Maria Julia Massimelli

Most biology undergraduates learn about mutations in multiple classrooms throughout their college career. Understanding personalised genome test results, genome editing controversies, and the appearance of new variants of viruses or antibiotic resistant bacteria all require foundational knowledge about mutations. However, the abstract nature of molecular processes surrounding mutations makes them one of the more difficult topics for students to understand and apply. Instructors need valid assessment tools to document student understanding and tailor their instructional methods to address student knowledge gaps. We describe here the development and validation of the Mutations Criterion Referenced Assessment (MuCRA). This formative assessment was developed through an iterative process involving expert feedback and student responses to both open-ended and multiple-choice questions. The final MuCRA is composed of 10 multiple-choice questions aligned with three learning objectives. The item difficulty for each question was between 0.32–0.65, while the discrimination index ranged from 0.31–0.75 and the reliability (KR20) for the MuCRA was 0.69. The congruence analyses demonstrated distractors are capturing student misconceptions in 9/10 questions. These data indicate that the MuCRA can be used to reliably assess student learning and common misconceptions about mutations.

Course-based undergraduate research experiences (CUREs) are an effective way to introduce students to contemporary scientific research. Research experiences have been shown to promote critical thinking, improve understanding and proper use of the scientific method, and help students learn practical skills including writing and oral communication. We aimed to improve scientific training by engaging students enrolled in an upper division elective course in a human microbiome CURE. The “Fiber Force” course is aimed at studying the effect of a wholesome high-fiber diet (40 to 50 g/day for two weeks) on the students’ gut microbiomes. Enrolled students participated in a noninvasive diet intervention, designed health surveys, tested hypotheses on the effect of a diet intervention on the gut microbiome, and analyzed their own samples (as anonymized aggregates). The course involved learning laboratory techniques (e.g., DNA extraction, PCR, and 16S sequencing) and the incorporation of computational techniques to analyze microbiome data with QIIME2 and within the R software environment. In addition, the learning objectives focused on effective student performance in writing, data analysis, and oral communication. Enrolled students showed high performance grades on writing, data analysis and oral communication assignments. Pre- and post-course surveys indicate that the students found the experience favorable, increased their interest in science, and heightened awareness of their diet habits. Fiber Force constitutes a validated case of a research experience on microbiology with the capacity to improve research training and promote healthy dietary habits.

Quantitative data analysis skills are basic competencies students in a STEM field should master. In this article, we describe a classroom activity using isolated figures from papers as a simple exercise to practice data analysis skills. We call this approach Just Figures. With this technique, instructors find figures from primary papers that address key concepts related to several of their course learning objectives. These figures are assigned as homework prior to class discussion. In class, instructors teach the lesson and include a 10- to 20-minute discussion of the figures assigned. Frequent and repeated discussion of paper figures during class increased students’ confidence in reading and analyzing data. The Just Figures approach also increased student accuracy when interpreting data. After six weeks of Just Figures practice, students scored, on average, three points higher on a 20-point data analysis assessment instrument than they had done before the Just Figures exercises. In addition, a course in which students consistently practiced Just Figures performed just as well on the data analysis assessment instrument and on a class exam dedicated to paper reading compared with courses where students practiced reading three entire papers. The Just Figures method is easy to implement and can effectively improve student data analysis skills in microbiology classrooms.

American Society for Microbiology (ASM) Curriculum Guidelines highlight the importance of instruction about informational flow in organisms, including regulation of gene expression. However, foundational central dogma concepts and more advanced gene regulatory mechanisms are challenging for undergraduate biology students. To increase student comprehension of these principles, we designed an activity for upper-level biology students centered on construction and analysis of physical models of bacterial riboswitches. Students manipulate an inexpensive bag of supplies (beads, pipe cleaners) to model two conformations of a riboswitch in a bacterial transcript. After initial pilot testing, we implemented the activity in three upper-level classes at one research-intensive and two primarily undergraduate institutions. To assess student perceptions of learning gains, we utilized a pre/post-activity 5-point Likert-type survey instrument to characterize student perceptions of confidence in both their understanding of riboswitches and their ability to apply the central dogma to riboswitches. Median post-test ranks were significantly higher than median pre-test ranks ( p < 0.0001) when compared by the Wilcoxon signed-rank test ( n = 31). Next, we assessed post-activity knowledge via use of a rubric to score student responses on exam questions. More than 80% of students could correctly describe and diagram examples of riboswitches; data from initial iterations were used to enhance curriculum materials for subsequent implementations. We conclude that this riboswitch activity leads to both student-reported increases in confidence in the ASM curriculum dimension of gene regulation, including central dogma concepts, and demonstrated student ability to diagram riboswitches, predict outcomes of riboswitches, and connect riboswitches to evolutionary roles.

