ABSTRACT
Chagas disease is caused by the parasite Trypanosoma cruzi, which is transmitted by insect-vectors in the taxonomic subfamily Triatominae and affects approximately 8,000,000 people world-wide. Current mitigation strategies for Chagas focus on insecticides, infrastructure improvements, and management of symptoms, which are largely unsustainable in underserved communities where the disease is widespread. Transmission patterns of vector-borne diseases are known to adaptively respond to habitat change; as such, the objective of our study was to evaluate how the physical characteristics of Triatoma dimidiata would vary in relation to land use in El Salvador. We hypothesized that the color and morphology of T. dimidiata would change with municipal levels of urban and natural green space, natural green space, and agricultural space, as well as municipal diversity, richness, and evenness of land use types. Our results characterize how T. dimidiata color and morphology vary directly with anthropogenic changes to natural and agricultural environments, which are reflective of a highly adaptable population primed to respond to environmental change. Mitigation studies of Chagas disease should exploit the relationships between anthropogenic land use and T. dimidiata morphology to evaluate how the transmission pattern of T. cruzi and Chagas disease symptomology are impacted.
ABSTRACT
Course-based undergraduate research experience (CURE) courses incorporate high-impact pedagogies that have been shown to increase undergraduate retention among underrepresented minorities and women. As part of the Building Infrastructure Leading to Diversity program at the University of Detroit Mercy, a CURE metagenomics course was established in the winter of 2019. Students investigated the bacterial community composition in a eutrophic cove in Lake Saint Clair (Harrison Township, MI, United States) from water samples taken in the summer and winter. The students created 16S rRNA libraries that were sequenced using next-generation sequencing technology. They used a public web-based supercomputing resource to process their raw sequencing data and web-based tools to perform advanced statistical analysis. The students discovered that the most common operational taxonomic unit, representing 31% of the prokaryotic sequences in both summer and winter samples, corresponded to an organism that belongs to a previously unidentified phylum. This result showed the students the power of metagenomics because the approach was able to detect unclassified organisms. Principal Coordinates Analysis of Bray-Curtis dissimilarity index data showed that the winter community was distinct from the summer community [Analysis of Similarities (ANOSIM) r = 0.59829, n = 18, and p < 0.001]. Dendrograms based on hierarchically clustered Pearson correlation coefficients of phyla were divided into a winter clade and a summer clade. The conclusion is that the winter bacterial population was fundamentally different from the summer population, even though the samples were taken from the same locations in a protected cove. Because of the small class sizes, qualitative as well as statistical methods were used to evaluate the course's impact on student attitudes. Results from the Laboratory Course Assessment Survey showed that most of the respondents felt they were contributing to scientific knowledge and the course fostered student collaboration. The majority of respondents agreed or strongly agreed that the course incorporated iteration aspects of scientific investigations, such as repeating procedures to fix problems. In summary, the metagenomics CURE course was able to add to scientific knowledge and allowed students to participate in authentic research.
ABSTRACT
The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker's yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain's cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale.IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.
