bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR/Cas9 in Yeast as a Tool for Teaching and Reinforcing Molecular Biology Concepts to Undergraduates
Authors: Ulbricht Randi J. 
Published: June 2021

Undergraduate students in an upper‐level molecular biology laboratory class used their training in biochemistry, cell biology and molecular biology to design and perform CRISPR/Cas9 genome editing on yeast. After an introduction to DNA repair and CRISPR/Cas9 in the lecture, the students took on the task to design an experiment using Cas9 to generate auxotrophic yeast mutants. They used readily available bioinformatics databases to retrieve yeast genome sequence and design CRISPR‐associated sgRNA to target the gene of interest. They cloned genes for the sgRNAs into a commercially available CRISPR/Cas9 vector, and then transformed it with a template for homology‐directed repair into yeast. Next, the students phenotyped and genotyped the potential auxotrophic mutants generated by CRISPR/Cas9. As the penultimate laboratory course in the program, this lab introduced new skills, provided opportunity to apply concepts discussed in lecture, and reinforced skills and knowledge from previous courses. A survey, exam and formal lab report assessed positive outcomes in learning and affect. The reagents in this laboratory course design are readily available and financially feasible for undergraduate laboratory courses, and the protocols/emphasis are adaptable to diverse program goals. Overall, incorporating the tractable, yet modern gene editing technology into undergraduate laboratory courses is both realistic and favorable to learning.Support or Funding InformationThis work is supported by Missouri State University, Department of Biomedical SciencesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FRESH – Freshman Research Engagement in Science: Early Results
Authors: Watters Michael K. , Bouyer Patrice , Clark Robert 
Published: June 2021

We describe here some early results of the FRESH (Freshman Research Engagement in the Sciences) program. A program with the goal to expose freshman to an ongoing research project during the academic year to promote student growth and improve retention in the STEM disciplines. Freshmen worked with a faculty mentor and were also chaperoned by a more senior student researcher in order that they learn lab techniques and the capacity to work independently. Participants were fully engaged in a research project (performing experiments, analyzing and discussing results), not a classic classroom projects, but discovery based projects. By bringing students into the research lab at this early stage, our aim was to improve retention by allowing science students to actually act as scientists, providing an enhanced experience over the usual freshman survey course content. Of the 13 students in two cohorts who joined the program as freshmen, 12 are still in their major and have co‐authored over 20 different papers and conference presentations to date. Based on these initial successes, we have modified our approach, tracking qualified applicants who we were unable to fund to serve as our control group in order to study the impact of the FRESH approach on student success.Support or Funding InformationThis work was supported by grants from the Indiana Space Grant Consortium (INSGC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion Of Muscle Acsl1 Caused Myopathy And Fiber Switch
Authors: Zhao Liyang , Bacudio Lawrence , Suchanek Amanda L. , Young Pamela A. , Pascual Florencia , Coleman Rosalind A. 
Published: June 2021

