Enhancing the Mentoring Experience for Underrepresented Students.

Undergraduate research is a valuable experience that increases the likelihood of a STEM major to continue on to postgraduate training in their field. For students from groups underrepresented in the biomedical sciences, a strong mentoring relationship during this undergraduate period is a key component in preparing them for the next stage of their education and can have a significant influence on their ability to persist in the pipeline. Although the ideal scenario to increase the diversity of the biomedical workforce is to provide more BIPOC (Black, Indigenous, People of Color) faculty mentors for our undergraduates, we also need to develop strategies to provide strong mentoring experiences for our BIPOC students when those mentors are not in great number. At Xavier University of Louisiana, we have used our NIH BUILD Project Pathways program to look more closely at the mentor matching process. Throughout the past seven years, we have moved from the traditional mentor, research-focused matching process to a student-centered process. The lessons learned here can be used by any University looking to craft an inclusive undergraduate research program to meet the needs of all students, but in particular a diverse student population.

BUILDING INTEGRATED PATHWAYS TO INDEPENDENCE FOR DIVERSE BIOMEDICAL RESEARCHERS: PROJECT PATHWAYS.

Diversity of backgrounds and life experiences on scientific teams is known to lead to more innovative ideas and better scientific products. However, in the United States, the percentages of individuals from underrepresented racial and ethnic groups who obtain doctoral degrees in the Sciences continue to be significantly lower than their percentages in the population. This has resulted in the need for nation-wide initiatives to remedy this inequality, and consequently produce more productive teams of scientific minds. Xavier University of Louisiana is a historically Black and Catholic university that is widely recognized in the US for the success of its undergraduate Science, Technology, Engineering, and Mathematics (STEM) programs. Project Pathways at Xavier is one of ten federally-funded Building Infrastructure Leading to Diversity (BUILD) programs with the overarching goal of diversifying the Biomedical research workforce. Project Pathways is designed as a holistic, integrated, and coordinated program across Biomedical academic departments, student academic and career support offices, and the University's faculty development center. The overall hypothesis of Project Pathways is that if individuals from groups underrepresented in scientific research careers are provided with a) early awareness and deepening exposure to Biomedical careers, b) supportive relationships as they move through the pathway, c) suitable infrastructure, and d) meaningful engagement in Biomedical research experiences and adequate research resources, then a higher number will succeed in entering and successfully completing graduate programs, leading to increased diversity in the Biomedical research workforce. Here, the significant strides of this program during its first five-year funding cycle are presented.

Baseline Characteristics of the 2015-2019 First Year Student Cohorts of the NIH Building Infrastructure Leading to Diversity (BUILD) Program.

Objective: The biomedical/behavioral sciences lag in the recruitment and advancement of students from historically underrepresented backgrounds. In 2014 the NIH created the Diversity Program Consortium (DPC), a prospective, multi-site study comprising 10 Building Infrastructure Leading to Diversity (BUILD) institutional grantees, the National Research Mentoring Network (NRMN) and a Coordination and Evaluation Center (CEC). This article describes baseline characteristics of four incoming, first-year student cohorts at the primary BUILD institutions who completed the Higher Education Research Institute, The Freshmen Survey between 2015-2019. These freshmen are the primary student cohorts for longitudinal analyses comparing outcomes of BUILD program participants and non-participants. Design: Baseline description of first-year students entering college at BUILD institutions during 2015-2019. Setting: Ten colleges/universities that each received <$7.5mil/yr in NIH Research Project Grants and have high proportions of low-income students. Participants: First-year undergraduate students who participated in BUILD-sponsored activities and a sample of non-BUILD students at the same BUILD institutions. A total of 32,963 first-year students were enrolled in the project; 64% were female, 18% Hispanic/Latinx, 19% African American/Black, 2% American Indian/Alaska Native and Native Hawaiian/Pacific Islander, 17% Asian, and 29% White. Twenty-seven percent were from families with an income <$30,000/yr and 25% were their family's first generation in college. Planned Outcomes: Primary student outcomes to be evaluated over time include undergraduate biomedical degree completion, entry into/completion of a graduate biomedical degree program, and evidence of excelling in biomedical research and scholarship. Conclusions: The DPC national evaluation has identified a large, longitudinal cohort of students with many from groups historically underrepresented in the biomedical sciences that will inform institutional/national policy level initiatives to help diversify the biomedical workforce.

