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Optimization of Electrofusion Condition for the Production of Korean Cattle Somatic Cell Nuclear Transfer Embryos

  • Kim, Se-Woong;Kim, Dae-Hwan;Jung, Yeon-Gil;Roh, Sang-Ho
    • Reproductive and Developmental Biology
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    • v.35 no.1
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    • pp.17-22
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    • 2011
  • This study was designed to determine the effect of electric field strength, duration and fusion buffer in fusion parameters on the rate of membrane fusion between the somatic cell and cytoplast for Korean cattle (HanWoo) somatic cell nuclear transfer (SCNT) procedure. Following electrofusion, effect of 5 or $10\;{\mu}M$ $Ca^{2+}$-ionophore of activation treatment on subsequent development was also evaluated. Cell fusion rates were significantly increased from 23.1% at 20 V/mm to 59.7% at 26 V/mm and 52.9% at 27 V/mm (p<0.05). Due to higher cytoplasmic membrane rupture or cellular lysis, overall efficiency was decreased when the strength was increased to 30 V/mm (18.5%) and 40 V/mm (6.3%) and the fusion rate was also decreased when the strength was at 25 V/mm or below. The optimal duration of electric stimulation was significantly higher in $25\;{\mu}s$ than 20 and $30\;{\mu}s$ (18.5% versus 9.3% and 6.3%, respectively, p<0.05). Two nonelectrolyte fusion buffers, Zimmermann's (0.28 M sucrose) and 0.28 M mannitol solution for cell fusion, were used for donor cell and ooplast fusion and the fusion rate was significantly higher in Zimmermann's cell fusion buffer than in 0.28 M mannitol (91.1% versus 48.4%, respectively, p<0.05). The cleavage and blastocyst formation rates of SCNT bovine embryos activated by $5\;{\mu}M$ $Ca^{2+}$-ionophore was significantly higher than the rates of the embryos activated with $10\;{\mu}M$ of $Ca^{2+}$-ionophore (70.0% versus 42.9% and 22.5% versus 14.3%, respectively; p<0.05). This result is the reverse to that of parthenotes which shows significantly higher cleavage and blastocyst rates in $10\;{\mu}M$ $Ca^{2+}$-ionophore than $5\;{\mu}M$ counterpart (65.6% versus 40.3% and 19.5% versus 9.7%, respectively; p<0.05). In conclusion, SCNT couplet fusion by single pulse of 26 V/mm for $25\;{\mu}s$ in Zimmermann's fusion buffer followed by artificial activation with $5\;{\mu}M$ $Ca^{2+}$-ionophore are suggested as optimal fusion and activation methods in Korean cattle SCNT protocol.

Effect of Adrenergic Receptors on the Nerve Conduction in Rat Sciatic Nerves (아드레날린 수용체가 백서 좌골신경의 신경전도에 미치는 영향)

  • Lee, Chung;Chung, Sung-Lyang;Choi, Yoon;Leem, Joong-Woo;Lim, Hang-Soo;Yang, Hyun-Cheol;Han, Sung-Min;Kong, Hyun-Seok;Lim, Seung-Woon
    • The Korean Journal of Pain
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    • v.12 no.2
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    • pp.177-182
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    • 1999
  • Background: Clonidine, an ${\alpha}_2$ adrenergic agonist blocks nerve conduction. However, in our previous experiment we found that adrenaline neither blocks nerve conduction by itself nor augment nerve conduction blockade by lidocaine near clinical concentrations. Possible explanations are: 1) there may be antagonism between some of adrenergic receptors, 2) clonidine may block nerve conduction via non-adrenergic mechanism. The purpose of this study is to obtain dose-response curves of several different forms of adrenergic receptor agonist to see the relative potencies of each adrenergic receptors to block nerve conduction. Methods: Recordings of compound action potentials of A-fiber components (A-CAPs) were obtained from isolated sciatic nerves of adult male Sprague-Dawley rats. Nerve sheath of the sciatic nerve was removed and desheathed nerve bundle was mounted on a recording chamber. Single pulse stimuli (0.5 msec, supramaximal stimuli) were repeatedly applied (2Hz) to one end of the nerve and recordings of A-CAPs were made on the other end of the nerve. Dose-response curves of epinephrine, phenylephrine, isoproterenol, clonidine were obtained. Results: $ED_{50}$ of each adrenergic agonist was: $4.51\times10^{-2}$ M for epinephrine; phenylephrine, $7.74\times10^{-2}$ M; isoproterenol, $9.61\times10^{-2}$ M; clonidine, $1.57\times10^{-3}$ M. Conclusion: This study showed that only clonidine, ${\alpha}_2$ adrenergic agonist, showed some nerve blocking action while other adrenergic agonists showed similar poor degree of nerve blockade. This data suggest that non-effectiveness of epinephrine in blocking nerve conduction is not from the antagonism between adrenergic receptors.