Skeletal muscle exhibits plasticity and complexity in the choice of fuel substrate. During starvation, muscle initiates protein degradation to provide amino acids for liver gluconeogenesis, during acute exercise, muscle uses glucose as the primary fuel, while during rest or prolonged exercise, muscle switches to use primarily fatty acids. Defective fatty acid metabolism is one of the leading causes of metabolic myopathy. Long chain acyl‐CoA synthetase isoform‐1 (ACSL1) converts long chain fatty acids to long chain acyl‐CoAs, before acyl‐CoAs enter mitochondria for β‐oxidation or are esterified to form complex lipids. Although 5 Acsl isoforms exist, deletion of Acsl1 reduces 90% of ACS activity and fatty acid oxidation (FAO) in gastrocnemius, indicating that ACSL1 is the predominant ACSL isoform in the muscle. The objective of this study was to examine how reduced FAO through deletion of Acsl1 affects muscle physiology. We hypothesized that ACSL1‐directed fatty acid metabolism is essential for energy balance in muscle, and that lack of ACSL1 causes myopathy. To test this hypothesis, we used a mouse model in which ACSL1 is specifically deleted in skeletal muscle (Acsl1M−/−). Compared to control mice (Acsl1flox/flox), Acsl1M−/− mice demonstrated 3.4‐ and 1.5‐fold increases in plasma creatine kinase and aspartate aminotransferase activities, indicating that deletion of Acsl1 led to muscle damage. Gastrocnemius from Acsl1M−/− mice also showed the presence of central nuclei, a marker of muscle regeneration. In addition, we detected expression of caspase 3 protein only in Acsl1M−/− muscle, suggesting that the apoptosis pathway was involved. Different muscle fiber types prefer to metabolize different substrates. To investigate the consequence of Acsl1 deletion on muscle fiber type, succinate dehydrogenase staining was performed and it showed a significantly increased percentage of oxidative fibers in Acsl1M−/− gastrocnemius compared to controls. In addition, gene expression of myosin heavy chain I, an oxidative muscle fiber marker, was 6 fold higher in Acsl1M−/− glycolytic fibers than in controls, suggesting a switch from glycolytic to oxidative fibers. We further hypothesized that the fiber switch in Acsl1M−/− gastrocnemius muscle was caused by increased damage to glycolytic fibers compared to oxidative fibers. In summary, deletion of Acsl1 caused muscle damage and regeneration and promoted a glycolytic to oxidative fiber switch. Ongoing studies are examining protein turnover to determine its contribution to muscle damage.Support or Funding InformationSupported by DK56598.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 Dose‐Related Effects of Doxorubicin Chemotherapy on Interstitial Amino Acid Concentrations in Skeletal Muscle
Authors: MacLean David , Fabris Sergio 
Published: June 2021

Doxorubicin (DOX) is a frontline anticancer therapeutic which has proven effective in the treatment of various cancers. However, very little is known regarding the effect that DOX has on SM metabolism, specifically its influence on interstitial amino acid (AA) concentrations. In SM, the interstitial space represents an important active compartment located between the vasculature and the tissue and it has been shown to play a functional role in the regulation and integration of various metabolic substances. The purpose of the current study was to determine the effect of DOX on interstitial AA concentrations in SM. Rats received a 1.5 or 4.5 mg/kg dose of DOX i.p. and grouped into endpoints every 24 hrs up to 192 hrs. A control group was administered a sham i.p. injection of saline. Total amino acids (TAA), essential amino acids (EAA) and branched‐chain amino acids (BCAA) were quantified in the interstitial space. The administration of DOX resulted in a dose‐related difference in interstitial AA. The 1.5 mg/kg dose resulted in a transient increase (P<0.05) in TAA, EAA and BCAA from 24–96 hrs as compared to control. Contrarily, TAA, EAA and BCAA decreased (P<0.05) 24–192 hours following the administration of 4.5 mg/kg DOX as compared to control. This study by way of the microdialysis technique, represents the first time that amino acid concentrations have been quantified in the interstitial space following the administration of a chemotherapeutic drug. In addition, the possibility of a dose‐related effect of DOX on amino acid transport mechanisms from the skeletal muscle compartment into the interstitium/vasculature is of particular interest as this may represent a regulatory mechanism that has not previously been considered.Support or Funding InformationSupported by NOSMFAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Catabolism of Nicotinic Acid and Nicotine and the Role of a Putative Monooxygenase in Bacillus niacini
Authors: Fine Sydney R. , Zoretich Kaeli M. , Snider Mark J. 
Published: June 2021

