bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integration of literature‐based activities to enhance the learning of content, scientific process, and quantitative analysis in biochemistry courses
Authors: Masuda Hisako 
Published: November 2023

Upper level biochemistry courses are a gateway to many different career paths. Students enroll in these courses in preparation for graduate school, professional school or immediately joining the work force. Regardless of their career interests, the application of knowledge into practice is important, but can be challenging to some students.At Indiana University Kokomo, Biochemistry I and II are the only major specific courses that students complete before their senior seminar course which requires students to prepare a mock research proposal. Therefore, one of the learning goals of the Biochemistry courses is to practice the higher order objectives in Bloom's taxonomy of learning, such as evaluation and synthesis of knowledge. In previous years, students expressed difficulties interpreting quantitative data, understanding experimental approaches and reading and finding appropriate research articles, which are all required skills in order to construct a proposal.Several in‐class and homework activities to practice such skills were developed. In collaboration with the science librarian at IUK, flipped lessons to effectively identify articles using NCBI and other databases were developed and tailored for students who lacked any experience in reading and writing scientific articles. Group discussions on a protein purification paper were followed by individual assignments to search for and comprehend articles describing methods such as protein purification, enzyme kinetics, and regulation. Students learned various aspects of a specific protein that they chose at the beginning of the semester. In particular, a protein with a known single amino acid substitution that leads to a genetic disease was suggested as a topic for selection to pique student's interest. Students presented their finding as a poster presentation at the end of semester. In the second semester, students were asked to formulate case study problems based on the reading to lead a discussion on each metabolic pathway. In parallel, students proposed and conducted a simple enzyme activity assay for the uncharacterized protein in laboratory sessions. These activities seemed to have reinforced the lecture material. Moreover, their understanding of the experimental approach and data analysis seemed to have improved. Positive effects of such activities on student learning, as well as current challenges will be discussed.
Modeling Interdisciplinary Collaborations Through a Course‐Based Undergraduate Research Experience (CURE)
Authors: Roberts Rebecca , Koeppe Julia , Price Simara , Allwein Benjamin , Anderson Trevor , Daubner Susan Colette , Irby Stefan , Mills Jeffrey , Pikaart Michael , Craig Paul 
Published: November 2023

Interdisciplinary collaborations are often essential to answer multi‐faceted questions, yet they present with many challenges such as understanding the information, methodologies, and norms of another field and effective communication between team members. We must begin to provide young scientists with the skills necessary for success as the call for more interdisciplinary science expands. We have modelled an interdisciplinary collaboration at the undergraduate level through a Course‐based Undergraduate Research Experience (CURE). Students in upper‐level Biochemistry and Structural Biology courses collaborated to assign functions to proteins “of unknown function” in the Protein Data Bank (PDB). The Structural Biology students used bioinformatics tools including the ProMOL plugin to the PyMOL molecular graphics environment, along with BLAST, Pfam, and Dali to predict protein functions. In parallel, the Biochemistry students expressed and purified the proteins and carried out in vitro testing, informed by the in silico‐predicted enzyme function. Students came together in cross‐disciplinary teams throughout the project to share progress, explain their discipline‐specific methodologies, and discuss next steps. They each presented an oral progress report on the entire project, which required that they not only understand their own work but that of their collaborators. Interdisciplinary teams presented final posters to the campus community and students prepared future directions documents. We had students fill out a pre‐ and post‐assessment. The assessment had two foci: the effectiveness of the cross‐course collaboration model and assessment of content learning. While a second iteration of the CURE will be necessary for a full statistical analysis, we see a trend that students learned the information and methodologies of both their own discipline and that of their collaborators. Moreover students appear to have increased their ability to communicate to and learn from their collaborators.Support or Funding InformationThis project is supported by NSF IUSE 1503811.
Transition to a Course‐based Undergraduate Research Experience (CURE)
Authors: Craig Paul Alfred , Mills Jeffrey L. , Roberts Rebecca , Pikaart Michael , Daubner Colette , Irby Stefan , Anderson Trevor 
Published: November 2023

