Peremyslov V.V.,Oregon State University |
Prokhnevsky A.I.,Fraunhofer Institute for Molecular Biotechnology |
Dolja V.V.,Oregon State University
Plant Cell | Year: 2010
The actomyosin system is conserved throughout eukaryotes. Although F-actin is essential for cell growth and plant development, roles of the associated myosins are poorly understood. Using multiple gene knockouts in Arabidopsis thaliana, we investigated functional profiles of five class XI myosins, XI-K, XI-1, XI-2, XI-B, and XI-I. Plants lacking three myosins XI showed stunted growth and delayed flowering, whereas elimination of four myosins further exacerbated these defects. Loss of myosins led to decreased leaf cell expansion, with the most severe defects observed in the larger leaf cells. Root hair length in myosin-deficient plants was reduced ∼10-fold, with quadruple knockouts showing morphological abnormalities. It was also found that trafficking of Golgi and peroxisomes was entirely myosin dependent. Surprisingly, myosins were required for proper organization of F-actin and the associated endoplasmic reticulum networks, revealing a novel, architectural function of the class XI myosins. These results establish critical roles of myosin-driven transport and F-actin organization during polarized and diffuse cell growth and indicate that myosins are key factors in plant growth and development. © 2010 American Society of Plant Biologists.
Licona-Limon P.,Yale University |
Henao-Mejia J.,Yale University |
Temann A.U.,Fraunhofer Institute for Molecular Biotechnology |
Gagliani N.,Yale University |
And 7 more authors.
Immunity | Year: 2013
Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4+ effector Tcells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection. © 2013 Elsevier Inc.
Kador K.E.,University of Nebraska - Lincoln |
Kador K.E.,University of Miami |
Mamedov T.G.,University of Nebraska - Lincoln |
Mamedov T.G.,Fraunhofer Institute for Molecular Biotechnology |
And 2 more authors.
Acta Biomaterialia | Year: 2011
In an effort to control the surface-mediated activation of thrombin and clot formation, proteins and molecules which mimic the anticoagulant properties of the vascular endothelial lining were immobilized on material surfaces. When immobilized on biomaterial surfaces, thrombomodulin (TM), an endothelial glycoprotein that binds thrombin and activates protein C (PC), was shown to generate activated PC (APC) and delay clot formation. However, TM-mediated activation of PC on biomaterial surfaces was shown to be limited by the transport of PC to the surface, with maximum activation obtained at a surface density of ∼40 fmole TM cm-2. This work investigates surface immobilized with TM and endothelial protein C receptor (EPCR), a natural cofactor to TM which increases the rate of activation of PC on the native endothelium. A sequential and ordered immobilization of TM and EPCR on polyurethane at an enzymatically relevant distance (<10 nm) resulted in higher amounts of APC compared with surfaces with immobilized TM or with TM and EPCR immobilized randomly and at TM surface densities (1400 fmole cm -2) which were previously shown to be transport limited. Ordered TM and EPCR samples also showed increased time to clot formation in experiments with platelet-poor plasma, as measured by thromboelastography. Surfaces immobilized with TM and its natural cofactor EPCR at an enzymatically relevant distance are able to overcome transport limitations, increasing anticoagulant activation and time to clot formation. © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Seid C.A.,Baylor College of Medicine |
Seid C.A.,The Texas Institute |
Curti E.,Baylor College of Medicine |
Curti E.,The Texas Institute |
And 20 more authors.
Human Vaccines and Immunotherapeutics | Year: 2015
Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamianawas evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purifiedNa-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP. © 2015, Christopher A Seid, Elena Curti, R Mark Jones, Elissa Hudspeth, Wanderson Rezende, Jeroen Pollet, Lori Center, Leroy Versteeg, Sonya Pritchard, Konstantin Musiychuk, Vidadi Yusibov, Peter J Hotez, and Maria Elena Bottazzi.
Zhu C.,PATH |
Shoji Y.,Fraunhofer Institute for Molecular Biotechnology |
McCray S.,Bend Research Inc. |
Burke M.,Bend Research Inc. |
And 6 more authors.
Pharmaceutical Research | Year: 2014
Purpose: Stable vaccines with long shelf lives and reduced dependency on the cold chain are ideal for stockpiling and rapid deployment during public emergencies, including pandemics. Spray drying is a low-cost process that has potential to produce vaccines stable at a wide range of temperatures. Our aim was to develop a stable formulation of a recombinant H1N1 influenza hemagglutinin vaccine candidate and take it to pilot-scale spray-drying production.Methods: Eight formulations containing different excipients were produced and assayed for antigen stability, powder characteristics, and immunogenicity after storage at a range of temperatures, resulting in the identification of four promising candidates. A pilot-scale spray-drying process was then developed for further testing of one formulation.Results: The pilot-scale process was used to reproducibly manufacture three batches of the selected formulation with yields >90%. All batches had stable physical properties and in vitro potency for 6 months at temperatures from -20°C to +50°C. Formulations stored for 3 months elicited immunogenic responses in mice equivalent to a frozen lot of bulk vaccine used as a stability control.Conclusions: This study demonstrates the feasibility of stabilizing subunit vaccines using a spray-drying process and the suitability of the process for manufacturing a candidate product. © 2014 Springer Science+Business Media New York.