bluuebunny/crossref_metadata_2025_split huggingface dataset:
Iterative Design of 3D Physical Serine Protease Models based on Biometric Data to Optimize Cognitive Load and Decrease Misconceptions in Undergraduate Biochemistry
Authors: Terrell Cassidy Renee , Calvert Jenifer , Randolph Adriane , Cortes Kimberly Linenberger 
Published: June 2021

Biochemistry students misconceptions related to enzyme‐substrate interactions can stem from prior knowledge in biology and chemistry where this and related concepts are inadequately represented visually, explained and/or assessed. Moreover, research has shown two‐dimensional representations not only fail to effectively convey biochemical concepts, but also propagate misconceptions. We hypothesize that three‐dimensional (3D) physical models used in conjunction with targeted active learning assessments will increase student understanding of shape, stereochemistry, and electrostatic interactions involved in enzyme‐substrate interactions. However, little research exists on how to design 3D physical models with corresponding assessments that optimize the cognitive load of the student which would enhance learning. This presentation will describe an iterative design process based on biometric, observational and rubric data analysis. The biometric data is collected using electroencephalographic (EEG) and eye tracking tools and voice recordings from simulated learning environments where biochemistry students completed a serine protease activity with corresponding physical models. In addition, observational and rubric analyses of student responses to activities from the simulated learning environment and real classroom sessions provide further data for the iterative activity design process. Here, we will offer suggestions for faculty interested designing 3D physical models with corresponding active learning assessments that optimize the cognitive load and target student misconceptions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Microbe for Ohio: A Service Learning Project That Swept the State
Authors: Bennett Jennifer A , Bowman Bridget , Hout Sokjita , Clinton Tim 
Published: June 2021

It is common for states across America to adopt official symbols that are used for representation. For example, states may have an official state tree, animal, flower, and song. Ohio has at least 25 state symbols, including a state beverage and a state fossil. At this time it does not yet have a state microbe. There is only one state that currently has an official microbe. Oregon has chosen Saccharomyces cerevisiae, commonly referred to as brewer's and baker's yeast, because of the state's brewing industry and many craft breweries. Three other states have selected a microbe and are pursuing legislation to have the microbe officially recognized. After learning of the stories surrounding the microbes selected in other states, I decided to introduce this as a unique service learning project into my upper‐level microbiology course at Otterbein University. This is a writing intensive course that serves as an upper‐level elective for Biology as well as Biochemistry and Molecular Biology majors. Each student was required to choose a microbe that they felt represented Ohio and write a persuasive paper on the topic. Six of these microbes were then selected for a vote by the general public. While the students in the course learned about many new microbes and fulfilled their writing requirements for the course, the major service goal of the project was to educate the public about microbiology, especially the fact that there are beneficial microbes. Microbes and their beneficial roles were presented to the public in a fun, educational way via social media. This multi‐disciplinary project involved many departments at Otterbein, including Biology and Earth Science, Communication, and History and Political Science, as well as the Biochemistry and Molecular Biology Program. The American Society for Microbiology and American Society for Biochemistry and Molecular Biology Student Chapters both played a vital role in relaying the information to the public and organizing events. Currently the “Microbe Term Election” is almost finished, and the next step is to write the winning “candidate microbe” into legislation. The “campaign” has been largely successful in its goals and has gained the support of various organizations, colleges and universities throughout Ohio and allowed the participation of the general public, including K‐12 classrooms in the voting process. The service learning initiative will continue throughout the year with a general education microbiology course in Spring 2019. The selection of an official state microbe is anticipated to continue to spark the public interest in microbiology and biochemistry, as people learn how microbes are beneficial and how they fulfill their important roles.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA Replication Genes Implicated in Heterochromatin Modulation
Authors: Moghul Tasmiya A. , Sharma Pragati , Reilly Eve , Zaratiegui Miguel A. 
Published: June 2021