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Performance Analysis of an Optical CDMA System for multi-user Environment (다중 사용자 환경에서의 광 CDMA 시스템 성능 분석)

  • 전상영;김영일;이주희
    • The Journal of Korean Institute of Communications and Information Sciences
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    • v.24 no.6B
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    • pp.1134-1141
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    • 1999
  • In this paper, we implement an optical code division multiple access(OCDMA) system and analyze the performance of the implemented system. In the implemented system, a transmitter encodes input data into optical pulses by using laser diode, and spreads the encoded pulses in an encoder which consists of 4 stage delay lines. The decoder which is the same structure as that of encoder delays and combines the spreaded pulses, and thus recovers the original data. At first, we discuss the auto- and cross-correlations of OCDMA signals under both environments of single user and multi-users, and then verify the simulation results with experimental results. We also evaluate the effect of a number of stages of delay line and code length on the system performance through computer simulations. As experimental results we can see that if the decoder have the same configuration as that of encoder, the peak auto-correlation characteristics can be achieved, and thus we can recover the original data from received data. As simulation results we can see that although bit error rate decreases as code length decreases or the number of stage of delay line increases, it is difficult to implement the system because the pulse width becomes narrow. From the results, we can apply CDMA technologies to optical communication networks.

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Comparison of In-Phase and Opposed-Phase FMPSPGR Images in Breath-hold T1-weighted MR IMaging of Liver (호흡정지 T1 강조 간 자기공명영상에서 동위상 역위상 FMPSPGR 영상의 비교)

  • 김명진;김만득;정재준;이종태;유형식
    • Investigative Magnetic Resonance Imaging
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    • v.1 no.1
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    • pp.142-147
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    • 1997
  • Purpose: To compare the effectiveness of the in-phase (IP) sequence and the opposed-phase (Op) sequence in the detection of focal hepatic lesions in the single breath-hold hepatic MR imaging with fast gradient T1-weighted pulse sequences. Materials and Methods: IP and OP T1-weighted breath-hold imaging was performed using fast gradient echo sequences in 45 patients referred for known focal hepatic lesions, in which 78 lesions were detected. Three blind readers independently reviewed the images for lesion detectability. The signal-to-noise ratio (SNR) of the liver, the lesion-to-liver contrast-to-noise ratio (CNR) and the liver-to-spleen CNR were also compared. A consensus was reached by three readers to determine which sequence is better in image quality. Results: On OP images, 61(78%), 61(78%), and 63(89%) lesions were correctly identified for reader 1, 2 and 3, respectively. On IP images, 66(85%), 65(83%), and 65(93%) lesions were detected for each reader, respectively. When two image sets were combined, 71(91 %), 69(88 %), and 76(97%) lesions respectively were detected for each reader. In cases of hepatocellular carcinoma, liver-to-Iesion CNR was greater on the OP images(p (0.05), but in other lesions significant difference was not demonstrated. Liver-to-spleen CNR was higher on OP images(p ( 0.1), but the SNR of the liver was higher on the IP images. Conclusion: Use of both IP and OP imaging can be helpful to avoid erroneous missing of some focal hepatic lesions.

