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Understanding the conditions leading to harmful algal blooms, especially those produced by toxic dinoflagellate species, is important for environmental and health safety. In addition to investigations into the environmental conditions necessary for the formation of toxic blooms, we postulate that investigating gene expression in proliferating cells is essential for understanding bloom dynamics. Expressed sequence tags were produced from cultured cells of the toxic dinoflagellate Alexandrium catenella sampled during the initiation phase of growth using Sanger's method and by 454 pyrosequencing. A significant proportion of identified genes (ca. 25%) represented enzymes and proteins that participate in a variety of cellular regulatory mechanisms that may characterize proliferating cells, e.g., control of the cell cycle and division, regulation of transcription, translation and posttranslational protein modifications, signaling, intracellular trafficking, and transport. All of the several genes selected for gene expression assays due to their involvement in metabolism and the cell cycle were overexpressed during exponential growth. These data will be useful for investigating the mechanisms underlying growth and toxin production in toxic Alexandrium species and for studying and monitoring the development of toxic blooms.

We have previously shown that mesenchymal stem cells (MSCs) reduced corneal inflammation and neovascularization in chemically-burned rat corneas in part through paracrine action. In order to identify the molecule(s) involved, we cocultured human MSCs in the following conditions, and examined the alterations in the cytokine secretion profile; (1) human peripheral blood mononuclear cells (hPBMCs), (2) chemically-damaged human corneal epithelial cells (hCECs), (3) hPBMCs/hCECs, (4) hMSCs, (5) hMSCs/hPBMCs, (6) hMSCs/hCECs, (7) hMSCs/hPBMCs/hCECs. We found that the levels of interleukin (IL)-6 and vascular endothelial growth factor (VEGF) by hMSCs markedly increased for hMSC/hCEC cocultures, and this elevation was further remarkable by the addition of hPBMCs. The hMSCs constitutively expressed transforming growth factor (TGF)-beta1, matrix metalloproteinase (MMP)-2, and thrombospondin-1. The secretion of MMP-9 by hCECs was significantly suppressed by hMSCs. Tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and IL-10 were not detectable in any cultures. The data presented herein provide the candidate molecules mediating the MSC-mediated modulation of inflammation and angiogenesis in cornea.

Purpose To compare corneal haze and transforming growth factor (TGF)-β expression in rat eyes with mechanical debridement of corneal epithelium or a chemically induced epithelial flap. Setting Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea. Methods Sixty corneas from 60 Sprague-Dawley rats were treated in 1 of 4 ways using a 2.0 mm trephine: corneal epithelium mechanically removed (Group 1), central cornea exposed to 20% ethanol for 30 seconds (Group 2), corneal epithelial flap made by applying 20% ethanol for 30 seconds and flap amputated (Group 3), corneal epithelial flap repositioned after ethanol-assisted detachment of epithelium as in Group 3 (Group 4). Corneal haze was graded. The TGF-β expression was measured in corneas and lacrimal glands using reverse transcription polymerase chain reaction and immunohistochemistry until day 30. Semiquantitative analysis was done in a density ratio of TGF-β/β-actin with image analyzer and densitometry. Results Corneal haze was more severe in Groups 1 and 3 than in Groups 2 and 4. By day 7, mRNA expression of TGF-β2 and type II receptor in corneas had increased more in Groups 1 and 3 than in Groups 2 and 4. In lacrimal glands, only TGF-β1 in Group 3 increased until day 7. In corneas, staining of both TGF-β1 and β2 increased, more prominently in Groups 1 and 3. Lacrimal gland staining was more intense in Groups 1 and 3. Conclusion Well-positioned corneal epithelial flaps may decrease corneal haze by reducing expression of TGF-β; inadvertent removal of an epithelial flap made by ethanol seems to exacerbate haze by increasing TGF-β.

