bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of the Pope Engagement Index to Measure the Cognitive Load of Biochemistry Students while Completing a 3D Physical Serine Protease Modeling Activity
Authors: Cortes Kimberly Linenberger , Terrell Cassidy , Calvert Jenifer , Randolph Adriane 
Published: June 2021

Research has shown students have significant difficulty understanding enzyme‐substrate relationships. One way instructors have suggested to support students while learning this content is through the use of 3D physical models of the enzyme and substrate. However, little research has been conducted on how the models impact the learning process and the cognitive load of the students. This study aims to measure the cognitive load of biochemistry students while completing a modeling activity focused on understanding the relationship between the substrates and active site of serine proteases. During a simulated learning environment, biochemistry students completed an activity related to how enzymes and substrates interact based on electronics, stereochemistry and geometric complementary using the serine protease physical models. During the activity electroencephalography was used to capture student cognitive processing in order to assign cognitive load of the tasks in the activity using the Pope Engagement Index. This presentation will specifically focus on how spatial ability of the student impacted the level of engagement and in what region of the brain was engaged when completing various tasks. Additional discussion will focus on how changes to the models impacted the cognitive load of the participants when completing the activity. Suggestions will be provided for faculty to think about when developing modeling activities to decrease extraneous cognitive load for students.Support or Funding InformationThis project is supported by the National Science Foundation under award number IUSE 1711402/1711425 to University of Minnesota, Rochester and Kennesaw State University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fungicide‐induced melanin loss causes changes the transfer RNA modification pool in the radiation resistant fungi Cladosporium sphaerospermum
Authors: Kelley Melissa , Yoshida George , Myers Ryan , Alem Kibret , Chati Prathamesh , Jora Manasses , Addepalli Balasubrahmanyam , Limbach Patrick A 
Published: June 2021

Fungi have been found in extreme environments all over the world. Of particular interest, Cladosporium sphaerospermum is a filamentous fungus found to grow around the nuclear reactor at Chernobyl. Thus far, little is known of the mechanistic details that allow these fungi to survive such harsh oxidative conditions. The current hypothesis is that the pigment melanin contributes to growth through enhancing energy production mechanisms. In this work, we aim to identify the effect melanin presence on post‐transcriptional modifications to transfer RNA (tRNA) in C. sphaerospermum. Post‐transcriptional modifications affect translation of response proteins and may be indicative of an underlying mechanism. Prior to this study, the tRNA genes of C. sphaerospermum were unknown. Through a bioinformatics approach, 195 tRNA genes have been identified throughout the genome. The identification of tRNA genes gives information on anticodon preference as well as composition of the tRNA pool. Furthermore, treatment with the fungicides pyroquilon and tricyclazole induce varying levels of melanin loss. Dose‐response curves were generated for optimal dosing. Both fungicides cause phenotype changes, growth rate differences, and tRNA modification profiles alterations. Next, the impact of melanin loss on C. sphaerospermum survival in the presence of ionizing radiation (IR) was determined through growth rates post‐IR. The effect of exposure to ionizing radiation on tRNA modifications has been qualitatively and quantitatively determined using a liquid chromatography coupled with mass spectrometry system.Support or Funding InformationFunding support was provided by the Defense Threat Reduction Agency (DTRA) and the University of Cincinnati.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ultradeep Palmitoyl‐proteomic Analysis Uncovers Over 1,300 Novel Human Palmitoyl‐proteins
Authors: Yang Wei , Zhou Bo , Wang Yang , Yan Yiwu , Freeman Michael R 
Published: June 2021