It will always remain a goal of an undergraduate biochemistry laboratory course to engage students hands‐on in a wide range of biochemistry laboratory experiences. In 2006, our research group initiated a project for in silico prediction of enzyme function based only on the 3D coordinates of the more than 3800 proteins “of unknown function” in the Protein Data Bank (PDB), many of which resulted from the Protein Structure Initiative. Students have used the ProMOL plugin to the PyMOL molecular graphics environment along with BLAST, Pfam, and Dali to predict protein functions. As young scientists, these undergraduate research students wanted to see if their predictions were correct and so they developed an approach for in vitro testing of predicted enzyme function that included literature exploration, selection of a suitable assay and the search for commercially available substrates. Over the past two years, a team of faculty members from seven different campuses (California Polytechnic San Luis Obispo, Hope College, Oral Roberts University, Rochester Institute of Technology, St. Mary's University, and Ursinus College) have transferred this approach to the undergraduate biochemistry teaching lab as a Course‐based Undergraduate Research Experience. A series of ten course modules and eight instructional videos have been created (www.promol.org/home/basil‐modules‐1) and the group is now expanding these resources, creating assessments and evaluating how this approach helps student to grow as scientists. The focus of this presentation will be the logistical implications of this transition on campuses that have different cultures, expectations, schedules and student populations.Support or Funding InformationThis project is supported by NSF IUSE 1503811.
Approaching Undergraduate Research with Students who are Deaf and Hard of Hearing
Authors: Gehret Austin U. , Trussell Jessica W. , Michel Lea V. 
Published: November 2023

An undergraduate research experience can provide a unique opportunity for students to learn and grow as scientists; when positive, this experience is often transformative in helping motivate students to pursue post‐graduate degrees and/or careers in STEM. Conversely, a negative research experience can sour a student's opinion of research, propagate misconceptions of graduate school, and turn students away from careers in STEM. Negative research experiences can be equally as devastating for faculty mentors and likely result in refusal to mentor future research students. Although d/Deaf and hard‐of‐hearing (DHH) students learn differently than their hearing peers, their experiences in undergraduate research mirror that of their hearing counterparts. Up until recently, most researchers have focused on how to understand and improve the learning environments for DHH students in the classroom. Here, we discuss several challenges and strategies derived from survey results associated with the undergraduate research experience for DHH students who were actively involved in undergraduate research. The challenges and strategies outlined aim to shape future mentoring and training efforts of DHH students with the goals of enriching their research experiences, increasing their engagement with research, and improving their pursuit of graduate STEM degrees and/or careers in STEM.Support or Funding InformationThis research was supported by the Camille and Henry Dreyfus Special Grant Program in the Chemical Sciences (Awarded to L.V. M.)
A Novel Protein Interaction Between The Cardiac Desmosome and COP9 Signalosome Complex Reveals A New Mechanism Underlying Sudden Death
Authors: Sheikh Farah , Liang Yan , Lyon Robert , Pellman Jason , Mezzano Valeria , Gu Yusu , Dalton Nancy , Lee Mong‐Hong , Iwakuma Tomoo , Nigam Vishal , Peterson Kirk 
Published: November 2023

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic based cardiomyopathy, characterized by ventricular dysfunction, fibrofatty replacement of the ventricle and ventricular arrhythmias leading to sudden cardiac death in young people. ARVC is termed a “disease of the desmosome”; however, limited information exists on how desmosomal protein dysregulation/loss triggers ARVC. To identify novel protein targets that impact desmosomal biology and the desmosomal disease, ARVC, we performed a yeast‐two‐hybrid screen using the desmosomal gene, desmoplakin (DSP) as bait to screen an adult human heart cDNA library. We identified subunit 6 of the COP9 signalosome (CSN6) as a novel DSP‐interacting protein, localizing at the cardiac intercalated disc (ICD) in both adult mouse and human heart. Well‐known functions of CSN are to regulate protein degradation via ubiquitination; however, the role of CSN6 in the heart and ARVC has not been defined. We show that CSN6 levels are dramatically reduced at the ICD in a mouse model of ARVC (cardiac‐specific DSP knockout mice (DSP‐cKO)) and a human ARVC patient harboring a novel DSP and known plakophilin‐2 mutation. Yeast‐two‐hybrid assays revealed that this novel DSP mutation in the ARVC patient specifically abrogated binding of DSP to CSN6. DSP‐cKO heart also displayed underlying protein degradation defects associated with loss of CSN6, including a striking accumulation of autophagy markers and autophagosomes at the ICD. Importantly, generation of novel cardiac‐specific CSN6 knockout models using the alpha‐myosin heavy chain (MHC)‐Cre (CSN6‐cKO) and alpha‐MHC‐MerCreMer (CSN6‐iKO) mouse lines revealed that loss of CSN6 in mice was sufficient to elicit premature death, which was associated with early ventricular depolarization delay and a biventricular form of cardiomyopathy, reminiscent of ARVC. Ultrastructurally, CSN6‐iKO hearts displayed loss of cardiac architecture associated with an accumulation of autophagosomes at the ICD, similar to our mouse model of ARVC. At the molecular level, CSN6‐iKO heart exhibited early loss of DSP (prior to heart dysfunction) and recapitulated the protein degradation defects observed in our mouse model of ARVC. We highlight the importance of CSN6 at the cardiac desmosome in maintaining desmosomal protein turnover (“desmophagy”), and suggest that loss of CSN6 and desmosomal protein turnover as new underlying mechanisms driving ARVC and sudden death.Support or Funding InformationF.S. is supported by grants from National Institutes of Health (NHLBI), American Heart Association and Tobacco Related Disease Research Program.
Understanding conformational changes during translesion synthesis: in silico studies of DinB
Authors: Perez Elevit , Benitez Benedetta Sampoli 
Published: November 2023

Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little fingers” and have a more solvent accessible active site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Recent hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the template strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. The CHARMM‐gui generator was used to generate a total of 3 systems: a binary system (with DNA and protein only) a ternary system (with DNA and correct incoming nucleotide), and a ternary system with a O6‐methylated guanine on the templating strand), a damage which DinB cannot bypass. Each system was run for a total of 10 ns using the NAMD software package. Visualization and analysis of the systems was performed using the Visual Molecular Dynamics (VMD) application. As predicted, there was little movement in the ternary system with the correct incoming nucleotide, suggesting the polymerase is in a closed conformation therefore active. While we observed some large‐scale movement in the binary system, suggesting that DinB prefers an open, non‐active conformation in the absence of the substrate, we did not see significant subdomain movements the O6‐methylated system, which was predicted to be similar to the binary system. The results point us toward a new direction, implying that instead large conformational changes, this polymerase achieves a catalytically competent state by small local movement occurring at specific sites.Support or Funding InformationThis work was funded by the Rose M. Badgeley Residuary Charitable Trust and the Sister Colette Mahoney Science Research Grant.
DNA Cytosine Methyltransferase Promotes Stationary Phase Fitness in Escherichia coli
Authors: Kambhampati Ooha , Finnerty‐Haggerty Lara , Huss Rebecca , Knapp Rachel , Militello Kevin 
Published: November 2023

