The Experts below are selected from a list of 176358 Experts worldwide ranked by ideXlab platform
Shinsuke Kikuchi - One of the best experts on this subject based on the ideXlab platform.
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surgical marking pen dye inhibits saphenous vein cell proliferation and migration in saphenous vein graft tissue
Journal of Vascular Surgery, 2016Co-Authors: Shinsuke Kikuchi, Richard D Kenagy, Thomas N Wight, Nobuyoshi Azuma, Michael Sobel, Alexander W ClowesAbstract:Objective Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Because little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting. Methods Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This cleavage plane was within the circular muscle layer of the media. Cell migration from Explants was measured daily as either (1) percentage of migration-positive Explants, which exclusively measures migration, or (2) number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from Explants as well as for proliferation, migration, and death of cultured outer layer cells. Dye was extracted from Explants with ethanol and quantified by spectrophotometry. Results There was significantly less cell migration from visibly blue compared with unstained outer layer Explants by both methods. There was no significant difference in migration from inner layer Explants adjacent to blue-stained or unstained sections of vein because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in nonblue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer Explants had 65.9 ± 8.0 ng dye/explant compared with 2.1 ± 1.3 for nonblue outer layer Explants. Dye applied in vitro to either outer or inner layer Explants dose dependently inhibited migration (IC 50 ∼10 ng/explant). The IC 50 s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 μg/mL, whereas the EC 50 for death was between 1 and 10 μg/mL. Conclusions Crystal violet inhibits venous cell migration and proliferation, indicating that alternative methods should be considered for marking vein grafts.
Alexander W Clowes - One of the best experts on this subject based on the ideXlab platform.
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surgical marking pen dye inhibits saphenous vein cell proliferation and migration in saphenous vein graft tissue
Journal of Vascular Surgery, 2016Co-Authors: Shinsuke Kikuchi, Richard D Kenagy, Thomas N Wight, Nobuyoshi Azuma, Michael Sobel, Alexander W ClowesAbstract:Objective Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Because little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting. Methods Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This cleavage plane was within the circular muscle layer of the media. Cell migration from Explants was measured daily as either (1) percentage of migration-positive Explants, which exclusively measures migration, or (2) number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from Explants as well as for proliferation, migration, and death of cultured outer layer cells. Dye was extracted from Explants with ethanol and quantified by spectrophotometry. Results There was significantly less cell migration from visibly blue compared with unstained outer layer Explants by both methods. There was no significant difference in migration from inner layer Explants adjacent to blue-stained or unstained sections of vein because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in nonblue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer Explants had 65.9 ± 8.0 ng dye/explant compared with 2.1 ± 1.3 for nonblue outer layer Explants. Dye applied in vitro to either outer or inner layer Explants dose dependently inhibited migration (IC 50 ∼10 ng/explant). The IC 50 s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 μg/mL, whereas the EC 50 for death was between 1 and 10 μg/mL. Conclusions Crystal violet inhibits venous cell migration and proliferation, indicating that alternative methods should be considered for marking vein grafts.
Randy D Dinkins - One of the best experts on this subject based on the ideXlab platform.
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agrobacterium tumefaciens mediated transformation of soybean glycine max l merrill using immature zygotic cotyledon Explants
Plant Cell Reports, 2000Co-Authors: M Srinivasa S Reddy, G B Collins, Randy D DinkinsAbstract:-mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon Explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the Explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon Explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon Explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus.
Abhinav Reddy Kethiri - One of the best experts on this subject based on the ideXlab platform.
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optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation an in vitro study
PLOS ONE, 2017Co-Authors: Virender S. Sangwan, Sayan Basu, Abhinav Reddy Kethiri, Sachin Shukla, Vivek SinghAbstract:Purpose Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven clinical techniques for treating limbal stem cell deficiency (LSCD). However, the ideal size and number of the limbal Explants required for transplantation has not been clearly elucidated. This in vitro study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion. Methods Limbal Explants obtained from both live and cadaveric biopsies were cultured on the denuded amniotic membrane. Explant size and the explant cell outgrowth (expansion) were measured using ImageJ software with respect to days. Cultures were characterized by assessing the rate of proliferation of cells with 5-bromo-2’-deoxyuridine (BrdU) assay along with the expression of different stem cell markers (ABCG2, p63α), corneal epithelial (CK3+12) and adherens junction molecules (E-Cadherin) by immunofluorescence. Results Explants from live biopsies had 80% growth potential in vitro whereas 40% of the cadaveric tissue failed to grow. Minimum explant sizes of 0.3 mm2 for live and ≥0.5 mm2 for cadaveric tissue had a mean expansion areas of 182.39±17.06 mm2 and 217.59±16.91 mm2 respectively suggesting adequate growth potential of the Explants. Mean total percentage of proliferative cells was 31.80±3.81 in live and 33.49±4.25 in cadaveric tissue expansion. The expression was noted to be similar in cells cultured from cadaveric compared to cells cultured from live limbal tissue with respect to ABCG2, p63α, CK(3+12) and E-cadherin. Conclusion Our findings show that a minimal amount of 0.3 mm2 live tissue would be sufficient for ample limbal cell expansion in vitro. Cadaveric Explants <0.5 mm2 had poor growth potential. However, larger Explants (≥ 0.5 mm2) had growth rate and proliferative potential similar to the live tissue. These findings could prove to be critical for clinical success especially while attempting cadaveric limbal transplantation. This study provides a novel clinical strategy for enhancing efficacy of the limbal transplantation surgery and opens the probability of even using the cadaveric tissue by considering the size of explant.
