Literatura académica sobre el tema "Host Cell Proteins (HCP)"
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Artículos de revistas sobre el tema "Host Cell Proteins (HCP)"
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Pilely, Katrine, Martin Rask Johansen, Rikke Raaen Lund, Thomas Kofoed, Thomas Kjærsgaard Jørgensen, Lars Skriver y Ejvind Mørtz. "Monitoring process-related impurities in biologics–host cell protein analysis". Analytical and Bioanalytical Chemistry 414, n.º 2 (1 de octubre de 2021): 747–58. http://dx.doi.org/10.1007/s00216-021-03648-2.
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AbstractDuring biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC–MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC–MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC–MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC–MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs.
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Gillespie, Paul F., Yanjie Wang, Kuo Yin, Emily Groegler, Nicholas Cunningham, Alyssa Q. Stiving, Jessica Raffaele et al. "Automated, Quantitative Capillary Western Blots to Analyze Host Cell Proteins in COVID-19 Vaccine Produced in Vero Cell Line". Vaccines 12, n.º 12 (5 de diciembre de 2024): 1373. https://doi.org/10.3390/vaccines12121373.
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Background/Objectives: Host cell protein (HCP) content is a major attribute for biological and vaccine products that must be extensively characterized prior to product licensure. Enzyme Linked Immunosorbent Assay (ELISA) and Mass Spectrometry (MS) are conventional methods for quantitative host cell protein analysis in biologic and vaccine products. Both techniques are usually very tedious, labor-intensive, and challenging to transfer to other laboratories. In addition, the ELISA methodology requires 2D SDS PAGE and 2D western blot antibody reagent validation to establish reagent coverage. This reagent coverage provides a rather weak link that is currently accepted, as the western blot is run under denaturing conditions and the ELISA is run under native conditions. Simple Western™ is a relatively new, automated, capillary western blot-based technology that allows for the separation, blotting, and detection of proteins. But, unlike traditional western blots, Simple Western™ is quantitative, allowing for the quantification of HCP content in biologic and vaccine samples. Antibody reagent validation is much more straightforward, as the reagent coverage can be directly linked between the 2D methodology and Simple Western™, as they are both run under denatured and reduced conditions. Methods: Herein we describe the development of a capillary western blot method to quantify the HCP content in samples generated using a Vero cell line for the production of an investigational live virus vaccine candidate (V590) for Coronavirus Disease-2019 (COVID-19). The HCP content in COVID-19 vaccine samples was evaluated using three methods: the new capillary western, the gold standard ELISA, and SDS-PAGE. Results/Conclusions: Strong agreement was observed in the HCP content data between the capillary western and SDS PAGE methods, whereas the ELISA HCP data were outliers, suggesting that the capillary western is generating HCP concentrations closer to the true concentration. This is the first report of using capillary western technology in analyzing HCP in vaccine samples.
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Lavoie, R., Alice di Fazio, R. Blackburn, Michael Goshe, Ruben Carbonell y Stefano Menegatti. "Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery". International Journal of Molecular Sciences 20, n.º 7 (8 de abril de 2019): 1729. http://dx.doi.org/10.3390/ijms20071729.
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The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.
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Van Manen-Brush, Kathleen, Jacob Zeitler, John R. White, Paul Younge, Samantha Willis y Marisa Jones. "Improving Chinese hamster ovary host cell protein ELISA using Ella®: an automated microfluidic platform". BioTechniques 69, n.º 3 (septiembre de 2020): 186–92. http://dx.doi.org/10.2144/btn-2020-0074.
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Chinese hamster ovary (CHO) cells are a mammalian cell line used in the production of therapeutic proteins. Host cell proteins (HCPs) are process-related impurities that are derived from the host cell expression system. During biopharmaceutical drug development, removal of HCPs is required. Enzyme-linked immunosorbent assay (ELISA) is a common technique to quantitate HCPs, but is a labor-intensive process that takes up to 7 h. Ella® is an automated instrument that utilizes microfluidics and glass nanoreactors to quantitate HCPs in 75 min using similar ELISA reagents. The antibodies and antigens are captured on three distinct glass nanoreactors, resulting in sensitive reproducible data. Our results indicate that Ella quantitates CHO HCPs with precision, accuracy, sensitivity and trends comparable with our traditional CHO HCP ELISA.
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Gonzaga, Zennia Jean C., Christina Merakou, Antonio DiGiandomenico, Gregory P. Priebe y Bernd H. A. Rehm. "A Pseudomonas aeruginosa-Derived Particulate Vaccine Protects against P. aeruginosa Infection". Vaccines 9, n.º 7 (20 de julio de 2021): 803. http://dx.doi.org/10.3390/vaccines9070803.
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Despite numerous efforts to develop an effective vaccine against Pseudomonas aeruginosa, no vaccine has yet been approved for human use. This study investigates the utility of the P. aeruginosa inherently produced polyhydroxyalkanaote (PHA) inclusions and associated host–cell proteins (HCP) as a particulate vaccine platform. We further engineered PHA inclusions to display epitopes derived from the outer membrane proteins OprF/OprI/AlgE (Ag) or the type III secretion system translocator PopB. PHA and engineered PHA beads induced antigen-specific humoral, cell-mediated immune responses, anti-HCP and anti-polysaccharide Psl responses in mice. Antibodies mediated opsonophagocytic killing and serotype-independent protective immunity as shown by 100% survival upon challenge with P. aeruginosa in an acute pneumonia murine model. Vaccines were stable at 4 °C for at least one year. Overall, our data suggest that vaccination with subcellular empty PHA beads was sufficient to elicit multiple immune effectors that can prevent P. aeruginosa infection.
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Wu, Hung-Yi, Pei-Che Chung, Hsiao-Wei Shih, Sy-Ray Wen y Erh-Min Lai. "Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System in Agrobacterium tumefaciens". Journal of Bacteriology 190, n.º 8 (8 de febrero de 2008): 2841–50. http://dx.doi.org/10.1128/jb.01775-07.
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ABSTRACT Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.
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Zoued, Abdelrahim, Eric Durand, Cecilia Bebeacua, Yannick R. Brunet, Badreddine Douzi, Christian Cambillau, Eric Cascales y Laure Journet. "TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System". Journal of Biological Chemistry 288, n.º 38 (6 de agosto de 2013): 27031–41. http://dx.doi.org/10.1074/jbc.m113.499772.
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The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.
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Alhuthali, Sakhr y Cleo Kontoravdi. "Population balance modelling captures host cell protein dynamics in CHO cell cultures". PLOS ONE 17, n.º 3 (23 de marzo de 2022): e0265886. http://dx.doi.org/10.1371/journal.pone.0265886.
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Monoclonal antibodies (mAbs) have been extensively studied for their wide therapeutic and research applications. Increases in mAb titre has been achieved mainly by cell culture media/feed improvement and cell line engineering to increase cell density and specific mAb productivity. However, this improvement has shifted the bottleneck to downstream purification steps. The higher accumulation of the main cell-derived impurities, host cell proteins (HCPs), in the supernatant can negatively affect product integrity and immunogenicity in addition to increasing the cost of capture and polishing steps. Mathematical modelling of bioprocess dynamics is a valuable tool to improve industrial production at fast rate and low cost. Herein, a single stage volume-based population balance model (PBM) has been built to capture Chinese hamster ovary (CHO) cell behaviour in fed-batch bioreactors. Using cell volume as the internal variable, the model captures the dynamics of mAb and HCP accumulation extracellularly under physiological and mild hypothermic culture conditions. Model-based analysis and orthogonal measurements of lactate dehydrogenase activity and double-stranded DNA concentration in the supernatant show that a significant proportion of HCPs found in the extracellular matrix is secreted by viable cells. The PBM then served as a platform for generating operating strategies that optimise antibody titre and increase cost-efficiency while minimising impurity levels.