N‐Heteroaromatic compounds (NHACs) comprise a class of molecules that are commonly found in pharmaceuticals, personal care products, and pesticides and are known to have toxic effects on the environment. Intriguingly, certain species of soil‐dwelling bacteria have the innate ability to degrade particular NHACs. A better understanding of how these soil bacteria degrade NHACs has important implications in the development of bioremediation techniques to remove toxic compounds from the environment. This investigation focuses on developing preexisting hypotheses in regards to how Bacillus niacini, a ubiquitous species of soil bacteria, degrades nicotinic acid (NA) and nicotine, both of which are NHACs. It has been hypothesized that the non‐canonical NA degradation pathway in B. niacini involves a reaction in which 2,6‐dihydroxynicotinic acid (2,6‐DHNA) undergoes oxidative decarboxylation to form 2,3,6‐trihydroxypyridine by an enzyme termed NicC‐like. Two methods have been utilized to establish the identity of the nicC‐like gene. First, a novel transformation technique was developed to generate a B. niacini knock out variant lacking the putative nicC gene. Second, the substrate for the NicC‐like enzyme, 2,6‐DHNA, was synthesized with a 60% yield. The substrate was utilized to assess the kinetic activity of the NicC‐like enzyme. Though there are hypotheses regarding the identity of the nicC‐like gene and its involvement in NA catabolism, sequence analysis indicates that the nicC‐like gene is only 21% similar to the nicC gene involved in NA degradation in Pseudomonas putida. In contrast, the nicC‐like gene is 24% similar to HspB, which encodes an enzyme involved in nicotine degradation. Additionally, the structure and the associated catabolic processes of nicotine and NA are similar, suggesting that the novel nicC‐like gene in B. niacini might play a role in either nicotine or NA degradation. Preliminary HPLC evidence indicates that B. niacini does not degrade nicotine. Furthermore, the NicC‐like enzyme is not kinetically active when incubated with the substrate of the HspB enzyme, 6‐Hydroxyl‐3‐succinoyl‐pyridine. Thus, the genes currently hypothesized to encode the enzymes necessary for NA catabolism do not code for nicotine degradation enzymes. Characterizing the reaction catalyzed by the putative NicC‐like enzyme and determining whether or not B. niacini catabolizes nicotine has led to an improved understanding about the genes necessary for NA degradation by this particular species of pervasive soil bacteria. Continued contributions to this field may eventually lead to the development of techniques to remove NHACs from the environment.Support or Funding InformationHenry J. Copeland Fund for Independent Study at the College of WoosterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cross‐talk among biochemical defense pathways in Arabidopsis thaliana
Authors: Peterson Alexandra , Bien Erica , Olson‐Manning Carrie , Matzner Steven 
Published: June 2021

Cellular metabolism is a complex set of interwoven interactions. That complexity challenges the prediction of how metabolism responds to change. To try to increase our ability to predict how single‐gene changes result in global metabolic shifts, we studied how changing expression in the indolic glucosinolate chemical defense pathway in Arabidopsis thaliana influences production in the related aliphatic glucosinolate pathway.Aliphatic glucosinolates are synthesized in a pathway parallel to the indolic pathway and share a number of enzymes (SUR1, GGP1). Research on the aliphatic pathway has shown aliphaticspecific and shared gene knockdowns have no control over the indolic pathway. However, by knocking down activity of each step in the indolic pathway and measuring the production of all glucosinolates via High Performance Liquid Chromatography (HPLC), our results suggest crosstalk between these pathways.Preliminary results reveal that when the gene GTSF9 of the indolic pathway is knocked down, the production of aliphatic glucosinolates is reduced, but the production of indolic glucosinolates is not affected. These results are puzzling given that GSTF9 is not thought to be shared between the pathways like the enzymes SUR1 and GGP1. Indeed, previous research suggests GSTF9 is predicted to act in the aliphatic pathway exclusively. To further study this pathway cross‐talk, we will extract and sequence RNA from single‐gene knockouts of the genes in the indolic pathway to track how changes in gene expression of individual enzymes change global patterns of expression. These results will allow us to begin to understand the genetic mechanisms that mediate cross‐talk between these pathways.Support or Funding InformationSD BRIN P20GM103443 SD EPSCOR IIA‐1355423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Transferrin‐1 from Drosophila melanogaster
Authors: Gorman Maureen J. , Brummett Lisa M. , LeSuer Leslie A. , Weber Jacob J. , Kanost MIchael R. 
Published: June 2021

