Reza Karimi

Novel Roles for Classical Factors at the Interface between Translation Termination and Initiation

The pathway of bacterial ribosome recycling following translation termination has remained obscure. Here, we elucidate two essential steps and describe the roles played by the three translation factors EF-G, RRF, and IF3. Release factor RF3 is known to catalyze the dissociation of RF1 or RF2 from ribosomes after polypeptide release. We show that the next step is dissociation of 50S subunits from the 70S posttermination complex and that it is catalyzed by RRF and EF-G and requires GTP hydrolysis. Removal of deacylated tRNA from the resulting 30S:mRNA:tRNA posttermination complex is then necessary to permit rapid 30S subunit recycling. We show that this step requires initiation factor IF3, whose role was previously thought to be restricted to promoting specific 30S initiation complex formation from free 30S subunits.

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl‐tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop‐off products of these events are hydrolysed by peptidyl‐tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop‐off of peptidyl‐tRNA, using a thermosensitive peptidyl‐tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl‐tRNA hydrolase mutant. In vitro measurements of peptidyl‐tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl‐tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12‐fold. It is suggested that RF4 may displace peptidyl‐tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

Perforin Lytic Activity Is Controlled by Calreticulin

The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 × 10−7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8.8 × 10−7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.

Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes.

We studied the dissociation rates of peptidyl‐tRNA from the P‐site of poly(U)‐programmed wild‐type Escherichia coli ribosomes, hyperaccurate variants altered in S12 (SmD, SmP) and error‐prone variants (Ram) altered in S4 or S5. The experiments were carried out in the presence and absence of streptomycin, and the effects of neomycin were tested in the wild‐type ribosomes. Binding of peptidyl‐tRNA to the P‐site of wild‐type ribosomes is much stronger than to their A‐site. Addition of streptomycin dramatically reduces its affinity for the P‐site. The S12 alternations make the P‐site binding of peptidyl‐tRNA much tighter, and the S4, S5 alterations make it weaker than in the case of the wild‐type. We find that when binding of peptidyl‐tRNA to the A‐site is weak, then the affinity for the P‐site is stronger, and vice versa. From these results, we formulate a hypothesis for the actions of streptomycin and neomycin based on deformations of the 16S rRNA tertiary structure. The results are also used to interpret some in vivo experiments on translational processivity.

Team-Based Learning in US Colleges and Schools of Pharmacy

Objective. To characterize the use of team-based learning (TBL) in US colleges and schools of pharmacy, including factors that may affect implementation and perceptions of faculty members regarding the impact of TBL on educational outcomes. Methods. Respondents identified factors that inhibit or enable TBL use and its impact on student learning. Results were stratified by type of institution (public/private), class size, and TBL experience. Results. Sixty-nine of 100 faculty members (69%) representing 43 (86%) institutions responded. Major factors considered to enable TBL implementation included a single campus and student and administration buy-in. Inhibiting factors included distant campuses, faculty resistance, and lack of training. Compared with traditional lectures, TBL is perceived to enhance student engagement, improve students’ preparation for class, and promote achievement of course outcomes. In addition, TBL is perceived to be more effective than lectures at fostering learning in all 6 domains of Bloom’s Taxonomy. Conclusions. Despite potential implementation challenges, faculty members perceive that TBL improves student engagement and learning.

Learning Bridge Tool to Improve Student Learning, Preceptor Training, and Faculty Teamwork

Objectives. To implement a Learning Bridge tool to improve educational outcomes for pharmacy students as well as for preceptors and faculty members. Design. Pharmacy faculty members collaborated to write 9 case-based assignments that first-year pharmacy (P1) students worked with preceptors to complete while at experiential sites. Assessment. Students, faculty members, and preceptors were surveyed about their perceptions of the Learning Bridge process. As in our pilot study,1 the Learning Bridge process promoted student learning. Additionally, the Learning Bridge assignments familiarized preceptors with the schools P1 curriculum and its content. Faculty teamwork also was increased through collaborating on the assignments. Conclusions. The Learning Bridge assignments provided a compelling learning environment and benefited students, preceptors, and faculty members.

Interface between problem-based learning and a learner-centered paradigm.