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Jeon, Hyung Jin, Bo Kyoung Choi, Seo In Hwang, Soo Hyun Kim, Gil Jung Kim, Jae Chan Park, Zung Yoon Yang y Kwang Yeon Hwang. "Optimization for Simultaneous Removal of Product/Process-Related Impurities of Peptide Fc-Fusion Protein Using Cation Exchange Chromatography". Processes 10, n.º 11 (11 de noviembre de 2022): 2359. http://dx.doi.org/10.3390/pr10112359.
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Fc fusion proteins are used as therapeutic agents with unique structures by combining the Fc domain of an antibody with other active proteins, cytokines, and enzymes. Peptide Fc-fusion proteins are complex fusion molecules that possess a structure different from that of monoclonal antibodies (mAbs) and are difficult to express, thereby affecting their quality. Many product/process-related impurities generated during the production of peptide Fc-fusion proteins pose a risk to the robustness of pre-existing three-column platforms for the purification of mAbs. Thus, we first evaluated the effect of pH, conductivity, and dynamic binding capacity (DBC; g of product per liter of resin) on the separation of host cell protein (HCP) and high molecular weight (HMW) and low molecular weight (LMW) proteins in strong cation exchange chromatography and then established an operating range using the design of experiments (DoE). Based on our studies, the optimal removal rates of HCP and HMW were achieved under the following conditions: 8 CV of wash buffer, 20–23 g/L of resin DBC, and an elution buffer conductivity of 63–66 mS/cm. The conductivity of the wash buffer used to remove the LMW was 50 mS/cm. In addition, reproducibility was confirmed by scaling up two batches using the Fractogel® EMD SO3− (M) resin. As a result of confirming with a validated test method in all batches, >55% yield, >98.2% purity, and >27% HCP reduction rate were satisfied. The cation exchanger exhibited an acceptable step yield and effectively reduced product/process-related impurities within the established range.
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Kiyonami, Reiko, Rafael Melani, Ying Chen, AI De Leon y Min Du. "Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs". International Journal of Molecular Sciences 25, n.º 17 (7 de septiembre de 2024): 9687. http://dx.doi.org/10.3390/ijms25179687.
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Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.
Tesis sobre el tema "Host Cell Proteins (HCP)"
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De, Lama Valderrama Noelia Milagros. "Development of new mass spectrometry methods for the characterization of protein impurities in therapeutic antibodies". Electronic Thesis or Diss., Strasbourg, 2025. http://www.theses.fr/2025STRAF008.
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Les protéines de cellule hôte (HCPs) sont des impuretés indésirables dans la production d’anticorps monoclonaux (mAbs), pouvant compromettre la sécurité, l’efficacité et la stabilité des traitements. Bien que l’ELISA soit couramment utilisée, elle présente des limites de couverture. Cette thèse explore des méthodes complémentaires basées sur la spectrométrie de masse. Une approche d’immuno-capture permet de détecter les HCPs non immune-réactifs, tandis que des workflows LC-MS/MS avancés avec des peptide standards offrent une quantification plus précise. Ces stratégies visent à améliorer le contrôle qualité dans la fabrication des mAbsHost cell proteins (HCPs) are unwanted by-products in the production of monoclonal antibodies (mAbs), and even at low levels, they can affect the safety, efficacy, and stability of biopharmaceuticals. While ELISA is widely used for HCP detection, it lacks full impurity coverage. This work explores complementary mass spectrometry-based methods to address these limitations. An immune-capture MS approach targets non-immunoreactive HCPs missed by ELISA, while advanced LC-MS/MS workflows using peptide standards enable more accurate and flexible quantification. These tools aim to improve impurity profiling and strengthen quality control in mAb manufacturing
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Carr, Sharon. "Adenovirus and its interaction with host cell proteins /". St Andrews, 2007. http://hdl.handle.net/10023/219.
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Qashqari, Fadi Saleh I. "Regulation of host cell proteins by adenovirus oncoproteins". Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/8020/.
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Adenovirus early region proteins, ElA, E1B-55K, E4orf3 and E4orf6 regulate host cell processes to facilitate viral replication. E4orf3 suppress host cell anti-viral activities through association with host cell proteins in E4orf3 nuclear-track structures, whilst E1B-55K, E4orf3 and E4orf6 are all recruited to viral replication centres during infection to promote viral DNA replication and inhibit host cell antiviral activities. Immunoprecipitation coupled to mass spectrometry identified Toplla as an Ad12 E4orf3-binding protein that localized with E4orf3 in adenovirus-infected cells. It was determined that Toplla expression was induced during infection, and that Toplla was required for the adenovirusdependent stabilization of p53. It was also established that, despite their ability to cooperate functionally, Toplla and p53 do not associate physically during infection. Immunoprecipitation coupled to mass spectrometry was also used to identify host cell proteins recruited to viral replication centres during adenovirus infection. The RP A-1 binding protein, Smarcall, and the FACT complex histone chaperone protein, SSRPl were identified as host cell proteins recruited to viral replication centres during infection. Following recruitment to viral replication centres Smarcall was found to be degraded in an E1B-55K and E4orf6 dependent manner, whilst SSRPl was found to be stable during infection and was not targeted for degradation.
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Bjertsjö, Rennermalm Anna. "Staphylococcal cell wall associated proteins : characteristics and host interactions /". Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-542-9/.
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Mohamed, Ahmed Attia Ali. "Interaction of hepatitis C virus polymerase with host cell proteins". Thesis, Durham University, 2009. http://etheses.dur.ac.uk/2107/.
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Hepatitis C virus (HCV) interacts with host cell proteins to modify cellular pathways creating a favourable environment that facilitates its replication and persistence. The purpose of the work presented in this thesis was to identify cellular proteins that can interact with NS5B, the virus's RNA-dependent RNA polymerase, that may contribute to the virus's biology. A number of cellular proteins were found to interact with NS5B using the yeast two-hybrid system. These proteins were involved in cellular pathways such as interferon signalling, lipid transport and metabolism, protein trafficking, cell proliferation and apoptosis. Of these, phospholipid scramblase 1 (PLSCR1) and zinc finger protein 143 (ZNF143) were selected for further investigation. The interactions were confirmed in vitro, and, for PLSCR1, the region that interacted with NS5B was determined to be within the amino-terminal region of the protein (61-137 a.a.). NS5B interacted with PLSCR1 and ZNF143 via a single interacting region localized in its N-terminus (1-153 a.a.).Expression of PLSCR1 or ZNF143 enhanced the ability of interferon to stimulate transcription from an interferon-stimulated response element (ISRE) reporter construct. Co-expression with NS5B was found to down-regulate this activity. Expression of a number of interferon-stimulated genes was investigated in the presence of NS5B, PLSCR1 or ZNF143 but no significant effect was observed. Overexpression of PLSCR1 had no effect on HCV sub-genomic replicon replication, while reduction of its expression by short hairpin RNA (shRNA) enhanced replication. Overexpression of ZNF143 was found to have a suppressive effect on replication but downregulating its expression did not enhance replication. In addition to using the yeast two-hybrid system to identify NS5B- interacting proteins, an in vitro pulldown assay coupled with mass spectrometry identified α- and β -tubulin associated with NS5B in vitro and in vivo. Subsequently this association was demonstrated to be an indirect interaction but the intermediatory partner was not identified. The domain that mediated the association with α- and β-tubulin was determined to be within the N-terminus of NS5B (1-153 a.a.). Nocodazole, an inhibitor of tubulin polymerization, had a marked effect on the association of α -tubulin with NS5B displacing it from the complex but had no effect on β -tubulin's association. Utilizing an HCV sub- genomic replicon, nocodazole was shown to have a significant inhibitory effect on replication. Taken together the data presented in this thesis showed that NS5B had a multitude of potential interactions with a variety of cellular proteins. The biological significance of some of these interactions on the cellular response to IFN and replicon replication was investigated. This work has generated a number of novel observations on the interaction between the virus and the cell that warrant future investigation
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Malinkevich, Anna. "MIRAGE DNA Transposon Silencing by C. elegans Condensin II Subunit HCP-6: A Masters Thesis". eScholarship@UMMS, 2014. http://escholarship.umassmed.edu/gsbs_diss/754.
