Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H(c)): antigen E) by Pichia pastoris

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.     

Using stable isotope analysis to examine fall armyworm (Lepidoptera: Noctuidae) host strains in a cotton habitat

Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), or fall armyworm, is an important agricultural pest of several crops in the Western Hemisphere, including cotton (Gossypium L.). Two morphologically identical host strains of fall armyworm exist that differ in plant host use and habitat distribution. The corn-strain is a primary pest of corn, Zea mays L., whereas the rice-strain is the majority population infesting rice (Oryza spp.) and turfgrass (Cynodon spp.). With the increased use of Bacillus thuringiensis (Bt) toxin-expressing cotton varieties and the necessity of ensuring adequate refuge areas to prevent the spread of Bt toxin resistance, it is crucial to identify the alternative plant hosts available for the fall armyworm population infesting cotton. Stable isotope analysis combined with the molecular analysis of strain-specific markers was used to investigate whether one or both strains routinely develop on cotton grown in the Mississippi delta. We found that the majority of fall armyworm adults present during the early cotton growing season arose from C4 plants (e.g., corn and sorghum, Sorghum vulgare Pers.) and that the only strain likely to be developing on cotton (a C3 plant) in substantial numbers was the corn-strain. The population distribution patterns observed were consistent with corn providing an important refuge for the fall armyworm strain infesting cotton and suggested that late season populations in the Mississippi delta may be migrants from more northern corn areas.     

Comparison of haplotype frequencies differentiate fall armyworm (Lepidoptera: Noctuidae) corn-strain populations from Florida and Brazil

Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a major economic pest throughout the Western Hemisphere. Populations can be subdivided into two morphologically identical but genetically distinct strains (corn-strain and rice-strain) that differ in their host plant preferences. These strains can be distinguished by using polymorphisms in the mitochondrial cytochrome oxidase 1 gene. Additional sequence analysis of this locus identified two sites that were highly polymorphic in the corn-strain population and that produced four different haplotype subgroups. Comparisons of the frequency distribution of these haplotypes found no seasonal or plant host specificities, but they did demonstrate that the Brazil corn-strain population is different from corn-strain fall armyworm found in Florida. The development of a rapid means of distinguishing fall armyworm populations originating from Brazil versus Florida provides an opportunity for investigating and comparing the genetic complexity and long-range movements of this important agricultural pest.     

Class-specific interaction of profilin and ADF isovariants with actin in the regulation of plant development

Two ancient and highly divergent actin-based cytoskeletal systems have evolved in angiosperms. Plant genomes encode complex actin and actin binding protein (ABP) gene families, most of which are phylogenetically grouped into gene classes with distinct vegetative or constitutive and reproductive expression patterns. In Arabidopsis thaliana, ectopic expression of high levels of a reproductive class actin, ACT1, in vegetative tissues causes severe dwarfing of plants with aberrant organization of most plant organs and cell types due to a severely altered actin cytoskeletal architecture. Overexpression of the vegetative class actin ACT2 to similar levels, however, produces insignificant phenotypic changes. We proposed that the misexpression of the pollen-specific ACT1 in vegetative cell types affects the dynamics of actin due to its inappropriate interaction with endogenous vegetative ABPs. To examine the functionally distinct interactions among the major classes of actins and ABPs, we ectopically coexpressed reproductive profilin (PRF4) or actin-depolymerizing factor (ADF) isovariants (e.g., ADF7) with ACT1. Our results demonstrated that the coexpression of these reproductive, but not vegetative, ABP isovariants suppressed the ectopic ACT1 expression phenotypes and restored wild-type stature and normal actin cytoskeletal architecture to the double transgenic plants. Thus, the actins and ABPs appear to have evolved class-specific, protein-protein interactions that are essential to the normal regulation of plant growth and development.     

Costa Rica's 'White legend': how racial narratives undermine its health care system

A dominant cultural narrative within Costa Rica describes Costa Ricans not only as different from their Central American neighbours, but it also exalts them as better: specifically, as more white, peaceful, egalitarian and democratic. This notion of Costa Rican exceptionalism played a key role in the creation of their health care system, which is based on the four core principles of equity, universality, solidarity and obligation. While the political justification and design of the current health care system does, in part, realize this ideal, we argue that the narrative of Costa Rican exceptionalism prevents the full actualization of these principles by marginalizing and excluding disadvantaged groups, especially indigenous and black citizens and the substantial Nicaraguan minority. We offer three suggestions to mitigate the self-undermining effects of the dominant national narrative: 1) encouragement and development of counternarratives; 2) support of an emerging field of Costa Rican bioethics; and 3) decoupling health and national successes.     

