bluuebunny/crossref_metadata_2025 huggingface dataset:
The Frequency of CHEK2 variant p.Ile157Thr in Men with Prostate Cancer: Towards Expanded Genetic Testing
Authors: Agubokwu Nnedi G. , Giri Veda N.
Published: June 2021

Prostate Cancer (PCA) is the most commonly diagnosed cancer in men in the United States; approximately 5% to 10% of PCAs are considered to be due to inherited mutations (pathogenic variants) that substantially raise the risk for the disease. In genetic testing, variants are classified on a spectrum from mutation, variant of uncertain significance (VUS) and benign. The Checkpoint Kinase 2 gene (CHEK2) variant p. Ile157Thr has had mixed classifications from various laboratories, with some laboratories classifying the variant as a VUS, while others classifying it as a mutation. The purpose of this project was to evaluate the rate of CHEK2 p. Ile157Thr in men affected with PCA. A further objective was to compare rates of the variant to men without a diagnosis of PCA. Primers for the variant were designed by Primer3Plus and obtained from Integrated DNA Technologies. To obtain the DNA samples, DNA extraction from mouthwash samples was performed, followed by quantification and a quality assessment of the DNA using the NanoDrop 2000. The samples were genotyped with the StepOnePlus Real‐Time PCR System. Finally, a 2% confirmation test was conducted to verify the genotypes that were obtained. According to the study, 1.4% of the men with PCA exhibited the CHEK2 variant p.Ile157Thr, particularly those with family histories of breast or colon cancers. The results supported the hypothesis that the rate of CHEK2 variant p.Ile157Thr would be higher among men affected by PCA. This study explores the role of CHEK2 variant p.Ile157Thr in prostate cancer risk, and may lead to expanded genetic testing for inherited PCAs.

Support or Funding Information

Department of Defense, Prostate Cancer Research Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pseudophosphatase MK‐STYX regulates neurite outgrowth and alters the morphology of primary neurons
Authors: Hinton Shantá D. , Banks Dallas , Dahal Arya , McFarland Alexander , Flowers Brittany , Stephens Christina , Swack Benjamin , Gugssa Ayele , Anderson Winston
Published: June 2021

Mitogen‐activated protein kinases (MAPKs) are essential players in important neuronal signaling pathways including neuronal development, plasticity, survival, learning, and memory. The inactivation of MAPKs is tightly controlled by MAPK phosphatases (MKPs), which also are important regulators of these neuronal processes. There is compelling evidence that a unique catalytically inactive member of the MKP family, MK‐STYX (mitogen activated kinase phosphoserine/threonine/tyrosine binding protein), is a regulator in neuronal processes such as neurite outgrowth. We previously reported that the MK‐STYX dramatically increases the number of primary neurites in rat pheochromocytoma (PC‐12) cells through the RhoA signaling pathway. MK‐STYX decreases RhoA activation, whereas knockdown of MK‐STYX increases RhoA activation. Furthermore, MK‐STYX increases phosphorylation of cofilin, a RhoA downstream effector, whereas phosphorylated cofilin decreases when MK‐STYX is knocked down with shRNA. Throughout the course of these experiments, we noticed that cells overexpressing MK‐STYX developed more neurites, which often branched. Here, we report that microtubules and microfilaments, components of the cytoskeleton that are involved in the formation of neurites, are present in MK‐STYX‐induced outgrowths. In addition, in response to nerve growth factor (NGF), MK‐STYX‐producing cells produced more actin growth cones than non‐MK‐STYX‐expressing cells. Transmission electron microscopy confirmed that MK‐STYX‐induced neurites form synapses. Furthermore, the expression of Tau‐1 and MAP2 (microtubule associated protein 2) in MK‐STYX‐induced neurites suggests that they have both axonal and dendritic properties. Further studies in hippocampal primary neurons demonstrated that MK‐STYX altered their morphology. A significant number of primary neurons expressing MK‐STYX had more than the normal number of primary neurites. Taken together these data provide evidence that MK‐STYX causes cytoskeletal rearrangement. It also highlights that this unique member of the MKP subfamily has the potential to have a major role in neuronal signaling.

Support or Funding Information

This work was supported by the National Science Foundation Grant MCB1113167 to S.D.H.; Coco Award to S.D.H.; Howard Hughes Medical Research Institute grant through the Undergraduate Science Education Program to the College of William and Mary; a DuPont Fund Student‐Faculty Research Team Stipend awarded to A.D. and S.D.H. through a grant by the Jessie Ball Grant Fund to the William and Mary Scholars for Undergraduate Research Experience program; Ferguson Awards to D.A.B., A.D., and C.S., and the Dintersmith Honors Fellowship to A.D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The First‐year Research Advancement Program (FRAP): Fostering Persistence of Underrepresented Students in Biology and Chemistry
Authors: Mills Kenneth Vincent , Paxson Julia
Published: June 2021