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Mobile genetic elements represent a large portion of the genome in many species. Posing a danger to the integrity of genetic information, silencing and structural machinery has evolved to suppress the mobility of foreign and transposable elements within the genome. Condensin proteins – which regulate chromosome structure to promote chromosome segregation – have been demonstrated to function in repetitive gene regulation and transposon silencing in several species. In model system Caenorhabditis elegans, microarray analysis studies have implicated Condensin II subunit HCP-6 in the silencing of multiple loci, including DNA transposon MIRAGE. To address the hypothesis that HCP-6 has a direct function in transcriptional gene silencing of the MIRAGE transposon, we queried MIRAGE expression and chromatin profiles in wild-type and hcp-6 mutant animals. Our evidence confirms that HCP-6 does indeed function during silencing of MIRAGE. However, we found no significant indication that HCP-6 binds to MIRAGE, nor that HCP-6 mediates MIRAGE enrichment of H3K9me3, the repressive heterochromatin mark observed at regions undergoing transcriptional silencing. We suggest that the silencing of MIRAGE, a newly evolved transposon and the only tested mobile element considerably derepressed upon loss of HCP-6, is managed by HCP-6 indirectly.
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Patel, Amit. "Interaction of enteropathogenic 'Escherichia coli' (EPEC) tir with host cell proteins". Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431869.
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Cowlishaw, Deborah Anne. "Identification of host proteins required for bacteriophage infection of Streptomyces sp". Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367589.
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Rytkönen, Anne. "Molecular studies of Neisseria - host cell interactions /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-018-4/.
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Dickey, Laura Leigh. "Respiratory synctial virus interactions with host-cell RNA-processing structures and proteins". Thesis, Boston University, 2013. https://hdl.handle.net/2144/10980.
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Thesis (Ph.D.)--Boston UniversityRespiratory syncytial virus (RSV) is a negative-strand RNA virus that causes significant pneumonia-related morbidity and mortality worldwide. There are currently neither vaccines nor effective therapies for RSV. As with other viruses, RSV mRNAs are translated using host-cell machinery, rendering the virus subject to cellular factors that regulate mRNA homeostasis. Stress granules (SGs) and processing bodies (p-bodies) are inter-dependent, stress-response cytoplasmic structures involved in mRNA triage and degradation, respectively. We hypothesized that RSV has evolved to manipulate cellular stress responses in order to facilitate optimal virus propagation. While wild-type (wt) RSV induced SGs in approximately 1% of infected cells, a mutant version of RSV whose Tr region was replaced with an inverted LeC sequence (LeC virus) induced SG formation in approximately 50% to 70% of infected cells. A 12U to A substitution relative to the 5' end of the LeC virus abrogated SG induction. Mixed-infection studies showed that wt RSV was able to prevent LeC-mediated SG induction. Unlike Sendai virus, RSV-mediated prevention of SG formation was independent of SG-associated t-cell intracellular antigen related (TIAR) protein. RSV infection altered neither the number nor distribution of p-bodies; however, p-body-associated decapping protein 1 (dcp1) was phosphorylated throughout RSV infection via the extracellular signal-regulated kinase (ERK) 1/2 pathway. RSV-mediated dcp1 phosphorylation was limited to serine 315, serine 319, and threonine 321. Dcp1 phosphorylation occurred in response to some, but not all, environmental stresses, and dcp1 was not phosphorylated during infection with HIV-1, measles, mumps, or canine distemper virus. Overexpression of dcp1 significantly attenuated RSV cytopathic effects, and preliminary data suggested that dcp1 phosphorylation regulated RSV-induced interleukin-8 production. Finally, an antibody toward cellular SG- and p-body-associated, RNA-binding protein p54 was able to recognize a subset of RSV nucleoprotein (N). p54 and RSV N contain a similar amino acid sequence motif, suggesting that they may have similar or competing activities that are important during RSV replication. Taken together, our results demonstrate that RSV can manipulate cellular RNA-processing structures and proteins to facilitate viral propagation.
Libros sobre el tema "Host Cell Proteins (HCP)"
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Spearman, Paul y Eric O. Freed, eds. HIV Interactions with Host Cell Proteins. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-642-02175-6.
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O, Freed Eric y SpringerLink (Online service), eds. HIV Interactions with Host Cell Proteins. Berlin, Heidelberg: Springer-Verlag Berlin Heidelberg, 2010.
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1942-, Cabello Felipe C., Pruzzo Carla y North Atlantic Treaty Organization. Scientific Affairs Division., eds. Bacteria, complement, and the phagocytic cell. Berlin: Springer-Verlag, 1988.
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M, McCrae y Society for General Microbiology, eds. Molecular aspects of host-pathogen interaction: Fifty-fifth Symposium of the Society for General Microbiology : held at Heriot-Watt University, Edinburgh, March 1997. Cambridge: Cambridge University Press, 1997.
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Spearman, Paul y Eric O. Freed. HIV Interactions with Host Cell Proteins. Springer Berlin / Heidelberg, 2012.
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Spearman, Paul y Eric O. Freed. HIV Interactions with Host Cell Proteins. Springer, 2010.
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Enterotoxins: Microbial Proteins and Host Cell Dysregulation. MDPI, 2016. http://dx.doi.org/10.3390/books978-3-03842-164-1.
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Laguette, Nadine y Monsef Benkirane, eds. Manipulation of the host cell by viral auxiliary proteins. Frontiers Media SA, 2015. http://dx.doi.org/10.3389/978-2-88919-484-1.
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(Editor), M. A. McCrae, J. R. Saunders (Editor), C. J. Smyth (Editor) y N. D. Stow (Editor), eds. Molecular Aspects of Host-Pathogen Interactions. Cambridge University Press, 1997.
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Vinod, Nikhra. COVID-19: Perspective, Patterns and Evolving strategies. Heighten Science Publications Inc., 2020. http://dx.doi.org/10.29328/ebook1003.
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The Global Virome: The viruses have a global distribution, phylogenetic diversity, and host specificity. They are obligate intracellular parasites with single- or double-stranded DNA or RNA genomes, and afflict bacteria, plants, animals, and human population. The infecting virus binds to receptor proteins on the host cell surface, followed by internalisation, replication, and cell lysis. Further, trans-species interactions of viruses with bacteria, small eukaryotes and host are linked with various zoonotic viral diseases and disease progression.
Capítulos de libros sobre el tema "Host Cell Proteins (HCP)"
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Hogwood, Catherine E. M., Lesley M. Chiverton y C. Mark Smales. "Characterization of Host Cell Proteins (HCPs) in CHO Cell Bioprocesses". En Methods in Molecular Biology, 243–50. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6972-2_16.
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Shah, Rahul, Bipin N. Savani y Shruti Chaturvedi. "Bleeding and Thrombotic Complications". En The EBMT Handbook, 355–63. Cham: Springer International Publishing, 2024. http://dx.doi.org/10.1007/978-3-031-44080-9_40.