All for One And One for All: Coordinating the Resources of Individual Student Research Training Initiatives in Biomedical Sciences at Xavier University of Louisiana.

Xavier University of Louisiana has a national reputation for producing science, technology, engineering, and mathematics (STEM) graduates who go on to obtain MD and PhD degrees. According to a 2013 National Science Foundation report, Xavier is ranked first in producing African American graduates who go on to receive life sciences PhD degrees, fifth in the nation in producing African American graduates who go on to receive science and engineering PhD degrees, and seventh in producing African American graduates who go on to receive physical sciences PhD degrees. Xavier is currently third among the nation's colleges and universities in the number of African American graduates enrolled in medical school, according to data compiled by the Association of American Medical Colleges, and ranked first in the number of African American alumni who successfully complete their medical degrees. The success of Xavier's graduates is due to a combination of university-based student support initiatives and externally funded programs, in particular, the Building Infrastructure Leading to Diversity (BUILD), Maximizing Access to Biomedical Research Careers (MARC) U*STAR, and Research Initiative in Scientific Enhancement (RISE) programs. These three programs, funded by the Training, Workforce Development, and Diversity (TWD) Division at the National Institutes of Health (NIH), offer select trainees undergraduate research opportunities, support mechanisms, and a variety of activities designed to improve their potential for success in graduate school. The BUILD, MARC U*STAR, and RISE programs work closely together and with the University to leverage the resources provided by each in order to provide the best experience possible for their students with a minimum of redundancy of effort. This chapter focuses on the program components and how the programs work together.

Novel flanking DNA sequences enhance FOXO1a DNA binding affinity but do not alter DNA bending.

FOXO1A, a member of the forkhead winged-helix family of proteins is a transcription factor with proapoptotic activities and plays a significant role in insulin and growth factor signaling. As such, FOXO1A is insulin responsive and binds to the insulin response element (IRE). However, multiple forkhead family members with diverse biological functions are also known to bind to the IRE. Therefore, additional DNA sequence elements may be required to provide increased binding affinity and specificity for FOXO1A. We have used the systematic evaluation of ligands by exponential enrichment (SELEX) to systematically identify additional DNA sequences important for FOXO1A binding. We demonstrate for the first time that, in addition to the IRE, two additional sequence elements are important for maximal FOXO1A binding: (1) the reverse complement (5'-GT(A/C)AACA-3') and (2) the flanking sequence (5'-ACAACA-3'). Although these additional elements do not contribute to the FOXO1A-induced DNA bending angle of 120 degrees , the presence of these additional elements does increase the affinity of FOXO1A DNA binding nearly 9-fold through a 1-to-1 binding stoichiometry. The increased binding affinity subsequently enhances the ability of FOXO1A to activate transcription from a luciferase reporter construct and from promoter regions of endogenous genes known to be direct transcriptional targets of FOXO1A.

Building integrated pathways to independence for diverse biomedical researchers: Project Pathways, the BUILD program at Xavier University of Louisiana.