We co founded the First Year Research Advancement Program (FRAP) in 2014, based on a workshop we attended by the Council on Undergraduate Research. Our goal is to increase persistence in STEM of academically promising students who belong to groups that traditionally do not persist at the same rate as majority students, including first‐generation students, PELL‐eligible students, and underrepresented minority students. Based on research that suggests that early exposure to research experiences increases discipline identity and persistence, we established FRAP. Two first‐year students per lab are paired with a senior student mentor, and each are paid for eight hours a week such that the experience replaces other work‐study. Students spend six hours a week in the lab, one hour in lab meeting, and one hour in a meeting with the cohort of 8–16 students in which faculty and students discuss college and STEM issues and form an academic professional network. Mentors are trained in coaching and mentorship. We have assessed the program in collaboration with the College's Office of Assessment and Research and found increases in self‐reported confidence and engagement. As our number of participants increases, we will study the effect on four‐year persistence versus matched sets of non‐participants. Over the last four years, 30 first year students have participated in FRAP. Our first cohort of FRAP students are now seniors; two current student mentors started as FRAP students themselves. Six of the eight FRAP students that started in the Mills lab are still part of the research group

Support or Funding Information

This work was supported by the National Science Foundation (MCB‐1517138 to KVM) and a Henry Dreyfus Teacher‐Scholar Award (KVM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Competency‐Based Approach to Developing Biomolecular Visual Literacy
Authors: Dries Daniel R. , Dean Diane M. , Franzen Margaret A. , Jakubowski Henry V. , Novak Walter R. P. , Procko Kristen , Roca Alberto I. , Terrell Cassidy R.
Published: June 2021

When you show a visual representation of a biomolecule, do your students “see” what you “see”? Are they able to interpret the model correctly and draw appropriate conclusions about the structure and atomic/molecular interactions? Do they grasp that cartoon and space‐filling models represent the same protein? Biochemistry instructors use visual representations of biomolecules to teach many concepts, yet there are few tools and frameworks to assess whether students can interpret these models. The BioMolViz.org group is a team of educators developing these tools. Over four years, we have identified a set of overarching themes that encompass biomolecular visualization skills with teachable objectives. We define proficiency levels for the objectives, describe student competency within the objective at the novice (first year) and amateur (junior/senior year) levels, and link these competencies with assessment questions. For example, the objective: “students will describe the significance of the monomer unit location within the three‐dimensional structure of a polymer” can be applied throughout the student's academic career. Novice students would be expected to identify the termini of a biological polymer, while more advanced students would catalog interactions of monomers that stabilize the macromolecule. Here, we present our current efforts on this Biomolecular Visualization Framework, including the results from four National Science Foundation‐funded 2018 workshops and our diversity SACNAS/ABRCMS outreach efforts. We propose targeted visual literacy development will enable students to understand the structure and associated function of biomolecules. The BioMolViz.org website makes resources widely available to help instructors facilitate this growth.

Support or Funding Information

This work was supported by the National Science Foundation under award numbers DUE‐1022793, ‐1323414, ‐1503811 and IUSE‐1712268. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the National Science Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pleiotropy and Flux Control in the Indolic Biochemical Pathways in Arabidopsis thaliana
Authors: Kack Jessa Joy , Leean Tori Kathryn
Published: June 2021

Metabolic pathways are a series of highly interconnected networks. Predicting how changes in one pathway influence the rest of metabolism is a challenge. Small changes in the expression of one gene in a single pathway may have far reaching pleiotropic effects across the genome or, alternatively, large changes in expression of one gene may be buffered and have only local effects. However, taking into account pathway properties of an enzyme (including the connectedness to other parts of metabolism or the amount of control an enzyme has over pathway production) may lead to predictable impacts on gene expression across the genome (e.g. perturbations in highly connected enzymes to influence a wide range of pathways). To address this question, we are studying the indolic glucosinolate pathway in Arabidopsis thaliana. Glucosinolates are herbivore defense compounds produced by relatives of Arabidopsis and protect the plant against generalist herbivores. The indolic glucosinolate pathway is also connected to the incredibly important plant hormone pathway, auxin, in that the pathways share a product (indole‐3‐acetaldoxime). Auxin is a growth hormone involved in nearly every step of plant development, thus we predict that some changes to the indolic pathway will produce only local effects while some changes may produce wide‐ranging effects. To investigate the influence of pathway position and connectedness on global gene expression and glucosinolate production we created gene expression knockdown lines in every gene in the indolic glucosinolate pathway. By monitoring indole glucosinolate pathway output with High Performance Liquid Chromatography (HPLC) and measuring global gene expression with RNAseq, we can study how changing gene expression within a pathway influences the output of that pathway and gene expression across the genome.