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Helicobacter pylori Strain 51 (Korean Isolate): Ordered Overlapping BAC Library, Combined Physical and Genetic Map, and Comparative Analysis with H. pylori Strain 26695 and Strain J99

  • KANG HYUNG-LYUN;LEE WOO-KON;SONG JAE-YOUNG;CHOI SANG-HAENG;PARK SEONG-GYU;RYU BOK-DEOK;LEE EUN-JOO;KIM JI-SUN;PARK JEONG-UCK;BAIK SEUNG-CHUL;CHOI MYOUNG-BUM;YOUN HEE-SHANG;KO GYUNG-HYUCK
    • Journal of Microbiology and Biotechnology
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    • v.15 no.4
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    • pp.844-854
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    • 2005
  • We constructed a defined physical and genetic map of H. pylori strain 51, previously isolated from a Korean patient with a duodenal ulcer, by combining a restriction analysis by pulse-field gel electrophoresis with the construction of a BAC library. A Notl-digest of H. pylori strain 51 genome yielded seven fragments, from which the genomic size was estimated to be 1,698$\pm$24 kb. The BAC library was constructed from 50 to 200 kb fragments of HindIII-digested genomic DNA. From 700 BAC clones, an ordered overlapping maxi-set of 82 BAC clones was assembled that covered the entire genome. The positions of 15 genes were localized in the strain 51 genome with 4-22 kb of resolution and were compared with their orthologues in strain 26695 and strain J99. The arrangement of the 15 genes was identical in strain 51 and strain J99, except for flaA and hpaA. The plasticity zone of strain 51, like that of strain J99, was located in the single region, and was shorter than those of strain 26695 and strain J99. The strain 51 plasticity zone consisted of ORFs common only to strain 51 and J99 or to strain 51 and 26695, as well as strain 51-specific ORFs. Three genetic translocations and/or inversions were found between orthologue ORFs in strain 51 and strain J99. These results show that the chromosomal organization of strain 51 differs from Western strains such as strain 26695 and strain J99.

Production of Second Generational Cloning Embryos with Activated Oocytes in Rabbits (토끼에서 수핵란의 세포질 활성화에 의한 제 2세대 복제수정란의 생산)

  • 이효정;윤희준;최창용;공일근;박충생;최상용
    • Journal of Embryo Transfer
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    • v.12 no.2
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    • pp.133-139
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    • 1997
  • Large scale production of cloned embryos requires the technology of multiple generational nuclear transfer(NT) by using NT embryos itself as the subsequent donor nuclei. In this work we investigated comparatively the effects of enucleated oocytes treated with ionomycin and 6-DMAP on the electrofusion rate and in vitro developmental potential in the first and second NT embryos. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 15 hours after hCG injection. The enucleated oocytes were pre-activated by 5 min incubation in 5$\mu$M ionomycin and 2 hours incubation in 2 mM 6-DMAP at 19~20 hours post-hCG before microinjection. In the first and second generation NT, the unsynchronized 16-cell stage embryos were used as nuclear donor. The separated donor blastomeres were injected into the enucleated activated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of single pulse for 60 $\mu$sec at 1.25kV/cm in $Ca^2$+, $Mg^2$+ - free 0.28 M mannitol solution. In the non-preactivation group, the electrofusion and electrical stimulation was given 3 pulses for 60 $\mu$sec at 1.25 kV/cm in 100$\mu$M $Ca^2$+, $Mg^2$+ 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in TCM-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The results obtained were summarized as follows: 1. In the first generational NT embryos, the electrofusion rate of preactivated and non-activated oocytes(80.4 and 87.8%) was not significantly different, but in the second generational NT embryos, the electrofusion rate was significantly(P<0.05) higher in the non-activated oocytes(85.7%) than in the preactivated oocytes(70.1%). 2) In the first and second generational NT embryos, the developmental potential to biastocyst stage was significantly(P<0.05) higher in the preactivated oocytes(39.3 and35.7%) than in the non-preactivated oocytes(16.0 and 13.3%). No significant difference in the developmental potential was shown between the first and second generational NT embryos derived from the preactivated oocytes. In conclusion, it may be efficient to use the oocytes preactivated with ionomycin and 6-DMAP for the multiple production of cloned embryos by recycling nuclear transfer.

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Transfer of Porcine Embryos Injected with Sperm Carrying with Exogenous DNA