Objective This study was performed to assess the prognostic value of serum hCG, progesterone, and inhibin A levels measured at 11 days post-ET for predicting pregnancy outcome in women participating in IVF. Methods Between May 2005 and April 2008, sera were obtained from 70 infertile women who underwent IVF-ET at 11 days post-ET and stored. HCG, progesterone, and inhibin A levels were measured by commercial enzyme-linked immunosorbent assay kits. The predictive accuracy of hCG, progesterone, and inhibin A levels for establishment of intrauterine pregnancy and ongoing pregnancy was calculated by receiver-operating characteristic curve analysis. Results For the prediction of intrauterine and ongoing pregnancy, serum hCG was better than progesterone and inhibin A. The predictive performance of progesterone and inhibin A was similar. The serum progesterone and inhibin A levels were significantly correlated each other (r=0.915, p=0.010). Conclusion A single measurement of the serum hCG level is sufficient to predict pregnancy outcome in IVF-ET patients.

Pterygium is an invasion of altered ocular tissue into the cornea. Bone marrow-derived stem cells have been reported to be involved in wound healing under chemotactic factors after pterygium removal and pain may act as a trigger signal. We evaluated the change of systemic and local chemotactic factors that could affect the mobilization and migration of BMSCs to the wound bed after conventional bare sclera pterygium excision. We also applied temporary amniotic membrane patch after pterygium removal, and compared the changes of cytokines with those of conventional bare sclera excision group. Substance-P (SP), vascular endothelial growth factor (VEGF), and stem cell factor (SCF) were measured in plasma and tear using ELISA and migrating CD34(+) cells by flow cytometry. The results showed that post-operative pain was much reduced (p0.05), and SP, VEGF and SCF kept consistently lower levels in plasma after temporary amniotic membrane application. Circulating CD34(+) cells increased slightly in the temporary amniotic membrane patch group compared with marked increase in the bare sclera group. Thus, the application of a temporary amniotic membrane after pterygium removal might be an effective therapeutic means by controlling pain and excessive infiltration of bone marrow-derived stem cells.

To investigate the effect of bevacizumab in a model of corneal inflammation.Epithelium from the cornea and limbus was completely removed using 100% alcohol in rats. Bevacizumab (1.25 mg/0.05 ml) or normal saline was subconjunctivally injected. One week later, corneas were examined and subjected to hematoxylin-eosin and immunofluorescent staining for VEGF. Expression of IL-2, IFN-gamma, IL-6, and TGF-beta 1 were analyzed by ELISA.Bevacizumab showed a borderline reduction in corneal neovascularization (11.0 +/- 4.8% and 18.0 +/- 10.0% in the bevacizumab and control groups, respectively; p = 0.054), while the extent of epithelialization was not affected (5.0 +/- 3.4% and 6.1 +/- 5.2% in the bevacizumab and control groups, respectively; p = 0.715). The infiltration of inflammatory cells was reduced (99.3 +/- 22.3 cells/x 400 in the bevacizumab-injected corneas and 182.3 +/- 58.0 cells/x 400 in the controls; p = 0.013). The levels of IL-2, IFN-gamma, and IL-6 were decreased in the rats with the bevacizumab injections (44 +/- 6 and 79 +/- 9 pg/ml for IL-2 in the bevacizumab-injected group and control, respectively, p = 0.025; 45 +/- 5 and 67 +/- 6 pg/ml for IFN-gamma in the bevacizumab-injected group and controls, respectively, p = 0.043; 45 +/- 6 and 75 +/- 8 pg/ml for IL-6 in the bevacizumab-injected group and controls, respectively, p = 0.030).Bevacizumab showed a reduction in inflammatory cell infiltration and cytokines in chemically burned rat corneas.

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.

