bluuebunny/crossref_metadata_2025_split huggingface dataset:
Methods of Mutation Efficiency Analysis for CRISPR/Cas9 in Fathead Minnow
Authors: Mielke Marissa J. , Blatz Donovan J. , Petros Mera T. , Kittelson Ashley , Via Zachary W. , Cavallin Jenna E. , Villeneuve Daniel L. , Ankley Gerald T. , Maki Jennifer A. 
Published: June 2021

CRISPR/Cas9 mutation of fathead minnow genes allows for assessment of the key event relationships highlighted in Adverse Outcome Pathways (AOPs), diagrammatic representations of biological data from chemical exposure experiments. An AOP developed to hypothesize how mercaptobenzothiazole (MBT) exposure results in decreased anterior swim bladder inflation in fathead minnow reasons that MBT inhibits thyroid peroxidase (TPO), leading to a decrease in T4 hormone synthesis and the swim bladder phenotype.In vitro fertilization and microinjection methods for fathead minnow were previously developed in collaboration with the Environmental Protection Agency research labs, and verified by successful CRISPR/Cas9 tyrosinase mutation. Tyrosinase mutant fish were identified by altered pigmentation and genotypically confirmed by Sanger sequencing and TIDE analysis (Tracking of Indels by Decomposition; tide.nki.nl). In a continued collaborative effort, mutation of TPO and additional AOP‐related targets have been attempted and assessment of possible mutant fish is ongoing.In this work, methods of mutation analysis were assessed, including T7 endonuclease I (T7EI) kit, TIDE analysis and assessment via DNA Bioanalyzer. Results of the T7EI protocol with TPO experimental fish provided evidence of mutations, however, both the TIDE and Bioanalyzer data showed that mutation efficiencies were low to nonexistent in these fish. This is consistent with the lack of observable phenotype. Since the CRISPR/Cas9 attempt was not successful in generating mutations, the validity of the TPO AOP can neither be supported nor refuted until guide strand design is optimized to induce a higher mutation efficiency.Current efforts are focused on guide strand redesign for a second attempt at TPO mutation. Additional targets are simultaneously being pursued for mutation with the in vitro fertilization and CRISPR/Cas9 system. Based on the data obtained here, TIDE analysis will be the primary technique utilized for assessment of mutation efficiency in subsequent studies. Though time‐consuming, TIDE analysis has proven reliable, provides clear results and is cost‐effective.Support or Funding InformationU.S. Environmental Protection Agency, Office of Research and Development, Mid‐Continent Ecology Division, Duluth, MN. The College of St. Scholastica Faculty Development Committee Research Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the role of ComFA ATPase in powering DNA uptake during bacterial transformation
Authors: Lin Xiaoxuan , Foster Hannah , Burton Briana 
Published: June 2021

Horizontal gene transfer (HGT) is a process that largely promotes bacterial diversity through lateral exchange of genetic materials. As one of the major forms of HGT, natural transformation has been observed in over 80 species. The process of transformation involves a membrane‐associated protein machinery that mediates the binding, uptake, and recombination of exogenous DNA. Despite its large impact, little is known about the mechanism, and particularly, the power source for DNA uptake across membranes. One of the most likely sources powering DNA uptake is a motor protein. It has been postulated that ComFA, a member of DEAD‐box helicase family and a ssDNA‐dependent ATPase, contributes to the force needed for DNA import. In this study, we made a series of mutants to analyze the role of highly conserved residues in multiple regions of Streptococcus pneumoniae ComFA. First, we show that several mutants exhibit a defect in transformability in vivo, with a 10‐ to 100‐fold decrease transformation efficiency. Second, we show that ATP hydrolysis is not required for ComFA‐DNA binding but may stimulate DNA release. Moreover, we present in vitro data suggesting the coupling mechanism between ATP hydrolysis and DNA binding of ComFA. Our results suggest the mechanism of ComFA in transformation and its possible DNA‐binding surfaces. Our current work focuses on examining the ComFA‐DNA interactions using p‐benzoyl‐L‐phenylalanine (pBpa)‐crosslinking. Overall results of this study support the hypothetical molecular structure of ComFA and its role in powering DNA uptake in natural transformation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Specific Ionic Interactions Contribute to Substrate Binding of Lysine Deacetylases
Authors: Toro Tasha B , Broussard Christian G , Swanier Jordan S , Watt Terry J 
Published: June 2021