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AbstractBleeding and thrombotic complications are an important cause of morbidity and mortality in patients undergoing hematopoietic cell transplantation (HCT). The major thrombotic complications include venous thromboembolism (VTE) including catheter-related thrombosis (CRT), sinusoidal obstruction syndrome (SOS), and transplant-associated thrombotic microangiopathy (TA-TMA), while bleeding commonly involves the gastrointestinal or respiratory tracts and is most common in thrombocytopenic patients or those with graft-versus-host disease (GVHD). HCT is associated with multiple risk factors for both thrombosis and bleeding including the underlying malignancy, thrombocytopenia, high-dose myeloablative chemotherapy (MAC) and immune-modulatory drugs, GVHD, infections, indwelling vascular catheters, and prolonged immobilization (Chiu and Lazo-Langner 2023; Gerber et al. 2008; Chaturvedi et al. 2016; Nadir and Brenner 2007). In addition, HCT is also associated with alterations in the coagulation system with activation of endothelium-dependent coagulation factors, increase in von Willebrand factor (vWF) and platelet adhesion, increased thrombin generation, decreased antithrombin levels, and decreased levels of anticoagulant proteins such as protein C (Vannucchi et al. 1994). Collectively, major patient-, disease-, and therapy-related factors contribute to hemostatic complications in HCT patients. Thrombotic and bleeding complications in HCT are discussed separately in the following section.
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Chen, Weibin, Catalin E. Doneanu, Matthew Lauber, Stephan Koza, Kesh Prakash, Martha Stapels y Kenneth J. Fountain. "Improved Identification and Quantification of Host Cell Proteins (HCPs) in Biotherapeutics Using Liquid Chromatography-Mass Spectrometry". En ACS Symposium Series, 357–93. Washington, DC: American Chemical Society, 2015. http://dx.doi.org/10.1021/bk-2015-1202.ch013.
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Schiesser, William E. "Host Cell Proteins with Cross Diffusion". En Virus Host Cell Genetic Material Transport, 35–60. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-030-68865-3_3.
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Honda, Tomoyuki y Keizo Tomonaga. "Host Molecular Chaperones: Cell Surface Receptors for Viruses". En Heat Shock Proteins, 293–307. Dordrecht: Springer Netherlands, 2013. http://dx.doi.org/10.1007/978-94-007-6787-4_19.
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Teter, Ken. "Cholera Toxin Interactions with Host Cell Stress Proteins". En Heat Shock Proteins, 323–38. Dordrecht: Springer Netherlands, 2013. http://dx.doi.org/10.1007/978-94-007-6787-4_21.
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Berry, A. M., U. Rasmussen, K. Bateman, S. Lindwall, K. Huss-Danell y B. Bergman. "Arabinogalactan-Protein Epitopes Are Host-Derived in Frankia-Alnus Symbiosis". En Cell and Developmental Biology of Arabinogalactan-Proteins, 281–82. Boston, MA: Springer US, 2000. http://dx.doi.org/10.1007/978-1-4615-4207-0_28.
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Doneanu, Catalin E. y Weibin Chen. "Analysis of Host-Cell Proteins in Biotherapeutic Proteins by LC/MS Approaches". En Methods in Molecular Biology, 341–50. Totowa, NJ: Humana Press, 2014. http://dx.doi.org/10.1007/978-1-62703-977-2_25.
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Henderson, Brian. "Cell Stress Proteins as Modulators of Bacteria-Host Interactions". En Novartis Foundation Symposia, 141–59. Chichester, UK: John Wiley & Sons, Ltd, 2008. http://dx.doi.org/10.1002/9780470754030.ch11.
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Diemer, Claudia, Martha Schneider, Hermann M. Schätzl y Sabine Gilch. "Modulation of Host Cell Death by SARS Coronavirus Proteins". En Molecular Biology of the SARS-Coronavirus, 231–45. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-642-03683-5_14.
Texto completoActas de conferencias sobre el tema "Host Cell Proteins (HCP)"
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Wang, Jianye, Kaixuan Zhu, Gang Zhao y Dayong Gao. "Effect of Temperature and Cryoprotectant Solutes on Water Permeability of SF21 Cell Membrane". En ASME 2013 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/sbc2013-14056.
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Insect cell as a host of viruses is extensively used for producing heterologous recombinant proteins. The eukaryotic proteins expressed by the insect cell is posttranslationally modified and harvested in a short period of time. The insect cell expression has been applied to both basic research and commercial applications. A large scale of proteins produced by the insect cell expression system are settled for researching the structure and function of the eukaryotic proteins, and the expression system integrated with routine biochemical techniques plays a significant role in diagnostic procedure and therapeutic approaches for the disease.
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Limper, Andrew H. y Theodore J. Kottom. "The Pneumocystis Carinii PcAce2 Transcription Factor Regulates Cell Wall Remodeling Induced By Organism Adhesion To Host Matrix Proteins". En American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California. American Thoracic Society, 2012. http://dx.doi.org/10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a3297.
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Utkina, A. A., A. A. Kudryavtseva y I. V. Manuhkov. "HIGH-YIELD PRODUCTION OF “DIFFICULT-TO-EXPRESS” PROTEINS ARDA AND ARDB WITH THE THERMO-INDUCIBLE VECTOR PIR-DPAL". En X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-379.
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The antirestriction proteins ArdA and ArdB inhibit type I restriction-modification (RM) systems. Such proteins are usually difficult to obtain in Escherichia coli cells in large quantities since they are toxic to the host cell. This work is devoted to the cloning of the ardA and ardB antirestrictase genes into the thermo-inducible vector pIR-DPAl vector, which made it possible to increase the efficiency of its expression and purification.
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Patwardhan, Anshuman V., Mohammad M. Ataai y Mansour Zenouzi. "Mathematical Modeling and Optimization of the Immobilized Metal Affinity Chromatography". En ASME 1996 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 1996. http://dx.doi.org/10.1115/imece1996-1195.
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Abstract Immobilized copper affinity chromatography experiments with E. coli cell extract have demonstrated that the cellular proteins of this most commonly used host organism elute from the column in a discontinuous fashion characterized by two major peaks. This result has interesting implications towards the mathematical modeling and subsequent optimization of IMAC. Namely, the elution behavior of the E. coli cell extract can be approximately simulated by simply two representative proteins that have a similar elution behavior as that of the peaks. We have identified the mixture of Bovine Serum Albumin and Tuna Heart Cytochrome-C to be excellent representatives of the E. coli cell extract. The equilibrium and transport parameters of these proteins were determined experimentally and used in a detailed mathematical model incorporating multicomponent Langmuir isotherm, axial dispersion, film mass transfer and intraparticle diffusion. Target proteins of varying affinities were also included in the model. Simulation results provided an excellent framework for identifying effective strategies for optimal column utilization as well as for attaining high resolution of the target protein.
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Zhang, Jun, Sheng Yan, Dan Yuan, Gursel Alici, Nam-trung Nguyen y Weihua Li. "High Throughput Cell-Free Extraction of Plasma by an Integrated Microfluidic Device Combining Inertial Microfluidics and Membrane". En ASME 2016 5th International Conference on Micro/Nanoscale Heat and Mass Transfer. American Society of Mechanical Engineers, 2016. http://dx.doi.org/10.1115/mnhmt2016-6717.
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Plasma is a host of various analytes such as proteins, metabolites, circulating nucleic acids (CNAs), pathogens. The key process of plasma extraction is to eliminate the contamination from blood cells. Conventional methods, such as centrifugation and membrane filtration, are generally lab-intensive, time consuming and even dangerous. In this study, we report an integrated microfluidic device that combines inertial microfluidics and membrane filter. The integrated microfluidic device was evaluated by the diluted (x1/10, x1/20) whole blood, and the quality of the extracted blood plasma was tested. It was found that quality of extracted blood plasma from integrated device was equivalent to that obtained by the centrifugation. This study demonstrates a significant progress towards the practical application of inertial microfluidics with membrane filter for high-throughput and high efficient blood plasma extraction.
