The Experts below are selected from a list of 2022 Experts worldwide ranked by ideXlab platform
Guangping Gao - One of the best experts on this subject based on the ideXlab platform.
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A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate.
Human gene therapy methods, 2017Co-Authors: Raed Ibraheim, Phillip W.l. Tai, Guangping GaoAbstract:Increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. Unfortunately, quality control measures for preparations during mid-production steps and economical means to assess the fidelity of multiple batches of rAAV preparations are lacking. Here we describe a scalable and accurate method for the direct quantitative polymerase chain reaction (qPCR) titration of rAAV genomes in Crude Lysate. Lysate samples are pretreated with DNase I to remove vector and packaging plasmid DNAs, followed by proteinase K to release endonuclease-resistant packaged viral genomes and to proteolyze factors inherent to Crude Lysates that can impinge upon quantitative PCR efficiencies. We show that this method is precise, scalable, and applicable for vector genome titrations of both single-stranded and self-complementary AAV genomes irrespective of serotype differences-a major limitation for standard Lysate transduction methods that indirectly screen for vector packaging efficiency. Our described method therefore represents a significant improvement to rAAV vector production in terms of alleviating time and cost burdens, in-process quality control assessment, batch/lot monitoring in large-scale preparations, and good manufacturing practices.
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290. A High-Throughput, Sensitive, Accurate and Reproducible Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate
Molecular Therapy, 2016Co-Authors: Raed Al-abbasee, Guangping GaoAbstract:Recombinant adeno-associated viruses (rAAVs) have become an attractive tool for the delivery of therapeutic gene products. Increasing interest and application of rAAV vectors in basic and clinical research has urged efforts to improve AAV production quality and yield. Currently, standard procedures call for genome titration of purified vectors to assess yield at the endpoint of vector production. Mid-production (in-process) methods to gauge AAV yield is severely lacking. In addition, the need to multiplex the production of small-scale AAV batches (e.g. novel serotypes/variants screening) may become cost-prohibitive if vector purification steps were required for hundreds of candidate viral genomes. The high cost of downstream processing and purification during large-scale AAV production can be significantly reduced if low-yield vector batches were identified with earlier monitoring steps during vector production. To meet this need, we have established an enhanced qPCR method for rAAV genome titration in Crude Lysate. In brief, critical parameters call for the elimination of inhibitory factors inherent to cellular Lysates that may reduce qPCR efficiency. These factors may include contaminating nucleic acids originating from packaging vector and helper plasmids and cellular genomes and proteins that are residual from the host cell. Lysate samples are thus directly treated with DNase I and proteinase K in 96-well plates to remove cellular/plasmid DNAs and abundant proteins, followed by TaqMan quantitative PCR analysis on Dnase I-resistant vector genomes. To determine treatment efficiencies, we also quantified the copy numbers of vector plasmids diluted in non-transfected Crude Lysate before and after treatment. We demonstrate that plasmid templates are completely removed by DNase I treatment and qPCR efficiency is markedly enhanced by proteinase K. This enhanced qPCR method is proven to be highly sensitive and reliable, as it can detect as low as 10 copies of plasmid in Crude Lysate diluent. Furthermore, we demonstrate that this method is scalable for simultaneous vector genome titration by testing the yields of 13 different serotypes of ssAAV and scAAV. Thus, this procedure can be applied to screen large panels of novel serotypes/variants for vector production. Importantly, we show across independent experiments that titer quantification has high inter-experimental reproducibility among multiple batches of samples. In summary, our described method fulfills unmet needs for quantifying vector genomes in Crude Lysates from both large- and small-scale AAV preparations in a high-throughput, sensitive, accurate, and reproducible manner. This will significantly improve in-process quality control, batch/lot monitoring in large-scale preparations, and good manufacturing practices (GMP) for AAV production - all key for vector manufacturing, as AAV continues to garner use and impact in both basic research and clinical applications.
Timo Glatter - One of the best experts on this subject based on the ideXlab platform.
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Crude-MS Strategy for in-Depth Proteome Analysis of the Methane-Oxidizing Methylocystis sp. strain SC2.
