William E. Tapprich

5′-Terminal Deletions Occur in Coxsackievirus B3 during Replication in Murine Hearts and Cardiac Myocyte Cultures and Correlate with Encapsidation of Negative-Strand Viral RNA

ABSTRACT Adult human enteroviral heart disease is often associated with the detection of enteroviral RNA in cardiac muscle tissue in the absence of infectious virus. Passage of coxsackievirus B3 (CVB3) in adult murine cardiomyocytes produced CVB3 that was noncytolytic in HeLa cells. Detectable but noncytopathic CVB3 was also isolated from hearts of mice inoculated with CVB3. Sequence analysis revealed five classes of CVB3 genomes with 5′ termini containing 7, 12, 17, 30, and 49 nucleotide deletions. Structural changes (assayed by chemical modification) in cloned, terminally deleted 5′-nontranslated regions were confined to the cloverleaf domain and localized within the region of the deletion, leaving key functional elements of the RNA intact. Transfection of CVB3 cDNA clones with the 5′-terminal deletions into HeLa cells generated noncytolytic virus (CVB3/TD) which was neutralized by anti-CVB3 serum. Encapsidated negative-strand viral RNA was detected using CsCl-purified CVB3/TD virions, although no negative-strand virion RNA was detected in similarly treated parental CVB3 virions. The viral protein VPg was detected on CVB3/TD virion RNA molecules which terminate in 5′ CG or 5′ AG. Detection of viral RNA in mouse hearts from 1 week to over 5 months postinoculation with CVB3/TD demonstrated that CVB3/TD virus strains replicate and persist in vivo. These studies describe a naturally occurring genomic alteration to an enteroviral genome associated with long-term viral persistence.

Structure of the 5′ Nontranslated Region of the Coxsackievirus B3 Genome: Chemical Modification and Comparative Sequence Analysis

ABSTRACT Coxsackievirus B3 (CVB3) is a picornavirus which causes myocarditis and pancreatitis and may play a role in type I diabetes. The viral genome is a single 7,400-nucleotide polyadenylated RNA encoding 11 proteins in a single open reading frame. The 5′ end of the viral genome contains a highly structured nontranslated region (5′NTR) which folds to form an internal ribosome entry site (IRES) as well as structures responsible for genome replication, both of which are critical for virulence. A structural model of the CVB3 5′NTR, generated primarily by comparative sequence analysis and energy minimization, shows seven domains (I to VII). While this model provides a preliminary basis for structural analysis, the model lacks comprehensive experimental validation. Here we provide experimental evidence from chemical modification analysis to determine the structure of the CVB3 5′NTR. Chemical probing results show that the theoretical model for the CVB3 5′NTR is largely, but not completely, supported experimentally. In combination with our chemical probing data, we have used the RNASTRUCTURE algorithm and sequence comparison of 105 enterovirus sequences to provide evidence for novel secondary and tertiary interactions. A comprehensive examination of secondary structure is discussed, along with new evidence for tertiary interactions. These include a loop E motif in domain III and a long-range pairing interaction that links domain II to domain V. The results of our work provide mechanistic insight into key functional elements in the cloverleaf and IRES, thereby establishing a base of structural information from which to interpret experiments with CVB3 and other picornaviruses.

MUC1 Regulates Expression of Multiple microRNAs Involved in Pancreatic Tumor Progression, Including the miR-200c/141 Cluster

MUC1 is a transmembrane glycoprotein that modulates transcription via its cytoplasmic domain. We evaluated the capacity of MUC1 to regulate the global transcription of microRNAs in pancreatic cancer cells expressing MUC1. Results indicated that MUC1 regulated expression of at least 103 microRNAs. We evaluated further regulation of the microRNA transcript cluster miR-200c/141, which was among the most highly regulated microRNAs. We found that MUC1 directly interacted with ZEB1, a known transcriptional repressor of the miR-200c/141 cluster, at the promoter of miR-200c/141, and further reduced transcript production. These data indicate that signaling through MUC1 influences cancer progression by regulating transcription of microRNAs that are associated with the process of metastasis.