Protein palmitoylation (more accurately known as protein S‐acylation) is a reversible lipid modification that plays critical roles in protein localization, activity, stability, and complex formation. Aberrant protein palmitoylation is associated with many human diseases such as neurodegenerative disorders and cancer. The past decade has witnessed striking progress in studying the enzymes, substrates, and functions of protein palmitoylation. However, it remains unclear whether protein palmitoylation is a widespread post‐translational modification comparable to protein phosphorylation and ubiquitination. Previously, we developed a palmitoyl‐proteomics method termed Palmitoyl‐Protein Identification and Site Characterization (PalmPISC) and applied it to profile the palmitoyl‐proteomes in human cancer cells and platelets (Yang et al., Mol Cell Proteomics, 2010; Dowal et al., Blood, 2011). Nevertheless, similar to other acyl‐biotinyl exchange (ABE)‐based palmitoyl‐proteomics methods, one shortcoming of the PalmPISC method is that many non‐palmitoylated proteins are co‐isolated with palmitoyl‐proteins, resulting in a high background that masks the detection of low‐abundance palmitoyl‐proteins and interferes with accurate quantification of palmitoyl‐proteins across different conditions. In this study, we coupled stable isotope labeling by amino acid in culture (SILAC) with PalmPISC to quantify the extent of co‐isolated proteins and found that ~65% of identified proteins were co‐isolated contaminants. To address this issue, we developed a low‐background ABE enrichment method and applied it to comprehensively profile the palmitoyl‐proteome of a human prostate cancer LNCaP cell line. Using stringent cutoffs (false discovery rate < 1% and fold change >2), a total of 2,895 proteins, including 1,571 known and 1,324 novel human palmitoyl‐proteins, were found to be significantly enriched. Western blotting analysis confirmed the palmitoylation of six novel palmitoyl‐protein candidates that play important roles in prostate cancer. In summary, our low‐background ABE enrichment method enables proteome‐wide analysis of protein palmitoylation and the study demonstrates that protein palmitoylation is more widespread than we previously thought.Support or Funding InformationThis work was supported by the Department of Defense W81XWH‐15‐1‐0167 (to W.Y.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Falling Apart: the Self‐Catalyzed Process of Protein Splicing
Authors: Chavez Angel , Minteer Christopher J. , Jaramillo Mario , Mills Kenneth V. 
Published: June 2021

Protein splicing is a self‐catalyzed process where an intervening intein is excised, with the concomitant ligation of the two flanking exteins to form a mature protein. The inteins we studied interrupt the DNA Pol II of Pyrococcus abyssi and Pyrococcus horikoshii. These extremophile inteins conditionally splice at high temperatures. The Hyperthermophilic and Thermophilic Hairpin (HTH) is only found in the structures of archaeal inteins. We hypothesize that the HTH increases stability by elongating the central beta‐sheet, and thus mutations that disrupt this hairpin would affect the stability toward unfolding and the reactivity at the active site. To further understand the protein splicing of the P. abyssi intein, an intein with an Arg40Glu mutation in the HTH was expressed, and we studied its activity. We also purified 15‐N labeled intein for NMR (HSQC) analysis of residue‐level changes induced by the mutation. We have also purified a variant of the P. horikoshii PolII intein in order to attempt to solve the structure of an unspliced precursor intein.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A structural investigation of NRZ mediated apoptosis regulation in zebrafish
Authors: Suraweera Chathura Dahamjith , Caria Sofia , Jarva Michael , Hinds Mark , Kvansakul Marc 
Published: June 2021