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Optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation: An in vitro study.
PLOS ONE, 2017Co-Authors: Abhinav Reddy Kethiri, Virender S. Sangwan, Sayan Basu, Sachin Shukla, Vivek SinghAbstract:Purpose Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven clinical techniques for treating limbal stem cell deficiency (LSCD). However, the ideal size and number of the limbal Explants required for transplantation has not been clearly elucidated. This in vitro study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion. Methods Limbal Explants obtained from both live and cadaveric biopsies were cultured on the denuded amniotic membrane. Explant size and the explant cell outgrowth (expansion) were measured using ImageJ software with respect to days. Cultures were characterized by assessing the rate of proliferation of cells with 5-bromo-2’-deoxyuridine (BrdU) assay along with the expression of different stem cell markers (ABCG2, p63α), corneal epithelial (CK3+12) and adherens junction molecules (E-Cadherin) by immunofluorescence. Results Explants from live biopsies had 80% growth potential in vitro whereas 40% of the cadaveric tissue failed to grow. Minimum explant sizes of 0.3 mm2 for live and ≥0.5 mm2 for cadaveric tissue had a mean expansion areas of 182.39±17.06 mm2 and 217.59±16.91 mm2 respectively suggesting adequate growth potential of the Explants. Mean total percentage of proliferative cells was 31.80±3.81 in live and 33.49±4.25 in cadaveric tissue expansion. The expression was noted to be similar in cells cultured from cadaveric compared to cells cultured from live limbal tissue with respect to ABCG2, p63α, CK(3+12) and E-cadherin. Conclusion Our findings show that a minimal amount of 0.3 mm2 live tissue would be sufficient for ample limbal cell expansion in vitro. Cadaveric Explants
Vivek Singh - One of the best experts on this subject based on the ideXlab platform.
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optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation an in vitro study
PLOS ONE, 2017Co-Authors: Virender S. Sangwan, Sayan Basu, Abhinav Reddy Kethiri, Sachin Shukla, Vivek SinghAbstract:Purpose Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven clinical techniques for treating limbal stem cell deficiency (LSCD). However, the ideal size and number of the limbal Explants required for transplantation has not been clearly elucidated. This in vitro study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion. Methods Limbal Explants obtained from both live and cadaveric biopsies were cultured on the denuded amniotic membrane. Explant size and the explant cell outgrowth (expansion) were measured using ImageJ software with respect to days. Cultures were characterized by assessing the rate of proliferation of cells with 5-bromo-2’-deoxyuridine (BrdU) assay along with the expression of different stem cell markers (ABCG2, p63α), corneal epithelial (CK3+12) and adherens junction molecules (E-Cadherin) by immunofluorescence. Results Explants from live biopsies had 80% growth potential in vitro whereas 40% of the cadaveric tissue failed to grow. Minimum explant sizes of 0.3 mm2 for live and ≥0.5 mm2 for cadaveric tissue had a mean expansion areas of 182.39±17.06 mm2 and 217.59±16.91 mm2 respectively suggesting adequate growth potential of the Explants. Mean total percentage of proliferative cells was 31.80±3.81 in live and 33.49±4.25 in cadaveric tissue expansion. The expression was noted to be similar in cells cultured from cadaveric compared to cells cultured from live limbal tissue with respect to ABCG2, p63α, CK(3+12) and E-cadherin. Conclusion Our findings show that a minimal amount of 0.3 mm2 live tissue would be sufficient for ample limbal cell expansion in vitro. Cadaveric Explants <0.5 mm2 had poor growth potential. However, larger Explants (≥ 0.5 mm2) had growth rate and proliferative potential similar to the live tissue. These findings could prove to be critical for clinical success especially while attempting cadaveric limbal transplantation. This study provides a novel clinical strategy for enhancing efficacy of the limbal transplantation surgery and opens the probability of even using the cadaveric tissue by considering the size of explant.
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Optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation: An in vitro study.
PLOS ONE, 2017Co-Authors: Abhinav Reddy Kethiri, Virender S. Sangwan, Sayan Basu, Sachin Shukla, Vivek SinghAbstract:Purpose Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven clinical techniques for treating limbal stem cell deficiency (LSCD). However, the ideal size and number of the limbal Explants required for transplantation has not been clearly elucidated. This in vitro study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion. Methods Limbal Explants obtained from both live and cadaveric biopsies were cultured on the denuded amniotic membrane. Explant size and the explant cell outgrowth (expansion) were measured using ImageJ software with respect to days. Cultures were characterized by assessing the rate of proliferation of cells with 5-bromo-2’-deoxyuridine (BrdU) assay along with the expression of different stem cell markers (ABCG2, p63α), corneal epithelial (CK3+12) and adherens junction molecules (E-Cadherin) by immunofluorescence. Results Explants from live biopsies had 80% growth potential in vitro whereas 40% of the cadaveric tissue failed to grow. Minimum explant sizes of 0.3 mm2 for live and ≥0.5 mm2 for cadaveric tissue had a mean expansion areas of 182.39±17.06 mm2 and 217.59±16.91 mm2 respectively suggesting adequate growth potential of the Explants. Mean total percentage of proliferative cells was 31.80±3.81 in live and 33.49±4.25 in cadaveric tissue expansion. The expression was noted to be similar in cells cultured from cadaveric compared to cells cultured from live limbal tissue with respect to ABCG2, p63α, CK(3+12) and E-cadherin. Conclusion Our findings show that a minimal amount of 0.3 mm2 live tissue would be sufficient for ample limbal cell expansion in vitro. Cadaveric Explants
