bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of an Assessment Tool for Evaluating Undergraduate Student Understanding of Pedigree Analysis
Authors: Grimes Zachary , Boury Nancy , Reid Joshua , Wasendorf Chloe , Couch Brock , James Olena , Seipelt‐Thiemann Rebecca 
Published: June 2021

Biology education is currently undergoing reform efforts to increase student retention and appreciation within the biological sciences. Both Vision and Change in Undergraduate Biology Education call for the increased use of evidence‐based pedagogical strategies to support learning in biology learning environments. However, integration of evidence‐based instructional strategies relies on biology instructors' knowledge of student pre‐conceptions and misconceptions. Genetics represents a critical area of biology education that presents many problems for student learning due to its abstract nature, the need to think through different spatial scales, and heavy reliance upon technical language. Pedigree analysis represents a convergence of topics in genetics and, therefore, has the potential to identify multiple student learning difficulties. Pedigree analysis requires an understanding of modes of inheritance, which requires a knowledge of the nature of both dominance and recessiveness of traits, as well as understanding of the connections between genotype and phenotype and use of the symbolic scale. This project sought to gain an understanding of students' misconceptions of pedigrees in order to promote the development of an assessment tool for students' misconceptions. The research team developed targeted questions and collected written responses to these open‐ended questions as well as conducting self‐selected student interviews. Student responses were coded to identify the most common misconceptions which were used as distractors a draft multiple choice concept inventory (CI). The draft CI was administered to freshman, sophomore, and upper division undergraduate students. These data were then used to further revise the finalized CI. Pre‐post data are currently being collected to ensure reliability and discriminatory power of this CI. Future studies will include analysis of the persistence of distinct misconceptions across the different student groups, as well as a more thorough analysis of student misconception types and their potential sources.Support or Funding InformationFunded by NSF Award #1710262This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improvements to Simplicity and Stability of the Cell‐Free Protein Synthesis Platform to Enable Applications in the Field and the Classroom
Authors: Gregorio Nicole E. , Kao Wesley Y. , Williams Layne C. , Oza Javin P. 
Published: June 2021

E. coli‐based cell‐free protein synthesis (CFPS) is a flexible platform technology for on‐demand protein production that allows users to express traditionally intractable proteins, perform high‐throughput screening, and, with augmentation, supports applications such as metabolic engineering and genetic code expansion. The broad utility of the CFPS platform arises from the elimination of the cellular membrane and capture of transcription and translation machinery in vitro. This obviates the need for living cells and creates an open system for direct manipulation of the environment of protein production. However, broad dissemination to field and classroom applications remains limited, as CFPS is dependent on technical scientific expertise for proper reaction setup and laboratory infrastructure for proper storage of reagents. Here, we report our efforts to improve broad accessibility of CFPS by 1) simplifying reaction setup and 2) improving shelf stability of the cell‐extract at various storage conditions. In order to simplify reaction setup, which currently requires precise pipetting of 10 reagents, we have combined the CFPS reagents into two stable premixes. These premixes can be stored at −20 °C for at least 4 months and allow the user to pipette fewer times with larger volumes for more accurate reaction setup by non‐experts. To improve the shelf stability of cell extract, which contains the sensitive transcription and translation machinery and is traditionally stored at −80 °C, we have characterized the functional effects of 10 protein stabilizing additives such as sugars, osmolytes, surfactants, and molecular crowding agents. Tests of single additives and combinations of two or three have allowed us to identify the landscape of small molecule additives that can support a variety of storage conditions ranging from −80 °C to room temperature to suit a variety of applications. When combined, these advances to reaction setup and cell extract composition provide the foundation for broad implementation of CFPS. Improved ease of reaction setup enables the use of CFPS by non‐experts, such as early career students, and improves efficiency for researchers and field applications. Additionally, increased storage stability diminishes reliance on the cold chain, making CFPS reagents easier to disseminate, commercialize, and use in the field. Together, these modifications provide a foundation for democratization of the CFPS platform.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology, Bill and Linda Frost Fund, and NSF‐1708919.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Study of Multidrug Efflux System Protein Degradation in E. coli Using Transposons Library
Authors: Rajapaksha Prasangi Irosha , Wei Yinan 
Published: June 2021

AcrB is a trimeric multidrug efflux pump in E. coli belonging to the Resistance‐Nodulation‐Division (RND) super family. We have previously found that the introduction of a small peptide tag, the ssrA tag, to the C‐terminus of AcrB lead to efficient degradation of the protein. Soluble proteases ClpP and ClpX have been found to be involved in the degradation. However, a significant reduction in the amount of AcrB‐ssrA could still be detected in knockout strains lacking functional ClpXP. We speculate that the reduction of AcrB‐ssrA level is due to another protease/proteases. This study focuses on the identification of other proteins capable of degrading AcrB‐ssrA. Transposons libraries containing random gene disruption were generated and screened for clones displaying increased expression of AcrB‐ssrA. Our hypothesis is that the increased activity of AcrB‐ssrA protein is due to a mutation or inactivation of a gene that is involved in the degradation of AcrB‐ssrA. The elevated level of AcrB‐ssrA expression was then confirmed using anti‐AcrB antibody and minimum inhibitory concentration assays. Several candidate genes have been identified, which we are currently characterizing for their potential role in AcrB‐ssrA degradation.Support or Funding InformationNSF grant CHE‐1709381This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydrophobic Mutations Promote UBQLN2 Oligomerization And Phase Separation
Authors: Castaneda Carlos A , Dao Thuy P , Yang Yiran , Jones Holly , Lei Yongna , Martyniak Brian 
Published: June 2021

Liquid‐liquid phase separation (LLPS) is hypothesized to be the dominant mechanism that underlies the formation of membraneless organelles, including stress granules formed under cellular stress containing sequestered RNA and proteins. Our lab has recently shown that Ubiquilin‐2 (UBQLN2), a protein involved in protein quality control through proteasomal degradation and autophagy pathways, is recruited to stress granules in vivo and undergoes LLPS in vitro under physiological conditions. Using NMR spectroscopy, we identified both intrinsically‐disordered and folded regions that are involved in UBQLN2 self‐association that also mediate UBQLN2 LLPS. One of these regions includes the proline‐rich (Pxx) segment, where known disease‐linked mutations have been shown to cause 1–2% of familial ALS or ALS/dementia cases. Using size‐exclusion chromatography, NMR spectroscopy, and microscopy, we demonstrated that Pxx mutations modulate UBQLN2 self‐association and phase separation in vitro. Pxx mutations at P497, P506 and P525 affected droplet morphology and dynamics to significantly different extents. Our data suggested that increased hydrophobicity of the amino acid promoted UBQLN2 LLPS and lowered the phase transition temperature at which UBQLN2 LLPS is first observed. To systematically investigate the molecular basis for how amino acid composition affects UBQLN2 phase separation, we generated all 19 possible amino acid replacements at residues P497, P506, P525, and V538. Our data indicate that the phase behavior of UBQLN2 is significantly modulated by mutations at positions 497 and 506, but weakly modulated by mutations at positions 525 and 538. These results suggest that positions 497 and 506 are ‘stickers’ whereas positions 525 and 538 are ‘spacers’ using the language of associative polymers. Consistent with our data above, hydrophobic amino acid substitutions promoted UBQLN2 LLPS. Our experiments suggest that UBQLN2 may be used as a model system to understand the sequence determinants of phase separation. Hydrophobic mutations modulate UBQLN2 oligomerization and LLPS, and potentially alter material properties of UBQLN2‐containing biomolecular condensates in the cell, promoting disease states.Support or Funding InformationC.A.C. graciously acknowledges funding from the ALS Association via grants 17‐IIP‐369 and 18‐IIP‐400, as well as from the National Science Foundation (CAREER award 1750462).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Elucidation of Engineered Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1) Variants with Improved Binding Affinity toward Matrix Metalloproteinase‐3 (MMP‐3)
Authors: Raeeszadeh‐Sarmazdeh Maryam , Sankaran Banumathi , Radisky Derek , Radisky Evette 
Published: June 2021

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes known for contributions to diseases including cancer and fibrosis. The TIMP N‐terminal domain, regarded as the “inhibitory domain”, has been the center of attention both for understanding natural regulation of MMPs by TIMPs, and for developing protein‐based MMP inhibitors for therapeutic applications. The role of the TIMP C‐terminal domain in MMP inhibition has not been well studied. I have used directed evolution via yeast surface display for protein engineering of full‐length human TIMP‐1 toward development of MMP‐3‐targeted ultra‐binders. TIMP‐1 variants with up to 10‐fold improvement in MMP‐3 binding (compared to WT‐TIMP‐1) were isolated after six rounds of library screening using fluorescence‐activated cell sorting (FACS). Analysis of individual and paired mutations from the selected TIMP‐1 variants revealed a cooperative effect between distant mutations on the N‐ and C‐terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP‐3. I have co‐crystallized two TIMP‐1 variants containing single and composite mutations in complex with the catalytic domain of MMP‐3 and solved the X‐ray crystal structures of these complexes. The protein structures reveal substantial conformational changes in the TIMP‐1 binding interfaces near the two cooperative mutations. Affinity appears to be strengthened by tighter cinching of a reciprocal tyrosine clasp formed between the N‐terminal loop of TIMP‐1 and the proximal MMP3 interface, and adoption of an alpha‐helical conformation by a segment of the TIMP‐1 C‐terminal domain, resulting in stabilized interactions between the two TIMP‐1 domains and improved hydrophobic interactions between TIMP‐1 and MMP‐3. Our protein engineering and structural studies provide novel insight into the cooperative function of N‐ and C‐terminal TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our results further point to new approaches for design of TIMP‐based protein therapeutics with improved affinity and selectivity.Support or Funding InformationThis work was supported by grants DOD W81XWH‐16‐2‐0030, and NIH R21 CA205471. Diffraction data were measured at the Berkeley National Synchrotron Light Source.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of an in Vitro CYP450 Induction Assay Using Cryopreserved Cell Lines
Authors: Lim Tae Young , Rizkallah Amy , Capacio Benedict 
Published: June 2021