BACKGROUND AND PURPOSE: Xavier University of Louisiana is a historically Black and Catholic university that is nationally recognized for its science, technology, engineering and mathematics (STEM) curricula. Approximately 73% of Xavier's students are African American, and about 77% major in the biomedical sciences. Xavier is a national leader in the number of STEM majors who go on to receive M.D. degrees and Ph.D. degrees in science and engineering. Despite Xavier's advances in this area, African Americans still earn about 7.5% of the Bachelor's degrees, less than 8% of the Master's degrees, and less than 5% of the doctoral degrees conferred in STEM disciplines in the United States. Additionally, although many well-prepared, highly-motivated students are attracted by Xavier's reputation in the sciences, many of these students, though bright and capable, come from underperforming public school systems and receive substandard preparation in STEM disciplines. The purpose of this article is to describe how Xavier works to overcome unequal education backgrounds and socioeconomic challenges to develop student talent through expanding biomedical training opportunities and build on an established reputation in science education. PROGRAM AND KEY HIGHLIGHTS: The National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS)-funded BUILD (Building Infrastructure Leading to Diversity) Program at Xavier University of Louisiana, Project Pathways, is a highly-innovative program designed to broaden the career interests of students early on, and to engage them in activities that entice them to continue their education towards biomedical research careers. Project strategies involve a transformation of Xavier's academic and non-academic programs through the redesign, supplementation and integration of academic advising, tutoring, career services, personal counseling, undergraduate research training, faculty research mentoring, and development of new biomedical and research skills courses. The Program also focuses on mentor training and providing faculty members with opportunities to improve their teaching skills as well as their research competitiveness. In addition to the wide range of activities supported by BUILD within the institution, Xavier University is partnering with a number of major research universities across the nation to achieve Project Pathways' goals. IMPLICATIONS: The strategies developed by Project Pathways are designed to address the challenges and barriers Xavier students face as they work towards graduate studies and entering the biomedical workforce. Xavier University of Louisiana has a long history of providing high quality, rigorous education to African American students in a very supportive environment with highly dedicated faculty and staff. The program highlighted here could be used by other institutions as a model program for assisting students in STEM and other biomedical fields of study to successfully matriculate through college and graduate school and develop their research careers.

Inquiry-based experiments for large-scale introduction to PCR and restriction enzyme digests.

Polymerase chain reaction and restriction endonuclease digest are important techniques that should be included in all Biochemistry and Molecular Biology laboratory curriculums. These techniques are frequently taught at an advanced level, requiring many hours of student and faculty time. Here we present two inquiry-based experiments that are designed for introductory laboratory courses and combine both techniques. In both approaches, students must determine the identity of an unknown DNA sequence, either a gene sequence or a primer sequence, based on a combination of PCR product size and restriction digest pattern. The experimental design is flexible, and can be adapted based on available instructor preparation time and resources, and both approaches can accommodate large numbers of students. We implemented these experiments in our courses with a combined total of 584 students and have an 85% success rate. Overall, students demonstrated an increase in their understanding of the experimental topics, ability to interpret the resulting data, and proficiency in general laboratory skills.

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.

Purification and characterization of enzymes from yeast: an extended undergraduate laboratory sequence for large classes.

Providing a project-based experience in an undergraduate biochemistry laboratory class can be complex with large class sizes and limited resources. We have designed a 6-week curriculum during which students purify and characterize the enzymes invertase and phosphatase from bakers yeast. Purification is performed in two stages via ethanol precipitation and anion exchange chromatography, and students perform both direct and coupled enzyme assays. By completion of the experimental series, students are able to identify which enzymes they have purified and have obtained kinetic parameters for one. This experimental series requires minimal instructor preparation time, is cost effective, and works with multiple sections of large groups of students. Students participating in this sequence showed increases in conceptual understanding of biochemical concepts as measured through in-class assessments and anonymous surveys.

Diamagnetic levitation changes growth, cell cycle, and gene expression of Saccharomyces cerevisiae.