Cellular DNA is stored compactly in the nucleus in complex with protein factors. The condensed structure or chromatin is categorized as euchromatin ‐ a relatively loose form consisting of frequently transcribed genes, and heterochromatin ‐ a denser, more compact form that covers mostly noncoding genes, transposable elements, and satellite repeats. Some of the functions of heterochromatin are: suppression of recombination of transposable elements, proper segregation of chromosomes and regulation of gene expression. Hence, heterochromatin is vital for maintaining genomic stability. Heterochromatin can be further classified as facultative (assembles and dissembles for gene expression regulation) and constitutive (condensed throughout the cell cycle). Although condensed, heterochromatin has to be unraveled during DNA replication. How does the cell know when to change heterochromatin for replication or when to return to its previous compact state? The overarching goal of this project is to understand the molecular factors that help modulate heterochromatin states to allow replication through silenced regions. Using fission yeast, we conducted a forward genetic screen with Hermes transposon as a random mutagen to obtain Position Effect Variegation (PEV) mutants of heterochromatic ade6 reporter gene. With whole genome sequencing, we identified the mutants that showed loss of silencing at the reporter gene; a number of these mutations were found to be in essential genes. Selection for loss of silencing at the pericentromeric ade6 reporter corresponded with an enrichment in mutants of DNA replication and repair genes. We hypothesize that these mutants can be recreated with CRISPR‐Cas9. The primary aim of my project is to generate mutant alleles of essential genes involved in DNA replication and repair, and validate their heterochromatin mutant phenotype. Using CRISPR ‐ Cas9 genome editing, we targeted five such genes in fission yeast: Vid21, Orc4, Cut1, Cut9, Drc1, and Dcr1 as a method control. The results we found show that we were able to generate viable mutant alleles that may be otherwise difficult to generate using conventional genetic manipulations. This study of the characterization of mutant alleles will enable us to understand their role(s) in the coordination of heterochromatin states and DNA replication and/or repair.Support or Funding InformationThis work was supported by RiSE at Rutgers, NIH grant K12GM093854‐07A1 to IRACDA at Rutgers University, NIH‐NIGMS T32 GM8339 New York for Science Partnerships in Research and Education ‐ Rutgers University Biotechnology Training Program and NIH NIGMS RO1 GM105831.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of RNA Modification Enzymes in Trypanosoma brucei
Authors: Schultz Will , Smith Xiane , Taber Cassandra Carol , Coffey Jordan , Militello Kevin 
Published: June 2021

RNA methylation is a type of posttranscriptional modification that plays an important role in controlling gene expression. The organism Trypanosoma brucei, the protozoan parasite responsible for Human African Trypanosomiasis, does not seem to have abundant promoter regions or transcriptional regulation machinery. Thus, RNA methylation may play an especially important role in regulating gene expression in this organism. We have identified the presence of 5‐methylcytosine in T. brucei RNA using both mass spectrometry and sodium bisulfite sequencing. Recently, we have identified seven putative cytosine RNA methyltransferase (CRMT) genes in T. brucei. All seven CRMTs are expressed in bloodstream and procyclic form parasites, as detected by qRT‐PCR. Two of the putative CRMTs, termed CRMT4 and CRMT5, are required for maximum parasite growth. Although we suspect these genes to be RNA methyltransferases, we do not have evidence for RNA methyltransferase activity. Both CRMT4 and CRMT5 were expressed in E. coli with N‐terminal 6x‐histidine tags. CRMT4 was produced in E. coli but was difficult to purify. SDS‐PAGE results for an N‐terminal His tagged protein indicate CRMT4 insolubility. Our next step will be to use PCR to create smaller fragments of CRMT4, which may be soluble. CRMT5 was produced in E. coli with an N‐terminal His tag and purified using a His‐affinity column. Purified CRMT5 was used in a series of methyltransferase assays using luciferase activity as a readout. CRMT5 addition results in luciferase activity in the presence of cytosine‐containing RNA (T. brucei total RNA and Poly‐IC RNA). There was little to no luciferase activity observed in the presence of RNA that lacks cytosine or when a mock purification from E. coli without the CRMT5 gene was used. Our next step will be to perform a methyltransferase reaction with CRMT5 and subsequently isolate the RNA for bisulfite sequencing to confirm the methylation of cytosine bases. Evidence for the presence of 5‐methylcytosine and RNA methyltransferases indicates the presence of a process to create an epitranscriptome in T. brucei.Support or Funding InformationNIH, 1R15AI133428‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Melanoma Antigen Genes (MAGEs): Drivers or Passengers in CARcinogenesis
Authors: Johnson Jodiene 
Published: June 2021