Transferrins are secreted proteins that bind iron. Mammalian serum transferrin is involved in iron transport, while mammalian lactoferrin functions as an immune protein. Both proteins are thought to protect against oxidative stress. Insect transferrin is similar to both serum transferrin and lactoferrin, and its functions are not well characterized. Previous studies have demonstrated that insect transferrin can function as an immune protein, and limited data suggest that it may have a role in protection against oxidative stress and in iron transport. The goal of this study was to identify the functions of Drosophila melanogaster transferrin‐1 (DmTsf1). Our approach was to observe changes in DmTsf1 concentration in response to immune challenge, oxidative stress, metal stress, and dietary iron availability; test the antimicrobial activity of apo and holo forms of DmTsf1; and evaluate phenotypes associated with RNAi‐mediated knockdown of DmTsf1. We estimated the concentration of DmTsf1 in hemolymph to be 0.5 μM in larvae, 4 μM in adult females and 20 μM in adult males. We found that DmTsf1 concentration increased after inoculation of bacteria, and that DmTsf1 inhibited bacterial growth by sequestering iron. These findings indicate that DmTsf1 can protect flies against bacterial infection. In contrast to its response to immune challenge, DmTsf1 was not upregulated in response to paraquat‐induced oxidative stress, metal stress, or changes in dietary iron availability. We used genetic crosses to generate insects with strong or weak knockdown of DmTsf1 and observed that strong knockdown resulted in mortality in the early larval stages, while 75% knockdown resulted in insects that eclosed as normal‐looking adults. We found that knockdown of DmTsf1 had no effect on an insect's susceptibility to paraquat; therefore, DmTsf1 does not seem to protect flies from paraquat‐induced oxidative stress. Likewise, knockdown of DmTsf1 had no effect on the susceptibility of insects to a high iron diet. Additional experiments to test the effect of knockdown on susceptibility to iron‐induced oxidative stress are ongoing. Our findings demonstrate that DmTsf1 inhibits bacterial growth by iron sequestration, that it does not protect against at least some types of oxidative stress, and that it plays an essential but still unknown role in development.Support or Funding InformationNIH NGMS R37 GM041247, NSF IOS 1656388This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Expression of Glutathione S‐Transferase isotopes, alpha, mu and pi, in Mice (Mus Musculus) Chronically Exposed to Atrazine
Authors: Schadler Analiese , Martin Debra 
Published: June 2021

Atrazine is one of the most heavily used herbicide in North America to control weeds in crops. The Environmental Protection Agency deemed a safe maximum level of atrazine to be 3 parts per billion (ppb) under the Safe Drinking Water Act. Many Midwest states in the United States have annual mean concentrations of atrazine far greater than 3 ppb. Numerous studies have concluded that atrazine negatively affects growth, development, immune and endocrine system functions in diverse organisms. Of interest is a family of detoxification enzymes found in hepatic tissues, glutathione S‐transferases (GST). There are three main isotypes of GST: μ, π and α. Previous research in fish showed chronic exposure to atrazine decreased the enzymatic activity for the μ isotype of GST. The current study utilized male and female CD‐1 mice (Mus musculus) (n = 20/group). Mice were chronically exposed to atrazine (0, 3, 30 ppb) via drinking water for 11 weeks. Analysis of GST enzyme activities was performed and for both the GSTμ and GSTα isotypes, statistically significant decreases in enzyme activity were observed.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of a Sulforaphane and Maitake Mushroom Extract Combination on the Expression of Glutamate Cysteine Ligase
Authors: Erwin Sharis A. , Ownby Stacy L. , Cornblatt Grace A. 
Published: June 2021