Staphylococcus aureus domain V functions in Escherichia coli ribosomes provided a conserved interaction with domain IV is restored

Domain V of Escherichia coli 23 S rRNA (residues 2023-2630) was replaced by that from Staphylococcus aureus, thereby introducing 132 changes in the rRNA sequence. The resulting ribosomal mutant was unable to support cell growth. The mutant was rescued, however, by restoring an interaction between domains IV and V (residues 1782 and 2586). Although the importance of this interaction, U/U in E. coli, C/C in S. aureus, is therefore demonstrated, it cannot be the only tertiary interaction important for ribosomal function as the rescued hybrid grew more slowly than the wild type. Additionally, although the single-site mutations U1782C and U2586C in E. coli are viable, the double mutant is lethal.

Complete Genome Sequences of Pseudomonas fluorescens Bacteriophages Isolated from Freshwater Samples in Omaha, Nebraska

ABSTRACT The complete genome sequences of four Pseudomonas fluorescens bacteriophages, UNO-SLW1 to UNO-SLW4, isolated from freshwater samples, are 39,092 to 39,215 bp long. The genomes are highly similar (identity, >0.995) but dissimilar from that of Pseudomonas phage Pf-10 (the closest relative, 0.685 to 0.686 identity), with 48 to 49 protein-coding genes and 66 regulatory sites predicted.

Barriers to Integration of Bioinformatics into Undergraduate Life Sciences Education

Bioinformatics, a discipline that combines aspects of biology, statistics, and computer science, is increasingly important for biological research. However, bioinformatics instruction is rarely integrated into life sciences curricula at the undergraduate level. To understand why, the Network for Integrating Bioinformatics into Life Sciences Education (NIBLSE, “nibbles”) recently undertook an extensive survey of life sciences faculty in the United States. The survey responses to open-ended questions about barriers to integration were subjected to keyword analysis. The barrier most frequently reported by the ~1,260 respondents was lack of faculty training. Faculty at associate’s-granting institutions report the least training in bioinformatics and the least integration of bioinformatics into their teaching. Faculty from underrepresented minority groups (URMs) in STEM reported training barriers at a higher rate than others, although the number of URM respondents was small. Interestingly, the cohort of faculty with the most recently awarded PhD degrees reported the most training but were teaching bioinformatics at a lower rate than faculty who earned their degrees in previous decades. Other barriers reported included lack of student interest in bioinformatics; lack of student preparation in mathematics, statistics, and computer science; already overly full curricula; and limited access to resources, including hardware, software, and vetted teaching materials. The results of the survey, the largest to date on bioinformatics education, will guide efforts to further integrate bioinformatics instruction into undergraduate life sciences education.

Major alteration in coxsackievirus B3 genomic RNA structure distinguishes a virulent strain from an avirulent strain

Coxsackievirus B3 (CV-B3) is a cardiovirulent enterovirus that utilizes a 5′ untranslated region (5′UTR) to complete critical viral processes. Here, we directly compared the structure of a 5′UTR from a virulent strain with that of a naturally occurring avirulent strain. Using chemical probing analysis, we identified a structural difference between the two 5′UTRs in the highly substituted stem-loop II region (SLII). For the remainder of the 5′UTR, we observed conserved structure. Comparative sequence analysis of 170 closely related enteroviruses revealed that the SLII region lacks conservation. To investigate independent folding and function, two chimeric CV-B3 strains were created by exchanging nucleotides 104–184 and repeating the 5′UTR structural analysis. Neither the parent SLII nor the remaining domains of the background 5′UTR were structurally altered by the exchange, supporting an independent mechanism of folding and function. We show that the attenuated 5′UTR lacks structure in the SLII cardiovirulence determinant.

