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Erin Easlon - Biology

Erin Easlon - Biology


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Erin Easlon

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Research output : Contribution to journal › Article › peer-review

T1 - The dihydrolipoamide acetyltransferase is a novel metabolic longevity factor and is required for calorie restriction-mediated life span extension

N2 - Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes.

AB - Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes.


The Effects of Practice-Based Training on Graduate Teaching Assistants’ Classroom Practices

Evidence-based teaching is a highly complex skill, requiring repeated cycles of deliberate practice and feedback to master. Despite existing well-characterized frameworks for practice-based training in K–12 teacher education, the major principles of these frameworks have not yet been transferred to instructor development in higher educational contexts, including training of graduate teaching assistants (GTAs). We sought to determine whether a practice-based training program could help GTAs learn and use evidence-based teaching methods in their classrooms. We implemented a weekly training program for introductory biology GTAs that included structured drills of techniques selected to enhance student practice, logic development, and accountability and reduce apprehension. These elements were selected based on their previous characterization as dimensions of active learning. GTAs received regular performance feedback based on classroom observations. To quantify use of target techniques and levels of student participation, we collected and coded 160 h of video footage. We investigated the relationship between frequency of GTA implementation of target techniques and student exam scores however, we observed no significant relationship. Although GTAs adopted and used many of the target techniques with high frequency, techniques that enforced student participation were not stably adopted, and their use was unresponsive to formal feedback. We also found that techniques discussed in training, but not practiced, were not used at quantifiable frequencies, further supporting the importance of practice-based training for influencing instructional practices.


The Best of Both Worlds: Building on the COPUS and RTOP Observation Protocols to Easily and Reliably Measure Various Levels of Reformed Instructional Practice

Researchers, university administrators, and faculty members are increasingly interested in measuring and describing instructional practices provided in science, technology, engineering, and mathematics (STEM) courses at the college level. Specifically, there is keen interest in comparing instructional practices between courses, monitoring changes over time, and mapping observed practices to research-based teaching. While increasingly common observation protocols (Reformed Teaching Observation Protocol [RTOP] and Classroom Observation Protocol in Undergraduate STEM [COPUS]) at the postsecondary level help achieve some of these goals, they also suffer from weaknesses that limit their applicability. In this study, we leverage the strengths of these protocols to provide an easy method that enables the reliable and valid characterization of instructional practices. This method was developed empirically via a cluster analysis using observations of 269 individual class periods, corresponding to 73 different faculty members, 28 different research-intensive institutions, and various STEM disciplines. Ten clusters, called COPUS profiles, emerged from this analysis they represent the most common types of instructional practices enacted in the classrooms observed for this study. RTOP scores were used to validate the alignment of the 10 COPUS profiles with reformed teaching. Herein, we present a detailed description of the cluster analysis method, the COPUS profiles, and the distribution of the COPUS profiles across various STEM courses at research-intensive universities.


Abstract

The yeast Saccharomyces cerevisiae produces two 14-3-3 proteins, Bmh1 and Bmh2, whose exact functions have remained unclear. Here, we performed a comprehensive proteomic analysis using multistep immunoaffinity purification and mass spectrometry and identified 271 yeast proteins that specifically bind to Bmh1 and -2 in a phosphorylation-dependent manner. The identified proteins have diverse biochemical functions and cellular roles, including cell signaling, metabolism, and cell cycle regulation. Importantly, there are a number of protein subsets that are involved in the regulation of yeast physiology through a variety of cell signaling pathways, including stress-induced transcription, cell division, and chitin synthesis at the cell wall. In fact, we found that a yeast mutant deficient in Bmh1 and -2 had defects in signal-dependent response of the MAPK (Hog1 and Mpk1) cascade and exhibited an abnormal accumulation of chitin at the bud neck. We propose that Bmh1 and -2 are common regulators of many cell signaling modules and pathways mediated by protein phosphorylation and regulate a variety of biological events by coordinately controlling the identified multiplex phosphoprotein components.

This work was supported in part by Grants-in-Aid for Scientific Research, Grants for the Integrated Proteomics System Project, Pioneer Research on the Genome Frontier from MEXT of Japan, and the MEXT program “Initiatives of Attractive Education in Graduate Schools”.

Tokyo Metropolitan University.

CREST, Japan Science and Technology Agency.

Mitsubishi Kagaku Institute of Life Sciences.

To whom correspondence should be addressed: Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa 239-8686, Japan. Telephone: 81 468 41 3810, ext. 3563. Fax: 81 468 44 5901. E-mail: [email protected]

Present address: Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa 239-8686, Japan.


Combining Peer Discussion with Instructor Explanation Increases Student Learning from In-Class Concept Questions

Use of in-class concept questions with clickers can transform an instructor-centered “transmissionist” environment to a more learner-centered constructivist classroom. To compare the effectiveness of three different approaches using clickers, pairs of similar questions were used to monitor student understanding in majors’ and nonmajors’ genetics courses. After answering the first question individually, students participated in peer discussion only, listened to an instructor explanation only, or engaged in peer discussion followed by instructor explanation, before answering a second question individually. Our results show that the combination of peer discussion followed by instructor explanation improved average student performance substantially when compared with either alone. When gains in learning were analyzed for three ability groups of students (weak, medium, and strong, based on overall clicker performance), all groups benefited most from the combination approach, suggesting that peer discussion and instructor explanation are synergistic in helping students. However, this analysis also revealed that, for the nonmajors, the gains of weak performers using the combination approach were only slightly better than their gains using instructor explanation alone. In contrast, the strong performers in both courses were not helped by the instructor-only approach, emphasizing the importance of peer discussion, even among top-performing students.


Expression, Purification, and Characterization of the Dihydrolipoamide Dehydrogenase-Binding Protein of the Pyruvate Dehydrogenase Complex from Saccharomyces cerevisiae

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The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate–aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1Δaat1Δ double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate–aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles.


Abstract

The yeast Saccharomyces cerevisiae produces two 14-3-3 proteins, Bmh1 and Bmh2, whose exact functions have remained unclear. Here, we performed a comprehensive proteomic analysis using multistep immunoaffinity purification and mass spectrometry and identified 271 yeast proteins that specifically bind to Bmh1 and -2 in a phosphorylation-dependent manner. The identified proteins have diverse biochemical functions and cellular roles, including cell signaling, metabolism, and cell cycle regulation. Importantly, there are a number of protein subsets that are involved in the regulation of yeast physiology through a variety of cell signaling pathways, including stress-induced transcription, cell division, and chitin synthesis at the cell wall. In fact, we found that a yeast mutant deficient in Bmh1 and -2 had defects in signal-dependent response of the MAPK (Hog1 and Mpk1) cascade and exhibited an abnormal accumulation of chitin at the bud neck. We propose that Bmh1 and -2 are common regulators of many cell signaling modules and pathways mediated by protein phosphorylation and regulate a variety of biological events by coordinately controlling the identified multiplex phosphoprotein components.

This work was supported in part by Grants-in-Aid for Scientific Research, Grants for the Integrated Proteomics System Project, Pioneer Research on the Genome Frontier from MEXT of Japan, and the MEXT program “Initiatives of Attractive Education in Graduate Schools”.

Tokyo Metropolitan University.

CREST, Japan Science and Technology Agency.

Mitsubishi Kagaku Institute of Life Sciences.

To whom correspondence should be addressed: Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa 239-8686, Japan. Telephone: 81 468 41 3810, ext. 3563. Fax: 81 468 44 5901. E-mail: [email protected]

Present address: Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa 239-8686, Japan.



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