Cancer Testis Antigens (CTAs) comprise a group of genes that are ordinarily expressed in the testis but abnormally expressed in a variety of cancers. The ability of CTAs to elicit an immune response makes them attractive targets for cancer immunotherapy. Melanoma Antigen Genes‐A (MAGE‐A) are Type I CTAs whose expression in cancers have been correlated with poor patient prognosis. The biological function of several members of the MAGE‐A gene family in cancer remains unclear. We have shown that MAGE‐A gene expression is regulated by epigenetic mechanisms such as CpG methylation. Our data indicate that several MAGE‐A genes can act as single gene drivers of cell proliferation and anchorage‐independent growth. We will investigate the molecular mechanisms that MAGE‐A5, ‐A8 and ‐A9 use to enhance cell proliferation by performing RT‐qPCR arrays for cancer‐associated genes and determine binding partners of these MAGE proteins using well‐established IP‐Mass/Spectrometry methods.Support or Funding InformationNSF Research Initiation Award to S.R. #HRD1764201; Department of Education Award # P382G170104 (TITLE‐VII)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A non‐enzymatic function associated with a putative histone demethylase regulates heterochromatin spreading and inheritance
Authors: Raiymbek Gulzhan , An Sojin , Khurana Nidhi , Gopinath Saarang , Biswas Saikat , Larkin Ajay , Trievel Raymond , Cho Uhn‐Soo , Ragunathan Kaushik 
Published: June 2021

Heterochromatin is characterized by histone hypoacetylation and an enrichment of methylation marks on histones which are associated with epigenetic silencing. In fission yeast, a single H3 methyltransferase Clr4 (Suv39h) directs all of the methylation of H3K9 i.e., H3K9me2/3 which enforces the establishment of transcriptionally silent regions in the genome referred to as heterochromatin. A putative histone demethylase, Epe1, is recruited to sites of heterochromatin via interaction with a chromo‐domain containing protein Swi6, homolog of HP1. The loss of Epe1 has been associated with the propagation of silent epigenetic states in a DNA or RNA sequence independent manner. How Epe1 carries out its function as an anti‐silencing factor remains unknown. Despite having sequence homology with known histone demethylases, Epe1 exhibits no detectable demethylase activity in vitro. Here, we demonstrate that mutations within the catalytic JmjC domain of Epe1 disrupt its interaction with Swi6. Using a combination of genetic and biochemical assays, we show that the interaction between Epe1 and Swi6 is direct, depends on the active conformation of the JmjC domain and is sensitive to mutations that affect co‐factor binding. Surprisingly, in cells the interaction between Epe1 and Swi6 requires functional heterochromatin. We reconstituted this dependence in vitro and demonstrate that the presence of an H3K9 tri‐methyl peptide enhances the interaction between the two proteins. We attribute this stimulatory effect of H3K9 methylation to an auto‐inhibitory role for the C‐terminal region of Epe1. Deleting this segment resulted in a dramatic increase in Swi6 binding and no further stimulation in the interaction between Epe1 and Swi6 upon addition of an H3K9me3 peptide. Finally, we demonstrate that this non‐enzymatic role of Epe1 regulates the binding of a histone deacetylase. Our work demonstrates that Epe1 selectively binds and inhibits heterochromatin bound Swi6 molecules which in turn impedes downstream heterochromatin assembly.Support or Funding InformationUniversity of MichiganThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Open complex stability regulates transcription initiation by E. coli RNA polymerase from the rrnB P1 promoter
Authors: Liao Guanyu , Henderson Kate , Wang Hao‐Che , Record M. Thomas 
Published: June 2021