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. Lysine deacetylases (KDACs) are enzymes that reverse this post‐translational modification, by catalyzing the hydrolysis of ɛ‐N‐acetyllysine residues in proteins via a conserved mechanism. Deacetylation is important for many cellular processes and aberrant KDAC activity has been linked to numerous diseases. While proper deacetylation is known to be critical for proper cellular function, details regarding substrates of particular KDACs and how substrate specificity is determined are lacking. We have observed that the activity of KDAC8 with charged substrates in vitro is sensitive to the ionic strength of the reaction buffer. This observation led us to hypothesize that charged residues near the acetylated lysine may be important for the interaction of KDAC8 with its substrates. To test this hypothesis, we have employed an in vitro assay which relies on the reaction of fluorescamine with lysine residues to produce a quantitative fluorescent signal upon deacetylation of a peptide substrate. Using this method, KDAC8 activity was measured with several substrates under conditions of varied ionic strength. Interestingly, for several positively charged peptide substrates, KDAC8 activity increases as ionic strength is decreased, but the opposite is often observed when negatively charged peptide substrates are present. Preliminary results with derivatives of a known peptide substrate suggest that a positively charged side chain adjacent to the acetylated lysine may make a specific contact with KDAC8, as the ionic strength effect varies with the position of the charge in the substrate sequence. Screening a larger set of peptides with charged residues at specific positions relative to the acetylated lysine for activity with KDAC8 provides insight into the contributions of charge to substrate specificity. Comparison of these data with molecular docking data of the same peptides with the active site of KDAC8 can predict specific interactions between charged residues in the KDAC8‐substrate pair. Together, these results will lead to a better understanding of KDAC8 substrate specificity. Extension of this approach to additional KDACs and substrate behavior in vivo will provide insight into substrate specificity of the KDAC family and the biological targets of each KDAC.Support or Funding InformationFunding was provided by NIH 5G12MD007595, R15GM129682, TL4GM118968, 5RL5GM118966, and the Louisiana Cancer Research Center.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating HSF1‐Mediated Interactions of Human Angiogenin with Chromatin
Authors: Vinopal Claire Grace , Dickson Kimberly A. 
Published: June 2021

Angiogenin (ANG) is a pancreatic‐type secretory ribonuclease that was identified for its ability to promote new blood vessel growth. More recently, ANG has been implicated in neurodegeneration, inflammation, and reproduction, revealing its broad biological roles. The goal of our work is to understand the molecular activities of human ANG. During sustained growth of cultured human cells, ANG interacts with chromatin to upregulate rDNA transcription. Recent work in our lab demonstrates that ANG interacts with heat shock transcription factor 1 (HSF1). Our work is aimed at expanding our knowledge of ANG interactions with the human genome and determining if ANG works cooperatively with HSF1 to regulate stress‐response genes. Using chromatin immunoprecipitation (ChIP) sequencing, we have identified genomic targets of ANG biding and characterized the co‐regulation of heat shock genes by ANG and HSF1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Protein‐Ligand Interactions in MsrA from E. coli
Authors: Winkie Mitchell L , So Joanne , Schlessman Jamie L , Smith Virginia F 
Published: June 2021