Bcl‐2 family proteins play a crucial role in regulating apoptosis, a process critical for development, eliminating damaged or infected cells, host‐pathogen interactions and in disease. Dysregulation of Bcl‐2 proteins elicits an expansive cell survival mechanism promoting cell migration, invasion and metastasis. Through a network of intrafamily protein– protein interactions Bcl‐2 family members regulate the release of cell death factors from mitochondria. NRZ is a novel zebrafish pro‐survival Bcl‐2 orthologue resident on mitochondria and the endoplasmic reticulum (ER). However, the mechanism of NRZ apoptosis inhibition has not yet been clarified. Here we examined the interactions of NRZ with pro‐apoptotic members of the Bcl‐2 family using a combination of isothermal calorimetry and mutational analysis of NRZ. We show that NRZ binds almost all zebrafish pro‐apoptotic proteins and displays a broad range of affinities. Furthermore, we define the structural basis for apoptosis inhibition of NRZ by solving the crystal structure of both apo ‐NRZ and a holo form bound to a peptide spanning the binding motif of the pro‐apoptotic zBad, a BH3‐only protein orthologous to mammalian Bad. The crystal structure of NRZ revealed that it adopts the conserved Bcl‐2 like fold observed for other cellular pro‐survival Bcl‐2 proteins and employs the canonical ligand binding groove to bind Bad BH3 peptide. NRZ engagement of Bad BH3 involves the canonical ionic interaction between NRZ R86 and Bad D104 and an additional ionic interaction between NRZ D79 and Bad R100, and substitution of either NRZ R86 or D79 to Ala reduces the binding to Bad BH3 tenfold or more. Our findings provide a detailed mechanistic understanding for NRZ mediated anti‐apoptotic activity in zebrafish by revealing binding to both Bad and Noxa, suggesting that NRZ is likely to occupy a unique mechanistic role in zebrafish apoptosis regulation by acting as a highly promiscuous pro‐apoptotic Bcl‐2 binder.Support or Funding InformationThis work was supported by the Australian Research Council (Fellowship FT130101349 to MK) and LaTrobe university (scholarship to CDS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PGE2 Augments Inflammasome Activation and M1 Polarization in Macrophages Infected with Salmonella Typhimurium and Yersinia enterocolitica
Authors: Sheppe Austin , Kummari Evangel , Walker Alyssa , Richards Angela , Hui Winnie , Lee Jung Hwa , Magnum Lauren , Borazjani Abdolsamad , Ross Matt , Edelmann Mariola 
Published: June 2021

Prostaglandin E2 (PGE2) is an important immunomodulatory molecule that is part of a larger group of bioactive lipids known as eicosanoids. PGE2 has long been reported to have conflicting roles in the regulation of inflammation. For example, some effects are primarily anti‐inflammatory and other roles that mediate inflammation. During intracellular infection with Gram‐negative bacteria, host macrophages form complexes called inflammasomes. Inflammasomes drive activation and secretion of inflammatory cytokines such as IL‐1β that signal nearby cells in the process of inflammatory cell death called pyroptosis. PGE2 is reported to inhibit NLRP3 inflammasome activation and enhance NLRC4 inflammasome activation. Since both NLRP3 and NLRC4 inflammasomes mediate IL‐1β secretion, the PGE2's function in the inflammatory processes does not seem to be clear. Previous studies relied on LPS stimulation of macrophages in place of bacterial infection. In this study, we used an infection model of THP‐1 human macrophages with Salmonella enterica Typhimurium (St) and Yersinia enterocolitica (Ye) that suggests PGE2 plays a vital role in enhancing the transcription and secretion of IL‐1β upon inflammasome activation. Interestingly, distinct virulence strategies between St and Ye are reflected in the alteration of COX‐2 and resulting PGE2 biosynthesis by these pathogens. Ye virulence factors inhibit COX‐2 expression and thus decrease the amount of PGE2 secreted during infection. St, in contrast, activates COX‐2 and enhances PGE2 production. In our studies, PGE2 enhances bacterial load of St inside macrophages 48 hours post infection, but it decreases intracellular Ye. Finally, our results suggest that PGE2 primarily enhances the transcription of pro‐inflammatory cytokines while repressing anti‐inflammatory processes such as resource scavenging and matrix remodeling.Support or Funding InformationThis study was supported by a fund no. 2016‐31200‐06012 from the National Institute of Food and Agriculture (NIFA), https://doi.org/10.13039/100005825, National Institutes of Health (NIH) fund no. 5P20GM103646‐02, https://doi.org/10.13039/100000002, as well as MS Center for Food Safety and Post‐harvest Technology Grant Program (USDA‐ARS SCA 58‐6066‐7081) and NIH1R15ES015348‐02. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancement of Algal Biofeedstocks in a Mixotrophic Batch Culture Supplemented with Exogenous Glycerol
Authors: Smythers Amanda L , Higginbotham Ethan , Holland Aaron , Stephenson Anthony , Kolling Derrick R.J. 
Published: June 2021