Initiation of transcription by E. coli RNA polymerase (RNAP) begins with specific binding to promoter DNA and ends with promoter escape. For the four T7A1 and λPR promoter/discriminator combinations, we found that the discriminator determined the lifetime and stability of the open complex and the escape point of RNAP in initiation from productive promoter complexes (1). Quantitative initiation studies with these promoter/discriminator combinations reveal that the RNA‐DNA hybrid length for promoter escape and the length distribution of short (abortive) RNA released before escape both increase with the lifetime or stability of the initiation‐competent open complex (OC). The escape point for productive complexes increases from the 7‐mer to 8‐mer step for T7A1 discriminators to the 10‐mer to 11‐mer step for λPR discriminators. Nonproductive complexes make a short RNA smaller than the escape length and stall, slowly releasing it and reinitiating. These findings led us to predict that escape from a promoter like the ribosomal rrnB P1 promoter, with an unstable OC, should occur at a very short RNA‐DNA hybrid length and without release of short RNAs (1). Currently, to obtain the experimental data to test this prediction, we are performing single round transcription assays for the rrnB P1 promoter and other promoters with the rrnB P1 discriminator. In preliminary studies, we have found NTP concentrations where a significant fraction of promoters initiate rapidly. For these NTP concentrations, we find that only the shortest RNAs (e.g. 3‐mer) are made by nonproductive complexes during the time period required for productive complexes to escape and begin elongation. In addition, initiation of λPR promoter with rrnB P1 discriminator shows similar pattern comparing to initiation of rrnB P1 promoter with its own discriminator. We conclude that the current results are consistent with the proposal of early escape of RNAP from promoters with unstable OC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermodynamic Analysis of a Bulged‐G Motif
Authors: Moore Ethan , Aman Zach , Grover Neena 
Published: June 2021

The bulged‐G motif has been recognized in the crystal structure of ribosomal RNA as it contains an S‐turn in the phosphodiester backbone. The S‐turn is formed by a G•U‐A base triple that has a non‐canonical A‐G base pair above the base triple, and an adenine bound to either a guanine or uracil below the base triple. The base triple contains the bulged G nucleotide and is a binding site for elongation factors which ensure that proper translation occurs; this same RNA also binds to sarcin and ricin that inhibit ribosomal function. A similar S‐shape in an RNA backbone was also found to occur in the bacterial c‐di‐GMP‐II riboswitch that contains a A•C‐G base triple. In this study, thermal denaturation and isothermal titration calorimetry (ITC) experiments were performed to delineate the role of specific nucleotides in the formation of the S‐turn and its interaction with magnesium ions. A wild type RNA construct was derived from the bulged‐G motif that contained the G•U‐A base triple flanked by the non‐canonical base pairs, and an RNA triple substitution construct was designed that replaced the base triple with a A•C‐G. The wild type RNA and the base‐triple‐substituted constructs had similar stabilities in 1 M KCl buffer at pH 7.0 and 5.5. RNA constructs are additionally stabilized in 10 mM magnesium, with 2.63 kcal/mol gain in stability for the wild type and 1.45 kcal/mol for A•G‐C constructs. ITC experiments were performed to measure the enthalpy, entropy, and the binding affinity of magnesium ions to RNA. Results for wild type and modified constructs will be presented.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐Function Analysis of the 3′ CITE from the Blackcurrant Reversion Nepovirus Genomic RNA 2 (BRV2)
Authors: Bean Scott‐Wesley M , Baquero Galvis Laura D. , Morrison Evan J. , Filbin‐Wong Megan E. 
Published: June 2021