The amino acid methionine is highly susceptible to oxidation via reactive oxygen species, resulting in loss of function in proteins containing oxidized methionines. This loss of protein function has been implicated in numerous neurodegenerative and age‐related diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehrig's Disease), emphysema, and arthritis. The enzyme methionine sulfoxide reductase (MsrA) catalyzes the stereospecific reduction of oxidized methionine in free amino acids, peptides, and proteins, restoring function in the latter. The catalytic mechanism of MsrA has been studied extensively, but less is known about how MsrA is able to recognize its large variety of substrates, from single amino acids or small molecules to oxidized methionines in the context of a protein. Earlier work from our lab using intrinsic and extrinsic fluorescence spectroscopy suggests that the presence of sulfoxide group on a methionine in a target protein enhances existing weak hydrophobic interactions with MsrA. The current study builds on these findings using fluorescence spectroscopy to characterize how MsrA of E. coli recognizes its substrates and repairs specific oxidized methionine residues in an intrinsically‐disordered form of staphylococcal nuclease and a nonapeptide derived from it.Support or Funding InformationThis work was supported by a Research Corporation award (grant number CC5482) to VFS. The Office of Naval Research supported Trident Scholar research by JS. MJW was supported by a Garrigues Program scholarship award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nitrite and NO Processing by CblC: A Human B12 Trafficking Chaperone
Authors: Mascarenhas Romila N , Gherasim Carmen , Banerjee Ruma 
Published: June 2021

Cobalamin (Cbl) or vitamin B12 derivatives are processed and escorted to the two known B12‐dependent enzymes: cytosolic methionine synthase (MS) and mitochondrial methylmalonyl CoA mutase (MCM). Mutations in the B12 trafficking genes lead to inborn errors of cobalamin metabolism belonging to the cblA‐G, J and mut complementation groups. Of these, cblC is the most common locus for mutations and patients present with both homocystinuria and methylmalonic aciduria. Functionally, CblC is involved in the early steps of the cytosolic B12 trafficking pathway and catalyzes divergent chemical reactions. Cobalamins entering the cytoplasm with various upper ligands are processed to a common cob(II)alamin intermediate by the CblC protein before being allocated to the methycobalamin or 5′‐deoxyadenosylcobalamin branches. Alkylcobalamins are cleaved via a nucleophilic displacement mechanism in the presence of glutathione (GSH) producing GSR and cob(I)alamin, which is subsequently oxidized to cob(II)alamin, and cyanocobalamin is cleaved via reductive elimination forming cob(II)alamin and cyanide. Herein, we report the discovery of a new activity catalyzed by CblC, i.e. nitrite reductase. The interactions between nitrite and NO with cobalamins have been characterized in solution, but the biological relevance of these reactions is largely unknown. It was previously shown that nitrosylcobalamin (NOCbl) has antitumor activity that preferentially targets cancer cells in which cobalamin receptor expression (TCII) is upregulated. The mechanism of action of NOCbl has been ascribed to its ability to release nitric oxide (NO•). However, the ready oxidation of NOCbl to nitritocobalamin (NO2Cbl) was not taken into consideration. In this study, we have investigated the reactivity of human CblC towards NOCbl and NO2Cbl. We show that CblC binds NOCbl with a konof 0.07 ± 0.01 mM−1s−1. The cobalamin is bound in a base‐off conformation as are the natural B12 derivatives and this is likely facilitated by the strongly σ‐donating NO‐ligand that promotes the dissociation of the coordinating dimethylbenzimidazole group. Surprisingly, neither thiols nor reductants remove the nitrosylgroup from CblC‐bound NOCbl. Addition of thiols to CblC‐bound NO2Cbl generates nitrite and cob(II)alamin (anaerobically) or aquocobalamin (aerobically). The nitrite that is release can potentially serve as an NO• source via the activity of cellular nitrite reductases. Collectively, our results show that the promiscuity of CblC towards cobalamins can be exploited to deliver small molecules or to inhibit intracellular B12 metabolism.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Heme Domain's Regulatory Role on Human Cystathionine β‐Synthase (hCBS) Product Formation
Authors: Ruiz Donovan 
Published: June 2021