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Tan Hui En, Glenda, Koay Tze Erhn y Shen Bingquan. "Anti-Virus Autobots: Predicting More Infectious Virus Variants for Pandemic Prevention through Deep Learning". En 4th International Conference on Machine Learning & Applications (CMLA 2022). Academy and Industry Research Collaboration Center (AIRCC), 2022. http://dx.doi.org/10.5121/csit.2022.121102.
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More infectious virus variants can arise from rapid mutations in their proteins, creating new infection waves. These variants can evade one’s immune system and infect vaccinated individuals, lowering vaccine efficacy. Hence, to improve vaccine design, this project proposes Optimus PPIme – a deep learning approach to predict future, more infectious variants from an existing virus (exemplified by SARS-CoV-2). The approach comprises an algorithm which acts as a “virus” attacking a host cell. To increase infectivity, the “virus” mutates to bind better to the host’s receptor. 2 algorithms were attempted – greedy search and beam search. The strength of this variant-host binding was then assessed by a transformer network we developed, with a high accuracy of 90%. With both components, beam search eventually proposed more infectious variants. Therefore, this approach can potentially enable researchers to develop vaccines that provide protection against future infectious variants before they emerge, preempting outbreaks and saving lives.
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ZOMER, G., M. HAMZINK, A. DE HAAN, G. KERSTEN y K. REUBSAET. "DEVELOPMENT AND OPTIMIZATION OF A QUANTITATIVE WESTERN BLOT AND DOT BLOT PROCEDURE FOR THE DETERMINATION OF RESIDUAL HOST CELL PROTEINS PRESENT IN INACTIVATED POLIO VACCINE USING A GZ11 BASED SIGNAL REAGENT". En Proceedings of the 15th International Symposium. WORLD SCIENTIFIC, 2008. http://dx.doi.org/10.1142/9789812839589_0066.
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Rickles, F. R., W. W. Hancock, K. Kobzik, N. Hogg y C. O’Hara. "THE DISTRIBUTION OF CROSS-LINKED FIBRIN IN HUMAN LUNG CARCINOMA PARALLELS THAT OF ACTIVATED HOST MONONUCLEAR LEUKOCYTES: IMMUNO-HISTOLOGIC STUDIES WITH MONOCLONAL ANTIBODIES". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643669.
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Previous immunohistologic studies of human lung carcinoma, using polyclonal antibodies to antigens shared between fibrinogen (FGN) and fibrin (FB), showed that FGN/FB were associated withtumor cells. These findings were interpreted as evidence of the presence of tumor-associated procoagulant activity(PCA). Wecompared the distribution of coagulation-associated proteins in 16 casesof human carcinoma of the lungofvarying histologic types, using polyclonal antibodies to FGN/FB and monoclonal antibodies (mAb) to cross-linked fibrin (XL-FB;UC-45), fibronectin (FN;PHM13), factor VIII (vWF:Ag)and a tissue factor-related antigen(TF:RAg;A1 -3)- Host mononuclear leukocytes were identifiedusing various mAb toT cells and macrophages, and studied for their expression of receptorsfor interleukin-2 (IL-2R). Positive resultsare summarizedIn addition, studies of the mononuclearcells adjacent to tumors in 12/12 casesshowed the presence of tumor-associated macrophages, 10/11 showed T cells,mainly T8+, and A/5 showed corresponding expression of IL-2R, suggesting cell activation.The use of highly specific mAb showed that XL-FB is actually more selectively distributed than is found using polyclonal antisera to FGN/FB, and indeed XL-FB was largely confined to those areas adjacent to tumors which are rich in mononuclearcells. These findings suggest that fibrin deposits in human carcinomasof the lung may be due todevelopment of PCA by activated host mononuclear cells, rather than tumor cells.This lack of XL-FB on tumor cellsinspite of A1- 3 binding suggests that TF:RAg may not be available on the tumor cell surface for the activation of clotting. Further studies are neededto define the functional capacity of PCA molecules on tumor cells and tumor-associated mononuclear cells in situ.
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Zhang, Xue-Qing, Mark Chen, Robert Lam, Xiaoyang Xu, Eiji Osawa y Dean Ho. "A Platform Approach to Gene Delivery via Surface Modified Nanodiamonds". En ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology. ASMEDC, 2010. http://dx.doi.org/10.1115/nemb2010-13340.
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The purpose of gene therapy is to introduce foreign genetic material into host cells to either supplement aberrant genes or to endow additional biological functions. To date, however, there has been only modest progress towards this goal, mainly due to the lack of safe, effective and broadly applicable delivery methods. Functional nanodiamonds (NDs) are rapidly emerging as promising platform carriers for next-generation therapeutics due to their innate biocompatibility, scalability, precise particle distribution, high surface area-to-volume ratio, near-spherical aspect ratio, and easily adaptable carbon surface for bioagent attachment. NDs have been functionalized with a range of therapeutics, proteins, antibodies, DNA, polymers, and other assorted biological agents. Furthermore, NDs are stable and dispersible in water, making them a promising and clinically important modality in improving the efficacy of the treatment of diseases and even some cancers at the molecular level. Mitochondrial function (MTT) and luminescent ATP production assays have demonstrated that NDs are not toxic to a wide variety of cell types. In this study, we functionalized NDs with amine groups via either covalent attachment of (3-aminopropyl) trimethoxysilane or surface immobilization of 800 Da low molecular weight polyethyleneimine (LMW PEI800) for plasmid DNA delivery. The latter delivery approach combines complementary characteristics of PEI800 and NDs to create a hybrid material that exhibits the high transfection efficiency of high molecular weight PEI, but without the inherent high cytotoxicity.
Informes sobre el tema "Host Cell Proteins (HCP)"
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Coplin, David L., Shulamit Manulis y Isaac Barash. roles Hrp-dependent effector proteins and hrp gene regulation as determinants of virulence and host-specificity in Erwinia stewartii and E. herbicola pvs. gypsophilae and betae. United States Department of Agriculture, junio de 2005. http://dx.doi.org/10.32747/2005.7587216.bard.