In Escherichia coli, DNA cytosine methyltransferase (Dcm) methylates the second cytosine in the sequence 5′CCWGG3, where W is A or T. Dcm is in an operon with the very short patch repair (vsr) gene, an endonuclease that repairs T‐G mismatches caused by the deamination of 5‐methylcytosine. The role of cytosine DNA methylation and the dcm operon in E. coli is unknown and remains an enigma. Growth curve analyses of wild‐type, dcm knockout and vsr knockout strains at both 37°C and 20°C indicated no effect of the dcm or vsr genes on growth rate. Our laboratory's microarray data comparing expression patterns between a wild‐type and dcm knockout strain indicated numerous gene expression changes occur at stationary phase. Similar results were obtained from microarray experiments comparing gene expression differences between untreated cells and cells treated with the cytosine DNA methylation inhibitor 5‐azacytidine. In summary, the microarray experiments suggest a role for Dcm in stationary phase but not logarithmic phase. Then, we tested whether there was an advantage of either the dcm or vsr genes during stationary phase. We performed competition experiments between the wild‐type strain and the dcm knockout strain or vsr knockout strain to determine which strain would persist during long term stationary phase. In the wild‐type/dcm knockout strain competition at 20°C, the wild‐type strain outcompeted the dcm knockout strain; thus the cells lacking the dcm gene have a disadvantage over wild‐type cells. The addition of a plasmid containing a functional copy of the dcm gene to the dcm knockout strain reversed the loss of fitness in the dcm knockout strain. In the wild‐type/vsr knockout strain competition experiment at 20°C, loss of the vsr gene resulted in a modest loss of fitness. Overall, our data indicate that dcm and the dcm operon promote stationary phase fitness. We aim to determine the mechanism for Dcm‐dependent increases in stationary phase fitness which may include changes in the composition of the transcriptome and/or mutation rate.Support or Funding InformationGeneseo Foundation
Mechanism of PWI Domain Binding to Nucleic Acids
Authors: Szymczyna Blair R , Chanzu Harry , Jandaghijafari Nafiseh , Lopez Elizabeth I , Flores Denise 
Published: November 2023

PWI and PWI‐like motifs are four‐helix bundles that are found in several proteins predicted or known to be involved in the regulation of pre‐messenger RNA processing, such as constitutive and alternative splicing and the 3′‐end modification of transcripts. Examples include SR‐related nuclear matrix protein at 160 kDa (SRm160) and RNA Binding Protein 25 (RBM25). The spliceosome also contains several proteins that contain PWI motifs, including Pre‐messenger RNA Processing proteins PRP2, PRP3 and PRP22. The PWI motif is named for a nearly invariant Proline(P)‐Tryptophan(W)‐Isoleucine(I) sequence, although PWI‐like motifs lack this tripeptide sequence and are homologous only in structure. Sub‐families of canonical PWI motifs can be defined by sequence homology, the position of the motif within the protein and the sequence of an adjacent region rich in positively charged (basic) amino acid residues. The PWI motif and the basic region constitute the PWI domain, and both elements are essential for optimal binding to all nucleic acid types. Although RNA is believed to be the natural target, PWI domains non‐specifically bind single‐ and double‐stranded DNA and RNA. The individual roles of the PWI motif and the basic region in the nucleic acid binding mechanism are still not clear. Several biophysical techniques, including electrophoretic mobility shift assays, gel filtration chromatography and fluorescence and nuclear magnetic resonance spectroscopies, are being employed to investigate the mechanism and stoichiometry of PWI domain binding to nucleic acid targets. Our investigation of several PWI domains reveal that both protein‐protein and protein‐nucleic acid interactions participate in the cooperative nucleic acid binding mechanism.Support or Funding InformationStart‐up funds from Western Michigan University to BRS.
Unique Structures in the 3′ UTR of Blackcurrant Reversion Nepovirus Genomic RNA 1 Promote Translation Initiation
Authors: Baquero‐Galvis Laura D. , Shields Elizabeth , Filbin‐Wong Megan E. 
Published: November 2023