Chlorella vulgaris is a fast growing, unicellular microalgae. With its limited nutritional requirements and ability to grow in adverse environments, it is a promising biodiesel feedstock that will not compete with agricultural resources.1 Published literature has shown C. vulgaris can produce up to 42% lipids per dry mass, the most desired of which are triacylglycerols (TAGs).2 TAGs consist of three fatty acids esterified to a glycerol backbone and are the most reduced, energy‐dense biomolecules. To generate drop‐in ready biodiesel, TAGs are transesterified to yield methyl‐ester alkyls and a glycerol byproduct.To realistically produce fuel on an industrial scale, microalgal TAG production must be increased beyond current levels. Previous work published by the Kolling Lab increased lipid accumulation in C. vulgaris by using an exogenous carbon source to form a heteromixotrophic culture, in which the cells are energetically supplemented by both exogenous carbon as well as photosynthetic light reactions.3 This project seeks to further increase TAG production by supplying cells with exogenous glycerol, thus providing a use for the transesterification byproduct while reducing the cellular energy investment for TAG production.While published studies have found 10 g/L glycerol to be optimal for lipid production in C. vulgaris, this was completed heterotrophically.4 While growing mixotrophically, we have increased lipid accumulation beyond previously published glycerol studies and have increased overall lipid productivity in glycerol concentrations of 31.5 and 63 g/L. We also monitored photosynthetic performance before and after glycerol addition and observed an increased reliance on cyclic electron transport (a mode of photosynthesis that does not result in the production of NADPH), indicating that the ATP need is greater than that of NADPH for TAG formation under photomixotrophic conditions. Finally, using isotopically labeled glycerol, we have used GC‐MS to determine the rate of glycerol uptake and that exogenously provided glycerol is directly used for the TAG backbone and not metabolized for the generation of fatty acids.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952.Amanda Smythers acknowledges a fellowship from the NASA West Virginia Space Grant Consortium, Grant #NNX15AK74A and Training Grant # NNX15AI01H.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Anoxia‐Tolerant Cell Line, AL4, Provides New Insight into Small Non‐Coding RNA Biology
Authors: Riggs Claire L , Podrabsky Jason E. 
Published: June 2021