Human cystathionine β‐synthase (hCBS) is a vital enzyme found within the human transulfuration pathway and is responsible for the biotransformation of serine and homocysteine into cystathionine. hCBS at high cysteine concentrations has been shown to release hydrogen sulfide as a byproduct via an alternate reaction, suggesting hCBS to be closely involved with regulating metabolic sulfur. Catalysis in both the primary and alternate reactions proceeds via a PLP dependent β‐replacement ping‐pong mechanism, with the last step before product dissociation from the internal aldimine (Schiff base) intermediate being the condensation of H2O or H2S, respectively. Consequently, mis‐regulation of substrate levels or loss of CBS activity has been causally linked to hyperhomocysteinemia, a biological condition signifying abnormally high levels of homocysteine within the circulatory system; this condition being one of many causes leading to cardiovascular disease. A highly conserved PLP catalytic and SAM regulatory domain can be seen when comparing yeast analogue (yCBS) to mammalian variants. However, the mammalian variants, specifically the human analog hCBS, differs by containing a unique heme binding domain not found in yCBS. Given that the heme prosthetic group is not required for catalytic activity, the role that this additional domain plays in regulating cystathionine production in hCBS is not yet clearly understood. Current methods for measuring CBS catalytic activity involve using radioactively labeled substrates and typically have focused only on yeast and truncated hCBS variants. We have adapted a previously established colorimetric coupled enzyme assay to measure hCBS kinetics, coupling CBS with cystathionine β‐lyase (CBL), and lactate dehydrogenase (LDH); where the stoichiometric oxidation of NADH, monitored spectrophotometrically at 340 nm, is correlated directly to the formation of cystathionine. Preliminary results suggest this coupled assay is a viable method for measuring product formation kinetics of full length wild type hCBS. Current and future work aims to 1) asses catalytic rate activity for wild type hCBS and 2) asses the activity of single point mutant variants with mutations localized within the heme binding domain. Through these experiments, we hope to elucidate a deeper understanding of how the heme binding domain affects cystathionine product formation.Support or Funding InformationNIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Cause of Peak Invertase Activity Delay using Isothermal Titration Calorimetry
Authors: Chan Dindi , Noh Jihoon , Mason Marco , Kenealey Jason 
Published: June 2021

Invertase catalyzes the hydrolysis of sucrose producing glucose and fructose. However, its mechanism of action is not well understood as other studies have shown that there is an initial delay before the enzyme reaches peak activity. This indicates that there may be another step for the active form of invertase to be activated. To better understand this mechanism, isothermal titration calorimetry (ITC) was used to measure the enzymatic activity of invertase. In this study, varying concentrations of invertase between 0.0125 to 0.125mg/mL were used to react with 1M and 2M sucrose solutions. Results show an exponential relationship between varying concentrations of invertase and the time to reach peak height. Increasing 0.0125mg/mL of invertase by tenfold, decreases the time required for the enzyme to reach peak activity by 85%. This suggests at increased invertase concentrations there is more active invertase, which may be because invertase forms a type of multimer for full activity. The effect of temperature was also explored, 1.2M sucrose was titrated into 0.016mg/mL invertase at temperatures from 25 to 55 °C. By comparing 25 and 45°C, we observed that the time required to reach peak activity decreased by 62%. However, at 55°C, the time increased by 24%. Also contributing to the delay is the viscosity of sucrose. Concentrations of sucrose ranging from 0.5 to 2M were titrated into 0.0125mg/mL of invertase. The time for the enzyme to reach peak activity increases by 23% when increasing sucrose concentration from 0.5M to 2M. However, this increase in time was also observed in the control (0.5 to 2M sucrose concentration titrated into 0.1M sodium acetate buffer). Therefore, the viscosity of sucrose does not completely account for the delay. In conclusion, the delay in peak maximal activity is in part due to increased sucrose concentration, probably due to inefficient mixing. Additionally, the delay in maximal activity is decreased in the presence of increasing invertase concentration indicating that there are inter‐invertase interactions that are required for maximal invertase activity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioanalytical Method Development for the Detection of the Rac/Cdc42 Inhibitor MBQ‐167 in Mouse Tissue
Authors: Rosado‐González Gabriela Tatiana , Del Mar Maldonado María , Bloom Joseph , Hernández‐O'Farril Eliud , Rodríguez‐Orengo José F , Dharmawardhane Suranganie 
Published: June 2021

