Recommendations for Effective Integration of Ethics and Responsible Conduct of Research (E/RCR) Education into Course-Based Undergraduate Research Experiences: A Meeting Report.

Advancement of the scientific enterprise relies on individuals conducting research in an ethical and responsible manner. Educating emergent scholars in the principles of ethics/responsible conduct of research (E/RCR) is therefore critical to ensuring such advancement. The recent impetus to include authentic research opportunities as part of the undergraduate curriculum, via course-based undergraduate research experiences (CUREs), has been shown to increase cognitive and noncognitive student outcomes. Because of these important benefits, CUREs are becoming more common and often constitute the first research experience for many students. However, despite the importance of E/RCR in the research process, we know of few efforts to incorporate E/RCR education into CUREs. The Ethics Network for Course-based Opportunities in Undergraduate Research (ENCOUR) was created to address this concern and promote the integration of E/RCR within CUREs in the biological sciences and related disciplines. During the inaugural ENCOUR meeting, a four-pronged approach was used to develop guidelines for the effective integration of E/RCR in CUREs. This approach included: 1) defining appropriate student learning objectives; 2) identifying relevant curriculum; 3) identifying relevant assessments; and 4) defining key aspects of professional development for CURE facilitators. Meeting outcomes, including the aforementioned E/RCR guidelines, are described herein.

Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation.

The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2-3 fold higher than of GlcCer. Although both activities follow Michaelis-Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production.

Evolution of the metabolic and regulatory networks associated with oxygen availability in two phytopathogenic enterobacteria.

BACKGROUND: Dickeya dadantii and Pectobacterium atrosepticum are phytopathogenic enterobacteria capable of facultative anaerobic growth in a wide range of O2 concentrations found in plant and natural environments. The transcriptional response to O2 remains under-explored for these and other phytopathogenic enterobacteria although it has been well characterized for animal-associated genera including Escherichia coli and Salmonella enterica. Knowledge of the extent of conservation of the transcriptional response across orthologous genes in more distantly related species is useful to identify rates and patterns of regulon evolution. Evolutionary events such as loss and acquisition of genes by lateral transfer events along each evolutionary branch results in lineage-specific genes, some of which may have been subsequently incorporated into the O2-responsive stimulon. Here we present a comparison of transcriptional profiles measured using densely tiled oligonucleotide arrays for two phytopathogens, Dickeya dadantii 3937 and Pectobacterium atrosepticum SCRI1043, grown to mid-log phase in MOPS minimal medium (0.1% glucose) with and without O2. RESULTS: More than 7% of the genes of each phytopathogen are differentially expressed with greater than 3-fold changes under anaerobic conditions. In addition to anaerobic metabolism genes, the O2 responsive stimulon includes a variety of virulence and pathogenicity-genes. Few of these genes overlap with orthologous genes in the anaerobic stimulon of E. coli. We define these as the conserved core, in which the transcriptional pattern as well as genetic architecture are well preserved. This conserved core includes previously described anaerobic metabolic pathways such as fermentation. Other components of the anaerobic stimulon show variation in genetic content, genome architecture and regulation. Notably formate metabolism, nitrate/nitrite metabolism, and fermentative butanediol production, differ between E. coli and the phytopathogens. Surprisingly, the overlap of the anaerobic stimulon between the phytopathogens is also relatively small considering that they are closely related, occupy similar niches and employ similar strategies to cause disease. There are cases of interesting divergences in the pattern of transcription of genes between Dickeya and Pectobacterium for virulence-associated subsystems including the type VI secretion system (T6SS), suggesting that fine-tuning of the stimulon impacts interaction with plants or competing microbes. CONCLUSIONS: The small number of genes (an even smaller number if we consider operons) comprising the conserved core transcriptional response to O2 limitation demonstrates the extent of regulatory divergence prevalent in the Enterobacteriaceae. Our orthology-driven comparative transcriptomics approach indicates that the adaptive response in the eneterobacteria is a result of interaction of core (regulators) and lineage-specific (structural and regulatory) genes. Our subsystems based approach reveals that similar phenotypic outcomes are sometimes achieved by each organism using different genes and regulatory strategies.

Cellular tolerance of prion protein PrP in yeast involves proteolysis and the unfolded protein response.

Secretory proteins undergo a stringent quality control process in the endoplasmic reticulum (ER). Misfolded ER proteins are returned to the cytosol and destroyed by the proteasome. Prion protein PrP is degraded by the proteasome in mammalian cells. However, the significance of proteolysis on PrP-induced cell death is controversial. Moreover, the specific pathway involved in PrP degradation remains unknown. Here, we demonstrate that the unglycosylated form of human PrP is subjected to the ER-associated protein degradation (ERAD) process in the yeast Saccharomyces cerevisiae. We also show that unglycosylated PrP is degraded by the Hrd1-Hrd3 pathway. Accumulation of misfolded proteins triggers the unfolded protein response (UPR), which promotes substrate refolding. Interestingly, we find that the expression of PrP leads to growth impairment in cells deficient in UPR and ERAD. These findings raise the possibility that decreased UPR activity and proteolysis may contribute to the pathogenesis of some prion-related diseases.

The Png1-Rad23 complex regulates glycoprotein turnover.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1-Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.

Analysis of ubiquitin chain-binding proteins by two-hybrid methods.

Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates. Distinct fates are bestowed on multi-Ub chains linked through different lysine residues. Ub contains seven conserved lysines, all of which could be used for multi-Ub chain formation. K29 and K48 are the signals for proteasome-mediated proteolysis. Multi-Ub chains linked through K63 have nonproteolytic functions. Studies of Ub-binding factors are likely the key to understanding diverse functions of the Ub molecule. Yeast two-hybrid assay can be a powerful approach to dissect the interaction between Ub and its binding proteins and also the function of these Ub-chain binding proteins in vivo.

Comparative sequence analysis of IS50/Tn5 transposase.

Comparative sequence analysis of IS50 transposase-related protein sequences in conjunction with known structural, biochemical, and genetic data was used to determine domains and residues that play key roles in IS50 transposase function. BLAST and ClustalW analyses have been used to find and analyze six complete protein sequences that are related to the IS50 transposase. The protein sequence identity of these six homologs ranged from 25 to 55% in comparison to the IS50 transposase. Homologous motifs were found associated with each of the three catalytic residues. Residues that play roles in transposase-DNA binding, protein autoregulation, and DNA hairpin formation were also found to be conserved in addition to other residues of unknown function. On the other hand, some homologous sequences did not appear to be competent to encode the inhibitor regulatory protein. The results were also used to compare the IS50 transposase with the more distantly related transposase encoded by IS10.

Chromosomal deletion formation system based on Tn5 double transposition: use for making minimal genomes and essential gene analysis.

In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes.