Increasing access for biochemistry research in undergraduate education: The malate dehydrogenase CURE community.

Integrating research into the classroom environment is an influential pedagogical tool to support student learning, increase retention of STEM students, and help students identify as scientists. The evolution of course-based undergraduate research experiences (CUREs) has grown from individual faculty incorporating their research in the teaching laboratory into well-supported systems to sustain faculty engagement in CUREs. To support the growth of protein-centric biochemistry-related CUREs, we cultivated a community of enthusiastic faculty to develop and adopt malate dehydrogenase (MDH) as a CURE focal point. The MDH CURE Community has grown into a vibrant and exciting group of over 28 faculty from various institutions, including community colleges, minority-serving institutions, undergraduate institutions, and research-intensive institutions in just 4 years. This collective has also addressed important pedagogical questions on the impact of CURE collaboration and the length of the CURE experience in community colleges, undergraduate institutions, and research-intensive institutions. This work provided evidence that modular or partial-semester CUREs also support student outcomes, especially the positive impact it had on underrepresented students. We are currently focused on expanding the MDH CURE Community network by generating more teaching and research materials, creating regional hubs for local interaction and increasing mentoring capacity, and offering mentoring and professional development opportunities for new faculty adopters.

Physiology of malate dehydrogenase and how dysregulation leads to disease.

Malate dehydrogenase (MDH) is pivotal in mammalian tissue metabolism, participating in various pathways beyond its classical roles and highlighting its adaptability to cellular demands. This enzyme is involved in maintaining redox balance, lipid synthesis, and glutamine metabolism and supports rapidly proliferating cells' energetic and biosynthetic needs. The involvement of MDH in glutamine metabolism underlines its significance in cell physiology. In contrast, its contribution to lipid metabolism highlights its role in essential biosynthetic processes necessary for cell maintenance and proliferation. The enzyme's regulatory mechanisms, such as post-translational modifications, underscore its complexity and importance in metabolic regulation, positioning MDH as a potential target in metabolic dysregulation. Furthermore, the association of MDH with various pathologies, including cancer and neurological disorders, suggests its involvement in disease progression. The overexpression of MDH isoforms MDH1 and MDH2 in cancers like breast, prostate, and pancreatic ductal adenocarcinoma, alongside structural modifications, implies their critical role in the metabolic adaptation of tumor cells. Additionally, mutations in MDH2 linked to pheochromocytomas, paragangliomas, and other metabolic diseases emphasize MDH's role in metabolic homeostasis. This review spotlights MDH's potential as a biomarker and therapeutic target, advocating for further research into its multifunctional roles and regulatory mechanisms in health and disease.

Exploring the uncharted territory of the potential protein-protein interactions of cytosolic malate dehydrogenase.

In this review, we examine the protein-protein interactions of cytosolic malate dehydrogenase (MDH), an under-studied area in cellular metabolism. We provide a comprehensive overview of MDH involvement in metabolism, especially its interactions with metabolic partners and dynamics of changing metabolism. We present an analysis of the biophysical nature of these interactions and the current methods used to study them. Our review includes an assessment of computational docking studies, which offer initial hypotheses about potential MDH interaction partners. Furthermore, we provide a summary of the sparse yet insightful experimental evidence available, establishing a foundation for future research. By integrating biophysical analysis and methodological advancements, this paper aims to illuminate the intricate network of interactions involving cytosolic MDH and their metabolic implications. This work not only contributes to our understanding of MDH's role in metabolism but also highlights the potential impact of these interactions in metabolic disorders.

Phosphorylation of mammalian cytosolic and mitochondrial malate dehydrogenase: insights into regulation.

