1. All the integrated position was not related with coding region and there was no significant difference in genomic variants between transgenic and non-transgenic cattle. To our knowledge, this is the first report of germ-line transmission through non-viral transgenic founder cattle. Those transgenic cattle will be valuable resource to many fields of biomedical research and agricultural science.

Lycorine, a natural alkaloid extracted from the Amaryllidaceae plant family, was reported to various physiological and pharmacological effects including anti-cancer activity. Nevertheless, there is no report of the anticancer effect of lycorine in oral cancer cells. The effects of lycorine on cell proliferation and apoptosis were examined through trypan blue exclusion assay, 4’-6-diamidino-2-phenylindole (DAPI) stain, Live/Dead assay, Western blot analysis and RT-PCR. Lycorine suppressed cell viability and induced apoptosis in MC3 and HSC-3 cell lines. Lycorine decreased survivin protein but did not affect its mRNA. It regulated survivin through accelerating protein degradation in a time-dependent manner although neither proteasome nor lysosome was not associated with lycorine-mediated protein degradation. Collectively, our results suggest that lycorine may be a potential therapeutic anti-cancer drug candidate for the treatment of human oral cancer.

목 적 : 자기공명영상(MRI)의 발달은 소아신경계의 병리학적 과정을 이해하고 진단하는데 중요한 역할을 하고 있으며 그 검사 건수는 나날이 증가하고 있다. 하지만 이같은 장점에도 불구하고 소아의 Brain MR 검사는 환자에게 주의를 주거나 Sedation을 하여 검사를 진행하지만 움직임, 소음 등으로 인하여 실패하는 경우가 발생한다. 고해상력의 영상 획득이 가능한 3.0T의 경우 1.5T에 비해 SAR가 약 4배 증가하게 되며, 최근 미국 소아과학회에서는 어린이가 어른보다 전자파에 더 큰 영향을 받기 때문에 더욱 주의하여 관리해야 한다고 주장하고 있다. 이때, 기존의 TSE가 아닌 다양한 Refocusing FA를 사용하는 Hypere-TSE은 동등한 SNR을 유지하면서 SAR를 감소시키기 때문에 그 해결책이 될 수 있다. 또한, MR 검사 시의 소음은 Gradient의 빠른 Switching을 통해 발생하는데, 이러한 기존의 소음 감소를 위한 Gradient mode인 Whisper mode는 Scan time이 증가하는 등의 단점이 있었으나 최근 E11 Upgrade를 통해 도입된 Acoustic noise reduction는 이를 개선하여 동등한 Scan time과 향상 된 소음 감소 효과를 가져왔다. 이에 본 연구에서는 소아 Brain MRI 검사 시 Hyperecho와 Acoustic Noise Reduction을 이용하여 Image quality를 동등하게 유지하면서 소아를 더욱 안전하고 안정된 환경에서 검사할 수 있는 방법을 제안하고자 한다. 대상 및 방법 : 장비는 SIEMENS사의 3.0T SKYRA(Germany)와 32ch Head coil을 사용하였고, Spherical Phantom D165(Siemens, Germany) 및 만 15세 이하의 소아 환자 20명(남자 12, 여자 8, 평균연령 6세)을 대상으로 하였다. Hyper-TSE와 Acoustic noise reduction을 사용했을 때와 사용하지 않았을 때의 T1 Dark fluid(TR: 2000, TE: 9, TI: 900, Matrix: 384*307, FOV: 200*200, NEX: 1) T2WI(TR: 6000 TE: 103, Matrix: 448*269, FOV: 200*200, NEX: 2), T2 FLAIR(TR: 9000, TE: 84, TI: 2500, Matrix: 320*192, FOV: 200*200, NEX: 1)에서 비에스코리아사의 디지털 소음 측정기를 통하여 Isocenter에서 150㎝ 거리에서의 소음을 측정하였고, SAR와 SNR은 Siemens NUMARIS/4 - syngo MR E11로 Gray matter, White matter, Eye ball을 관심 영역 0.02㎠ 측정하여 IBM SPSS Statistics v22.0을 통해 정량적 분석을 하였다. 결 과 : 정량분석 결과 Hyper-TSE와 Acoustic noise reduction을 사용했을 때는 사용하지 않았을 때에 비해 SAR가 T1 Dark fulid에서 -26%, T2에서 -33%, T2 FLAIR에서 –33%로 감소되었으며, 소음은 T1 Dark fulid에서 -14%, T2에서 -16.6%, T2 FLAIR에서 –16.1% 감소하였고, SNR은 T1 Dark fulid에서 GM: 17%, WM: 15%, Eye ball: 12%, T2에서 GM: 10%, WM: 14%, Eye ball: 14%, T2 FLAIR에서 GM: 3%, WM: 9%, Eye ball: 7%로 증가한 것으로 나타났다. CNR은 T1 Dark fulid에서 14%, T2에서 3%, T2 FLAIR에서 –19%로 증감된 것으로 나타났다. 비모수 대응표본 Wilcoxon T검정을 실시한 결과 대부분의 값에서 유의한 차이(p<.05)가 있는 것으로 나타났다. 결 론 : Hyperecho를 사용하게 되면 SNR을 유지하면서 SAR를 상당히 감소시킬 수 있다는 것과 Acoustic noise reduction을 사용할 경우 동일하거나 약간의 검사 시간 증가와 함께 많이 조용하게 된다는 이론적 사실을 바탕으로 안전하고 안정된 환경을 만들기 위해 노력하였다. 이번 연구를 통해 Hyperecho 및 Acoustic noise reduction을 사용할 경우 기존의 Sequence와 비교하여 SAR와 소음은 감소시키면서 SNR과 CNR 값은 동등하거나 증가된 영상을 얻을 수 있고 이를 통하여 소아 환자에게 더욱 안전하고 안정된 환경을 제공하고 효과적이며 진단 가치 높은 영상을 얻을 수 있으리라 사료된다.

