• Title/Summary/Keyword: In-vitro techniques

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Generation of Full-Length Infectious cDNA Clones of Middle East Respiratory Syndrome Coronavirus

  • Lee, Jeong Yoon;Bae, Sojung;Myoung, Jinjong
    • Journal of Microbiology and Biotechnology
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    • v.29 no.6
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    • pp.999-1007
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    • 2019
  • Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 and related infection cases have been reported in over 20 countries. Roughly 10,000 human cases have so far been reported in total with fatality rates at up to 40%. The majority of cases have occurred in Saudi Arabia with mostly sporadic outbreaks outside the country except for the one in South Korea in 2015. The Korean MERS-CoV strain was isolated from the second Korean patient and its genome was fully sequenced and deposited. To develop virus-specific protective and therapeutic agents against the Korean isolate and to investigate molecular determinants of virus-host interactions, it is of paramount importance to generate its full-length cDNA. Here we report that two full-length cDNAs from a Korean patient-isolated MERS-CoV strain were generated by a combination of conventional cloning techniques and efficient Gibson assembly reactions. The full-length cDNAs were validated by restriction analysis and their sequence was verified by Sanger method. The resulting cDNA was efficiently transcribed in vitro and the T7 promoter-driven expression was robust. The resulting reverse genetic system will add to the published list of MERS-CoV cDNAs and facilitate the development of Korean isolate-specific antiviral measures.

Influence of Autophagy Induction after Hormone Treatment on Oocytes Maturation of Porcine

  • Kim, Sang Hwan;Yoon, Jong Taek
    • Journal of Embryo Transfer
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    • v.33 no.4
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    • pp.271-280
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    • 2018
  • Here, we evaluated the mode of programmed cell death during porcine oocyte maturation by comparing the two major pathways associated with programmed cell death, apoptosis (type I), and autophagy (type II). We investigated the expression and localization of major genes involved in autophagy and apoptosis at mRNA and protein levels. Furthermore, the effect of hormonal stimulation on autophagy and apoptosis was analyzed. We found that the activity of autophagy-associated genes was increased in the cumulus-oocyte complexes (COCs) following follicle-stimulating hormone (FSH) treatment, while the addition of luteinizing hormone (LH) reversed this effect. The expression of proteins associated with autophagy was the highest in FSH-treated COCs. On the other hand, caspase-3 protein level was maximum in COCs cultured with LH. The treatment with rapamycin resulted in the effect similar to that observed with FSH treatment and increased autophagy activity. Thus, hormonal stimulation of pig oocytes resulted in distinct patterns of maturation. The high-quality oocytes majorly relied on the type II pathway (autophagy), while the type I pathway (apoptosis) was more prominent among poor-quality oocytes. Further investigation of this distinction may allow the development of techniques to produce high-quality oocytes in porcine in vitro fertilization.

Comparison of complication rates after transvaginal ultrasound-guided oocyte pick-up procedures with respect to ovarian response

  • Gungor, Nur Dokuzeylul;Gurbuz, Tugba;Onal, Murat
    • Clinical and Experimental Reproductive Medicine
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    • v.49 no.2
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    • pp.142-148
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    • 2022
  • Objective: The aim of this study was to compare the complication rates of oocyte pick-up (OPU) procedures via transvaginal ultrasonography in patients with different levels of ovarian reserve. Methods: In total, 789 patients who underwent OPU procedures for in vitro fertilization (IVF) were included in the study. Results: Individuals with normal ovarian reserve had a 2.947-fold higher risk of complications in OPU procedures than individuals with low ovarian reserve, and individuals with high ovarian reserve had a 7.448-fold higher risk of complications than individuals with low ovarian reserve. In addition, a higher number of IVF trials was associated with an increased risk of complications. Conclusion: The results of this study show that OPU has a higher risk of complications, particularly severe pain, in patients with high ovarian reserve. It is thought that complications can be reduced by preferring mild stimulation in patients with high ovarian reserve. Collecting fewer oocytes is also associated with a lower risk of complications from OPU. Even if a patient's reserve is very good, fewer and higher-quality oocytes should be targeted with the use of the lowest possible dose of drugs.