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Gram-negative plant pathogenic bacteria employ specialized type-III secretion systems (TTSS) to deliver an arsenal of pathogenicity proteins directly into host cells. These secretion systems are encoded by hrp genes (for hypersensitive response and pathogenicity) and the effector proteins by so-called dsp or avr genes. The functions of effectors are to enable bacterial multiplication by damaging host cells and/or by blocking host defenses. We characterized essential hrp gene clusters in the Stewart's Wilt of maize pathogen, Pantoea stewartii subsp. stewartii (Pnss; formerly Erwinia stewartii) and the gall-forming bacterium, Pantoea agglomerans (formerly Erwinia herbicola) pvs. gypsophilae (Pag) and betae (Pab). We proposed that the virulence and host specificity of these pathogens is a function of a) the perception of specific host signals resulting in bacterial hrp gene expression and b) the action of specialized signal proteins (i.e. Hrp effectors) delivered into the plant cell. The specific objectives of the proposal were: 1) How is the expression of the hrp and effector genes regulated in response to host cell contact and the apoplastic environment? 2) What additional effector proteins are involved in pathogenicity? 3) Do the presently known Pantoea effector proteins enter host cells? 4) What host proteins interact with these effectors? We characterized the components of the hrp regulatory cascade (HrpXY ->7 HrpS ->7 HrpL ->7 hrp promoters), showed that they are conserved in both Pnss and Fag, and discovered that the regulation of the hrpS promoter (hrpSp) may be a key point in integrating apoplastic signals. We also analyzed the promoters recognized by HrpL and demonstrated the relationship between their composition and efficiency. Moreover, we showed that promoter strength can influence disease expression. In Pnss, we found that the HrpXY two-component signal system may sense the metabolic status of the bacterium and is required for full hrp gene expression in planta. In both species, acyl-homoserine lactone-mediated quorum sensing may also regulate epiphytic fitness and/or pathogenicity. A common Hrp effector protein, DspE/WtsE, is conserved and required for virulence of both species. When introduced into corn cells, Pnss WtsE protein caused water-soaked lesions. In other plants, it either caused cell death or acted as an Avr determinant. Using a yeast- two-hybrid system, WtsE was shown to interact with a number of maize signal transduction proteins that are likely to have roles in either programmed cell death or disease resistance. In Pag and Pab, we have characterized the effector proteins HsvG, HsvB and PthG. HsvG and HsvB are homologous proteins that determine host specificity of Pag and Pab on gypsophila and beet, respectively. Both possess a transcriptional activation domain that functions in yeast. PthG was found to act as an Avr determinant on multiple beet species, but was required for virulence on gypsophila. In addition, we demonstrated that PthG acts within the host cell. Additional effector genes have been characterized on the pathogenicity plasmid, pPATHₚₐg, in Pag. A screen for HrpL- regulated genes in Pnsspointed up 18 candidate effector proteins and four of these were required for full virulence. It is now well established that the virulence of Gram-negative plant pathogenic bacteria is governed by Hrp-dependent effector proteins. However; the mode of action of many effectors is still unresolved. This BARD supported research will significantly contribute to the understanding of how Hrp effectors operate in Pantoea spp. and how they control host specificity and affect symptom production. This may lead to novel approaches for genetically engineering plants resistant to a wide range of bacterial pathogens by inactivating the Hrp effectors with "plantabodies" or modifying their receptors, thereby blocking the induction of the susceptible response. Alternatively, innovative technologies could be used to interfere with the Hrp regulatory cascade by blocking a critical step or mimicking plant or quorum sensing signals.
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Coplin, David, Isaac Barash y Shulamit Manulis. Role of Proteins Secreted by the Hrp-Pathways of Erwinia stewartii and E. herbicola pv. gypsophilae in Eliciting Water-Soaking Symptoms and Initiating Galls. United States Department of Agriculture, junio de 2001. http://dx.doi.org/10.32747/2001.7580675.bard.
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Many bacterial pathogens of plants can inject pathogenicity proteins into host cells using a specialized type III secretion system encoded by hrpgenes. This system deliver effector proteins, into plant cells that function in both susceptible and resistant interactions. We have found that the virulence of Erwinia stewartii(Es; syn. Pantoea stewartii) and Erwinia herbicola pv. gypsophilae (Ehg, syn. Pantoea agglomerans), which cause Stewart's wilt of corn and galls on Gypsophila, respectively, depends on hrpgenes. The major objectives of this project were: To increase expression of hrpgenes in order to identify secreted proteins; to identify genes for proteins secreted by the type-III systems and determine if they are required for pathogenicity; and to determine if the secreted proteins can function within eukaryotic cells. We found that transcription of the hrp and effector genes in Es and Ehg is controlled by at least four genes that constitute a regulatory cascade. Environmental and/or physiological signaling appears to be mediated by the HrpX/HrpY two component system, with HrpX functioning as a sensor-kinase and HrpY as a response regulator. HrpYupregulateshrpS, which encodes a transcriptional enhancer. HrpS then activates hrpL, which encodes an alternate sigma factor that recognizes "hrp boxes". All of the regulatory genes are essential for pathogenicity, except HrpX, which appears only to be required for induction of the HR in tobacco by Es. In elucidating this regulatory pathway in both species, we made a number of significant new discoveries. HrpX is unusual for a sensor-kinase because it is cytoplasmic and contains PAS domains, which may sense the redox state of the bacterium. In Es, a novel methyl-accepting protein may function upstream of hrpY and repress hrp gene expression in planta. The esaIR quorum sensing system in Es represses hrp gene expression in Es in response to cell-density. We have discovered six new type III effector proteins in these species, one of which (DspE in Ehg and WtsE in Es) is common to both pathogens. In addition, Es wtsG, which is a homolog of an avrPpiB from P. syringae pv. pisi, and an Ehg ORF, which is a homolog of P. syringae pv. phaseolicola AvrPphD, were both demonstrated to encode virulence proteins. Two plasmidborne, Ehg Hop proteins, HsvG and PthG, are required for infection of gypsophilia, but interestingly, PthG also acts as an Avr elicitor in beets. Using a calmodulin-dependent adenylate cyclase (cyaA) reporter gene, we were successful in demonstrating that an HsvG-CyaA fusion protein can be transferred into human HeLa cells by the type-III system of enteropathogenic E. coli. This is a highly significant accomplishment because it is the first direct demonstration that an effector protein from a plant pathogenic bacterium is capable of being translocated into a eukaryotic cell by a type-III secretion system. Ehg is considered a limiting factor in Gypsophila production in Israel and Stewart’s Wilt is a serious disease in the Eastern and North Central USA, especially on sweet corn in epidemic years. We believe that our basic research on the characterization of type III virulence effectors should enable future identification of their receptors in plant cells. This may lead to novel approaches for genetically engineering resistant plants by modifying their receptors or inactivating effectors and thus blocking the induction of the susceptible response. Alternatively, hrp gene regulation might also provide a target for plant produced compounds that interfere with recognition of the host by the pathogen. Such strategies would be broadly applicable to a wide range of serious bacterial diseases on many crops throughout the USA and Israel.
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Grumet, Rebecca y Benjamin Raccah. Identification of Potyviral Domains Controlling Systemic Infection, Host Range and Aphid Transmission. United States Department of Agriculture, julio de 2000. http://dx.doi.org/10.32747/2000.7695842.bard.
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Potyviruses form one of the largest and most economically important groups of plant viruses. Individual potyviruses and their isolates vary in symptom expression, host range, and ability to overcome host resistance genes. Understanding factors influencing these biological characteristics is of agricultural importance for epidemiology and deployment of resistance strategies. Cucurbit crops are subject to severe losses by several potyviruses including the highly aggressive and variable zucchini yellow mosaic virus (ZYMV). In this project we sought to investigate protein domains in ZYMV that influence systemic infection and host range. Particular emphasis was on coat protein (CP), because of known functions in both cell to cell and long distance movement, and helper component-protease (HC-Pro), which has been implicated to play a role in symptom development and long distance movement. These two genes are also essential for aphid mediated transmission, and domains that influence disease development may also influence transmissibility. The objectives of the approved BARD project were to test roles of specific domains in the CP and HC-Pro by making sequence alterations or switches between different isolates and viruses, and testing for infectivity, host range, and aphid transmissibility. These objectives were largely achieved as described below. Finally, we also initiated new research to identify host factors interacting with potyviral proteins and demonstrated interaction between the ZYMV RNA dependent RNA polymerase and host poly-(A)-binding protein (Wang et al., in press). The focus of the CP studies (MSU) was to investigate the role of the highly variable amino terminus (NT) in host range determination and systemic infection. Hybrid ZYMV infectious clones were produced by substituting the CP-NT of ZYMV with either the CP-NT from watermelon mosaic virus (overlapping, but broader host range) or tobacco etch virus (TEV) (non- overlapping host range) (Grumet et al., 2000; Ullah ct al., in prep). Although both hybrid viruses initially established systemic infection, indicating that even the non-cucurbit adapted TEV CP-NT could facilitate long distance transport in cucurbits, after approximately 4-6, the plants inoculated with the TEV-CPNT hybrid exhibited a distinct recovery of reduced symptoms, virus titer, and virus specific protection against secondary infection. These results suggest that the plant recognizes the presence of the TEV CP-NT, which has not been adapted to infection of cucurbits, and initiates defense responses. The CP-NT also appears to play a role in naturally occurring resistance conferred by the zym locus in the cucumber line 'Dina-1'. Patterns of virus accumulation indicated that expression of resistance is developmentally controlled and is due to a block in virus movement. Switches between the core and NT domains of ZYMV-NAA (does not cause veinal chlorosis on 'Dina-1'), and ZYMV-Ct (causes veinal chlorosis), indicated that the resistance response likely involves interaction with the CP-NT (Ullah and Grumet, submitted). At the Volcani Center the main thrust was to identify domains in the HC-Pro that affect symptom expression or aphid transmissibility. From the data reported in the first and second year report and in the attached publications (Peng et al. 1998; Kadouri et al. 1998; Raccah et al. 2000: it was shown that: 1. The mutation from PTK to PAK resulted in milder symptoms of the virus on squash, 2. Two mutations, PAK and ATK, resulted in total loss of helper activity, 3. It was established for the first time that the PTK domain is involved in binding of the HC-Pro to the potyvirus particle, and 4. Some of these experiments required greater amount of HC-Pro, therefore a simpler and more efficient purification method was developed based on Ni2+ resin.