Cytochrome P450 (CYP450) enzymes are responsible for the majority for Phase I metabolism. Induction occurs when a drug increases the rate of CYP450 enzyme production. As a result, the rates of metabolism of subsequent drugs increase, posing concerns for dosing and safety. To further expand the USAMRICD Absorption, Distribution, Metabolism, Excretion, and Toxicity Center of Excellence (ADMET CoE) profiling capabilities, a CYP450 induction assay is being developed using alternative cell lines to primary human hepatocytes to measure changes in gene expression of CYP1A2 and CYP3A4 isoenzymes.Primary human hepatocytes are the gold standard for evaluating P450 induction in vitro; however, they have inherent limitations such as high costs, fastidious nature, and significant lot‐to‐lot variability. Induction assessments using cryopreserved hepatocytes and two immortalized cell lines, HepaRG and HepatoCells, are being conducted to determine a more robust, cost‐effective alternative model. Each cell line was exposed to the standard inducers rifampicin and omeprazole to assess CYP3A4 and CYP1A2 activity, respectively. Following a 72‐hour incubation period, RNA from the cell cultures was extracted and purified. Fold changes of gene expression were measured using real‐time quantitative PCR (RT‐qPCR). Hepatocytes displayed an average fold change of 92.7 for CYP3A4 and 49.6 for CYP1A2, whereas HepaRG displayed changes of 28.6 and 179.5, respectively. Average values for CYP3A4 and CYP1A2 for HepatoCells were 58.7 and 43.9. Based on the data, HepatoCells most closely mimicked cryopreserved hepatocytes in comparison to HepaRG cells. Additional data on assay parameters and technique optimization will be presented.Support or Funding InformationOpinion disclaimer: The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of the Army, Department of Defense, or the U.S. Government. This project was generously supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. Army Medical Research and Materiel Command. Support is also provided by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Differential Effects of Cisplatin and Oxaliplatin on Nucleolar Stress using Platinum ‘Liaison’ Compounds
Authors: Prochneau Jack , McDevitt Christy , Sutton Emily , Cunningham Rachael , DeRose Victoria 
Published: June 2021

Platinum(II) compounds are a versatile and effective class of chemotherapy drugs, employed extensively worldwide in single and combination therapy treatments. In one accepted mechanism of action these compounds bind to DNA, interfere with replication events and stimulate apoptosis. However, these small platinum compounds have multiple cellular targets and the three FDA‐approved platinum agents, cisplatin, carboplatin, and oxaliplatin have different activity, resistance and side effect profiles. Two of the FDA‐approved agents, cisplatin and oxaliplatin, exemplify this phenomenon with the former inducing more severe forms of ototoxicity and nephrotoxicity. One marked difference is that unlike cisplatin, oxaliplatin treatment induces markers of nucleolar stress response, including relocalization of nucleolar phosphoprotein NPM1 in mammalian cell culture. It is presently unknown which structural features of these square‐planar molecules, including aquation‐labile chloride/oxalate or non‐labile ammine/1,2‐diaminocyclohexane ligands, give rise to such different effects. To investigate how platinum ligands may influence the nucleolar stress response we have synthesized a series of ‘liaison’ Pt(II) compounds with different ligand combinations, and measured the effects of this series of compounds on NPM1 relocalization, DNA damage markers, and cell death. The results of this study will lead to an understanding of the structural features and molecular interactions underlying the ability of oxaliplatin to influence nucleolar processes.Support or Funding InformationSupport from the National Institutes of Health R25HD0708 (JP) and T32 GM007759 (ES), the National Science Foundation CHE 1710721 (VJD) and MRI 1531189 (for shared instrumentation), and the University of Oregon is gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome Dynamics of Coevolving Podoviruses and Marine Cyanobacteria
Authors: Fenderson Brooke A , Marston Marcia F 
Published: June 2021