Canonical eukaryotic translation initiation is enhanced by interactions between the 5′ 7‐methylguanosine cap (m7G) and the 3′ poly(A) tail, which exist on opposite ends of the mRNA molecule in the untranslated regions (UTRs). Viruses, however, hijack translation components using non‐canonical methods; typically differing in the structure of their 5′ and/or 3′ UTRs. Blackcurrant reversion nepovirus (BRV), a virus that infects the blackcurrant plant, has a genome consisting of two positive‐sense RNAs (RNA1 and RNA2). Neither RNA has a 5′ m7G cap, yet they both have a poly(A) tail. Structures within the 3′ UTRs of RNA1 and RNA2 called cap‐independent translation enhancers (3′ CITEs), are hypothesized to interact with a region of their 5′ UTRs. These distinct structural interactions facilitate the recruitment of essential initiation factors needed for translation, but this exact mechanism is not well understood. It is thought that the 5′ and 3′ UTRs possess conserved and complementary sequences which facilitate interaction. This study seeks to elucidate the specific secondary structure of the RNA2 3′ CITE and to correlate such to its role in translation initiation. Using in silico modeling of folded RNA2 and in vitro RNA structural probing, we determined a preliminary secondary structure for the 3′ CITE. We also identified structural changes when the putative 5′ ‐ 3′ UTR complementarity is disrupted. Using a luciferase reporter gene flanked by the RNA2 3′ CITE and wild‐type BRV2 5′ UTR, we show how the putative 5′ ‐ 3′ UTR complementarity is required for the quantity of and the rate at which luciferase protein is made. We conclude that the 3′ CITE is important for promoting translation initiation and the putative 5′ ‐ 3′ UTR complementarity between the ends of the viral RNA is necessary but not sufficient for protein production. The structure‐function relationship of the BRV RNA2 3′ CITE will lead to a clearer understanding of how BRV can be controlled to prevent Blackcurrant Reversion Disease (BRD) and shed light on vital processes that govern this unique class of nepoviruses.Support or Funding InformationColorado‐Wyoming Alliance for Minority Participation in STEMMSU Denver College of Letters, Arts and Sciences Dean's Seed GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring Clustered Regularly Interspaced Short Palindromic Repeats Diversity in Soil Bacteria
Authors: Hassan Sherouk , Shah Rameen , Wei Yufeng 
Published: June 2021

Clustered regularly interspaced short palindromic repeats (CRISPR) is a defense system found in bacteria (and archaea) against bacteriophages, viruses that infect bacteria. CRISPR DNA sequence consists of short, identical nucleotide repeats that are interspaced by spacers. The spacer sequences are records of viral infections experienced by the bacteria and play the role of an “immunization card”: if the spacer sequences match the infecting viral DNA, the bacteria will cut the viral DNA with CRISPR‐associated (Cas) proteins, eliminating the threat of viral infection. We speculate that the spacer sequences reflect the history of bacteriophage infections, which are unique to the environment bacteria reside in. Environmental factors, such as location and time, weather, local ecosystems, and pollutions, will alter the spacer sequences found in bacteria. This research focuses on investigating and discovering new spacer sequences from soil samples collected around NY/NJ metropolitan area, a historically heavily polluted urban ecosystem, at various locations, timepoints, and weather conditions. To accomplish this goal, the genomic DNA was extracted and purified from the collected soil samples. The CRISPR locus was amplified by using polymerase chain reaction (PCR) and then purified via gel electrophoresis. The purified CRISPR DNA was then ligated into a p‐Drive vector and transformed into M15 competent cells. The cells were cultured to amply the vector containing ligated CRISPR sequence, which was subsequently analyzed by aligning against CRISPR spacer database to identify new space sequence. We expect that the newly discovered spacer sequences reflect the history of viral infections unique to the regions investigated, which can potentially inform the understanding of health risks related to environmental bacteria and viruses.Support or Funding Information‐ U.S. Education Department Minority Science & Engineering Improvement Program grant # P120A160084‐ NIH AREA Grant # CA179410‐ USED Title III Part F, HSI‐STEM & Articulation Grant #P03C160155This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.