Oxidative stress contributes to numerous health issues including neurodegenerative diseases and cancer. Reduced glutathione (GSH) is a major antioxidant that neutralizes reactive oxygen species (ROS) occurring both endogenously through respiration and exogenously during exposure to environmental stressors. Increasing levels of GSH to combat ROS is critical to reducing oxidative stress. The rate‐limiting step of glutathione synthesis is catalyzed by the enzyme glutamate cysteine ligase, a heterodimer consisting of a Glutamate‐Cysteine Ligase Catalytic Subunit (GCLC) and a Glutamate‐Cysteine Ligase Modifier Subunit (GCLM). In this study, we focus on the effect the combination of sulforaphane and maitake mushroom extract has in elevating the production of GSH as a means of preventing cellular damage caused by oxidative stress. HepG2 liver cancer cells were treated over a time course of 24h with maitake mushroom extract (250 μg/mL) and a physiologically achievable level of sulforaphane (0.5 μM). The combination induced gene expression of both GCLC and GCLM an average of 2‐fold as early as 6h. Additionally, we examined the levels of GSH in HepG2 cells treated with the combination.Previously, our laboratory demonstrated that the combination induced the expression of Phase 2 detoxifying enzymes NAD(P)H quinone dehydrogenase 1(NQO‐1), UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1), and heme oxygenase 1 (HMOX‐1/HSP32) as well as reduced the expression of pro‐inflammatory markers interleukin‐6 (IL‐6) and cyclooxygenase‐2 (COX‐2). These results show that the sulforaphane and maitake mushroom extract combination possesses anti‐oxidative properties that may promote cellular health.Support or Funding InformationResearch supported by Nutramax LaboratoriesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of Yeast Nem1‐Spo7 Protein Phosphatase Complex by Protein Kinase C
Authors: Dey Prabuddha , Su Wen‐Min , Han Gil‐Soo , Carman George M. 
Published: June 2021

Pah1 phosphatidate phosphatase in the yeast Saccharomyces cerevisiae catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol. This is the penultimate step in the synthesis of triacylglycerol in yeast and higher eukaryotes. Pah1 is primarily found in the cytosol as a physiologically inactive phosphoprotein, but becomes active through its translocation to the nuclear/ER membrane, where its substrate phosphatidate resides. The membrane translocation of Pah1 occurs via its dephosphorylation by the membrane‐associated protein phosphatase complex Nem1 (catalytic subunit)‐Spo7 (regulatory subunit). In this work, we showed that both Nem1 and Spo7 were phosphorylated by protein kinase C on serine residues. The phosphorylations of each subunit were dependent on their concentrations, phosphatidylserine, diacylglycerol, the amount of protein kinase C, ATP, and the time of the reactions. Peptides derived from Nem1 and Spo7 that contained putative protein kinase C target sites were tested as substrates for the kinase. The Nem1 peptide containing Ser‐201 and the Spo7 peptide containing Ser‐22 and Ser‐28 were substrates for protein kinase C. That Ser‐201 and Ser‐22 and Ser‐28, respectively, were targets in the Nem1 and Spo7 peptides were confirmed by a combination of mass spectrometry and mutagenesis to alanine residues. Full‐length Nem1 S201A and Spo7 S22A and S28A mutant proteins isolated from yeast were defective in their phosphorylations by protein kinase C.Support or Funding InformationSupported by NIH grant GM050679.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 phosphorylation state on perilipin 5 localization and trafficking.
Authors: Bailey Hannah M. , Tansey John T. 
Published: June 2021

Perilipins are a family of five proteins found on the surface of lipid storage droplets in nearly all tissues. These proteins act as cofactors for lipases and scaffolding for other proteins involved in lipid metabolism. In addition to the lipid droplet surface, members of the perilipin family have been found in the cytosol, endoplasmic reticulum, plasma membrane and mitochondria. The localization of these proteins is in part determined by their phosphorylation state. Recently perilipin 5 has been shown to localize to the nucleus when phosphorylated by PKA on serine 155 and interact with the peroxisome proliferator‐activated receptor gamma coactivator alpha (PGC‐1a). In silico analysis of a phosphomimetic mutant (Ser155Asp) indicate that this site may contain a nuclear localization sequence. Using a CHO model cell line we observed that translocation to the nucleus could be stimulated by treatment with forskolin and isobutylmethylxanthine (IBMX) and occurred independent of treating cells with oleic acid to promote triacylglycerol deposition. We are currently investigating the affects of stimulating other phosphorylation pathways to determine if they also phosphorylate perilipin 5 and affect subcellular localization. These data have the potential to gain insights to the regulation of perilipin 5 trafficking into the nucleus and therefore interacting with PGC‐1a regulating mitochondrial biogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.