Malate dehydrogenase (MDH) is a key enzyme in mammalian metabolic pathways in cytosolic and mitochondrial compartments. Regulation of MDH through phosphorylation remains an underexplored area. In this review we consolidate evidence supporting the potential role of phosphorylation in modulating the function of mammalian MDH. Parallels are drawn with the phosphorylation of lactate dehydrogenase, a homologous enzyme, to reveal its regulatory significance and to suggest a similar regulatory strategy for MDH. Comprehensive mining of phosphorylation databases, provides substantial experimental (primarily mass spectrometry) evidence of MDH phosphorylation in mammalian cells. Experimentally identified phosphorylation sites are overlaid with MDH's functional domains, offering perspective on how these modifications could influence enzyme activity. Preliminary results are presented from phosphomimetic mutations (serine/threonine residues changed to aspartate) generated in recombinant MDH proteins serving as a proof of concept for the regulatory impact of phosphorylation. We also examine and highlight several approaches to probe the structural and cellular impact of phosphorylation. This review highlights the need to explore the dynamic nature of MDH phosphorylation and calls for identifying the responsible kinases and the physiological conditions underpinning this modification. The synthesis of current evidence and experimental data aims to provide insights for future research on understanding MDH regulation, offering new avenues for therapeutic interventions in metabolic disorders and cancer.

Length of course-based undergraduate research experiences (CURE) impacts student learning and attitudinal outcomes: A study of the Malate dehydrogenase CUREs Community (MCC).

Course-based undergraduate research experiences (CUREs) are laboratory courses that integrate broadly relevant problems, discovery, use of the scientific process, collaboration, and iteration to provide more students with research experiences than is possible in individually mentored faculty laboratories. Members of the national Malate dehydrogenase CUREs Community (MCC) investigated the differences in student impacts between traditional laboratory courses (control), a short module CURE within traditional laboratory courses (mCURE), and CUREs lasting the entire course (cCURE). The sample included approximately 1,500 students taught by 22 faculty at 19 institutions. We investigated course structures for elements of a CURE and student outcomes including student knowledge, student learning, student attitudes, interest in future research, overall experience, future GPA, and retention in STEM. We also disaggregated the data to investigate whether underrepresented minority (URM) outcomes were different from White and Asian students. We found that the less time students spent in the CURE the less the course was reported to contain experiences indicative of a CURE. The cCURE imparted the largest impacts for experimental design, career interests, and plans to conduct future research, while the remaining outcomes were similar between the three conditions. The mCURE student outcomes were similar to control courses for most outcomes measured in this study. However, for experimental design, the mCURE was not significantly different than either the control or cCURE. Comparing URM and White/Asian student outcomes indicated no difference for condition, except for interest in future research. Notably, the URM students in the mCURE condition had significantly higher interest in conducting research in the future than White/Asian students.

External Collaboration Results in Student Learning Gains and Positive STEM Attitudes in CUREs.

The implementation of course-based undergraduate research experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to be authentic, they should reflect the increasingly collaborative nature of research. While some CUREs have expanded, involving multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no-CURE, 2) single-institution CURE (CURE), and 3) external collaborative CURE (ec-CURE), and assessed academic and attitudinal outcomes. The ec-CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypotheses and discuss their data. The sharing of ideas, data, and materials with an external faculty member allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec-CURE had the greatest gains in experimental design; self-reported course benefits; scientific skills; and science, technology, engineering, and mathematics (STEM) importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2- and 4-year institutions, illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.

Sodium hydrogen exchanger (NHE1) palmitoylation and potential functional regulation.

AIMS: Determine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family. MAIN METHODS: NHE1 expressed in native rat tissues or in heterologous cells was assessed for palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with [3H]palmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP. KEY FINDINGS: In this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pHi), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pHi which is dependent on the presence of NHE1. SIGNIFICANCE: Palmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions.

COVID-360: A Collaborative Effort to Develop a Multidisciplinary Set of Online Resources for Engaging Teaching on the COVID-19 Pandemic.

The COVID-19 pandemic has challenged undergraduate instructors and students in an unprecedented manner. Each has needed to find creative ways to continue the engaged teaching and learning process in an environment defined by physical separation and emotional anxiety and uncertainty. As a potential tool to meet this challenge, we developed a set of curricular materials that combined our respective life science teaching interests with the real-time scientific problem of the COVID-19 pandemic in progress. Discrete modules were designed that are engaging to students, implement active learning-based coursework in a variety of institutional and learning settings, and can be used either in person or remotely. The resulting interdisciplinary curriculum, dubbed "COVID-360," enables instructors to select from a menu of curricular options that best fit their course content, desired activities, and mode of class delivery. Here we describe how we devised the COVID-360 curriculum and how it represents our efforts to creatively and effectively respond to the instructional needs of diverse students in the face of an ongoing instructional crisis.