Small non‐coding RNAs (ncRNAs) can rapidly, reversibly, and specifically regulate gene expression. These characteristics make them compelling candidates for regulating gene expression in anoxia‐tolerant vertebrates, as survival of anoxia requires dramatic yet reversible changes in physiology. Recent small ncRNA sequencing in anoxia‐tolerant vertebrates revealed groups of candidate small ncRNAs for supporting anoxia tolerance. The expression of mitosRNAs, small ncRNAs derived from the mitochondria, was particularly striking in the most anoxia‐tolerant vertebrate, embryos of the annual killifish Austrofundulus limnaeus, and inspired further study. Cell culture is a particularly useful tool for detailed study of small ncRNA biology, due to the ease of subcellular imaging and chemical and genetic manipulation. Therefore, we aimed to establish and characterize an anoxia‐tolerant cell line from A. limnaeus to use as a tool to probe the biology of small ncRNAs of interest. The AL4 cell line was established from embryonic tissue of A. limnaeus using a tissue explant method. Proteomic phenotyping suggested a neuroepithelial identity. The cells can survive for over 40 d without oxygen or replenishment of growth medium, compared to only 3 d of anoxic survival for green monkey kidney epithelial cells (Vero) and mouse neuroblastoma cells (N2A). Additionally, AL4 cells accumulate lactate during anoxia, which is consistent with whole embryos, as well as anoxia‐sensitive mammals. AL4 express many of the same small noncoding RNAs as the whole annual killifish embryos, including anoxia‐responsive mitosRNAs. Known mammalian stress‐responsive small ncRNAs also respond to anoxia in AL4. Therefore AL4 both reflects the biology of whole embryos and can be used in comparative studies with anoxia‐sensitive mammalian species. We used the cell line to investigate highly anoxia‐responsive mitosRNAs derived from mitochondrial tRNA cysteine. We found that these mitosRNAs are enriched in the mitochondria, particularly near the nucleus, and appear to be present in the cytoplasm as well. They also appear to be de novo transcribed in the mitochondria during anoxia. This body of work provides the first evidence for mitochondrial de novo transcription and cytoplasmic localization of mitosRNAs in response to stress. The establishment of the AL4 cell line provides a unique tool for investigating cellular mechanisms of vertebrate anoxia tolerance, including mitosRNAs, and has the potential to transform our understanding of the role of oxidative metabolism in cell biology.Support or Funding InformationThis work was supported by NSF GRFP DGE‐1057604 to Claire L. Riggs and NSF IOS‐1354549 to Jason E. Podrabsky.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The extracellular matrix signals through protein kinase MEKK‐3 and transcription factor ATF‐7 to activate cytoprotective genes
Authors: Wimberly Keon , Choe Keith 
Published: June 2021

Animal cells secrete proteins and carbohydrates to create an extracellular matrix (ECM) that provides mechanical support for tissues. Rather than being an inert scaffold, the ECM is a dynamic, physiologically active component in living tissues. Barrier ECMs stand at the surface of animals and are the first of line defense against many environmental stressors such as osmotic pressure changes, toxins, and biological pathogens. Activation of cytoprotective genes is the second line of defense against environmental stress. Cytoprotective genes are conserved and influence adaptation to climate change, disease progression and pathogenesis, and aging. Therefore, a molecular understanding of fundamental signaling mechanisms that control cytoprotective genes has broad relevance. The molecular signaling pathways that sense and regulate cytoprotective responses within cells have been studied in detail, but far less is known about how barrier ECMs interact with environmental stress to influence cytoprotective gene regulation. The nematode model organism Caenorhabditis elegans is covered by a complex barrier ECM termed the cuticle. Given that the cuticle directly interacts with the environment, we hypothesize that it may include a sensor for stress. We recently used RNAi screening, collagen mutations, and RNAseq in C. elegans to demonstrate that disruption of specific bands of collagen, called annular furrows, significantly induce organic osmolyte, detoxification, and immunity cytoprotective genes (p<0.05) (Dodd et al., 2018 Genetics 208:1467). To identify regulators of cytoprotective gene transcription downstream of annular furrows, we conduced an RNAi screen of all C. elegans' protein kinases and transcription factors in annular furrow mutants expressing fluorescent cytoprotective gene reporters. This screen and subsequent RT‐qPCR analysis identified protein kinase MEKK‐3 and transcription factor ATF‐7 as significantly required for expression of cytoprotective genes* in annular furrow mutants (p>0.001; *atf‐7 knockdown did not significantly repress immunity gene induction). Previous C. elegans' literature indicates that MEKK‐3 regulates longevity with dietary restriction and that ATF‐7 regulates innate immunity. Importantly, both of these proteins are conserved in humans where they regulate diverse stress responses and are implicated in tumorigenesis. We are now leveraging the experimental tractability of C. elegans to determine how these two conserved signaling genes function to confer protection to environmental stress in this novel ECM‐initiated context.Support or Funding InformationNSF, UF Graduate School Fellowship, McKnight Doctoral Fellowship, William Townsend Porter Predoctoral Fellowship from the American Physiological SocietyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.