Structure of the 5′ Untranslated Region of Enteroviral Genomic RNA

Enteroviral RNA genomes share a long, highly structured 5= untranslated region (5= UTR) containing a type I internal ribosome entry site (IRES). The 5= UTR is composed of stably folded RNA domains connected by unstructured RNA regions. Proper folding and functioning of the 5= UTR underlies the efficiency of viral replication and also determines viral virulence. We have characterized the structure of 5= UTR genomic RNA from coxsackievirus B3 using selective 2=-hydroxyl acylation analyzed by primer extension (SHAPE) and base-specific chemical probes in solution. Our results revealed novel structural features, including realignment of major domains, newly identified long-range interactions, and an intrinsically disordered connecting region. Together, these newly identified features contribute to a model for enteroviral 5= UTRs with type I IRES elements that links structure to function during the hierarchical processes directed by genomic RNA during viral infection. IMPORTANCE: Enterovirus infections are responsible for human diseases, including myocarditis, pancreatitis, acute flaccid paralysis, and poliomyelitis. The virulence of these viruses depends on efficient recognition of the RNA genome by a large family of host proteins and protein synthesis factors, which in turn relies on the threedimensional folding of the first 750 nucleotides of the molecule. Structural information about this region of the genome, called the 5= untranslated region (5= UTR), is needed to assist in the process of vaccine and antiviral development. This work presents a model for the structure of the enteroviral 5= UTR. The model includes an RNA element called an intrinsically disordered RNA region (IDRR). Intrinsically disordered proteins (IDPs) are well known, but correlates in RNA have not been proposed. The proposed IDRR is a 20-nucleotide region, long known for its functional importance, where structural flexibility helps explain recognition by factors controlling multiple functional states

Fostering Curiosity, Inquiry, and Scientific Thinking in Elementary School Students: Impact of the NE STEM 4U Intervention

In this qualitative study, we assessed the impact of the NE STEM 4U intervention on elementary school youth in terms of excitement, curiosity, and STEM concepts. The NE STEM 4U intervention incorporates a problem-based learning theoretical framework in an after-school time, weekly or twice-weekly intervention. We assessed student performance over two academic years of participation in the intervention using the Dimensions of Success observational tool. Ultimately, we link this mentor-led program with increases in curiosity, inquiry, and STEM concept gains. Taken together, these findings support after-school interventions in STEM areas as key encouraging excitement in youth and motivation to pursue a career in a STEM content area

NE STEM 4U: an out-of-school time academic program to improve achievement of socioeconomically disadvantaged youth in STEM areas

Background The Nebraska Science, Technology, Engineering, and Mathematics 4U (NE STEM 4U) program was initiated at the University of Nebraska at Omaha (UNO) in 2013. NE STEM 4U is a student-run, faculty-led program facilitating problem-based learning (PBL) sessions in science, technology, engineering, and mathematics (STEM) for socioeconomically disadvantaged kindergarten through grade 8 (K-8) students. PBL sessions are provided throughout the academic year in a twice-weekly, after-school, informal education program. The instructional material provided after school builds upon the curricula of the school day. Importantly, this program is a partnership between faculty members and administrators in higher education at UNO with community partners of Omaha including Collective for Youth, Beyond School Bells, and Omaha Public Schools. We focus on engaging K-8 youth in after-school immersion experiences in STEM fields using undergraduate students as mentors and facilitators using a model of problem-based learning. Results This program fosters an educational pipeline for students with hands-on experience in problem-solving and critical thinking. The partnerships among the community provide the foundation for success for students across the K-16 pipeline. Conclusions Herein, we describe the model of this program as documented by demonstrated successes to date in an effort to guide others in developing such a model in their city or region. We also provide models for implementation of assessment instruments

Enhancing the STEM Ecosystem through Teacher-Researcher Partnerships

STEM faculty at the University of Nebraska at Omaha (UNO) have partnered with teachers and administrators in the Omaha Public Schools (OPS) to implement a Teacher-Researcher Partnership Program. This program establishes resources and infrastructure that engage K-12 science teachers in scientific research experiences. In the first implementation of this program, eleven UNO faculty mentors, drawn from several STEM disciplines, were matched with eleven OPS teachers to conduct genuine research projects in support of their teaching

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

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, confidence in process and content, and the perception of translatability to the classroom

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

NE STEM 4U afterschool intervention leads to gains in STEM content knowledge for middle school youth

Afterschool interventions in STEM are linked to learning gains during the school day. These opportunities engage and excite students about STEM concepts since they observe a more hands-on, project-oriented approach. Often these opportunities for afterschool interventions are infrequent in nature and leave gaps for students in their maturation and understanding. Herein we describe the first report of an afterschool intervention, named NE STEM 4U, targeting socioeconomically disadvantaged middle school youth via a twice weekly, year-long intervention, studied across two years. We assessed the impact of this program on i.) short-term, individual student gains in STEM content knowledge and ii.) delivery of the program in terms of appropriateness for age group and content using the DoS observation tool. We observed statistically significant gains in STEM content knowledge over short-term assessment using a multiple-group, pre-test post-test research design comparing scores in content before and after the intervention. In this report, we highlight the impact of this nascent program in Omaha Public Schools

RNA Structure in the 5′ Untranslated Region of Enterovirus D68 Strains with Differing Neurovirulence Phenotypes

Enterovirus-D68 (EV-D68) is a positive-sense single-stranded RNA virus within the family Picornaviridae. EV-D68 was initially considered a respiratory virus that primarily affected children. However, in 2014, EV-D68 outbreaks occurred causing the expected increase in respiratory illness cases, but also an increase in acute flaccid myelitis cases (AFM). Sequencing of 2014 outbreak isolates revealed variations in the 5′ UTR of the genome compared to the historical Fermon strain. The structure of the 5′ UTR RNA contributes to enterovirus virulence, including neurovirulence in poliovirus, and could contribute to neurovirulence in contemporary EV-D68 strains. In this study, the secondary and tertiary structures of 5′ UTR RNA from the Fermon strain and 2014 isolate KT347251.1 are analyzed and compared. Secondary structures were determined using SHAPE-MaP and TurboFold II and tertiary structures were predicted using 3dRNAv2.0. Comparison of RNA structures between the EV-D68 strains shows significant remodeling at the secondary and tertiary levels. Notable secondary structure changes occurred in domains II, IV and V. Shifts in the secondary structure changed the tertiary structure of the individual domains and the orientation of the domains. Our comparative structural models for EV-D68 5′ UTR RNA highlight regions of the molecule that could be targeted for treatment of neurotropic enteroviruses

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

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