Journal of Proteome Research, 2018Co-Authors: Anna Hakobyan, Werner Liesack, Timo GlatterAbstract:Methylocystis sp. strain SC2 is a representative of the alphaproteobacterial methane oxidizers or type IIa methanotrophs. These microorganisms play a crucial role in methane cycling. Here, we developed an efficient analytical proteomics workflow for strain SC2. It tackles the major challenges related to the high amount of integral membrane proteins that need to be efficiently solubilized and digested for downstream analysis. Each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized. Our new Crude-Lysate-MS approach proved to increase protein quantification accuracy and proteome coverage of strain SC2. It captured 62% of the predicted SC2 proteome, with up to 10-fold increase in membrane-associated proteins relative to less effective conditions. The use of Crude cell Lysate for downstream analysis showed to be highly efficient for SC2 and other members of the family Methylocystaceae. Using two contrasting nitrogen conditions, we further validated our workflow efficiency by analyzing the SC2 proteome for differentially expressed proteins involved in methane and nitrogen metabolism. Our Crude-MS approach may be applied to a variety of proteomic workflows incorporating cell types with challenging solubilization properties. Data are available via ProteomeXchange with identifier PXD009027.
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Crude-MS Strategy for in-Depth Proteome Analysis of the Methane-Oxidizing Methylocystis sp. strain SC2
2018Co-Authors: Anna Hakobyan, Werner Liesack, Timo GlatterAbstract:Methylocystis sp. strain SC2 is a representative of the alphaproteobacterial methane oxidizers or type IIa methanotrophs. These microorganisms play a crucial role in methane cycling. Here, we developed an efficient analytical proteomics workflow for strain SC2. It tackles the major challenges related to the high amount of integral membrane proteins that need to be efficiently solubilized and digested for downstream analysis. Each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized. Our new Crude-Lysate-MS approach proved to increase protein quantification accuracy and proteome coverage of strain SC2. It captured 62% of the predicted SC2 proteome, with up to 10-fold increase in membrane-associated proteins relative to less effective conditions. The use of Crude cell Lysate for downstream analysis showed to be highly efficient for SC2 and other members of the family Methylocystaceae. Using two contrasting nitrogen conditions, we further validated our workflow efficiency by analyzing the SC2 proteome for differentially expressed proteins involved in methane and nitrogen metabolism. Our Crude-MS approach may be applied to a variety of proteomic workflows incorporating cell types with challenging solubilization properties. Data are available via ProteomeXchange with identifier PXD009027
Anna Hakobyan - One of the best experts on this subject based on the ideXlab platform.
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Crude-MS Strategy for in-Depth Proteome Analysis of the Methane-Oxidizing Methylocystis sp. strain SC2.
Journal of Proteome Research, 2018Co-Authors: Anna Hakobyan, Werner Liesack, Timo GlatterAbstract:Methylocystis sp. strain SC2 is a representative of the alphaproteobacterial methane oxidizers or type IIa methanotrophs. These microorganisms play a crucial role in methane cycling. Here, we developed an efficient analytical proteomics workflow for strain SC2. It tackles the major challenges related to the high amount of integral membrane proteins that need to be efficiently solubilized and digested for downstream analysis. Each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized. Our new Crude-Lysate-MS approach proved to increase protein quantification accuracy and proteome coverage of strain SC2. It captured 62% of the predicted SC2 proteome, with up to 10-fold increase in membrane-associated proteins relative to less effective conditions. The use of Crude cell Lysate for downstream analysis showed to be highly efficient for SC2 and other members of the family Methylocystaceae. Using two contrasting nitrogen conditions, we further validated our workflow efficiency by analyzing the SC2 proteome for differentially expressed proteins involved in methane and nitrogen metabolism. Our Crude-MS approach may be applied to a variety of proteomic workflows incorporating cell types with challenging solubilization properties. Data are available via ProteomeXchange with identifier PXD009027.