Blood Compatibility of Artificial Blood-Contacting Surface Seeded with Cultured Bovine Endothelial Cells (소폐동맥 내피세포를 이용한 인조혈액접촉표면의 혈액 접합성)

  • 김원곤;곽영태;유세영
    • Journal of Chest Surgery
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    • v.26 no.2
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    • pp.80-85
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    • 1993
  • Synthetic and biosynthetic vascular grafts of small diameter have long been considered to be prone to thrombosis, ultimately leading to the complete graft occlusion. Endothelial cell seeding onto synthetic blood-contacting surfaces has been suggested to be an ideal means to solve this problem. This study described a culture method of bovine endothelial cells and evaluated blood-compatibility and seeding efficiency of cultured endothelial cells. Bovine pulmonary artery endothelial cells were harvested enzymatically and grown to confluence on polystyrene culture flask surfaces using established techniques. The identification of endothelial cells was made through the demonstration of expression of factor VIII R:Ag by immunofluorescent technique. To quantitate the effect of improvement in blood-compatibility of viable endothelial cells, endothelial monolayers were exposed to blood containing $\^$111/In-oxine labeled platelets. Viable endothelial monolayers retained less labeled platelets than control surfaces. The Indium-labeled endothelial cells were seeded onto three different blood-contacting surfaces of Dacron vascular graft immobilized in specially equipped wells and incubated for specific time intervals (t=15, 30, 60, 120 minutes). Longer incubation times showed improved cell adherence in collagen-coated and fibrin-coated Dacron vascular graft groups. However in untreated Dacron grafts, no direct relationship was observed between incubation time and endothelial cell seeding efficiency. This may be due to leakage of endothelial cells through porosity of Dacron grafts in this in-vitro experimental condition.

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Could Natural Products Confer Inhibition of SARS-CoV-2 Main Protease? In-silico Drug Discovery

  • Mohamed-Elamir F Hegazy
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2020.12a
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    • pp.14-14
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    • 2020
  • In December 2019, the COVID-19 epidemic was discovered in Wuhan, China, and since has disseminated around the world impacting human health for millions. Herein, in-silico drug discovery approaches were utilized to identify potential candidates as Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) inhibitors. We investigated several databases including natural and natural-like products (>100,000 molecules), DrugBank database (10,036 drugs), major metabolites isolated from daily used spices (32 molecules), and current clinical drug candidates for the treatment of COVID-19 (18 drugs). All tested compounds were prepared and screened using molecular docking techniques. Based on the calculated docking scores, the top ones from each project under investigation were selected and subjected to molecular dynamics (MD) simulations followed by molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations. Combined long MD simulations and MM-GBSA calculations revealed the potent compounds with prospective binding affinities against Mpro. Structural and energetic analyses over the simulated time demonstrated the high stabilities of the selected compounds. Our results showed that 4-bis([1,3]dioxolo)pyran-5-carboxamide derivatives (natural and natural-like products database), DB02388 and Cobicistat (DB09065) (DrugBank database), salvianolic acid A (spices secondary metabolites) and TMC-310911 (clinical-trial drugs database) exhibited high binding affinities with SARS-CoV-2 Mpro. In conclusion, these compounds are up-and-coming anti-COVID-19 drug candidates that warrant further detailed in vitro and in vivo experimental estimations.

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Studies on Pharmaceutical Quality of Oriental Medicinal Preparations (I) - Studies on Decoction of Nokyong-Sagunja-Tang - (한방방제의 제제학적 연구(1) - 추출방법에 따른 녹용사군자탕의 비교 -)

  • Choi, Hyuck-Jae;Kim, Eun-Jin;Kim, Sung-Soo;Kim, Nam-Jae
    • Korean Journal of Pharmacognosy
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    • v.37 no.3
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    • pp.143-150
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    • 2006
  • Decoction of oriental medicinal preparation is prepared in various manners, and changes of chemical constituents might be occurred depending on the processing techniques. The present study was undertaken to investigate the phγsio-chemical and pharmacological equivalence between two extraction methods of Nokyong-sagunja-Tang. Samples were Prepared as follows ; Sample-I was prepared by simultaneously extracting Sagunja-Tang and velvet antler in one vessel. Sample-lI was prepared by adding velvet antler exact to the water extract of Sagunja-Tang. Both sanples showed similar results of physiochemical parameters such as pH, yield, TLC and HPLC chromatogram, and contents of ginsenoside $Rb_1$ and glycyrrhizin. Also, there were little different between two samples in pharmacological effects such as DPPH free radical scavenging effect, and inhibitory effects on xanthine oxidase, hyaluronidase, trypsin, TBA-Rs formation and hemolysis in vitro. And both samples showed no significant difference in antifatigue activities in mice. These results suggest that there might be little difference between two extraction process when velvet antler added to Sagunia-Tang.

