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Use of transient expression for the rapid, large-scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale-up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha-1-antitrypsin (rAAT) by co-infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 +/- 8.7)% and (290 +/- 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD(600) of 0.5 and a 0.25-min vacuum infiltration, and multiple 1-min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 +/- 0.09)% and (4.1 +/- 0.29)% of the total soluble protein, respectively, or (90 +/- 1.7) and (140 +/- 10) mg per kg fresh weight leaf tissue at 6 days post-induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale-up.
Use of transient expression for the rapid, large-scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale-up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha-1-antitrypsin (rAAT) by co-infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 +/- 8.7)% and (290 +/- 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD(600) of 0.5 and a 0.25-min vacuum infiltration, and multiple 1-min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 +/- 0.09)% and (4.1 +/- 0.29)% of the total soluble protein, respectively, or (90 +/- 1.7) and (140 +/- 10) mg per kg fresh weight leaf tissue at 6 days post-induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale-up.
Abstract. Potential inhalation exposure of agricultural workers and bystanders to aerosolized particles emitted by typical agricultural spray nozzles is influenced by the particle size distribution (PSD) of the spray. However, inhalation risk assessments do not currently factor in the human-relevant PSD that may be inhaled during pesticide handling activities. This study was conducted to characterize the PSD of aerosols collected with OSHA Versatile Sampler (OVS) tubes, which are monitoring devices commonly used for inhalation risk assessment in worker exposure studies. An Oxford Lasers N60V particle size analyzer was used for characterizing the spray PSD emitted from various agricultural nozzles. Side-by-side air sampling with OVS tubes and Respicon TM particle samplers was conducted to characterize the size distribution of aerosols collected on the OVS tubes during spraying of a diluted chlorothalonil formulation. Based on this comparison, OVS tubes captured the inhalable fraction (mass median diameter (D50) = 100 µm), with approximately 40% of the total inhalation concentration contributing to systemic exposure (D50 = 10 µm) regardless of nozzle spray quality. In addition, nozzles with fine and medium spray produced higher airborne concentrations compared to nozzles with coarse spray. Thus, the use of modern low-drift nozzles (e.g., air-induction nozzles) that emit larger spray droplets can substantially reduce the airborne concentration levels within inhalable particle size fractions. While the concentrations within these airway fractions (e.g., respirable, thoracic, inhalable) increased from extremely coarse to very fine spray nozzles, the relative proportion of each fraction within the total inhalable concentration remained constant regardless of spray quality or nozzle type. Such information on the PSD of pesticide applications can be used to refine inhalation risk assessments for agricultural workers and bystanders. Keywords: Chlorothalonil, Exposure monitoring, Inhalation exposure, Particle size distribution, OVS tube, Respicon TM particle sampler.
-The objective of this work was to develop an experimental kit for assessments of repellency, deterrence for oviposition, and insecticidal activity on adults of the whitefly Bemisia tabaci biotype B. The kit, which consisted of arenas and nebulizer, was effective for conducting bioassays, and the application of aqueous extracts by inhaler was adequate. The techniques are simple, cheap, and may contribute to research on this insect.
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