CRISPR/Cas9-induced knock-out/-in can be occurred at specific locus in the genome by non-homologous end joining (NHEJ) or homology directed repair (HDR). Here, we demonstrate the targeted insertion into the specific loci of embryo fertilized by semen from transgenic cattle via CRISPR/Cas9 system. Recently, we published on the efficient generation of transgenic cattle using the DNA transposon system (Yum et al. Sci Rep. 2016 Jun 21;6:27185). In the study, eight transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon system) via microinjection. In the analysis of their genome stability using next-generation sequencing, there was no significant difference in the number of genetic variants between transgenic and non-transgenic cattle. All the transgenic cattle have grown up to date (the oldest age: 33 months old, the youngest age: 15 months old) without any health issue. One of transgenic male cattle expressing GFP reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for in vitro fertilization (IVF). On seven days after IVF, expression of GFP was observed at blastocyst stage and was seen in 80% of the embryos. Another application is to edit the GFP locus of the transgenic cattle because long-term and ubiquitous expression of transgene didn’t affect their health. In one cell stage embryos produced using GFP frozen-thawed semen, microinjection of sgRNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of sgRNA target site into fertilized eggs resulted in expression of RFP. This indicated that the GFP locus of transgenic cattle shows potential candidates for stable insertion of the functional transgene. Knock-out/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we demonstrated P-112 that transgenic cattle via transposon system are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the potential target site for future gene engineering via genome-editing technology. Finally, all those animals could be a valuable agricultural and veterinary science resource for studying the effects of gene manipulation on biomedical research and medicine. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550-20160004).

The CRISPR/Cas9 system is proved to be a powerful tool for knock-out and knock-in in various species. By introducing genetic materials of two components (Cas9 and small guide (sg) RNA) into cells or pronuclear of the fertilized embryo, gene editing occurs. Some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals since Cas9 is indispensable in the CRISPR/Cas9 system. Accordingly, the production of Cas9 expressing cattle may provide the broadly used gene editing platform in cattle. For this study, Cas9 and RFP genes were cloned into PiggyBac (PB) transposon system. PB-Cas9-RFP and transposase were microinjected into 1436 in vitro fertilized embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounting for 14.1% of total formed blastocysts were selected and transferred into 5 recipient cow. After gestation periods, four transgenic cattle were delivered without any veterinary assistance. From a transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNAs in DNA form for various genes such as PRNP, RB1 and BLG were transfected as 2ug of sgRNA per 5x105 cells using Nucleo factor system (Neon®, invitrogen, program#16). As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1assay. Those data demonstrated that for the first time, transgenic cattle with Cas9 expression were born, grown up to date and will be avaluable resource for genome-editing in cattle. This work was supported by BK21PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU550-20160004).

Antibiotic Detection Kit (Combination I), a lateral flow immunoassay (LFIA) developed for detecting antibiotic residues in milk, was utilized for the analysis of antibiotic residues in the muscle tissue of olive flounder. After 5-h treatment of samples by placing them in water dosed with sulfadimethoxine (SDM; 200 g/ton water), the residue depletion of SDM was investigated in 25 cultured olive flounders (Paralichthys olivaceus). Muscles from fish were sampled before treatment and on the 1st, 2nd, 3rd, 4th and 5th days after treatment. The concentration of SDM in the muscle was then determined by LFIA. The absorbance ratio of the sample to the control blank (Bs/Bo) was employed as an index to determine the residue in olive flounder muscle. To investigate the recovery rate, standard solutions were added to muscle samples to obtain final concentrations of 25 and 50 ng/mL in the muscle. The recovery rates of all spiked samples were >96.6% of the spiked value. SDM was detectable in the muscle of fish treated with the drug until the 1st day of the withdrawal period. The present study shows that the LFIA can be easily adopted to detect SDM residues in the tissue of farmed fish.