Support or Funding Information

SD BRIN P20GM103443SD EPSCOR IIA‐1355423

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mtDNMT1 and DNMT3b cooperate to methylate mitochondrial DNA and regulate mitochondrial transcription
Authors: Shock Lisa , Burton Elliot , Robinson Jason , Taylor Shirley
Published: June 2021

Mitochondria are dynamic modulators of cell physiology, rewiring their metabolic and signaling pathways to allow for rapid and flexible adaptions to environmental changes. Mitochondrial DNA (mtDNA) is packaged into DNA‐protein complexes called nucleoids, and the DNA is epigenetically modified (1,2). We, and others, have demonstrated that mtDNA methylation represents a powerful mechanism for regulating transcription of mtDNA‐encoded oxidative phosphorylation (OXPHOS) genes under conditions of cellular stress (1,3). We identified a mitochondrial (mt) isoform of DNA methyltransferase 1 (mtDNMT1) that translocates to the mt matrix, and binds to mtDNA in critical control regions (1). Our new data demonstrates that mtDNMT1 interacts with several proteins essential for proper functioning of mitochondria, including core components of the nucleoid. These findings place mtDNMT1 at the heart of mitochondrial nucleoid organization, and suggest that mtDNMT1 plays a critical role in regulating nucleoid structure and mitochondrial output, either as a consequence of, or independently from, its methylation activity. We now have evidence that mtDNMT1 is not working alone: DNMT3b works in concert with mtDNMT1 to methylate mtDNA and regulate mt transcription. CRISPR/Cas9‐mediated inactivation of mtDNMT1 and/or DNMT3b activity resulted in significant decrease in mitochondrial methylation across the heavy and light strand promoters of mtDNA, with a concomitant reduction in transcription of several mtDNA‐encoded OXPHOS genes. Cells lacking mtDNMT1 and/or DNMT3b exhibited a striking morphological change, undertaking an elongated, fibroblast‐like appearance with increased migration, reminiscent of cells undergoing epithelial‐to‐mesenchymal transition (EMT), a hallmark of cancer metastasis. These observations implicate mtDNA methylation in the phenotypic plasticity that occurs in response to changing cellular growth conditions, driving the initiation and progression of numerous pathologies. We propose that the mitochondrial DNA methylation machinery may constitute a novel target for therapeutic intervention.

Support or Funding Information

MCB 1122018 (NSF) to Taylor, Start‐up funds (VCU Dept of Microbiology and Immunology) to Shock, IRG‐14‐192‐40 (ACS) to Shock

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Accumulation of Gut Bacteria May Cause the Age‐Related Decline of Anoxia Tolerance in Adult Drosophila melanogaster
Authors: Sargent James , Campbell Jacob , Harrison Jon
Published: June 2021

Cell death occurring from anoxia is the major pathology during heart attack, stroke and multiple other diseases. Humans vary substantially in their ability to survive anoxia, especially across ages, and the basis to this variation is not well‐understood. Drosophila melanogaster have similar metabolic pathways to humans but have much better capacities to tolerate anoxia, suggesting that understanding mechanisms of anoxia tolerance in flies may provide insight for the development of new medical treatments. We exposed adult Drosophila, ages 1, 3, 5, 7, 9, and 12 days old, to six hours of anoxia and assessed survival 24‐hours post‐treatment. Seventy‐nine percent of adults one day past eclosion survived; while only 10% of twelve‐day‐old adults survived; thus Drosophila show age‐related decline in anoxia tolerance like humans. In anoxia, ATP levels declined rapidly (< 30 min) to near‐zero levels in both 1 and 12 day old adults; thus the better anoxia‐tolerance of young adults is not due to a better capacity to maintain cellular energetic status. The concentration of bacteria in the gut is known to increase strongly with age in Drosophila. To test whether declining anoxia tolerance might be due to this increasing bacterial load, we replaced their food daily, every third day, or every sixth day, allowing us to vary gut bacterial load from low to high, respectively. At 12 days of age, each treatment group was exposed to six hours of anoxia and assayed for gut bacterial load. Anoxia tolerance was strongly and negatively affected by bacterial load. These data suggest that increasing bacterial load may play an important role in the age‐related decline of anoxia tolerance in Drosophila.

Support or Funding Information

This research was supported by NSF IOS 1256745 and the SOLUR program at ASU.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intestinal proteome response to salinity stress in three tilapiine spp
Authors: Root Larken , Kültz Dietmar , Cnaani Avner , Con Pazit
Published: June 2021

Chronic salinity stress is likely to have significant impacts on organismal performance, especially near the upper bounds of the Critical Salinity maximum (CSmax), the “zone of resistance”. Some euryhaline fish species maintain stable internal osmolality over great salinity ranges, while close relatives tolerate much narrower ranges. Greater tolerance ranges have also been linked to reduced growth under optimum conditions. For example, breeding for improved growth rates in the tilapia species Oreochromis niloticus has inadvertently resulted in decreased tolerance to hypersaline water conditions. To examine changes in growth and stress tolerance at the cellular and tissue levels, high salinity acclimation challenges were conducted for three cichlid species, including the previously mentioned O. niloticus with reduced salinity tolerance, wild‐type (WT) O. mossambicus which exhibit tolerance to salt concentrations greater than seawater, and WT Astatotilapia burtoni which exhibit low salinity tolerance similar in range to the O. niloticus strain. Three intestinal sections were sampled from salinity‐challenged fish at 90% CSmax, and from control fish maintained in freshwater, and used to establish a standardized data‐independent acquisition (DIA) assay for label‐free quantitative proteomics for a large number of proteins in each species. Spectral libraries created by data‐dependent acquisition (DDA) and annotated using multiple search engines were used for DIA assay construction. Skyline and a sample training set were used for automated and manual assay curation to select and validate reproducible transitions and proteotypic peptides for protein quantitation. Salinity effects on intestinal protein expression patterns in each species were statistically evaluated and corresponding protein sets analyzed for gene ontology enrichment. The resulting data on salinity‐dependent intestinal proteome dynamics provide insight into biochemical processes controlling growth and stress tolerance.

Support or Funding Information

Funded by BARD and NSF Grant IOS‐1355098

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prematurity and postnatal development regulate the expression of the FXR‐FGF19 axis in neonatal pigs
Authors: Smith Victoria Alice , Jiang Yanjun , Thymann Thomas , Sangild Per , Manjarin Rodrigo , Burrin Douglas
Published: June 2021

The farnesoid X receptor (FXR) is a nuclear bile acid receptor that regulates bile acid homeostasis. A key FXR target is fibroblast growth factor 19 (FGF19), which functions as an endocrine hormone. FGF19 is produced in both the intestine and liver in humans and pigs, whereas mice and rats express the human ortholog FGF15 only in the intestine, thus making pigs a more relevant model of human FGF19 biology. Gut bile acids activate small intestinal FXR triggering FGF19 secretion which circulates via the portal blood to inhibit hepatic bile acid synthesis by suppressing the rate limiting enzyme in bile acid synthesis, CYP7A1, via activation of extracellular‐signal‐regulated kinases (ERK) and c‐Jun N‐terminal kinase (JNK‐1) MAP kinase pathways and the orphan nuclear receptor, small heterodimer partner (SHP). Thus, enterohepatic FXR‐FGF19 signaling functions as a negative feedback signal to regulate bile acid homeostasis. Bile acid secretion has been shown to increase with gestational age. We hypothesized that expression and activation of the intestinal FXR‐FGF19 axis would be diminished in premature vs. term pigs and increased with postnatal age. We studied piglets delivered by cesarean‐section at 10 days preterm and full term. All pigs were fitted with orogastric and umbilical arterial catheters and fed minimal enteral nutrition with bovine colostrum for 5 d and then bovine mature milk until 26 d‐of‐age. Plasma and tissue samples were collected at days 0, 5, 11, and 26 d. We developed a porcine FGF19 specific sandwich ELISA assay by cloning the porcine FGF19 gene, overexpressing the protein using a baculovirus expression system, and developing monoclonal antibodies. We quantified the plasma FGF19 concentration using the ELISA and assayed distal intestinal gene expression of FGF19 and FXR by RT qPCR. We found that plasma FGF19 levels are strikingly lower (P<0.05) in premature vs. term newborn pigs. In addition, postnatal age markedly altered activity of the FXR‐FGF19 axis, where plasma FGF19 levels increased significantly 5 d after birth in preterm, but not term pigs. Plasma FGF19 levels then decreased (P<0.05) significantly between 5 and 26 d‐of‐age in both preterm and term pigs. Intestinal FXR and FGF19 expression were lower (P<0.05) in premature vs term newborn pigs and decreased (P<0.05) between day 5 and 26 d‐of‐age. We conclude that both prematurity and postnatal age regulate the FXR‐FGF19 axis in neonatal pigs. These findings have implications for the bile acid homeostasis, FGF19 function and growth of preterm and term infants during early postnatal life.

Support or Funding Information

Agricultural Research Institute, USDA‐ARS, NIH, CalPoly Summer Undergraduate Research Program (SURP), NEOMUNE

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PABP and eIF4B Effects Binding Affinity and Kinetic Rates of Genome Linked Viral Protein (VPg) with Eukaryotic Initiation Factor 4F
Authors: Khan Mateen A. , Goss Dixie J.
Published: June 2021

Translation initiation is a critical early step in the viral replication cycle. Interaction of eukaryotic translational initiation factors with VPg of Turnip mosaic virus (TuMV) plays an important role in the cap‐independent initiation of protein synthesis. To understand this process in potyviruses, we investigated the equilibrium binding, thermodynamic parameters and rate constants for the interaction of VPg with eIF4F in the presence of PABP and eIF4B using fluorescence and stopped‐flow spectroscopy. Equilibrium studies showed that PABP increased VPg binding to eIF4F about 3‐fold; however, both PABP and eIF4B together enhanced VPg binding to eIF4F about 4‐fold, showing an additive effect rather than the large increase in affinity shown for cap binding. Temperature dependent studies showed the increase in VPg binding to eIF4F in the presence of PABP and eIF4B was enthalpy‐driven and entropy‐favorable. PABP and eIF4B decreased the entropic contribution for VPg binding to eIF4F. The decrease in entropy for the formation of eIF4F·4Bmiddot;PABP‐VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. Rate constant for eIF4F interaction with VPg in the presence of PABP and eIF4B was 4‐fold faster; and dissociation rate was 3‐fold slower as compared to eIF4F alone. The temperature dependence of the VPg binding to eIF4F was significantly reduced in activation energy in the presence of PABP and eIF4B. When both PABP and eIF4B were present, not only was the energy barrier reduced but the binding rate was faster and dissociation rate was slower for the formation of eIF4F‐4B‐PABP‐VPg complex suggests more stable platform for further initiation complex formation. These results further support the importance of PABP and eIF4B interaction to eIF4F with VPg for efficient viral translation.

Support or Funding Information

National Science Foundation MCB‐1513737 (to D.J.G.) and Alfaisal University Research Support IRG‐16414 (to M.A.K.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential protein splicing of salt‐dependent inteins from Haloquadratum walsbyi
Authors: Lynch Abigeal Kathleen , Amunya Shirley , Gomez Alvin , Reitter Julie , Mills Kenneth
Published: June 2021

Protein splicing is a self‐catalyzed, post‐translational process in which an intervening polypeptide sequence, or an intein, catalyzes its removal from two flanking polypeptide sequences, or exteins, as well as the ligation of the exteins. We are interested in the splicing capabilities of two inteins that interrupt the cdc21 protein from Haloquadratum walsbyi (Hwa). Hwa is a halophilic archaeon found in high salt environments such as the Red Sea. For this reason, we hypothesize that protein splicing may be salt dependent. We are interested in studying the splicing capabilities of both inteins separately and when expressed as part of the same protein. One intein, Cdc21a, does not splice on overexpression in E. coli, but can be induced to splice in vitro on incubation with 2.5 M sodium chloride at 28°. The other intein, Cdc21d, splices in vivo, and can be induced to splice further with higher concentrations of salt. When expressed in the same precursor fusion protein, the inteins continue to show differential activity conditional on salt concentration, leading to alternative spliced products.

Support or Funding Information

This work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM), the Dreyfus Foundation (KVM), and Diane D. Brink P12 to the Alumni/Parent Summer Research Scholarship Fund. AL

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aerobiotics: Toward the Discovery of New Antibiotics From Airborne Actinomycetes
Authors: Hudgens Zachary , Wickard Aaron , Nealon Christopher , Sweet Charles
Published: June 2021

In the last decade the rising prevalence of multi‐drug resistant (MDR) infections such as Staphylococcus, Acinetobacter, Klebsiella, Mycobacterium, and others have made bacterial illnesses increasingly difficult to treat. Resistant bacterial infection could kill millions in the coming decades ‐ surpassing cancer in mortality ‐ and in September 2016 a woman in the United States died from Klebsiella pneumoniae resistant to all known antibiotics. The need is dire for new antimicrobial therapies, but developmental approaches over the last few decades have been slow to yield new drugs for reasons both scientific and economic. In response to this crisis we propose a return to the original well of natural product discovery, isolating new antibiotics from actinomycete bacteria in the aerobiome. To facilitate the discovery of novel antibiotic metabolites in these organisms, a collection of air‐harvested isolate cultures within the order Actinomycetales has been curated at the US Army Edgewood Chemical Biological Center (ECBC). This collection is sourced from lower atmosphere biosampling conducted by several institutions, including the United States Naval Academy (USNA). To date the ECBC screen has identified 43 organisms with novel antimicrobial bioactivity, and promising members of this collection are under study at USNA in a target‐agnostic isolation and characterization scheme using HPLC and affinity column purification, bioactivity assays, and spectral characterization. In this work we demonstrate the isolation and partial purification of antimicrobial activities secreted into liquid culture by several of these bacteria, including a broad‐spectrum activity against both Gram‐positive and Gram‐negative tester strains.

Support or Funding Information

Funding for this study was provided by the Office of Naval Research Midshipman Research Program and the Defense Threat Reduction Agency grant CB10171.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Proteins Interacting with Fyn Kinase at Fertilization in Patiria miniata
Authors: Bates Lauren Shea , Carroll David
Published: June 2021

Release of Ca2+ from the endoplasmic reticulum is the integral molecular mechanism responsible for initiating development at fertilization. The components of the signaling pathway controlling this Ca2+ release are not fully known in any organism. Eggs and sperm of the starfish, Patiria miniata, present an attractive model to study the molecular aspects of fertilization because of the ability to obtain millions of viable sperm and eggs, along with the ease of in vitro fertilization. Two Src family kinases (SFKs) and the lipase PLCγ have been shown to be involved in this signaling pathway and are necessary for Ca2+ release. However, additional interacting proteins and the receptor(s) initiating this pathway have not been identified or characterized. In this study, GST fusion protein constructs of the SH2 domains of Fyn (a SFK) were prepared and affinity interactions were performed at different time points before and after sperm addition to eggs. The affinity complexes were subjected to Western blotting to visualize the interacting proteins. Using an anti‐phosphotyrosine antibody, 5 distinct bands indicate that tyrosine phosphorylated egg proteins of 250, 150, 100, ~80, and ~65kDa bind to the fusion proteins as fast as 1 minute post sperm addition, while the same 5 bands plus two additional bands at ~200kDa and ~125kDa are seen in eggs at 2, 3, 5, 10, and 20 minutes post sperm addition, with varying intensities. Furthermore, tyrosine phosphorylated proteins from ~70kDa to ~45kDa are seen to bind with varying intensity at 5, 10, and 20 minutes post sperm addition. These findings indicate that multiple proteins are interacting with the SFK Fyn in a fertilization‐specific manner. In future experiments, mass spectrometry and mRNA knock down strategies will be used to identify these proteins and to discover their function in regulating Ca2+ release at fertilization.

Support or Funding Information

This work was funded by the NSF and Florida Institute of Technology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measuring cognitive load & impact of modeling activities in undergraduate biochemistry
Authors: Terrell Cassidy R. , Aleuy Lana , Calvert Jenifer , Hampton‐Ashford Aana , Prat‐Resina Xavier , Randolph Adriane , Cortes Kimberly
Published: June 2021

Among the STEM disciplines, biochemistry presents a particular challenge to students with abstract concepts that can lead to rampant, resistant misconceptions that impede learning. The development of undergraduate students' conceptual understanding of abstract structure‐function relationships in biochemistry a the long‐term goal of the collaborative research team from University of Minnesota, Rochester (UMR) and Kennesaw State University (KSU). The objective of this study is to facilitate this process by implementing three‐dimensional (3D) virtual and physical modeling activities across the (bio)chemistry curriculum that optimize cognitive load and thereby enhance student learning in (bio)chemistry. As such, we aim to characterize the cognitive load of the 3D physical and virtual modeling activities by defining what and how many cognitive tasks and concepts are accessed using the 3D modeling activities. To this end, a UMR cohort participated in a simulated learning environment completing the modeling activities while tracking cognition by means of eye‐tracking and electroencephalogram (EEG) technologies. Here, we will present the first set of data from fall 2017 interviews and inclass activities from a biochemistry course. Ultimately this project would provide evidence to more intelligently design and execute modeling activities that optimize the cognitive load, deepen students' ability to understand biochemistry concepts, and overcome misconceptions; thus creating better consumers of (bio)chemistry education.

Support or Funding Information

This 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 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fecal fermentation products of common bean‐derived fiber inhibit C/EBPα and PPARγ expression and lipid accumulation but stimulate PPARδ and UCP2 expression in the adipogenesis of 3T3‐L1 cells
Authors: Lu Hsin‐Yu , Cheng Wen‐Hsing
Published: June 2021

Clinical and animal studies have suggested efficacies of common bean (Phaseolus vulgaris) consumption on weight loss. Fermentation of common bean‐derived dietary fiber by gut microbiota is proposed to mitigate obesity; however, the mechanism of actions are unclear. This study was conducted to investigate whether and how fecal fermentation of common bean‐derived dietary fiber impacts adipogenesis in a cell model. Dietary fiber was generated by in vitro digestion of cooked, lyophilized common bean flour, followed by anaerobic fermentation with the use of fresh feces from healthy mice without antibiotics treatment. The murine 3T3‐L1 cells were induced to differentiate in the presence of the fermentation products. Cell viability, lipid accumulation, and protein and mRNA levels of key transcriptional factors and activators were determined during the 8‐day time course. Data were analyzed by 1‐factor or 2‐factor (bean ‘ time) ANOVA. Treatment of the fecal fermentation products inhibited adipocyte differentiation and lipid accumulation in a dose‐ and time‐dependent manner. The fermentation products decreased (P < 0.05) protein levels of two key transcription factors for adipogenesis, CCAAT/enhancer binding protein α and peroxisome proliferator‐activated receptor γ by 79–92% and 78–90%, respectively, and their downstream target fatty acid binding protein 4 by 49–86% and 63–98% at protein and mRNA levels, respectively, during the time course. In contrast, the fermentation products increased (P < 0.05) levels of two proteins promoting energy expenditure, peroxisome proliferator‐activated receptor δ (41–58%) at days 2–4 and mitochondrial uncoupling protein 2 (1.1–1.2 fold), at days 4–8. Altogether, fecal fermentation of dietary fiber derived from in vitro digestion of common bean temporally and dose‐dependently inhibits adipogenesis and key adipogenic transactivators but activates two energy expenditure proteins in 3T3‐L1 cells.

Support or Funding Information

National Institute of Food and Agriculture (Multistate W3150, accession no. 1007904, project no. MIS‐141030)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Caveolin‐1 Positive Microvesicles and Apoptotic Bodies as Early Biomarkers of Acute Lung Injury and Possible Mediators of TGF‐β‐mediated Repair/Remodeling
Authors: Oliveira Suellen DArc Santos , Castellon Maricela , Machado Roberto , Bonini Marcelo G. , Minshall Richard
Published: June 2021

Endothelial cell injury is a hallmark of Acute Lung Injury and Acute Respiratory Distress Syndrome (ALI/ARDS). We and others have shown endothelial cell caveolin‐1 (Cav‐1) expression is critical for maintaining vascular homeostasis. The aim of this study was to determine how Cav‐1 is depleted during ALI/ARDS and how this may lead to defective vascular repair. ALI was induced in C57BL6 WT mice (male; 2.5–3 months old) by nebulized Escherichia coli lipopolysaccharide (LPS; 10 mg over 1 hr daily for 1 or 4 days). Lung pathology and Cav‐1+ EVs in platelet‐free plasma were assessed by immunohistochemistry, quantification of immune cell number in bronchoalveolar lavage fluid, and western blot. In vitro, human pulmonary artery endothelial cells (HPAECs) and isolated bone marrow‐derived macrophages (BMDMs) were treated with LPS or EVs from control or LPS‐treated animals. At 24 hs, LPS‐induced ALI increased the total number of EVs in murine plasma by 50%. Characterization of isolated EVs indicated Cav‐1 is abundantly expressed in large microvesicles and apoptotic bodies when compared with smaller vesicles, suggesting Cav1+ EVs may be an endothelial cell damage‐associated factor. By western blot, we observed EV‐Cav‐1 expression was increased by 38% after 24 hrs of LPS exposure (0.61 ± 0.05 to 0.98 ± 0.16 a.u.) in control and LPS‐treated mice, respectively (n= 8–10 mice; P<0.05) and decreased below basal level after 96 hrs indicating endothelial Cav‐1 depletion may be partially due to release Cav‐1+ EVs into the circulation. In vitro, LPS‐treated HPAECs released Cav1+ EVs in a concentration‐dependent manner and EVs‐derived from LPS‐treated mice induced TGF‐β release from BMDMs. Thus, EC‐Cav‐1 depletion may be due in part to EV shedding as a result of EC damage which in vivo may be a mechanism of macrophage‐dependent TGF‐β‐induced vascular repair/remodeling. These novel observations may provide an important link to new ways of preventing the onset or progression of ALI/ARDS.

Support or Funding Information

CNPq Fellowship‐CsF/Brazil (SDO) and HL125356 and DOD W911NF‐15‐R‐002 (RDM, MGB).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Associations between Early Pregnancy Maternal Body Mass Index (BMI) and Offspring Sex with Placental DNA Methylation at Term
Authors: Thakali Keshari M. , Zhong Ying , Andres Aline , Shankar Kartik
Published: June 2021

Background

The placenta serves as the definitive maternal‐fetal interface and mediates exchange of nutrients, gases, and waste between mother and the developing fetus. The placenta integrates signals from both mother and baby, coordinating maternal nutrient supply with fetal demand and development. In epidemiological studies, maternal obesity and obesogenic diets are associated with fetal overgrowth and greater risk of obesity and metabolic dysfunction in later‐life. Animal and human studies have shown maternal obesity‐associated alterations in placental gene expression, as well as sex‐dependent structural and functional placental differences. Whether maternal obesity and offspring sex are important contributors to placental epigenetic alterations is yet to be determined.

Hypothesis

We hypothesized that placental epigenetic patterns are associated with maternal early pregnancy body composition (BMI or percent body fat) and offspring sex.

Methods

Using Reduced Representation Bisulfite Sequencing (RRBS), we assessed genome scale DNA methylation at approximately 300,000 CpGs in 150 placenta from uncomplicated full‐term pregnancies (N=72 normal weight mothers, N=78 overweight/obese mothers). Maternal BMI and percent body fat were assessed at <10 weeks of gestation.

Results

In multivariable linear regression models adjusted for multiple testing (q<0.2) and covariates (maternal age at delivery, infant sex, and delivery mode), maternal early pregnancy BMI was significantly associated with placental DNA methylation of 54 specific CpGs while maternal early pregnancy body fat percentage was significantly associated with methylation of 1 CpG. Likewise, in multivariable linear regression, male infant sex was significantly associated with methylation of 34 specific CpGs. Of the 54 CpGs significantly associated with maternal early pregnancy BMI, gene‐set enrichment analysis of the proximal genes associated with these differentially‐methylated CpGs showed biological processes related to lipid metabolism, DNA repair, and vesicle exocytosis to be differentially methylated. Of the 34 CpGs significantly associated with male offspring sex, gene‐set enrichment analysis of the proximal genes associated with these differentially‐methylated CpGs showed biological processes related to corticotropin secretion and protein localization to the nucleolus to be differentially methylated.