Light-emitting diodes (LEDs) are considered to be effective in skin rejuvenation. We investigated the clinical efficacy of LED phototherapy for skin rejuvenation through the comparison with three different treatment parameters and a control, and also examined the LED-induced histological, ultrastructural, and biochemical changes. Seventy-six patients with facial wrinkles were treated with quasimonochromatic LED devices on the right half of their faces. All subjects were randomly divided into four groups treated with either 830nm alone, 633nm alone, a combination of 830 and 633nm, or a sham treatment light, twice a week for four weeks. Serial photography, profilometry, and objective measurements of the skin elasticity and melanin were performed during the treatment period with a three-month follow-up period. The subject's and investigator's assessments were double-blinded. Skin specimens were evaluated for the histologic and ultrastructural changes, alteration in the status of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and the changes in the mRNA levels of IL-1ss, TNF-alpha, ICAM-1, IL-6 and connexin 43 (Cx43), by utilizing specific stains, TEM, immunohistochemistry, and real-time RT-PCR, respectively. In the results, objectively measured data showed significant reductions of wrinkles (maximum: 36%) and increases of skin elasticity (maximum: 19%) compared to baseline on the treated face in the three treatment groups. Histologically, a marked increase in the amount of collagen and elastic fibers in all treatment groups was observed. Ultrastructural examination demonstrated highly activated fibroblasts, surrounded by abundant elastic and collagen fibers. Immunohistochemistry showed an increase of TIMP-1 and 2. RT-PCR results showed the mRNA levels of IL-1ss, TNF-alpha, ICAM-1, and Cx43 increased after LED phototherapy whereas that of IL-6 decreased. This therapy was well-tolerated by all patients with no adverse effects. We concluded that 830 and 633nm LED phototherapy is an effective approach for skin rejuvenation.

Objective To evaluate sperm nuclear DNA fragmentation and chromatin structure after 18 hours'incubation at room temperature. Methods Twenty-eight male partners who participating IVF treatment were prospectively included in this study. Ejaculated sperm count and motility were assessed. The sperm was then immediately processed by the conventional swim-up method. After utilization of some of the sample for routine clinical use, the remainder of each of the samples was divided into two aliquots. One aliquot was immediately assessed for sperm nuclear DNA fragmentation (TUNEL assay) and chromatin structure (toluidine blue [TB] staining). The other aliquot was incubated at room temperature for 18 hours and then assessed by two methods. Only dark-TB sperms were considered as having abnormal chromatin structure. Data before and after extended incubation were compared using a paired Student's t-test. Results Before and after extended culture, nuclear DNA fragmentation assessed by TUNEL was 4.9±4.7% and 7.0±6.4%, respectively (p=0.008). The proportion of abnormal chromatin structure (dark-TB sperm) was 8.2±5.6% and 10.3±6.5% (p

Clear cell tumor of borderline malignancy has been reported to account for 5 to 8% of all clear cell neoplasms, and

임신과 합병된 골반장기탈출은 10,000~15,000 분만 당 1건의 빈도로 드문 질환이다. 경산, 낮은 사회경제적 계층, 부적절한 산전 관리, 영양 실조, 이전 복부 수술력, 골반 구조의 인종적 차이, 골반부 지지 근육과 결합조직의 약화, 섬유조직 반응과 관련있으며, 자궁경부 난산으로 인한 자궁경부 열상, 자궁 파열, 모체 및 태아 사망 등의 합병증을 일으킬 수 있다. 산통 (분만 진통) 중 자궁경부 부종으로 인한 3기의 골반장기탈출에 대해 제왕절개 분만 후 황산마그네슘 국소 치료한 국내 첫 증례를 경험하였기에 간단한 문헌고찰과 함께 보고하는 바이다.

현재의 분류에는 포함되지 않는 드문 형태의 뮐러관 기형이 최근 보고되고 있다. 30세의 미산부 여성이 2년간의 불임을 주소로 내원하였다. 복강경상 자궁 저부 및 난관의 상태는 정상이었다. 자궁경상 중복 자궁경관 및 질중격을 확인하였고 자궁 하단부에서 교통이 있는 완전 중격 자궁을 확인하였다. 오른쪽 자궁경관을 통해 자궁경하 중격제거술을 성공적으로 시행하였다. 자궁협부에 교통이 있는 중복 자궁경관 및 질중격을 가진 완전 중격 자궁의 변형된 예로서 자궁경을 통한 수술을 통해 치료될 수 있었다.

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.