IUBMB/PSBMB 2019 Conference/Plenary: National Program Accreditation. Does it help drive change and support faculty and student or does it limit our creativity?

Accreditation of academic programs and recognition of student degrees provide academic institutions a measure of a set of community agreed upon standards. These can aid pedagogical change, support faculty to successfully engage students in their discipline and to provide a mechanism to maintain standards. Several professional scientific societies from engineering, chemistry, and biochemistry and molecular biology have developed standards by which departments can be recognized for accreditation. As one of the members of the American Society for Biochemistry and Molecular Biology who helped develop the accreditation and standardized exams and a committee member of the American Chemical Society's Committee on Professional Training I will present the evolution of the accreditation process, discuss the benefits and challenges with being an accreditation. How these programs serve their communities and at times can hinder or be used to support potential creativity and teaching pedagogies will also be discussed.

Tools for teaching biochemistry and molecular biology: A parallel session at the IUBMB/PSBMB 2019 "Harnessing Interdisciplinary Education in Biochemistry and Molecular Biology" conference.

Approaches to learning and teaching have been undergoing massive changes. Technology has enabled many innovations while other methods have embedded authentic research approaches or looked to other disciplines. The tools in education session of the conference looked at tools being used to teach biochemistry and molecular biology ranging from online platforms, authentic research experiences to the use of music.

Sodium hydrogen exchanger and phospholipase D are required for alpha1-adrenergic receptor stimulation of metalloproteinase-9 and cellular invasion in CCL39 fibroblasts.

Matrix metalloproteinase 9 (MMP-9) plays a critical role in digesting the extracellular matrix and has a vital function in tumor metastasis and invasion; this protease activity is significantly increased in non-small cell lung cancers. The sodium hydrogen exchanger isoform 1 (NHE1) functions as a focal point for signal coordination and cytoskeletal reorganization. NHE1 is thought to play a central role in establishing signaling components at the leading edge of a migrating cell. Therefore, we studied the relationship between NHE1 and MMP-9 activity in Chinese hamster lung fibroblasts (CCL39) stimulated with phenylephrine (PE). We show that PE increases MMP-9 gelatinolytic activity in CCL39 cells. The inhibition of phospholipase D (PLD) signaling abrogated PE-induced MMP-9 activity. The role of PLD as an essential signaling intermediate was confirmed when the addition of permeable phosphatidic acid increased MMP-9 activity in the same cells. PE-induced invasion was increased 1.9-fold over controls and the PE response was lost when 1-butanol was used to block PLD signaling. Cells pre-treated with the NHE1 inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) prior to PE addition resulted in a notable decrease in MMP-9 activation and cell invasion as compared to untreated PE-stimulated cells. CCL39 NHE1 null cells demonstrated no increase in MMP-9 protease activity or cell invasion in response to PE treatment. Reconstitution of NHE1 expression recovered the PE-induced activation of protease activity and cell invasion. MMP-9 processing was altered in cells expressing a proton transport defective NHE1 but retained the ability to respond to PE. Conversely, cells expressing an ezrin, radixin, moesin (ERM)-binding deficient NHE1 had a lower MMP-9 activity and the protease did not respond to PE addition. Parallel studies on NCI-H358 non-small cell lung cancer (NSCL) cells showed that PE stimulated both MMP-9 activity and cell invasion in an NHE1 dependent manner. This work describes for the first time a PE-induced relationship between NHE1 and MMP-9 and a new potential mechanism by which NHE1 could promote tumor formation and metastasis.

Assessing stakeholder perceptions of the american society for biochemistry and molecular biology accreditation program for baccalaureate degrees.

The American Society for Biochemistry and Molecular Biology (ASBMB) began an accreditation program in 2013. The criteria for accreditation of undergraduate programs include sufficient infrastructure - number and expertise of faculty, physical space and equipment, support for faculty and students - and incorporation of core concepts in the curriculum - structure and function of biomolecules; information storage; energy transfer; and quantitative skills. Students in accredited programs are able to have their degrees ASBMB certified by taking an exam focused on knowledge or skills across the four core concept areas. Members of the accreditation committees administered a survey to key stakeholders in the BMB community: undergraduate programs, both those that have applied for accreditation and those that have not; alumni/ae of accredited programs; graduate and professional programs; and employers. The goals of the study were to gauge the success of the program and determine necessary areas of improvement. The results indicate that the major benefits of applying for accreditation are the impetus to gather data and analysis not generally collected, and access to assessment data via the exam. However, stakeholders outside of the undergraduate community showed little awareness of the accreditation program. Additionally, the application process itself was seen to be very time consuming. This feedback will be used to improve the process and engage in further outreach. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):464-471, 2018.

