Small Science: A Tool and Tips for Converting Food Science Demonstrations into Public Inquiry Experiences

Small-scale science activities provide an opportunity for engagement of diverse, large audiences at settings such as 4-H fairs. We present practical information for implementation of small-scale food science experiences in the Extension education context. Our focus is description of a tool for adaptation of activities for inquiry-based learning and tips for miniaturization of activities to save on costs and resources

Riboflavin Riboswitch Regulation:Hands-On Learning about the Role of RNA Structures in the Control of Gene Expression in Bacteria

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

Relationships between PrPSc stability and incubation time for United States scrapie isolates in a natural host system.

Transmissible spongiform encephalopathies (TSEs), including scrapie in sheep (Ovis aries), are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP(C)) into a â-rich conformer (PrP(Sc)) that accumulates into higher-order structures in the brain and other tissues. Distinct strains of TSEs exist, characterized by different pathologic profiles upon passage into rodents and representing distinct conformations of PrP(Sc). One biochemical method of distinguishing strains is the stability of PrP(Sc) as determined by unfolding in guanidine hydrochloride (GdnHCl), which is tightly and positively correlated with the incubation time of disease upon passage into mice. Here, we utilize a rapid, protease-free version of the stability assay to characterize naturally occurring scrapie samples, including a fast-acting scrapie inoculum for which incubation time is highly dependent on the amino acid at codon 136 of the prion protein. We utilize the stability methodology to identify the presence of two distinct isolates in the inoculum, and compare isolate properties to those of a host-stabilized reference scrapie isolate (NADC 13-7) in order to assess the stability/incubation time correlation in a natural host system. We demonstrate the utility of the stability methodology in characterizing TSE isolates throughout serial passage in livestock, which is applicable to a range of natural host systems, including strains of bovine spongiform encephalopathy and chronic wasting disease

Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA

Previous studies have come to conflicting conclusions about the requirement for the ω subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking ω does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking ω, likely explaining why strains lacking ω display a stringent response in vivo. These results demonstrate that ω plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of ω in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the β′ subunit that physically link ω to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function

Clinical information for sheep samples.

†<p>Sheep #3 was inoculated with x124 intralingually (IL) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043060#pone.0043060-Bett1" target="_blank">[34]</a>.</p>‡<p>IN = Intranasal/nasopharyngeal route of inoculation, resulting in contact with nasal mucosa and near immediate swallowing with no need for insertion of an esophageal tube.</p

Comparison of 136-VDEP and 13-7 Scrapie Isolates.

<p>Curves were prepared as described in Fig. 2, with pH-adjusted GdnHCl. (A). <i>Comparison of 13-7 scrapie stability in AA₁₃₆ and VV₁₃₆ hosts</i>. Red curve–13-7- infected PrP^Sc from AA₁₃₆ hosts (average of sheep #14, #15, #20); Blue curve–13-7-infected PrP^Sc from VV₁₃₆ hosts (average of sheep #13, 14). The results from three technical replicates from each animal were averaged before averaging biological replicates; red error bars represent SEM and blue error bars represent the range. (B, C). <i>Serial passage of 13-7 inoculum in Suffolk sheep.</i> (C) depicts the process of serial passage, with incubation times of disease in the hosts indicated below the animal in months. Colored symbols in (C) correspond to the colors used to depict the unfolding curve of PrP^Sc from the same sheep in (B). In (B), the results from four independent curves from each animal were averaged, and error bars reflect the SD for technical replicates. (D). <i>Comparison of stability between 136-VDEP and 13-7 isolates</i>. Red curve–136-VDEP-infected PrP^Sc; Black curve–13-7-infected PrP^Sc. Curves are averaged across 4 (red) or 8 (black) biological replicates of each isolate, and error bars reflect SEM of biological replicates. To allow for statistical analysis, the 136-VDEP curve includes AV₁₃₆ and VV₁₃₆ animals, and the 13-7 curve includes VV₁₃₆ and AA₁₃₆ animals (since Fig. 3A demonstrates that genotype does not have an inherent effect on stability). The same pattern is observed with averaged curves of two 136-VDEP-infected VV₁₃₆ sheep (blue curve) and two 13-7-infected VV₁₃₆ sheep (purple curve). [GdnHCl]_1/2 values for the individual animals were also calculated and averaged, with a mean [GdnHCl]_1/2 for 136-VDEP of 2.36 M±0.06 (SEM) and a mean [GdnHCl]_1/2 for 13-7 of 2.80 M±0.10 (SEM). Means of the two populations were significantly different by an unpaired student’s T-test (<i>P</i>-value = 0.0003).</p