Genetic variation in <Emphasis Type="Italic">Delonix s.l.</Emphasis> (Leguminosae) in Madagascar revealed by AFLPs: fragmentation,conservation status and taxonomy

The distribution of genetic diversity has potential to inform conservation efforts but is rarely incorporated when conservation status is assigned to a species. These data can be beneficial to the conservation assessment process by providing information on subpopulations, gene flow and effective population sizes, thus achieving more successful assessments. In order to obtain a better understanding of the patterns of genetic variation and their relationship to conservation in the fragmented flora of Madagascar, this study assessed genetic diversity among and within Delonix s.l. (Leguminosae) using AFLP markers. The genetic diversity of eight species of Delonix s.l. (covering 79 sample sites and 254 individuals) showed a range of values (25–61% for polymorphic loci, and 0.076–0.192 Shannon’s Index). Results from an analysis of molecular variance (AMOVA) suggest that a majority of the genetic variance is attributed to variation within species (87%), which is also supported by a principle coordinate analysis of genetic distances between sites. The results were used to compare the genetic difference between species of different threat status and show that even closely related species with the same IUCN threat status differ in their genetic structure, probably arising from differences in life history traits, pollen and seed dispersal, and fragmentation. Species that are recently affected by habitat destruction and fragmentation are likely to be at high potential risk of genetic erosion contributing to their ongoing decline. Thus, genetic variation should be taken into consideration in conservation assessments, whenever possible, to provide accurate and targeted conservation recommendations in order to achieve more successful conservation outcomes.     

RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata) - a species with color-based sexual selection and 11 visual-opsin genes

Background  

PCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.     

Maximization of production of secreted recombinant proteins in Pichia pastoris fed-batch fermentation

Pontryagin's Maximum Principle has been applied for optimization of secreted proteins from Pichia pastoris fed-batch fermentation. The objective of this work is to maximize the total accumulated product per unit operation time under different given conditions and system constraints. To obtain optimal solutions, an automated curve-fitting software, Table Curve 2D, was employed to construct the necessary mathematical models and solve the complicated functions. In the solution processes, the end of the glycerol batch phase was defined as the initial state of the system, the end of the methanol fed-batch phase as the final state, the cell mass produced along with product accumulated as state variables, and the specific growth rate (mu) as the control variable. Initially, a relationship between the specific production rate (rho) and mu was established. Then, according to Pontryagin's Maximum Principle, the admissible range of mu and its trajectories for the optimal operations were determined. Four representative cases with different combinations of the operation time along with the initial and final states were evaluated. A close correlation was obtained between the predicted values of the model equation with the experimental results from the Pichia pastoris fed-batch fermentations producing secreted alpha-galactosidase. The approaches proposed here greatly simplify the computational processes and validate the optimization strategy as a generalized approach to maximize the yield from fed-batch fermentations.     

Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau

It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.     

New restriction fragment length polymorphisms in the cytochrome oxidase I gene facilitate host strain identification of fall armyworm (Lepidoptera: Noctuidae) populations in the southeastern United States

Several restriction sites in the cytochrome oxidase I gene of fall armyworm, Spodoptera frugiperda (J.E. Smith), were identified by sequence analysis as potentially being specific to one of the two host strains. Strain specificity was demonstrated for populations in Florida, Texas, Mississippi, Georgia, and North Carolina, with an AciI and SacI site specific to the rice (Oryjza spp.)-strain and a BsmI and HinfI site joining an already characterized MspI site as diagnostic of the corn (Zea mays L.)-strain. All four of these sites can be detected by digestion of a single 568-bp polymerase chain reaction-amplified fragment, but the use of two enzymes in separate digests was found to provide accurate and rapid determination of strain identity. The effectiveness of this method was demonstrated by the analysis of almost 200 adult and larval specimens from the Mississippi delta region. The results indicated that the corn-strain is likely to be the primary strain infesting cotton (Gossypium spp.) and that an unexpected outbreak of fall armyworm on the ornamental tree Paulownia tomentosa (Thunb.) Sieb. & Zucc. ex Steud. was due almost entirely to the rice-strain.