  • Cho, Seong-Keun;Cho, Hwang-Yun;Park, Mi-Ryung;Park, Jong-Sik;Yoo, Jae-Gyu;Kim, Jin-Hoi
    • Proceedings of the KSAR Conference
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    • 2001.03a
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    • pp.61-61
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    • 2001
  • The main goal of this study was to produce transgenic piglets by the method of injection of sperm-mediated exogenous DNA. Spermatozoa (1$\times$106 sperm of final concentration) obtained from caudal epididymis were mixed with pBC1-hEPO (20 ng/${mu}ell$) or pcDNA3 LAC Z (20 ng/${mu}ell$), and followed by electroporation (500 V, 25 ㎌). Matured oocytes having the first polar body and dense cytoplasm were selected and centrifuged at 12,000g for 6 min. After sperm injection, the oocytes were activated electrically (1.7 ㎸/cm, 30 $\mu$ sec, single pulse) in 0.3 M mannitol solution. Eggs injected sperm were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air for 192 h. This study were comprised 3 experiments. Experiment 1 compared the developmental efficiencies between the sperm-injected oocytes (Group 1) and further activated electrically (Group 2). Experiment 2 compared the expression of pcDNA3 LAC Z in the embryos produced by Group 1 and Group 2. Finally, experiment 3 carried out transfer of embryos (1-8 cell stage) transfected with pBC1 -hEPO into surrogate recipients synchronized by injection of combination of PG600 with hCG. The rates of cleavage and development into blastocyst stage in Group 2 were significantly higher than those of Group 1 (71.3% and 28.1% vs. 43.3% and 10.3%, respectively, p<0.05). Thirty (24.2%) out of 124 embryos analyzed in Group 2 were positive by X-gal. Similarly, in Group 1, 16.3% (8/49) were positive. After transfer of 789 embryos to 7 recipient gilts, three out of them examined by ultrasound became pregnant. One recipient is in day 50 pregnancy. On day 54 of gestation, two were carried out uterotomy in order to confirm the pregnancy One had 7 and another had 2 fetuses. We conclude that injection of sperm-mediated gene transfer will be used as a valuable tool for the production of transgenic piglets.

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A Research on The Pulse & Disease-patterns and Diagnostic Theory of Exogenous Febrile Disease in the "Sanghanjeonsaengjip(傷寒全生集)" ("상한전생집(傷寒全生集).변상한발열례(辨傷寒發熱例)" 등에 대한 연구(硏究))

  • Choi, Dong-Su;Sheen, Yeong-Il1
    • Journal of Korean Medical classics
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    • v.23 no.4
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    • pp.103-153
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    • 2010
  • "Sanghanjeonsaengjip(傷寒全生集)" is a classic medical work, written by the outstanding physician Dohwa(陶華) in the Ming Dynasty. The characteristic of "SangHanJeonSaengJip" is that this book succeeded to the spirit of pattern identification and treatment of Treatise on Cold Damage Diseases, newly changed a table of contents by symptoms, and together with this indicated the prescriptions in accordance with diswase-pattern at "YujeunghwalInseo(類證活人書)", "Hwajegookbang(和劑局方)" etc. Also because this kept the existing ephedra decoction, cinnamom twig decoction, minor decoction of bupleurum, decoction for reinforcing middle-energizer and replenishing qi etc.'s name on and unlikely indicated the medicine composition, it caused confusion, but at the later ages "Euhakipmun(醫學入門)" the so-called 'Doci(陶氏)' was added to the prescription name, so we are able to distinguish. Together with this, this book dose not indicate the dosage of medicine and indicates the first, the second, and the third classes[上中下] below medicine. As this dose not mean the three grades of quality"good, fair, and poor[上中下] of "Shennong's Classic of Materia Medica" but expresses the sovereign medicinal as the first class[上], minister medicinal as the second class[中] and assistant and courier medicinal as the third class[下], doctors can voluntarily decide the dosage of medicine in accordance with the degree of disease. At this thesis, I single out ten chapters in contents of 2nd volume named Hyeong(亨) corresponding to the details, among "Sanghanjeonsaengjip(傷寒全生集)". I discussed superficial fever types of exogenous febrile disease in chapter 1, aversion to cold types of exogenous febrile disease in chapter 2, syndrome caused un-sufficient sweating in chapter 3, organic fever types of exogenous febrile disease in chapter 4, aversion to wind types of exogenous febrile disease in chapter 5, Tidal fever types of exogenous febrile disease in chapter 6, Alternative attacts of chills and fever in chapter 7, Dysphoria with smothery sensation in chapter 8, Fidgetiness of exogenous febrile disease in chapter 9, and Headache of exogenous febrile disease in chapter 10, and together with this I discussed, in detail, which influence the prescriptions which are listed on each chapter have caused on future generations In accordance with this, I think that the above-mentioned symptoms and prescriptions are important when I research cold damage and warm disease study. So I orderly research revision, annotation, rendering and an investigation.

