A cross-institutional analysis of the effects of broadening trainee professional development on research productivity.

PhD-trained scientists are essential contributors to the workforce in diverse employment sectors that include academia, industry, government, and nonprofit organizations. Hence, best practices for training the future biomedical workforce are of national concern. Complementing coursework and laboratory research training, many institutions now offer professional training that enables career exploration and develops a broad set of skills critical to various career paths. The National Institutes of Health (NIH) funded academic institutions to design innovative programming to enable this professional development through a mechanism known as Broadening Experiences in Scientific Training (BEST). Programming at the NIH BEST awardee institutions included career panels, skill-building workshops, job search workshops, site visits, and internships. Because doctoral training is lengthy and requires focused attention on dissertation research, an initial concern was that students participating in additional complementary training activities might exhibit an increased time to degree or diminished research productivity. Metrics were analyzed from 10 NIH BEST awardee institutions to address this concern, using time to degree and publication records as measures of efficiency and productivity. Comparing doctoral students who participated to those who did not, results revealed that across these diverse academic institutions, there were no differences in time to degree or manuscript output. Our findings support the policy that doctoral students should participate in career and professional development opportunities that are intended to prepare them for a variety of diverse and important careers in the workforce.

Increased filamentous growth of Candida albicans in simulated microgravity.

Knowledge of simulated microgravity (SMG)-induced changes in the pathogenicity of microorganisms is important for success of long-term spaceflight. In a previous study using the high aspect ratio vessel bioreactor, we showed that the yeast species Saccharomyces cerevisiae underwent a significant phenotypic response when grown in modeled microgravity, which was reflected in the analysis of gene expression profiles. In this study, we establish that Candida albicans responds to SMG in a similar fashion, demonstrating that there is a conserved response among yeast to this environmental stress. We also report that the growth of C. albicans in SMG results in a morphogenic switch that is consistent with enhanced pathogenicity. Specifically, we observed an increase in filamentous forms of the organism and accompanying changes in the expression of two genes associated with the yeast-hyphal transition. The morphological response may have significant implications for astronauts' safety, as the fungal pathogen may become more virulent during spaceflight.

Yeast genomic expression patterns in response to low-shear modeled microgravity.

UNLABELLED: The low-shear microgravity environment, modeled by rotating suspension culture bioreactors called high aspect ratio vessels (HARVs), allows investigation in ground-based studies of the effects of microgravity on eukaryotic cells and provides insights into the impact of space flight on cellular physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) causes significant phenotypic changes of a select group of Saccharomyces cerevisiae genes associated with the establishment of cell polarity, bipolar budding, and cell separation. However, the mechanisms cells utilize to sense and respond to microgravity and the fundamental gene expression changes that occur are largely unknown. In this study, we examined the global transcriptional response of yeast cells grown under LSMMG conditions using DNA microarray analysis in order to determine if exposure to LSMMG results in changes in gene expression. RESULTS: LSMMG differentially changed the expression of a significant number of genes (1372) when yeast cells were cultured for either five generations or twenty-five generations in HARVs, as compared to cells grown under identical conditions in normal gravity. We identified genes in cell wall integrity signaling pathways containing MAP kinase cascades that may provide clues to novel physiological responses of eukaryotic cells to the external stress of a low-shear modeled microgravity environment. A comparison of the microgravity response to other environmental stress response (ESR) genes showed that 26% of the genes that respond significantly to LSMMG are involved in a general environmental stress response, while 74% of the genes may represent a unique transcriptional response to microgravity. In addition, we found changes in genes involved in budding, cell polarity establishment, and cell separation that validate our hypothesis that phenotypic changes observed in cells grown in microgravity are reflected in genome-wide changes. This study documents a considerable response to yeast cell growth in low-shear modeled microgravity that is evident, at least in part, by changes in gene expression. Notably, we identified genes that are involved in cell signaling pathways that allow cells to detect environmental changes, to respond within the cell, and to change accordingly, as well as genes of unknown function that may have a unique transcriptional response to microgravity. We also uncovered significant changes in the expression of many genes involved in cell polarization and bud formation that correlate well with the phenotypic effects observed in yeast cells when grown under similar conditions. These results are noteworthy as they have implications for human space flight.

Identification of a novel mitogen-activated protein kinase in Toxoplasma gondii.

Toxoplasma gondii is an Apicomplexan parasite causing significant morbidity and mortality in immunocompromised hosts. Mitogen activated protein kinases regulate diverse biologic processes including proliferation, differentiation, survival and stress responses. We searched a new T. gondii genomic database to identify a 1.6 kilobase pair (kbp) coding region with features suggesting a mitogen activated protein kinase. This gene is predicted to encode a 58kDa protein with a threonine, aspartic acid, tyrosine (TDY) activation loop, similar to parasite and plant mitogen activated protein kinases, but distinct from mammalian mitogen activated protein kinases (with threonine, glycine, tyrosine (TGY) motifs). The predicted protein shares 45% amino acid identity with human stress-activated p38alpha mitogen activated protein kinase. Expression of the cloned gene in Escherichia coli produced a protein with an apparent molecular weight of 63kDa and which exhibited kinase activity. Following osmotic stress, the abundance of the mRNA encoding this T. gondii mitogen activated protein kinase, which we name TgMAPK-1, increased in tachyzoites. Its expression rescued hog1-deficient yeast grown under osmotic stress. These data confirm that the gene product is a stress-response mitogen activated protein kinase. Upon conversion of T. gondii tachyzoites to the latent bradyzoite form in vitro, tgMAPK-1 transcript accumulation increased, suggesting a role in parasite proliferation or stage differentiation. We previously demonstrated that pyridinylimidazole p38 mitogen activated protein kinase inhibitors block T. gondii replication. These inhibitors also blocked TgMAPK-1 autophosphorylation, suggesting that TgMAPK-1, or other parasite mitogen activated protein kinases are novel drug development targets.

Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture.

This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.

Spaceflight enhances cell aggregation and random budding in Candida albicans.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans-induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.

Modeled microgravity increases filamentation, biofilm formation, phenotypic switching, and antimicrobial resistance in Candida albicans.

Candida albicans is an opportunistic fungal pathogen responsible for a variety of cutaneous and systemic human infections. Virulence of C. albicans increases upon exposure to some environmental stresses; therefore, we explored phenotypic responses of C. albicans following exposure to the environmental stress of low-shear modeled microgravity. Upon long-term (12-day) exposure to low-shear modeled microgravity, C. albicans transitioned from yeast to filamentous forms at a higher rate than observed under control conditions. Consistently, genes associated with cellular morphology were differentially expressed in a time-dependent manner. Biofilm communities, credited with enhanced resistance to environmental stress, formed in the modeled microgravity bioreactor and had a more complex structure than those formed in control conditions. In addition, cells exposed to low-shear modeled microgravity displayed phenotypic switching, observed as a near complete transition from smooth to "hyper" irregular wrinkle colony morphology. Consistent with the presence of biofilm communities and increased rates of phenotypic switching, cells exposed to modeled microgravity were significantly more resistant to the antifungal agent Amphotericin B. Together, these data indicate that C. albicans adapts to the environmental stress of low-shear modeled microgravity by demonstrating virulence-associated phenotypes.

The role of FLO11 in Saccharomyces cerevisiae biofilm development in a laboratory based flow-cell system.

A role of the FLO11 in Saccharomyces cerevisiae biofilm development in a flow cell system was examined. We carried out an ectopic FLO11 expression in the wild type (wt) BY4741 strain that has low levels of endogenous FLO11 transcript. In contrast to the nonadhesive wt, the FLO11 overexpression strain (BY4741 FLO11(+)) readily adhered to both liquid-hydrophobic and liquid-hydrophilic solid interfaces and was able to grow as a biofilm monolayer in a flow system. Cellular features associated with FLO11 were examined and found to be consistent with the previous studies conducted in different strains of S. cerevisiae. When grown in suspended liquid culture, BY4741 FLO11(+) formed larger cellular aggregates (clumps), consisting of from five to 60 cells, and displayed an increased cell surface hydrophobicity, without changes in the cell size or growth rate, compared to wt. However, the invasive growth associated with FLO11 expression was not observed in BY4741 FLO11(+). The significance of these findings is discussed in the context of clinically and industrially relevant biofilms.

GRS1, a yeast tRNA synthetase with a role in mRNA 3' end formation.

Transcription termination and 3' end formation are essential processes necessary for gene expression. However, the specific mechanisms responsible for these events remain elusive. A screen designed to identify trans-acting factors involved in these mechanisms in Saccharomyces cerevisiae identified a temperature-sensitive mutant that displayed phenotypes consistent with a role in transcription termination. The complementing gene was identified as GRS1, which encodes the S. cerevisiae glycyl-tRNA synthetase. This result, although unusual, is not unprecedented given that the involvement of tRNA synthetases in a variety of cellular processes other than translation has been well established. A direct role for the synthetase in transcription termination was determined through several in vitro assays using purified wild type and mutant enzyme. First, binding to two well characterized yeast mRNA 3' ends was demonstrated by cross-linking studies. In addition, it was found that all three substrates compete with each other for binding to GlyRS enzyme. Next, the affinity of the synthetase for the two mRNA 3' ends was found to be similar to that of its "natural" substrate, glycine tRNA in a nitrocellulose filter binding assay. The effect of the grs1-1 mutation was also examined and found to significantly reduce the affinity of the enzyme for the three RNA substrates. Taken together, these data indicate that not only does this synthetase interact with several different RNA substrates, but also that these substrates bind to the same site. These results establish a direct role for GRS1 in mRNA 3' end formation.

Binding to Elongin C inhibits degradation of interacting proteins in yeast.

Elongin C is a highly conserved, low molecular weight protein found in a variety of multiprotein complexes in human, rat, fly, worm, and yeast cells. Among the best characterized of these complexes is a mammalian E3 ligase that targets proteins for ubiquitination and subsequent degradation by the 26 S proteasome. Despite its crucial role as a component of such E3 ligases and other complexes, the specific function of Elongin C is unknown. In yeast, Elongin C is a non-essential gene and there is no obvious phenotype as associated with its absence. We previously reported that in Saccharomyces cerevisiae Elongin C (Elc1) interacts specifically and strongly with a class of proteins loosely defined as stress response proteins. In the present study, we examined the role of yeast Elc1 in the turnover of two of these binding partners, Snf4 and Pcl6. Deletion of Elc1 resulted in decreased steady-state levels of Snf4 and Pcl6 as indicated by Western blot analysis. Northern blot analysis of mRNA prepared from elc1 null and wild type strains revealed no difference in mRNA levels for Snf4 and Pcl6 establishing that the effects of Elc1 are not transcriptionally mediated. Reintroduction of either yeast or human Elongin C into Elc1 null strains abrogated this effect. Taken together, these data document that the levels of Snf4 and Pcl6 are dependent on the presence of Elc1 and that binding to Elc1 inhibits the degradation of these proteins. The results suggest a new function for yeast Elongin C that is distinct from a direct role in targeting proteins for ubiquitination and subsequent proteolysis.