Background Problem-based learning (PBL) has made a major shift in support of student learning for many medical school curricula around the world. Since curricular development of PBL in the early 1970s and its growth in the 1980s and 1990s, there have been growing numbers of publications providing positive and negative data in regard to the curricular effectiveness of PBL. The purpose of this study was to explore supportive data for the four core objectives of PBL and to identify an interface between the objectives of PBL and a learner-centered paradigm. Methods The four core PBL objectives, ie, structuring of knowledge and clinical context, clinical reasoning, self-directed learning, and intrinsic motivation, were used to search MEDLINE, the Education Resources Information Center, the Educator’s Reference Complete, and PsycINFO from January 1969 to January 2011. The literature search was facilitated and narrowed if the published study included the following terms: “problem-based learning”, “medical education”, “traditional curriculum”, and one of the above four PBL objectives. Results Through a comprehensive search analysis, one can find supportive data for the effectiveness of a PBL curriculum in achieving the four core objectives of PBL. A further analysis of these four objectives suggests that there is an interface between PBL objectives and criteria from a learner-centered paradigm. In addition, this review indicates that promotion of teamwork among students is another interface that exists between PBL and a learner-centered paradigm. Conclusion The desire of medical schools to enhance student learning and a need to provide an environment where students construct knowledge rather than receive knowledge have encouraged many medical schools to move into a learner-centered paradigm. Implementation of a PBL curriculum can be used as a prevailing starting point to develop not only a learner-centered paradigm, but also to facilitate a smooth curricular transition from a teacher-centered paradigm to a learner-centered paradigm.

Developing and Implementing an Interprofessional Course and Identifying Its Strengths and Challenges

An interprofessional course (IPC) was developed to provide basic guidance in developing essential knowledge, skills, attitudes, and values in order to function effectively in an interprofessional healthcare community. A total of 357 first-year students from seven professional programs: Dental Health Science, Masters of Healthcare Administration, Occupational Therapy, Physical Therapy, Physician Assistant Studies, Pharmacy, and Professional Psychology, were enrolled in the IPC. Sixteen faculty members from the above programs participated to deliver four didactic topics: teambuilding and communication, diversity, professionalism, and community resources. A fifth topic was assigned to summarize didactic and experiential experiences. Pre and post surveys were implemented to evaluate the effectiveness of the entire IPC. Our results indicated that students appreciated the value of an IPC for developing interprofessional skills and that an interprofessional course is a meaningful curricular activity to be offered to students. Steps taken to establish and implement the IPC and identify strengths and challenges are discussed.

Implementation of an Integrated Longitudinal Curricular Activity for Graduating Pharmacy Students

Objectives. To evaluate whether a novel integrated longitudinal curricular activity to prepare graduating doctor of pharmacy (PharmD) students for 2 comprehensive examinations was successful, and to assess whether it engaged other pharmacy students in curricular discussion and learning. Design. Thirty-eight of 91graduating third-year (P3) students in a PharmD program formed 11 teams to create and present pharmacotherapeutic posters to their peers. The impact of the novel activity on graduating students’ performance on the North American Pharmacist Licensure Examination (NAPLEX) and a comprehensive commercial examination was assessed. All first-year (P1), second-year (P2), and P3 students reviewed and discussed the content of each poster. Assessment. Participants in the integrated longitudinal curricular activity performed better than nonparticipants on the commercial examination (p=0.023) and NAPLEX (p=0.033). However, regardless of participation, commercial examination scores predicted a significant amount of variance (ie, 34%) in NAPLEX scores. The P3 participants (83%) believed the curricular activity assisted them in their NAPLEX preparation, while 75% of P1 students, 79% of P2 students, and 80% of P3 students agreed that poster review provided an effective summary of different disease states. Ninety percent of faculty poster evaluators reported that the posters were professional, and all evaluators agreed that participants effectively conveyed their message to the intended audience. Conclusion. The integrated longitudinal curricular activity provided a positive learning environment for all pharmacy students and may have better prepared graduating students’ for the NAPLEX.

Forecasting academic success through implementation of an online prerequisite review tutorials program for first year pharmacy students

OBJECTIVE Online prerequisite review (OPR) tutorials were designed and implemented to reinforce foundational scientific material in order to protect in-class time, foster self-directed learning, and ensure all students have similar baseline knowledge. METHODS Twenty-one tutorials covering undergraduate prerequisite material were developed by faculty and organized into six core modules, comprising basic biology, chemistry, and physiology topics. A quiz on this material was given on the first day of each course. This score was correlated with the final exam score at course completion. Additional student and faculty feedback was collected through surveys. RESULTS 2372 quiz-exam pairings were collected over three consecutive fall semesters. A one point increase in the quiz score was associated with a 3.6 point (95% confidence interval 3.1-4.0) higher exam score, as well as a greater probability of passing the exam (P<0.0001). Furthermore, simple linear regression revealed a positive correlation between quiz and exam scores (P<0.0001). Three full years of student survey data revealed an overwhelmingly positive perception of the OPR tutorials, and surveyed faculty reported better use of class time and improved student competency and participation. CONCLUSIONS Implementation of OPR tutorials may give faculty more efficient use of class time, and their associated quizzes serve as an early indicator for students at-risk of not passing who are candidates for early interventions. Furthermore, the OPR tutorial design gives it great transferability to biomedical post-graduate programs.