Canonical translation initiation is promoted and regulated by an interaction between proteins bound to a cap structure (a methylated guanosine triphosphate, m7G, as the 5′ nucleotide) at the 5′ end and those bound to a polyadenylate [poly(A)] tail at the 3′ end of a messenger RNA (mRNA). This end‐to‐end interaction contributes to the recruitment, positioning, and activation of the translation machinery. Blackcurrant reversion nepovirus (BRV) has two bipartite genomic RNAs (RNA 1 and RNA 2) that do not contain 5′ cap structures, but do have 3′ poly(A) tails. Hence, the mechanism of end‐to‐end interaction in BRV has not been clearly defined. In other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 5′ and 3′ untranslated regions (UTRs) bind via proposed RNA‐RNA kissing‐loop interactions between a 5′ hairpin loop and a 3′ cap independent translation enhancer (CITE). Likewise, the mechanism by which the 5′ and 3′ UTRs recruit protein synthesis machinery is directly related to their primary, secondary and higher‐order structure. Considering that the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our main goal focuses on determining the secondary and possible tertiary structures of the 3′ CITE structure in the genomic RNAs found in the BRV. Toward that goal, we have in vitro transcribed the minimal BRV RNA 1 CITE sequence, and probed our purified RNA with a series of covalent nucleobase, sugar and backbone modifiers. Combining our results, we have found a unique secondary structure for the BRV RNA 1 CITE that differs from a previously proposed structure based upon in silico thermodynamic folding predictions. This structure likely plays an important role regulating protein synthesis of viral proteins encoded by the RNA 1 message by either facilitating end‐to‐end interaction with the 5′ UTR and/or directly recruiting important translation protein machinery.Support or Funding InformationMSU Denver College of Letters, Arts & Sciences Mini‐grant, MSU Denver Undergraduate Research Grant, MSU Denver Center for Advanced STEM Education Undergraduate Research Grant
Analysis of Eukaryotic Translation Initiation Factor (eIF) Phosphorylation by Mass Spectrometry
Authors: Beglinger Katherine , Villa Nancy , Andaya Armann , Leary Julie , Fraser Christopher 
Published: November 2023

Translation initiation is the rate‐limiting step of protein synthesis and is regulated by eukaryotic initiation factors (eIFs), which work together to recruit a mRNA to the ribosome and locate the initiation codon. An additional layer of regulation of this pathway is likely influenced by post‐translational modifications of initiation factors, specifically phosphorylation. While several global studies have identified extensive numbers of phosphorylation sites, it is still unclear which sites are important in regulating translation. The objective of this study to to identify which phosphorylation sites are key in regulating translation initiation and to determine how phosphorylation regulates the function and structure of translation initiation factors.We have started to determine which phosphorylation sites on the cap binding complex, eIF4F, are important in regulating translation. To build on our previous work, we are recombinantly expressing and purifying this complex from HeLa cells that have been treated with various kinase inhibitors. We are quantifying site‐specific phosphorylation stoichiometries using a novel cerium oxide dephosphorylation and tandem mass tagging (TMT) method followed by nanoLC‐MS/MS. Identified phosphorylation sites that change in response to kinase inhibitors will be characterized using functional and structural assays.The translation initiation factor eIF4B has been shown to have an important role in translation initiation by stimulation the unwinding of mRNA in preparation of recruitment to the ribosome. Prior studies have identified a phosphorylation site at Serine 422 of eIF4B. This site is known to be important in stimulating translation initiation as well as increasing its affinity to eIF3, however the mechanism in which this site works is poorly understood. To study the role of this phosphorylation site, we expressed and purified recombinant phosphomimetic (Ser422Glu) and null phosphorylation (Ser422Ala) mutants from SF9 cells. We are employing ion mobility mass spectrometry to study the structural changes that occur when eIF4B is phosphorylated. In addition, we are using a reconstituted translation initiation system to understand the effect of eIF4B phosphorylation on eIF4A‐dependent helicase activity and mRNA recruitment.Support or Funding InformationNIH T32 Training Grant (T32CM113770) in Chemical Biology, NIGMS (NIH) RO1 GM092927, The Floyd and Mary Schwall Fellowship in Medical Research