Bioinformatics core competencies for undergraduate life sciences education

Although bioinformatics is becoming increasingly central to research in the life sciences, bioinformatics skills and knowledge are not well integrated into undergraduate biology education. This curricular gap prevents biology students from harnessing the full potential of their education, limiting their career opportunities and slowing research innovation. To advance the integration of bioinformatics into life sciences education, a framework of core bioinformatics competencies is needed. To that end, we here report the results of a survey of biology faculty in the United States about teaching bioinformatics to undergraduate life scientists. Responses were received from 1,260 faculty representing institutions in all fifty states with a combined capacity to educate hundreds of thousands of students every year. Results indicate strong, widespread agreement that bioinformatics knowledge and skills are critical for undergraduate life scientists as well as considerable agreement about which skills are necessary. Perceptions of the importance of some skills varied with the respondent’s degree of training, time since degree earned, and/or the Carnegie Classification of the respondent’s institution. To assess which skills are currently being taught, we analyzed syllabi of courses with bioinformatics content submitted by survey respondents. Finally, we used the survey results, the analysis of the syllabi, and our collective research and teaching expertise to develop a set of bioinformatics core competencies for undergraduate biology students. These core competencies are intended to serve as a guide for institutions as they work to integrate bioinformatics into their life sciences curricula.

Genuine Faculty-Mentored Research Experiences for In-Service Science Teachers: Increases in Science Knowledge, Perception, and Confidence Levels

ABSTRACT The overall purpose of this multifocused study was to explore how participation in genuine mentored scientific research experiences impacts in-service science teachers and the knowledge and skills needed for their own science teaching. The research experiences resulted from a partnership between the University of Nebraska at Omaha and the Omaha Public School District. This Teacher-Researcher Partnership Program facilitated opportunities in inquiry, science content, interaction with laboratory instrumentation and technologies, critical discussion of literature, and dissemination of findings for participating in-service science teacher professional development utilizing an inquiry-based theoretical framework wherein we examined science teacher preparation via inquiry-based methods in the research laboratory. A mixed-methods approach with a convergent typology (i.e., qualitative and quantitative analyses conducted separately and integrated) was used to investigate the impact of the program on teachers. Our research question was as follows: How do teachers define and approach scientific research before and after a genuine research experience? We observed 3 emergent nodes or themes by which teachers indicated significant gains: science content knowledge, confidence, and perception. Moreover, we determined that participation by science teachers in a mentored research experience using current scientific technologies and tools improved teacher confidence in science and inquiry as well as an ongoing commitment to provide similar types of experiences to their students. These data support the need for the participation of in-service science teachers in genuine research experiences to boost technological and pedagogical content knowledge, con fidence in process and content, and the perception of translatability to the classroom.

How do undergraduate STEM mentors reflect upon their mentoring experiences in an outreach program engaging K-8 youth?

BackgroundMany university students are becoming involved in mentoring programs, yet few studies describe the impact of mentoring on the mentor. Additionally, many studies report that students graduating from college are not prepared to enter the workforce in terms of key career skills and/or content knowledge. Herein, we examine the impact of our program, NE STEM 4U (Nebraska Science, Technology, Engineering and Math for You), in which undergraduate (UG) mentors engage K-8 youth in after-school STEM experiments. The UGs reflected upon their experiences using post-mentoring evaluations, 12- and 24-week interviews, and exit surveys. Many of the questions asked of the mentors related directly to their own professional development, such as self-evaluation of communication, organization, and problem-solving skills, while other questions related to content knowledge and reflection.ResultsPost-mentoring, UGs reflected on the delivery/teaching significantly more (p ≤ 0.001 for each) than other variables (i.e., their own content knowledge gains, the students’ content knowledge gains, scaffolding the lessons, or overall professional growth). By analyzing the evaluations and interviews together, some significant, self-reported gains emerged. For example, 94.15% of the UG reported that the experience was beneficial to their education. Additionally, UG mentors self-reported significant gains (p ≤ 0.01 for each) moving from 12- to 24-weeks in the program in the categories of organization, STEM content knowledge, preparedness to teach, and engagement in the program. However, UG did not report significant gains in dependability. Importantly, when mentors ranked themselves at 24-weeks, they were blinded to (unaware of) the ranking they gave themselves at 12-weeks.ConclusionsThis study helps to fill a gap in the literature by providing insight into the gains UG mentors report attaining after mentoring to K-8 students. These data suggest that participation by UGs in this program promoted self-reflection as well as self-reported gains related to career preparedness and STEM content knowledge.