If we are to teach effectively, tools are needed to measure student learning. A widely used method for quickly measuring student understanding of core concepts in a discipline is the concept inventory (CI). Using the American Society for Microbiology Curriculum Guidelines (ASMCG) for microbiology, faculty from 11 academic institutions created and validated a new microbiology concept inventory (MCI). The MCI was developed in three phases. In phase one, learning outcomes and fundamental statements from the ASMCG were used to create T/F questions coupled with open responses. In phase two, the 743 responses to MCI 1.0 were examined to find the most common misconceptions, which were used to create distractors for multiple-choice questions. MCI 2.0 was then administered to 1,043 students. The responses of these students were used to create MCI 3.0, a 23-question CI that measures students’ understanding of all 27 fundamental statements. MCI 3.0 was found to be reliable, with a Cronbach’s alpha score of 0.705 and Ferguson’s delta of 0.97. Test item analysis demonstrated good validity and discriminatory power as judged by item difficulty, item discrimination, and point-biserial correlation coefficient. Comparison of pre- and posttest scores showed that microbiology students at 10 institutions showed an increase in understanding of concepts after instruction, except for questions probing metabolism (average normalized learning gain was 0.15). The MCI will enable quantitative analysis of student learning gains in understanding microbiology, help to identify misconceptions, and point toward areas where efforts should be made to develop teaching approaches to overcome them.

KSHV ORF57 (MTA) promotes RNA stability of ORF59, a viral DNA polymerase processivity factor. Here, we show that the integrity of both ORF59 RNA ends is necessary for ORF57-mediated ORF59 expression and deletion of both 5’ and 3’ regions, or one end region with a central region, of ORF59 RNA prevents ORF57-mediated translation of ORF59. The ORF59 sequence between nt 96633 and 96559 resembles other known MTA-responsive elements (MREs). ORF57 specifically binds to a stem-loop region from nt 96596–96572 of the MRE, which also binds cellular RBM15. Internal deletion of the MRE from ORF59 led to poor export, but accumulation of nuclear ORF59 RNA in the presence of ORF57 or RBM15. Despite of being translatable in the presence of ORF57, this deletion mutant exhibits translational defect in the presence of RBM15. Together, our results provide novel insight into the roles of ORF57 and RBM15 in ORF59 RNA accumulation and protein translation.

Polyadenylate-binding protein cytoplasmic 1 (PABPC1) is a cytoplasmic-nuclear shuttling protein important for protein translation initiation and both RNA processing and stability. We report that PABPC1 forms a complex with the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein, which allows ORF57 to interact with a 9-nucleotide (nt) core element of KSHV p oly a denylated n uclear (PAN) RNA, a viral long noncoding RNA (lncRNA), and increase PAN stability. The N-terminal RNA recognition motifs (RRMs) of PABPC1 are necessary for the direct interaction with ORF57. During KSHV lytic infection, the expression of viral ORF57 leads to a substantial decrease in overall PABPC1 expression, along with a shift in the cellular distribution of the remaining PABPC1 to the nucleus. Interestingly, PABPC1 and ORF57 have opposing functions in modulating PAN steady-state accumulation. The suppressive effect of PABPC1 specific to PAN expression is alleviated by small interfering RNA knockdown of PABPC1 or by overexpression of ORF57. Conversely, ectopic PABPC1 reduces ORF57 steady-state protein levels and induces aberrant polyadenylation of PAN and thereby indirectly inhibits ORF57-mediated PAN accumulation. However, E1B-AP5 (heterogeneous nuclear ribonucleoprotein U-like 1), which interacts with a region outside the 9-nt core to stimulate PAN expression, does not interact or even colocalize with ORF57. Unlike PABPC1, the nuclear distribution of E1B-AP5 remains unchanged by viral lytic infection or overexpression of ORF57. Together, these data indicate that PABPC1 is an important cellular target of viral ORF57 to directly upregulate PAN accumulation during viral lytic infection, and the ability of host PABPC1 to disrupt ORF57 expression is a strategic host counterbalancing mechanism.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.