Metastatic breast cancer is the second most common cancer in US women. The mortality rate has decreased approximately 40% in the last 25 years due to advancement in treatment and diagnostics. However, it is estimated that 20–30% of all breast cancer patients will develop metastatic breast cancer. Therefore, there is a need to develop targeted therapeutics to reduce cancer cell migration and subsequent metastasis. The novel small molecule MBQ‐167 inhibits Rac and Cdc‐42, proteins involved in cancer cell growth, migration and invasion. In vivo studies have shown that MBQ‐167 inhibits mammary tumor growth and metastasis in immunocompromised mice by ~90% (Humphries‐Bickley, et al., 2017). However, to continue validating this drug for FDA approval, it is necessary to study the pharmacokinetic profile of the drug, which includes elucidating the tissue distribution of the compound in the body. Hence, it is necessary to develop a rapid and sensitive method to quantify MBQ‐167 in tissues. The purpose of this study was to develop and validate a method for the quantification of MBQ‐167 in mouse tissues using Supercritical fluid chromatography coupled with electrospray ionization tandem mass spectrometry (SFC‐MS/MS). Mouse livers were homogenized, spiked with MBQ‐167, and liquid‐liquid extractions were performed with different organic solvents (heptane, ethyl acetate, 2‐propanol, toluene, and acetonitrile), in order to determine the optimal sample preparation method to selectively separate the drug MBQ‐167 from endogenous material in mouse tissue. Separation was performed on an ACQUITY UPC2BEH (3.0 × 100 mm, 1.7 μm) column at 40 °C with a mobile phase consisting of CO2 and methanol 0.1 % formic acid (95:5, v/v) at a flow rate of 1 mL/min. A tandem triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode. High recovery of MBQ‐167 (> 65%) and lower matrix effects were observed upon extraction with heptane:ethyl acetate (1:1). Consistent recovery was observed in other tissues such as kidney, lungs and heart. The method was sensitive with a lower limit of quantification (LLOQ) of 10 ng/mL. Linearity was observed over the concentration range of 10–1000 ng/mL. The data obtained supports the use of SFC‐MS/MS as a highly specific method to evaluate the tissue distribution of MBQ‐167 in mice.Support or Funding InformationThis study is supported by MARC grant NIH/NIGMS 5T34GM007821‐39 (to GRG) and NIGMS‐RISE 25 GM061838 (to MM).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Accuracy of Canine Scent Detection of Lung Cancer in Blood Serum
Authors: Junqueira Heather , Quinn Thomas , Biringer Roger , Hussein Mohamed , Smeriglio Courtney , Barrueto Luisa , Finizio Jordan , Huang Michelle 
Published: June 2021

Canines are able to differentiate between serum samples taken from cancer patients and samples taken from normal controls. This study further supports the use of dogs as biomedical research tools for detection of cancer biomarkers. In particular, this study was designed to determine the accuracy of canines' ability to detect, by scent alone, lung cancer biomarkers in blood serum.Operant conditioning was used in the form of clicker training to train four beagles to distinguish, by scent alone, blood serum from malignant lung cancer patients when presented along with healthy controls in a double‐blind format. Non‐small cell lung cancer and healthy control blood serum samples were presented to 2‐year‐old beagles.Three dogs were able to correctly identify the cancer samples with a sensitivity of 96.7%, specificity of 97.5%, positive predictive value (PPV) of 90.6% and negative predictive value (NPV) of 99.2%. One of the dogs, Snuggles, was unmotivated to perform during training and tested with 80% specificity and 60% sensitivity. This study paves the way for a larger scale research project designed to explore the use of canine scent detection as a tool for detecting cancer biomarkers, ultimately leading to their identification.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.