Conclusions

These data suggest that maternal obesity, as assessed by early pregnancy BMI, and male offspring sex have modest but unique effects on placental DNA methylation patterns.

Support or Funding Information

This work was supported by USDA ARIS Project 6026‐51000‐010‐05S.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Milk Fat Globule Membrane on Lymphocyte Gene Expression and Markers of Metabolism and Inflammation in the Postprandial Period
Authors: Beals Elizabeth , Demmer Elieke , Rivera Nancy , Rogers Tara S. , Gertz Erik R. , Van Loan Marta D. , German J. Bruce , Smilowitz Jennifer T. , Zivkovic Angela M.
Published: June 2021

Objective

To test for a difference in lymphocyte gene expression in response to a high fat dairy challenge with or without milk fat globule membrane (MFGM).

Methods

Data was analyzed from twenty individuals with BMI > 25.0 kg/m2. On two separate occasions, each participant consumed a dairy‐based meal high in saturated fat with or without the addition of MFGM, following a 12‐hour fasting blood draw. Inflammatory markers including interleukin‐6 (IL‐6) and C‐reactive protein (CRP), lipid and metabolic panels, and lymphocyte gene expression fold changes were measured using multi‐plex assays, clinical lab services, and TaqMan RT‐PCR, respectively. Fold changes were determined using the Pfaffl method, tested for differences through Wilcoxon Signed Rank, and corrected for multiple comparisons. Postprandial responses were quantified using incremental area under the curve (iAUC). Data was analyzed in Microsoft Excel and JMP Pro 13. Correlation analysis was used to determine associations between markers.

Results

The median postprandial insulin response was found to be significantly lower following the meal containing MFGM (p=0.0003). Out of 56 genes analyzed, EPHX2 was shown to be more up‐regulated in the absence of MFGM (p=0.009). The median gene expression was lower and had a narrower response range when MFGM was added to the high‐fat test meal.

Conclusion

The protein and lipid composition of MFGM is thought to be anti‐inflammatory. These exploratory analyses suggest that addition of MFGM to a high saturated fat meal may attenuate the upregulation of soluble epoxide hydrolase gene expression. Changes in inflammatory markers are important for determining a patient's inflammatory status, which is useful for the development of personalized nutrition and medicine, and disease prevention.

This study is registered on ClinicalTrials.gov Identifier: NCT01811329

Support or Funding Information

This project was made possible by support from the National Dairy Council, Rosemont, IL. and the USDA, Agricultural Research Service, Western Human Nutrition Research Center

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Global Profiling of the Oxidative Stress Induced Effects on RNA Modifications by Liquid Chromatography‐Tandem Mass Spectrometry
Authors: Jora Manasses , Sun Congliang , Limbach Patrick A. , Addepalli Balasubrahmanyam
Published: June 2021

The goal of this work was to profile the oxidative damage exerted by ultraviolet‐A radiation (UVA, 370 nm) or H2O2 on RNA modifications by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Oxidative damage to RNA impacts biological systems due to its association with metabolic, mitochondrial, neurological diseases and cancer.1,2 In the presence of photosensitizer (e.g., riboflavin), UVA radiation induces the production of reactive oxygen species (ROS, O2•, HOO•, HO•) in vivo.3 Likewise, H2O2 generates ROS under physiological conditions, and formation of radicals is accelerated via Fenton reactions in the presence of catalysts (e.g., Fe2+, Cu2+).4

Oxidation pathways of canonical nucleosides are well documented.5 However, the effect of ROS on modified ribonucleosides is not well known, and information about RNA damage is largely extrapolated from studies on DNA. More than 160 modified nucleosides have been reported in various types of RNA of different organisms.6 E. coli with 46 modifications in its tRNA serve as a good model system to investigate the stress‐induced effects. Apart from the known oxidative products arising from canonical nucleosides, we have documented a list of modified nucleosides that are adversely affected by UVA or H2O2. To understand the oxidative environment generated by UVA exposure, we exposed E. coli total tRNA with or without a photosensitizer for defined period of time. In a similar manner, the effect of H2O2 on RNA modifications was investigated by incubating E. coli total tRNA and H2O2, with or without the presence of transition metal ion catalysts. The oxidative damage observed on various modifications was measured by subjecting it to LC‐MS/MS following complete hydrolysis of tRNA. We will present data that describes the susceptibility of various functional groups of these modifications and underlying patterns. Further, these studies also document a range of RNA photoproducts and chemical alterations arising due to the oxidative conditions.

Support or Funding Information

Financial support of this work was provided by the NSF (CHE1507357) and DTRA (HDTRA1‐15‐1‐0033), both to Patrick A. Limbach, and the University of Cincinnati.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.