Podoviruses are dsDNA bacteriophage with ~45 kb genomes that are abundant in coastal marine ecosystems. Many of these podoviruses infect cyanobacteria and are thought to undergo antagonistic coevolution with their host. During coevolution, the host evolves resistance to the virus and the viruses evolve to overcome this resistance. Coevolution can impact the fitness and genetic diversity of both host and virus. In this study, whole‐genome sequencing was used to examine the genome dynamics of two closely‐related podoviral strains coevolving with cyanobacteria Synechococcus WH7803 in continuous culture. For each podoviral strain, replicate chemostats containing Synechococcus WH7803 were inoculated with the virus and maintained for 24 weeks. Two additional chemostats of Synechococcus without virus served as the controls. Monthly samples were taken from all chemostats and used to isolate individual viral plaques and Synechococcus colonies. The genomes of viral and host isolates from various time points were then fully sequenced and compared to the genomes of the ancestral strains. Viruses in all four chemostats accumulated mutations as time progressed. These mutations consisted mostly of single nucleotide polymorphisms leading to amino acid changes in the tail fiber region of the viral genome. In addition, large deletions, ranging from 500 to 1600 nt occurred in isolates from later time points in three of the four chemostats. Interestingly, these deletions occurred in regions of the genome that are not shared by the two podoviral strains. In contrast to the viruses, host cells accumulated far fewer mutations and no gene deletions were observed. Only a few mutations were needed for the host to become resistant to the podoviral strains. One of these mutations was located in an intergenic region likely involved in gene regulation and provided broad‐spectrum resistance to both podoviruses and myoviruses. Studying the genome dynamics that occur during coevolution will lead to a better understanding of how viruses and their hosts interact and coexist in natural ecosystems.Support or Funding InformationThis research was supported by the National Science Foundation (OCE‐0314523, OCE‐1029684 and OIA‐1736030) to M. Marston.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Illuminating DYRK1A Substrates Through a Multi‐omic Drug Based Approach
Authors: Guard Steven , Poss Zachary , Ebmeier Chris , Webb Kristofor , Old William 
Published: June 2021

Protein kinases comprise only ~2.5% of human protein coding genes yet are responsible for the regulation of nearly every cellular process through their catalytic activity. The kinome thus represents one of the most druggable classes of regulatory proteins for human disease. However, despite major advances in protein identification through mass‐spectrometry based techniques, it remains difficult to uncover substrate profiles for individual kinases using pharmacological manipulation. Undesirable off‐target inhibition and diverse secondary events muddle the phosphorylation landscape that can be detected using phosphoproteomic methods. Further, phosphorylation events and target engagement are often cell type specific confounding our ability to discern contributions of off‐target kinases. Using the protein kinase DYRK1A and inhibitor harmine as a model, we utilize orthogonal mass spectrometry‐based approaches to distinguish kinase‐substrates. Thermal proteome profiling revealed that harmine differentially stabilizes proteins in a cell type and dose dependent manner. Drug target stabilization in combination with substrate motif analysis were used to elucidate high‐confidence DYRK1A substrates from a SILAC‐based harmine phosphoproteome. The substrates revealed in this study will be crucial to understanding DYRK1A function and contribution to human health.Support or Funding InformationDARPA cooperative agreement 13‐34‐RTA‐FP‐007 to W.M.OLinda Crnic Institute for Down syndrome‐ Grand Challenge GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of the C‐terminal domain of MtlA and its role in cell wall metabolism in the fungus Aspergillus nidulans
Authors: Nichols Stewart , Cannavo Matthew M. , Jackson‐Hayes Loretta M. , Loprete Darlene M. 
Published: June 2021

The Mid2‐like protein MtlA is a putative sensor that affects cell wall integrity in the filamentous fungus Aspergillus nidulans. Plasmid‐based, overexpression of MtlA can suppress the calC2 mutation in the A. nidulans orthologue of protein kinase C (PkcA), which results in hypersensitivity to the chitin‐binding agent Calcofluor White. In filamentous fungi, as in yeasts, hypersensitivity to CFW correlates with defects in cell wall integrity. MtlA is predicted to have a single transmembrane domain, an extracellular domain rich in serine and threonine amino acids residues, and a C‐terminal domain of 59 amino acids. A MtlA ‐GFP hybrid localizes to the plasma membrane of vegetative hyphae. In the cell wall integrity pathway in yeast, the C‐terminal domain of the stress receptors, Wsc1 and Mid2, have been shown to play an important role in the signal transduction pathway. We have shown that the C‐terminal domain of MtlA plays an important role in cell wall integrity. We have created and analyzed C‐terminal truncation mutants of MtlA and have shown that complete removal of the C‐terminal domain obliterates its function. We have also shown that amino acids 241–251 are critical for the function of MtlA. Site directed mutagenesis of key amino acids will be elucidated.Support or Funding InformationRhodes CollegeNational Science FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.