Survey of case study users during pandemic shift to remote instruction.

Before COVID-19, the use of case studies to learn science was well established in high school and postsecondary classrooms. Once the pandemic ensued, many faculty continued to use the method as a way to infuse elements of active learning remotely. The results of a survey taken by 600 faculty reveal how they accomplished this feat. Respondents to the survey found that the case method readily transferred to online learning. Most used a mixture of synchronous and asynchronous classrooms. Serious challenges were encountered, primarily due to the difficulty instructors had in keeping track of learner participation. Many obstacles were overcome by creative strategies such as using Google Forms. Some semblance of a normal classroom was achieved by using online conferencing tools and using small groups in synchronous breakout rooms. Cases were commonly broken into chunks and spread over several days. This worked especially well with cases that were already structured this way, including interrupted cases and problem-based learning exercises. Assessment of student performance largely followed the traditional path of exams, projects, and essays, although a third of the faculty attempted to evaluate participation. Classes conducted via an asynchronous approach were largely lecture based, with cases given to learners to complete as homework either individually or as groups. The greatest challenge in this setting was that answers to case questions were often readily available to learners on the internet. This was avoided by faculty modifying questions or creating their own.

Preparation of the Escherichia coli RNase E protein and reconstitution of the RNA degradosome.

The RNA degradosome is a multienzyme complex that plays a key role in the processing of stable RNAs, the degradation of mRNAs, and the action of small regulatory RNAs. Initially discovered in Escherichia coli, similar or related complexes are found in other bacteria. The core of the RNA degradosome is the essential endoribonuclease, RNase E. The C-terminus of this enzyme serves as a scaffold to which other components of the RNA degradosome bind. These ligands include the phosphorolytic 3'-exonuclease, polynucleotide phosphorylase, the DEAD-box RNA helicase, RhlB, and the glycolytic enzyme, enolase. In addition, the DEAD-box RNA helicases CsdA and RhlE and the RNA binding protein, Hfq, may bind to RNase E in place of one or more of the prototypical components. This chapter describes purification of RNase E (the Rne protein), reconstitution of a minimal degradosome that recapitulates the activity of authentic degradosomes, and methods for the assay of the reconstituted complex.

Role of RNA structure and susceptibility to RNase E in regulation of a cold shock mRNA, cspA mRNA.

Degradation of the cspA mRNA in vivo is very rapid at temperatures greater than 30 degrees C and is moderately dependent on RNase E. Investigations in vitro show that degradosomes prepared from normal or cold-shocked cultures cleave the cspA mRNA preferentially at a single site in vitro between two stem-loops approximately 24 residues 3' to the termination codon and approximately 31 residues from the 3' end. The site of cleavage is independent of the temperature and largely independent of the phosphorylation status of the 5' end of cspA mRNA. A 5' stem-loop, potential occlusion of the initiation and termination codons, temperature-dependent translational efficiency, and the position of the RNase E cleavage site can explain the differential stability of the cspA mRNA.

Physical and functional interactions among RNase E, polynucleotide phosphorylase and the cold-shock protein, CsdA: evidence for a 'cold shock degradosome'.

Escherichia coli contains at least five ATP-dependent DEAD-box RNA helicases which may play important roles in macromolecular metabolism, especially in translation and mRNA decay. Here we demonstrate that one member of this family, CsdA, whose expression is induced by cold shock, interacts physically and functionally with RNase E. Three independent approaches show that after a shift of cultures to 15 degrees C, CsdA co-purifies with RNase E and other components of the RNA degradosome. Moreover, functional assays using reconstituted minimal degradosomes prepared from purified components in vitro show that CsdA can fully replace the resident RNA helicase of the RNA degradosome, RhlB. In addition, under these conditions, CsdA displays RNA-dependent ATPase activity. Taken together, our data are consistent with a model in which CsdA accumulates during the early stages of cold acclimatization and subsequently assembles into degradosomes with RNase E synthesized in cold-adapted cultures. These findings show that the RNA degradosome is a flexible macromolecular machine capable of adapting to altered environmental conditions.