This study investigated the antibacterial effects of Galla rhois extract (GRE) against Campylobacter jejuni and Campylobacter coli. The minimum inhibitory concentrations (MICs) of GRE against C. jejuni and C. coli were 0.28 and 0.55 mg/mL, respectively, and the corresponding minimum bactericidal concentrations (MBCs) were 4.4 and 5.5 mg/mL. C. jejuni treated with the MIC, MBC or 2×MBC of GRE showed significant inhibition of growth compared with that of the control group during the incubation period, and no viable bacteria were detected at 24 h after incubation. C. coli treated with MIC, MBC or 2×MBC of GRE also showed inhibition of growth compared with that of the control group during the incubation period, and in the C. coli cultures treated with MBC and 2×MBC of GRE, no viable bacteria were detected at 24 h after incubation. In conclusion, GRE is a candidate antibacterial agent against C. jejuni and C. coli, and may have applications for the control of Campylobacter infection in poultry.

본 연구는 오배자 에탄올 추출물 (GRE), 염소산나트륨 (SC) 그리고 오배자 에탄올 추출물과 염소산나트륨 합제(GS)의 B. abortus에 대한 항균효과를 확인하기 위해 수행 되었다. GRE, SC 그리고 GS를 B. abortus에 처리하여 배양한 후, B. abortus의 생존수를 확인하였으며, 마우스 탐식세포 내 감염된 B. abortus의 증식 억제효과를 경시별 (2, 24, 48시간)로 조사하였다. GRE, SC 그리고 GS는 각각 400 μg/mL 이하, 15 mM 그리고 0.6GS (GS 1, GRE 1,000 μg/mL + SC 30 mM) 이하의 농도에서 세포독성을 나타나지 않았다. 모든 처리구에서 B. abortus의 생존율은 용량- 의존적으로 현저하게 감소하는 결과를 나타내었다. 또한, GRE (400 μg/mL), SC (15 mM) 그리고 0.5GS (GRE 500 μg/mL + SC 15 mM)를 처리한 세포에서 배양 48시간 후에, B. abortus의 증식이 통계적으로 유의성 있게 감소하였으며 (GRE, p < 0.01; SC and 0.5GS, p < 0.001), 특히, GS를 처리한 경우, B. abortus의 세포내 증식이 GRE와 SC의 상승작용에 의한 강력한 항균효과를 나타내었다. 결론적으로, GS는 B. abortus에 대한 항균물질로서 유용할 뿐만 아니라, 식육과 우유 위생 분야에 적용할 수 있을 것으로 생각된다.

This study investigated the antibacterial effects of GR ethanol extracts (GRE), sodium chlorate (SC) and a combination of GRE and SC (GS) on Brucella abortus (B. abortus). The antibacterial activities of GRE, SC and GS towards B. abortus were evaluated by incubating B. abortus with GRE, SC and GS. Following treatment with GRE, SC and GS, B. abortus survival and intracellular proliferation in macrophages were monitored. In the cellular cytotoxicity assay, GRE, SC and GS are not cytotoxic at concentrations less than 400 μg/ml, 15 mM and 0.6GS (1 of GS, GRE 1,000 μg/ml + SC 30 mM), respectively. The viability of B. abortus was markedly decreased in a dosedependent manner in all treatment groups. In addition, B. abortus intracellular proliferation within macrophages was significantly reduced in cells treated with GRE (400 μg/mL), SC (15 mM) and 0.5GS (GRE 500 μg/mL + SC 15 mM) after 48 hr-incubation (GRE, p < 0.01; SC and 0.5GS, p < 0.001). Especially, in the treatment of GS, the synergistic effect of GRE and SC treatment on B. abortus in macrophage was observed. In conclusion, GS is useful as an antibacterial candidate against B. abortus, and can be applied in the field of meat and milk hygiene.

