Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems

Identification of suitable housing system for dairy cattle in North East Zone of Tamil Nadu, India, with respect to microclimate

Aim: To identify the suitable roofing pattern for dairy cattle in North East Zone of Tamil Nadu, India, based on micro climatic conditions. Materials and Methods: Initially, survey was conducted to identify and categorize the major housing patterns existing in the region for further detailed investigation. In total, 30 farmers/farms consisting of five housing types with six replicates were selected. Temperature and temperature humidity index (THI) were recorded using the maximum-minimum thermometer and digital thermo-hygrometers. The study was conducted for 1 year covering four seasons namely South West monsoon (June-August), North East monsoon (September-November), cold season (December-February), and summer season (April-May). The data were statistically analyzed using statistical package SPSS 17. Results: Animal shelters with cement sheets recorded the highest temperature (26.71±1.13°C) and THI (77.23±1.76) at 8.00 am, whereas the lowest temperature (24.83±1.17°C) and THI (74.54±1.72) were recorded in the thatched shed. There was significant difference (p<0.01) in temperature and THI at 8.00 am during South West monsoon and North East monsoon seasons between the housing types. During cold and summer seasons, there was no significant difference (p≥0.05) in the environmental variables among various shelter systems. Conclusion: Thatched housing is found to be the suitable one with respect to the climatic variables, followed by tile roof and metal roof. The cement sheet roofed housing is found to be the most unsuitable one in the region for dairy cattle

Generation and characterization of induced pluripotent stem cells in domestic Asian water buffalo (Bubalus bubalis)

Induced pluripotent stem cells (iPSCs) have numerous applications in livestock to improve production traits, disease resistance, biopharming, conservation of germplasm, disease modeling, regenerative medicine etc. Here, we have described the derivation of iPSCs from buffalo fetal fibroblasts (BuiPSCs) by lentivirus based transduction of mouse derived pluripotency marker genes Oct4, Sox2, Klf4 and c-Myc. The BuiPSCs showed typical buffalo embryonic stem cells like colony morphology which were alkaline phosphatase (AP) positive and expressed pluripotency markers Oct4, Nanog, SOX2, KLF4, FoxD3 and SSEA1, TRA-1-60, TRA-1-81. The cells were carrying normal karyotype and were able to differentiate into cell of all three germ layers in vitro. The BuiPSCs could be propagated beyond 20th passages. To the best of our knowledge, this is the first report on generation of buiPSCs in domestic water buffalo (Bubalus bubalis) using mouse derived transcription factors and the reprogrammed cells could self renew more than 20th passage

Generation of transgenic mesenchymal stem cells expressing green fluorescent protein as reporter gene using no viral vector in caprine

502-509Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. While there are many ways to label and track cells each with strengths and weakness, the green fluorescent protein (GFP) is a reporter gene commonly employed. In the present study, caprine MSC were collected from bone marrow and cells were characterised with MSC specific markers. Passage 10 (P10) MSC cells were transfected using plasmid vector containing GFP as reporter gene with different concentrations of DNA and lipofectamine. Six different concentrations of DNA and lipofectamine as 1 µg DNA: 2 µL lipofectamine, 1 µg DNA: 2.5 µL lipofectamine, 1.2 µg DNA: 2.2 µL lipofectamine, 1.2 µg DNA: 2.5 µL lipofectamine, 1.5 µg DNA: 2.5 µL lipofectamine, 1.5 µg DNA: 3 µL lipofectamine were used. After 24 h and 48 h of transfection, caprine MSC were observed under florescent microscope. Highest transfection rate indicating green flourecscent MSC were found when the cells were transfected with 1.2 µg DNA: 2.2 µL lipofectamine and 1.5 µg DNA: 2.5 µL lipofectamine than other combinations. These cells have been propagated beyond 4th passage maintaining GFP expression. The results indicated that stable GFP positive MSC cells can be generated using the above protocol. These cells are being used for transplantation studies

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Not AvailableThe present work was carried out to study the ability of avian "Extract Egg" (EE) for reprogramming caprine fetal cells. The isolated caprine fetal cells were cultured in stem cell media supplemented with different percentages of either EE or FBS. The results indicated that the supplementation of 2-4% EE formed lesser but larger size stem cell like cell colonies as compared to 6% or 10% EE. The expression of pluripotent genes were comparatively higher in colonies developed in 2% or 4% as compared to 6% or 10% EE. Further, immunocytochemistry revealed that the colonies developed in all percentage of EE expressed pluripotent markers like Oct4, Nanog, TRA-1-60 and TRA-1-81. Our findings indicated that avian EE has the potentiality to reprogram caprine fetal cells into embryonic state which may help in generation of pluripotent stem cells without using viral vector.Not Availabl

Not Available

Not AvailableThis study was designed to observe the effect of cytochalasin B (CCB) concentrations on ploidy and early development of parthenogenetic embryos in a caprine species. Caprine oocytes were matured in the presence of different concentrations of CCB (5, 10, 15, and 20 μg/ml) and activated by 7% ethanol followed by incubation with 2 mM DMAP. For embryos fertilized in vitro, oocytes were matured in maturation medium without CCB. The cleavage rate and further embryo development were significantly higher (P < 0.05) when oocytes were treated in this way. The percentage of embryos showed higher diploid values in 15 μg/ml CCB (83.66 ± 1.13), followed by 20 (72.22 ± 1.22), 10 (68.57 ± 1.17), and 5 μg/ml (62.00 ± 2.48). These results indicate that CCB with a concentration of 15 μg/ml in maturation medium can be used for the production of diploid parthenogenetic embryos in the caprine species.Not Availabl

Generation and characterization of induced pluripotent stem cells in domestic Asian water buffalo (Bubalus bubalis)

Induced pluripotent stem cells (iPSCs) have numerous applications in livestock to improve production traits, disease resistance, biopharming, conservation of germplasm, disease modeling, regenerative medicine etc. Here, we have described the derivation of iPSCs from buffalo fetal fibroblasts (BuiPSCs) by lentivirus based transduction of mouse derived pluripotency marker genes Oct4, Sox2, Klf4 and c-Myc. The BuiPSCs showed typical buffalo embryonic stem cells like colony morphology which were alkaline phosphatase (AP) positive and expressed pluripotency markers Oct4, Nanog, SOX2, KLF4, FoxD3 and SSEA1, TRA-1-60, TRA-1-81. The cells were carrying normal karyotype and were able to differentiate into cell of all three germ layers in vitro. The BuiPSCs could be propagated beyond 20th passages. To the best of our knowledge, this is the first report on generation of buiPSCs in domestic water buffalo (Bubalus bubalis) using mouse derived transcription factors and the reprogrammed cells could self renew more than 20th passage