Inhomogeneous magnetic fields are used in magnetic traps to levitate biological specimens by exploiting the natural diamagnetism of virtually all materials. Using Saccharomyces cerevisiae, this report investigates whether magnetic field (B) induces changes in growth, cell cycle, and gene expression. Comparison to the effects of gravity and temperature allowed determination of whether the responses are general pathways or stimulus specific. Growth and cell cycle analysis were examined in wild-type (WT) yeast and strains with deletions in transcription factors Msn4 or Sfp1. Msn4, Sfp1, and Rap1 have been implicated in responses to physical forces, but only Msn4 and Sfp1 deletions are viable. Gene expression changes were examined in strains bearing GFP-tagged reporters for YIL052C (Sfp1-dependent), YST-2 (Sfp1/Rap1-dependent), or SSA4 (Msn4-dependent). The cell growth and gene expression responses were highly stimulus specific. B increased growth only following Msn4 or Sfp1 deletion, associated with decreased G1 and G2/M and increased S phase of the cell cycle. In addition, B suppressed expression of both YIL052C and YST2. Gravity decreased growth in an Sfp1 but not Msn4-dependent manner, in association with decreased G2/M and increased S phase of the cell cycle. Additionally, gravity decreased expression of SSA4 and YIL052C genes. Temperature increased cell growth in an Msn4- and Sfp1-dependent manner in association with increased G1 and G2/M with decreased S phase of the cell cycle. In addition, temperature increased YIL052C gene expression. This study shows that B has selective effects on cell growth, cell cycle, and gene expression that are stimulus specific.

Gene expression and survival changes in Saccharomyces cerevisiae during suspension culture.

This study explores the connection between changes in gene expression and the genes that determine strain survival during suspension culture, using the model eukaryotic organism, Saccharomyces cerevisiae. The Saccharomyces cerevisiae homozygous diploid deletion pool (HDDP), and the BY4743 parental strain were grown for 18 h in a rotating wall vessel (RWV), a suspension culture device optimized to minimize the delivered shear. In addition to the reduced shear conditions, the RWVs were also placed in a static position or in a shaker in order to change the amount of shear stress on the cells. Using simple linear regression, it was found that there were 140 differentially expressed genes for which >70% of the variation can be explained by shear stress alone. A significant number of these genes are involved in catalytic activity. In the HDDP, shear stress was associated with significant survival changes in 15 deletion strains (R(2>) > 0.7) Interestingly, both analyses uncovered changes in the ribosomal protein machinery. Comparing the changes in gene expression and strain survival under the different shear conditions allows for the insights into the molecular mechanisms behind the cells response to shear stress. This in turn can provide information for the optimization of suspension culture.

Megalin binds and internalizes angiotensin-(1-7).

Megalin is a multiligand receptor heavily involved in protein endocytosis. We recently demonstrated that megalin binds and mediates internalization of ANG II. Although there is a strong structural resemblance between ANG II and ANG-(1-7), their physiological actions and their affinity for the angiotensin type 1 receptor (AT(1)R) are dissimilar. Therefore, the hypothesis of the present work was to test whether megalin binds and internalizes ANG-(1-7). The uptake of ANG-(1-7) was determined by exposure of confluent monolayers of BN/MSV cells (a model representative of the yolk sac epithelium) to fluorescently labeled ANG-(1-7) (100 nM) and measurement of the amount of cell-associated fluorescence after 4 h by flow cytometry. Anti-megalin antisera and an AT(1)R blocker (olmesartan) were used to interfere with uptake via megalin and the AT(1)R, respectively. ANG-(1-7) uptake was prevented by anti-megalin antisera (63%) to a higher degree than olmesartan (13%) (P < 0.001). In analysis by flow cytometry of binding experiments performed in brush-border membrane vesicles isolated from kidneys of CD-1 mice, anti-megalin antisera interfered with ANG-(1-7) binding more strongly than olmesartan (P < 0.05 against positive control). Interactions of megalin with ANG-(1-7) at a molecular level were studied by surface plasmon resonance, demonstrating that ANG-(1-7) binds megalin dose and time dependently and with an affinity similar to ANG II. These results show that the scavenger receptor megalin binds and internalizes ANG-(1-7).

Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance.

This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

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.