Embryo transfer in Korean Native Black Goat;Embryo recovery and transfer for the production of transgenic goat (한국재래흑염소 수정란의 이식;형질전환 흑염소 생산을 위한 수정란의 채취와 이식)

  • Shin, Sang-Tae
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2000.05a
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    • pp.64-75
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    • 2000
  • During the last three decades considerable advances has been made in goat embryo production and transfer technology. The Korean native black goat is the most useful domestic ruminant in this country for biological investigation and application because it has a lot of merits such as relatively short generation period (1 vs 2 year for a cow), easy of handling, well adaptation, high fertility, convenient and inexpensive. This article covers the methods of superovulation, estrus synchronization, embryo collection and transfer techniques, pregnancy diagnosis and subsequent pregnancy and kidding rates for the production of transgenic Korean native black goats. More than one hundred goat kids have been produced as a result of our transgenic goat project via microinjection of foreign gene into pronuclei, in vitro culture, transfer of various stages of fresh and frozen-thawed microinjected embryos into oviducts or uteri of recipient does. We have got two transgenic goats carrying a transgene targeting the expression of recombinant human granulocyte colony stimulating factor (hG-CSF) to the mammary gland so far. Since collection and transfer of embryos in this species is usually accomplished by laparotomy, exteriorization of the reproductive tract for surgical embryo collection leads to the formation of post-operative adhesions. Nonsurgical or laparoscopic technique to reduce adhesions from repeated surgeries has great advantages in improving embryo production and transfer especially from valuable donors. We will discuss this later.

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Effect of Mequitazine on the Muscarinic Receptors (Mequitazine의 Muscarine수용체에 대한 작용)

  • 이신웅;장태수
    • Biomolecules & Therapeutics
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    • v.3 no.3
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    • pp.192-198
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    • 1995
  • The affinity of mequitazine, a non-sedating antihistamine, for muscarinic receptors was evaluated in the guinea-pig ventricle and ileum by in vitro binding techniques and functional studies. In binding studies, [$^3$H]quinuclidinyl benzilate (QNB) identified a single class of muscarinic receptors with similar apparent $K_{D}$ value of about 100 pM in two tissues. Mequitazine inhibited [$^3$H]QNB binding to muscarinic receptors competitively. Analysis of the mequitazine inhibition curve of [$^3$H]QNB binding to ventricular microsome and ileal homogenate indicated the presence of a single homogeneous binding site with Ki value of 25 nM and 18 nM, respectively. In functional studies, mequitazine caused parallel rightward shifts of concentration-response curves for carbachol and histamine in the isolated guinea-pig ileum. The slope values obtained from Schild plot analysis for the antagonistic action of mequitazine on muscarinic and histamine $H_1$-receptors were not significantly different from unity. The p $A_2$values of mequitazine for muscarinic and histamine $H_1$-receptors were about 7.6 ( $K_{M}$= 25.1 nM) and 8.88 ( $K_{H}$= 1.32 nM), respectively. These results indicate that the muscarinic receptor blocking action of mequitazine is 15 times less potent than the $H_1$receptor blocking action, but high concentration of this drug may cause the peripheral muscarinic receptor blocking effect.t.t.t.

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Embryo transfer in Korean Native Black Goat: Embryo recovery and transfer for the production of transgenic goat (한국재래흑염소 수정란의 이식: 형질전환 흑염소 생산을 위한 수정란의 채취와 이식)

  • 신상태
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2000.06a
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    • pp.64-75
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    • 2000
  • During the last three decades considerable advances has been made in goat embryo production and transfer technology. The Korean native black goat is the most useful domestic ruminant in this country for biological investigation and application because it has a lot of merits such as relatively short generation period(1 vs 2 year for a cow), easy of handling, well adaptation, high fertility, convenient and inexpensive. This article covers the methods of superovulation, estrus synchronization, embryo collection and transfer techniques, pregnancy diagnosis and subsequent pregnancy and kidding rates for the production of transgenic Korean native black goats. More than one hundred goat kids have been produced as a result of our transgenic goat project via microinjection of foreign gene into pronuclei, in vitro culture, transfer of various stages of fresh and frozen-thawed microinjected embryos into oviducts or uteri of recipient does. We have got two transgenic goats carrying a transgene targeting the expression of recombinant human granulocyte colony stimulating factor(hG-CSF) to the mammary gland so far. Since collection and transfer of embryos in this species is usually accomplished by laparotomy, exteriorization of the reproductive tract for surgical embryo collection leads to the formation of post-operative adhesions. Nonsurgical or laparoscopic technique to reduce adhesions from repeated surgeries has great advantages in improving embryo production and transfer especially from valuable donors. We will discuss this later.

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Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces

  • Jepson, Tyler A.;Chung, Jean K.
    • BMB Reports
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    • v.54 no.3
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    • pp.157-163
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    • 2021
  • The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions-the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton's tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions.