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

Recovery of Medium-Chain-Length Polyhydroxyalkanoates (PHAs) Through Enzymatic Digestion Treatments and Ultrafiltration

Medium-chain-length (mcl) polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated intracellularly as energy resources by bacterial species such as Pseudomonas putida. The most popular method for PHA recovery in the downstream processing is solvent extraction using chloroform and methanol. An alternate method is bioseparation using enzymatic digestion process which eliminates the need for hazardous solvents. This research focuses on an attempt to optimize the recovery of PHAs by solubilisation of non-PHA granules through enzymatic treatments such as; Alcalase (to digest the denatured proteins), sodium dodecyl sulfate (SDS) to assist solubilisation, ethylene diamine tetra acetic acid (EDTA) to complex divalent cations and lysozyme to digest the peptidoglycan wall enveloping the cell. The experiment was designed through Taguchi's design of experiment (DOE) using Qualitek-4 software. The results show that Alcalase enzyme used had the most significant effect on the treatment process and contributed to about 71.5 in terms of process factor importance among the different factors on treatment performance for PHA recovery. It is desired to recover the PHA granules in water suspension after the enzymatic treatment by removing the solubilised non-PHA cell material through crossflow ultrafiltration system and purified through continuous diafiltration process. Final purity of PHA in water suspension obtained using GC analysis is 92.6, with a nearly 90 recovery, thus concluding that this method is indeed a suitable alternative. (c) 2006 Elsevier B.V. All rights reserved

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

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

Oxidative CO2 reforming of methane in La0.6Sr 0.4Co0.8Ga0.2O3-δ (LSCG) hollow fiber membrane reactor

10.1021/es403158kEnvironmental Science and Technology472414510-14517ESTH