CUREs in biochemistry-where we are and where we should go.

Integration of research experience into classroom is an important and vital experience for all undergraduates. These course-based undergraduate research experiences (CUREs) have grown from independent instructor lead projects to large consortium driven experiences. The impact and importance of CUREs on students at all levels in biochemistry was the focus of a National Science Foundation funded think tank. The state of biochemistry CUREs and suggestions for moving biochemistry forward as well as a practical guide (supplementary material) are reported here. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):7-12, 2017.

RhoA Kinase (Rock) and p90 Ribosomal S6 Kinase (p90Rsk) phosphorylation of the sodium hydrogen exchanger (NHE1) is required for lysophosphatidic acid-induced transport, cytoskeletal organization and migration.

The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show that each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.

Integrating standard operating procedures and industry notebook standards to evaluate students in laboratory courses.

To enhance the preparedness of graduates from the Biochemistry and Biotechnology (BCBT) Major at Minnesota State University Moorhead for employment in the bioscience industry we have developed a new Industry certificate program. The BCBT Industry Certificate was developed to address specific skill sets that local, regional, and national industry experts identified as lacking in new B.S. and B.A. biochemistry graduates. The industry certificate addresses concerns related to working in a regulated industry such as Good Laboratory Practices, Good Manufacturing Practices, and working in a Quality System. In this article we specifically describe how we developed a validation course that uses Standard Operating Procedures to describe grading policy and laboratory notebook requirements in an effort to better prepare students to transition into industry careers.

Inside out: targeting NHE1 as an intracellular and extracellular regulator of cancer progression.

The sodium hydrogen exchanger isoform one is a critical regulator of intracellular pH, serves as an anchor for the formation of cytoplasmic signaling complexes, and modulates cytoskeletal organization. There is a growing interest in the potential for sodium hydrogen exchanger isoform one as a therapeutic target against cancer. Sodium hydrogen exchanger isoform one transport drives formation of membrane protrusions essential for cell migration and contributes to the establishment of a tumor microenvironment that leads to the rearrangement of the extracellular matrix further supporting tumor progression. Here, we focus on the potential impact that an inexpensive, $100 genome would have in identifying prospective therapeutic targets to treat tumors based upon changes in gene expression and variation of sodium hydrogen exchanger isoform one regulators. In particular, we will focus on the ezrin, radixin, moesin family proteins, calcineurin B homologous proteins, Ras/Raf/MEK/ERK signaling, and phosphoinositide signaling as they relate to the regulation of sodium hydrogen exchanger isoform one in cancer progression.

Bringing the excitement and motivation of research to students; Using inquiry and research-based learning in a year-long biochemistry laboratory: Part I-guided inquiry-purification and characterization of a fusion protein: Histidine tag, malate dehydrogenase, and green fluorescent protein.

A successful laboratory experience provides the foundation for student success, creating active participation in the learning process. Here, we describe a new approach that emphasizes research, inquiry and problem solving in a year-long biochemistry experience. The first semester centers on the purification, characterization, and analysis of a novel fusion protein within a guided research experience. Throughout the semester, students gradually acquire skills as they are allowed to work independently. A fusion protein, malate dehydrogenase-green fluorescent protein with a histidine affinity tag (MGH), is used throughout the semester. The fusion protein allows for a high throughput analysis and is stable for duration of the semester. Students start with the purification and analysis of the plasmid DNA and end with an enzymatic analysis of MGH. As students take ownership of their experiments and choose two different chromatographic resins, they make many choices throughout the semester. Skills, motivation, confidence levels, and attitudes were assessed before and after the semester. Students achieved high levels of critical biochemical laboratory skills and critical thinking while increasing their confidence and motivation for working in a biochemical research setting.