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Blum, Abraham, Henry T. Nguyen y N. Y. Klueva. The Genetics of Heat Shock Proteins in Wheat in Relation to Heat Tolerance and Yield. United States Department of Agriculture, agosto de 1993. http://dx.doi.org/10.32747/1993.7568105.bard.
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Fifty six diverse spring wheat cultivars were evaluated for genetic variation and heritability for thermotolerance in terms of cell-membrane stability (CMS) and triphenyl tetrazolium chloride (TTC) reduction. The most divergent cultivars for thermotolerance (Danbata-tolerant and Nacozari-susceptible) were crossed to develop an F8 random onbred line (RIL) population. This population was evaluated for co-segragation in CMS, yield under heat stress and HSP accumulation. Further studies of thermotolerance in relations to HSP and the expression of heterosis for growth under heat stress were performed with F1 hybrids of wheat and their parental cultivars. CMS in 95 RILs ranged from 76.5% to 22.4% with 71.5% and 31.3% in Danbata and Nacozari, respectively. The population segregated with a normal distribution across the full range of the parental values. Yield and biomass under non-stress conditions during the normal winter season at Bet Dagan dit not differ between the two parental cultivar, but the range of segregation for these traits in 138 RILs was very high and distinctly transgressive with a CV of 35.3% and 42.4% among lines for biomass and yield, respectively. Mean biomass and yield of the population was reduced about twofold when grown under the hot summer conditions (irrigated) at Bet Dagan. Segregation for biomass and yield was decreased relative to the normal winter conditions with CV of 20.2% and 23.3% among lines for biomass and yield, respectively. However, contrary to non-stress conditions, the parental cultivars differed about twofold in biomass and yield under heat stress and the population segregated with normal distribution across the full range of this difference. CMS was highly and positively correlated across 79 RILs with biomass (r=0.62**) and yield (r=0.58**) under heat stress. No such correlation was obtained under the normal winter conditions. All RILs expressed a set of HSPs under heat shock (37oC for 2 h). No variation was detected among RILs in high molecular weight HSP isoforms and they were similar to the patterns of the parental cultivars. There was a surprisingly low variability in low molecular weight HSP isoforms. Only one low molecular weight and Nacozari-specific HSP isoform (belonging to HSP 16.9 family) appeared to segregate among all RILs, but it was not quantitatively correlated with any parameter of plant production under heat stress or with CMS in this population. It is concluded that this Danbata/Nacozari F8 RIL population co-segregated well for thermotolerance and yield under heat stress and that CMS could predict the relative productivity of lines under chronic heat stress. Regretfully this population did not express meaningful variability for HSP accumulation under heat shock and therefore no role could be seen for HSP in the heat tolerance of this population. In the study of seven F1 hybrids and their parent cultivars it was found that heterosis (superiority of the F1 over the best parent) for CMs was generally lower than that for growth under heat stress. Hybrids varied in the rate of heterosis for growth at normal (15o/25o) and at high (25o/35o) temperatures. In certain hybrids heterosis for growth significantly increased at high temperature as compared with normal temperature, suggesting temperature-dependent heterosis. Generally, under normal temperature, only limited qualitative variation was detected in the patterns of protein synthesis in four wheat hybrids and their parents. However, a singular protein (C47/5.88) was specifically expressed only in the most heterotic hybrid at normal temperature but not in its parent cultivars. Parental cultivars were significantly different in the sets of synthesized HSP at 37o. No qualitative changes in the patterns of protein expression under heat stress were correlated with heterosis. However, a quantitative increase in certain low molecular weight HSP (mainly H14/5.5 and H14.5.6, belonging to the HSP16.9 family) was positively associated with greater heterosis for growth at high temperature. None of these proteins were correlated with CMS across hybrids. These results support the concept of temperature-dependent heterosis for growth and a possible role for HSP 16.9 family in this respect. Finally, when all experiments are viewed together, it is encouraging to find that genetic variation in wheat yield under chronic heat stress is associated with and well predicted by CMS as an assay of thermotolerance. On the other hand the results for HSP are elusive. While very low genetic variation was expressed for HSP in the RIL population, a unique low molecular weight HSP (of the HSP 16.9 family) could be associated with temperature dependant heterosis for growth.
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Tzfira, Tzvi, Michael Elbaum y Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, diciembre de 2005. http://dx.doi.org/10.32747/2005.7695881.bard.
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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.
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Dickman, Martin B. y Oded Yarden. Characterization of the chorismate mutase effector (SsCm1) from Sclerotinia sclerotiorum. United States Department of Agriculture, enero de 2015. http://dx.doi.org/10.32747/2015.7600027.bard.
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Sclerotinia sclerotiorum is a filamentous fungus (mold) that causes plant disease. It has an extremely wide range of hosts (>400 species) and causes considerable damage (annual multimillion dollar losses) in economically important crops. It has proven difficult to control (culturally or chemically) and host resistance to this fungus has generally been inadequate. It is believed that this fungus occurs in almost every country. Virulence of this aggressive pathogen is bolstered by a wide array of plant cell wall degrading enzymes and various compounds (secondary metabolites) produced by the fungus. It is well established that plant pathogenic fungi secrete proteins and small molecules that interact with host cells and play a critical role in disease development. Such secreted proteins have been collectively designated as “effectors”. Plant resistance against some pathogens can be mediated by recognition of such effectors. Alternatively, effectors can interfere with plant defense. Some such effectors are recognized by the host plant and can culminate in a programmed cell death (PCD) resistant response. During the course of this study, we analyzed an effector in Sclerotiniasclerotiorum. This specific effector, SsCM1 is the protein chorismatemutase, which is an enzyme involved in a pathway which is important in the production of important amino acids, such a Tryptophan. We have characterized the Sclerotiniaeffector, SsCM1, and have shown that inactivation of Sscm1 does not affect fungal vegetative growth, development or production of oxalic acid (one of this fungus’ secondary metabolites associated with disease) production. However, yhis does result in reduced fungal virulence. We show that, unexpectedly, the SsCM1 protein translocates to the host chloroplast, and demonstrated that this process is required for full fungal virulence. We have also determined that the fungal SsCM1 protein can interact with similar proteins produced by the host. In addition, we have shown that the fungal SsCM1 is able to suppress at least some of the effects imposed by reactive oxygen species which are produced as a defense mechanism by the host. Last, but not least, the results of our studies have provided evidence contradicting the current dogma on at least some of the mechanist aspects of how this pathogen infects the host. Contrary to previousons, indicating that this pathogen kills its host by use of metabolites and enzymes that degrade the host tissue (a process called necrotrophy), we now know that at least in the early phases of infection, the fungus interacts with live host tissue (a phenomenon known as biotrophy). Taken together, the results of our studies provide novel insights concerning the mechanistic aspects of Sclerotinia-host interactions. We hope this information will be used to interfere with the disease cycle in a manner that will protect plants from this devastating fungus.