Growth and Structural Properties of Fe Thin Films Electrodeposited on n-Si(111) (n-Si(111) 기판 위에 전기증착에 의한 Fe 박막의 성장과 구조적 특성)

  • Kim Hyun-Deok;Park Kyeong-Won;Lee Jong-Duk
    • Journal of the Korea Institute of Information and Communication Engineering
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    • v.10 no.9
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    • pp.1663-1670
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    • 2006
  • Single crystal Fe thin films were grown directly onto n-Si(111) substrates by pulsed electrodeposition. Cyclic Voltammogram(CV) indicated that the $Fe^{2+}/n-Si(111)$ interface shows a good diode behavior by forming a Schottky barrier. From Mott-Schottky (MS) relation, it is found that the flat-band potential of n-Si(111) substrate and equilibrium redox potential of $Fet^{2+}$ ions are -0.526V and -0.316V, respectively. The nucleation and growth kinetics at the initial reaction stages of Fe/n-Si(111) substraste was studied by current transients. Current transients measurements have indicated that the deposition process starts via instantaneous nucleation and 3D diffusion limited growth. After the more deposition, the deposition flux of Fe ions was saturated with increase of deposition time. from the as-deposited sample obtained using the potential pulse of 1.4V and 300Hz, it is found that Fe nuclei grows to three dimensional(3D) islands with the average size of about 100nm in early deposition stages. As the deposition time increases, the sizes of Fe nuclei increases progressively and by a coalescence of the nuclei, a continuous Fe films grow on the Si surface. In this case, the Fe films show a highly oriented columnar structure and x-ray diffraction patterns reveal that the phase ${\alpha}-Fe$ grows on the n-Si(111) substrates.

Clinical Observation on Hyperthyroidism (갑상선기능항진증(甲狀腺機能亢進症)의 임상적(臨床的) 관찰(觀察))

  • Lee, Kyu-Bo;Kang, Bann;Song, Suk-Ho;Park, Hi-Myung;Whang, Kee-Suk
    • The Korean Journal of Nuclear Medicine
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    • v.3 no.2
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    • pp.39-47
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    • 1969
  • A clinical analysis was made on 161 cases of hyperthyroidism seen at the Radioisotope Laboratory of Kyungpook National University Hospital. This series consisted of 144 cases of diffuse goiter and 17 cases of nodular goiter. 1) Hyperthyroidism was most prevalent in the 4th decade and male to female ratio was 1:4.6. 2) Cardinal symptoms in the order of frequency were weakness, easy fatigability, palpitation, weight loss, nervousness, perspiration, heat intolerance, increased appetite, insomnia and dysmenorrhea. 3) Major physical findings in the order of frequency were goiter, fine tremor, tachycardia, wide pulse pressure, emaciation, warm moist skin, exophthalmos, systolic hypertension and atrial fibrillation. 4) The complications were ophthalmopathy (34.2%), thyrotoxic heart disease (5.6%), thyroid crisis (1 case), pretibial myxedema (1 case) and thyrotoxic myopathy (1 case). 5) Mean values of the six hour and twenty-four hour $^{131}I$ uptakes by the thyroid glands were 67.5% and 71.6%, respectively, in diffuse goiter and 64.5% and 65.0%, respectively, in nodular goiter. 6) Mean values of twenty-four hour $PB^{131}I$ conversion ratio were 76.3% in diffuse goiter and 70.2% in nodular goiter and those of the basal metabolic rate was +51% in the former and +41% in the latter. Mean serum cholesterol level was 152mg% in diffuse goiter and that in nodular goiter was 175mg%. 7) Among the 134 cases treated with $^{131}I$, 66 cases (49.3%) were successful1y controlled with single dose and in the majority of the cases the initial therapeutic dose required was $4.1{\sim}5.0mC$ in diffuse goiter and $5.1{\sim}6.0mC$ in nodular goiter. 8) With $^{131}I$ treatment the symptoms improved in the following order: heat intolerance, emaciation, nervousness, insomnia, easy fatigability, weakness, fine tremor, goiter, perspiration, exertional dyspnea and palpitation. And in a few cases improvement of even exophthalmos was seen. 9) Following $^{131}I$ treatment myxedema occurred in 4 cases (3%) and reccurrence in 9 cases (6.8%).

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