This study investigated the therapeutic effects of Galla rhois (GR) ethanol extract (GRE), sodium chlorate (SC), and a combination of GRE and SC on mice infected with Brucella abortus (B. abortus). Mice were infected intraperitoneally with B. abortus and then treated with GRE, SC, and a combination GRE and SC in drinking water for 14 days. Then, serum antibodies were used in a tube agglutination test (TAT), after which the weight and CFUs from each spleen were measured. In addition, histopathological changes in each liver were examined at 14 days post-infection. At 14 days post-infection, negative reactions of serum antibodies in PC (positive control), SCT (SC 1.6 g/L drinking water), GRT (GRE 200 mg/L drinking water), and GST (GRE 200 mg + SC 1.6 g/L drinking water) were 0, 40, 60, and 80%, respectively. The average spleen weight was not significantly different between the groups. At 14 days post-infection, bacterial numbers in all treated groups were significantly lower compared to to that of the PC (GRT and SCT, P<0.05; GST, P<0.001). In terms of histopathological changes in the livers, there were numerous multifocal microgranulomas in the PC, whereas this number successively decreased in the SCT, GRT, and GST groups. Conclusively, a combination of GRE and SC exhibits therapeutic effects on mice infected with B. abortus. These results suggest the potential efficacy of a mixture of GRE and SC in the treatment of brucellosis.

The biodiesel production characteristics in a pulsed-corona plasma reactor has been investigated through parametric tests. Transesterification of rapeseed oil together with camelina oil was done with the change of such variables as voltage of power, molar ratio, KOH catalyst and temperature. The energetic electrons emitted from pulsed-corona plasma has contributed to the enhancement of yield on rapeseed oil in short time (15 min). The higher yield on camelina oil was observed in 5 min. The optimal parameters were shown as the voltage of 23 kV, the molar ratio of 5/1, the content of KOH catalyst of 0.6 wt% and the temperature of 28℃ under the rotating rate of spark gap of 900 rpm.

To understand the evolution and speciation of closely related species, a multiple approach encompassing morphological, behavioral, and genetic analyses is necessary. In Korea, three species of Loxoblemmus crickets occur widely. L. campestris and L. equestris are morphologically indistinguishable, whereas males of L. doenitzi are different from the other two species in head morphology. The genetic analyses using the partial mitochondrial COI sequences showed that L. doenitzi diverged off earlier than L. campestris and L. equestris. The analyses of laboratory recordings revealed that distributions of calling song characters generally overlapped among three cricket species. However, the number of pulses in a chirp was two in L. doenitzi and four in L. campestris, but it was greater than or equal to six in L. equestris. Provided that females make mate choice based on this calling song character, the differentiation in this character may lead to premating reproductive isolation and may have evolved during the speciation proccess in these closely related species.

To provide a basis for studying the pharmacological actions of tetracaine HCl, we analyzed the membrane activities of this local anesthetic. The n-(9-anthroyloxy) stearic and palmitic acid (n-AS) probes (n = 2, 6, 9, 12 and 16) have been used previously to examine fluorescence polarization gradients. These probes can report the environment at a graded series of depths from the surface to the center of the membrane bilayer structure. In a dosedependent manner, tetracaine HCl decreased the anisotropies of 6-AS, 9-AS, 12-AS and 16-AP in the hydrocarbon interior of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV), and liposomes derived from total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. However, this compound increased the anisotropy of 2-AS at the membrane interface. The magnitude of the membrane rotational mobility reflects the carbon atom numbers of the phospholipids comprising SPMV, SPMVTL and SPMVPL and was in the order of the 16, 12, 9, 6, and 2 positions of the aliphatic chains. The sensitivity of the effects of tetracaine HCl on the rotational mobility of the hydrocarbon interior or surface region was dependent on the carbon atom numbers in the descending order 16-AP, 12-AS, 9-AS, 6-AS and 2-AS and on whether neuronal or model membranes were involved in the descending order SPMV, SPMVPL and SPMVTL.

At present, most of web-based applications are on the basis of HTML-based form documents. These HTML-based form documents only play a role as user interfaces, but they don"t involve the procedures or rules of processing business works that aim to documents. However, form documents imply methods for treating documents, and these procedural knowledge embedded can utilize to actively perform business work processes. Procedural knowledge is defined in ERML(Executable Rule Markup Language), which is a XML-based rule markup language that has a format of Prolog rules. The ERML is not only executable in an inference engine, but also can be geared with WfMS (Workflow Management System) and can be used in a heterogeneous rule-based application. Thus, this paper proposes a ERML-based scheme for processing active documents as device for automating a workflow that involve a process of treating documents. Our active documents themselves involve declarative knowledge to support the process and control of business rules embedded in documents and the automation of processing documents. Also, we propose a framework for processing the defined active documents. In order to demonstrate the usefulness of the proposed framework, a prototype called ActiveWebForm is designed and implemented for a case of processing a request for requisition of IP(Internet Procurement) system. As a result, our approach is expected to contribute to intelligence and automation of Internet applications as processing documents by embedding declarative knowledge.