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Banai, Menachem y Gary Splitter. Molecular Characterization and Function of Brucella Immunodominant Proteins. United States Department of Agriculture, julio de 1993. http://dx.doi.org/10.32747/1993.7568100.bard.
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The BARD project was a continuation of a previous BARD funded research project. It was aimed at characterization of the 12kDa immunodominant protein and subsequently the cloning and expression of the gene in E. coli. Additional immunodominant proteins were sought among genomic B. abortus expression library clones using T-lymphocyte proliferation assay as a screening method. The 12kDa protein was identified as the L7/L12 ribosomal protein demonstrating in the first time the role a structural protein may play in the development of the host's immunity against the organism. The gene was cloned from B. abortus (USA) and B. melitensis (Israel) showing identity of the oligonucleotide sequence between the two species. Further subcloning allowed expression of the protein in E. coli. While the native protein was shown to have DTH antigenicity its recombinant analog lacked this activity. In contrast the two proteins elicited lymphocyte proliferation in experimental murine brucellosis. CD4+ cells of the Th1 subset predominantly responded to this protein demonstrating the development of protective immunity (g-IFN, and IL-2) in the host. Similar results were obtained with bovine Brucella primed lymphocytes. UvrA, GroE1 and GroEs were additional Brucella immunodominant proteins that demonstrated MHC class II antigenicity. The role cytotoxic cells are playing in the clearance of brucella cells was shown using knock out mice defective either in their CD4+ or CD8+ cells. CD4+ defective mice were able to clear brucella as fast as did normal mice. In contrast mice which were defective in their CD8+ cells could not clear the organisms effectively proving the importance of this subtype cell line in development of protective immunity. The understanding of the host's immune response and the expansion of the panel of Brucella immunodominant proteins opened new avenues in vaccine design. It is now feasible to selectively use immunodominant proteins either as subunit vaccine to fortify immunity of older animals or as diagnostic reagents for the serological survaillance.
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Citovsky, Vitaly y Yedidya Gafni. Viral and Host Cell Determinants of Nuclear Import and Export of the Tomato Yellow Leaf Curl Virus in Tomato Plants. United States Department of Agriculture, agosto de 2002. http://dx.doi.org/10.32747/2002.7585200.bard.
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Tomato yellow leaf curl geminivirus (TYLCV) is a major pathogen of cultivated tomato, causing up to 100% crop loss in many parts of the world. In Israel, where TYLCV epidemics have been recorded since the 1960' s, this viral disease is well known and has been of economic significance ever since. In recent years, TYLCV outbreaks also occurred in the "New World" - Cuba, The Dominican Republic, and in the USA, in Florida, Georgia and Louisiana. Thus, TYLCV substantially hinders tomato growth throughout the world. Surprisingly, however, little is known about the molecular mechanisms of TYLCV interaction with the host tomato cells. The present proposal, a continuation of the project supported by BARD from 1994, expanded our understanding of the molecular mechanisms by which TYLCV enters the host cell nucleus for replication and transcription and exits it for the subsequent cell-to-cell spread. Our project sought two objectives: I. To study the roles of the viral capsid protein (CP) and host cell factors in TYLCV nuclear import. II. To study the roles of CP and host cell factors in TYLCV nuclear export. Our research toward these goals have produced the following major achievements: . Developed a one-hybrid assay for protein nuclear export and import (#3 in the List of Publications). . Identified a functional nuclear export signal (NES) in the capsid protein (CP) of TYLCV (#3 in the List of Publications). . Discovered homotypic interactions between intact TYLCV CP molecules and analyzed these interactions using deletion mutagenesis of TYLCV CP (#5 in the List of Publications). . Showed developmental and tissue-specific expression of the host factor required for nuclear import of TYLCV CP, tomato karyopherin alpha 1, in transgenic tomato plants (#14 in the List of Publications). . By analogy to nuclear import of TYLCV ,identified an Arabidopsis VIPI protein that participates in nuclear import of Agrobacterium T -complexes via the karyopherin alpha pathway (#4,6, and 8 in the List of Publications). These research findings provided significant insights into (i) the molecular pathway of TYLCV entry into the host cell nucleus, and (ii) the mechanism by which TYLCV is exported from the nucleus for the cell-to-cell spread of infection. Furthermore, the obtained knowledge will help to develop specific strategies to attenuate TYLCV infection, for example, by blocking viral entry into and/or exit out of the host cell nucleus. Also, as much of our findings is relevant to all geminiviruses, new anti- TYLCV approaches developed based on the results of our research will be useful to combat other members of the Geminivirus family. Finally, in addition to the study of TYLCV nuclear import and export, our research contributed to our understanding of general mechanisms for nucleocytoplasmic shuttling of proteins and nucleic acids in plant cells.
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Sadot, Einat, Christopher Staiger y Zvi Kam Weizmann. functional genomic screen for new plant cytoskeletal proteins and the determination of their role in actin mediated functions and guard cells regulation. United States Department of Agriculture, enero de 2003. http://dx.doi.org/10.32747/2003.7587725.bard.
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The original objectives of the approved proposal were: 1. To construct a YFP fused Arabidopsis cDNA library in a mammalian expression vector. 2. To infect the library into a host fibroblast cell line and to screen for new cytoskeletal associated proteins using an automated microscope. 3. Isolate the new genes. 4. Characterize their role in plants. The project was approved as a feasibility study to allow proof of concept that would entail building the YFP library and picking up a couple of positive clones using the fluorescent screen. We report here on the construction of the YFP library, the development of the automatic microscope, the establishment of the screen and the isolation of positive clones that are plant cDNAs encoding cytoskeleton associated proteins. The rational underling a screen of plant library in fibroblasts is based on the high conservation of the cytoskeleton building blocks, actin and tubulin, between the two kingdoms (80-90% homology at the level of amino acids sequence). In addition, several publications demonstrated the recognition of mammalian cytoskeleton by plant cytoskeletal binding proteins and vice versa. The major achievements described here are: 1. The development of an automated microscope equipped with fast laser auto-focusing for high magnification and a software controlling 6 dimensions; X, Y position, auto focus, time, color, and the distribution and density of the fields acquired. This system is essential for the high throughput screen. 2. The construction of an extremely competent YFP library efficiently cloned (tens of thousands of clones collected, no empty vectors detected) with all inserts oriented 5't03'. These parameters render it well representative of the whole transcriptome and efficient in "in-frame" fusion to YFP. 3. The strategy developed for the screen allowing the isolation of individual positive cDNA clones following three rounds of microscopic scans. The major conclusion accomplished from the work described here is that the concept of using mammalian host cells for fishing new plant cytoskeletal proteins is feasible and that screening system developed is complete for addressing one of the major bottlenecks of the plant cytoskeleton field: the need for high throughput identification of functionally active cytoskeletal proteins. The new identified plant cytoskeletal proteins isolated in the pilot screen and additional new proteins which will be isolated in a comprehensive screen will shed light on cytoskeletal mediated processes playing a major role in cellular activities such as cell division, morphogenesis, and functioning such as chloroplast positioning, pollen tube and root hair elongation and the movement of guard cells. Therefore, in the long run the screen described here has clear agricultural implications.
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Epel, Bernard y Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, noviembre de 2005. http://dx.doi.org/10.32747/2005.7695874.bard.
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To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.