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Crude-MS Strategy for in-Depth Proteome Analysis of the Methane-Oxidizing Methylocystis sp. strain SC2
2018Co-Authors: Anna Hakobyan, Werner Liesack, Timo GlatterAbstract:Methylocystis sp. strain SC2 is a representative of the alphaproteobacterial methane oxidizers or type IIa methanotrophs. These microorganisms play a crucial role in methane cycling. Here, we developed an efficient analytical proteomics workflow for strain SC2. It tackles the major challenges related to the high amount of integral membrane proteins that need to be efficiently solubilized and digested for downstream analysis. Each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized. Our new Crude-Lysate-MS approach proved to increase protein quantification accuracy and proteome coverage of strain SC2. It captured 62% of the predicted SC2 proteome, with up to 10-fold increase in membrane-associated proteins relative to less effective conditions. The use of Crude cell Lysate for downstream analysis showed to be highly efficient for SC2 and other members of the family Methylocystaceae. Using two contrasting nitrogen conditions, we further validated our workflow efficiency by analyzing the SC2 proteome for differentially expressed proteins involved in methane and nitrogen metabolism. Our Crude-MS approach may be applied to a variety of proteomic workflows incorporating cell types with challenging solubilization properties. Data are available via ProteomeXchange with identifier PXD009027
Ramnath Misra - One of the best experts on this subject based on the ideXlab platform.
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recombinant salmonella typhimurium outer membrane protein a is recognized by synovial fluid cd8 cells and stimulates synovial fluid mononuclear cells to produce interleukin il 17 il 23 in patients with reactive arthritis and undifferentiated spondylo
Clinical and Experimental Immunology, 2016Co-Authors: Smriti Chaurasia, A K Shasany, Amita Aggarwal, Ramnath MisraAbstract:: In developing countries, one-third of patients with reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) are triggered by Salmonella typhimurium. Synovial fluid mononuclear cells (SFMCs) of patients with ReA and uSpA proliferate to low molecular weight fractions (lmwf) of outer membrane proteins (Omp) of S. typhimurium. To characterize further the immunity of Omp of Salmonella, cellular immune response to two recombinant proteins of lmwf, OmpA and OmpD of S. typhimurium (rOmpA/D-sal) was assessed in 30 patients with ReA/uSpA. Using flow cytometry, 17 of 30 patients' SF CD8(+) T cells showed significant intracellular interferon (IFN)-γ to Omp Crude Lysate of S. typhimurium. Of these 17, 11 showed significantly more CD8(+) CD69(+) IFN-γ T cells to rOmpA-sal, whereas only four showed reactivity to rOmpD-sal. The mean stimulation index was significantly greater in rOmpA-sal than rOmpD-sal [3·0 (1·5-6·5) versus 1·5 (1·0-2·75), P 15-241·52) pg/ml, P < 0·05] and IL-6 [59·78 (2·03-273·36) versus 10·17 (0·004-190·19) ng/ml, P < 0·05]. The rOmpA-sal-specific CD8(+) T cell response correlated with duration of current synovitis (r = 0·53, P < 0·05). Thus, OmpA of S. typhimurium is a target of SF CD8(+) T cells and drives SFMC to produce increased cytokines of the IL-17/IL-23 axis which contribute to the pathogenesis of Salmonella-triggered ReA.
Raed Ibraheim - One of the best experts on this subject based on the ideXlab platform.
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A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate.
Human gene therapy methods, 2017Co-Authors: Raed Ibraheim, Phillip W.l. Tai, Guangping GaoAbstract:Increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. Unfortunately, quality control measures for preparations during mid-production steps and economical means to assess the fidelity of multiple batches of rAAV preparations are lacking. Here we describe a scalable and accurate method for the direct quantitative polymerase chain reaction (qPCR) titration of rAAV genomes in Crude Lysate. Lysate samples are pretreated with DNase I to remove vector and packaging plasmid DNAs, followed by proteinase K to release endonuclease-resistant packaged viral genomes and to proteolyze factors inherent to Crude Lysates that can impinge upon quantitative PCR efficiencies. We show that this method is precise, scalable, and applicable for vector genome titrations of both single-stranded and self-complementary AAV genomes irrespective of serotype differences-a major limitation for standard Lysate transduction methods that indirectly screen for vector packaging efficiency. Our described method therefore represents a significant improvement to rAAV vector production in terms of alleviating time and cost burdens, in-process quality control assessment, batch/lot monitoring in large-scale preparations, and good manufacturing practices.