bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA‐binding specificity of TBX5 and its coding variants associated with Holt‐Oram Syndrome
Authors: Cesar Brenda I , Carrasquillo Emmanuel , Rodriguez Jose A 
Published: June 2021

Transcription factors (TFs) are important sequence‐specific DNA binding proteins that regulate gene expression and control cellular state. Evaluation of the DNA‐binding properties of transcription factors is fundamental for understanding their possible roles in controlling gene regulatory networks. TBX5 is a transcription factor that has been identified as an important regulator in organ development. TBX5 mutations have been associated to Holt‐Oram Syndrome, a genetic disorder that affects upper limb formation and often the heart. We aim to determine the specificity of DNA‐binding of TBX5 and its T‐Box DNA‐binding domain and how it changes when the protein mutates. We have cloned TBX5 and its T‐Box domain that contains a His‐ and Strep‐Tag affinity tags that we plan to use for purifying after we overexpress it in E. coli cells. We will then analyze the DNA‐binding specificity of TBX5 and the changes it conveys when it mutates using Cognate Site Identification by High‐Throughput Systematic Evolution of Ligands by Exponential enrichment (HT‐SELEX), as this will allow us to better understand the impact of TBX5 mutations on its function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isolation and characterization of lipase inhibitory activity from Solanum tuberosum L
Authors: Katimbwa Dorsilla Anono , Lim Jinkyu 
Published: June 2021

Previous studies using 14 different cultivars of Solanum tuberosum L.showed that patatins, lipid acyl enzymes, have varied activities and are regulated by lipase inhibitory compound. In this study we isolated the lipase inhibitory compound by serial solvent extraction and using several chromatographic methods. The inhibitory compound showed high solubility in water and weak solubility in butanol and ethyl acetate. This solubility property suggests that the molecule may have both hydrophilic and hydrophobic moieties. Inhibition kinetics study showed that the compound may have mixed type inhibition as it may interact with the substrate and is markedly influenced by the structure of ester bond or lipid emulsion. The compound also exhibited high potency with an estimated IC50 of 216 ng/mL in the impure extract. The potency of the lipase inhibition of the compound is comparable to the commercially available anti‐obesity drug, Orlistat, whose LC50 was 0.4 ng/mL. Taken together with stability of the inhibitory compound during storage, high temperature and low pH stability, it presents many possibilities in developing applications in anti‐obesity remedies with less likelihood of side‐effects related with hydrophilic lipase inhibitors currently in use in the global market.Support or Funding InformationKorea National Research FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrochemical Investigation of R2lox, a Novel Class of Heterobimetallic Mn/Fe Protein
Authors: Schneider Camille R. , Miller Effie K. , Smith Zachary R. , Shafaat Hannah S. 
Published: June 2021

In order to sustain life, nature accomplishes a diverse array of chemical reactions; one such way this diversity is achieved is through the use of metalloenzymes. In Mycobacterium tuberculosis, a unique Mn/Fe heterobimetallic cofactor has recently been discovered in the R2‐like ligand binding oxidase metalloprotein, R2lox.1 While the function of this protein remains elusive, the cofactor is similar to that of the ribonucleotide reductase Ic subclass, which holds the one‐electron oxidizing equivalent necessary for enzymatic function in the form of a Mn(IV)/Fe(III) metal cofactor.2 R2lox, following oxygen activation, forms an unprecedented tyrosine‐valine ether crosslink in its active site, indicative of two‐electron chemistry, ultimately giving a stable Mn(III)/Fe(III) resting state.1–3 However, the means by which these virtually identical metal cofactors accomplish these different types of chemistry is unknown. In addition, while R2lox can also incorporate a Fe/Fe cofactor when incubated with exclusively Fe(II), the Mn/Fe cofactor is proposed to be the biologically relevant cofactor, despite going against the well‐established Irving‐Williams series.4 To begin to probe potential reactivity in this system, R2lox was investigated using electrochemistry techniques. Both the Fe/Fe and Mn/Fe cofactors were studied in three different R2lox variants. For all three variants tested, only the Mn/Fe cofactor displayed redox activity under the conditions tested with an oxidation process at biologically accessible potentials. Following this initial characterization, a pH dependence was conducted to investigate potential proposed states.5 This study represents the first electrochemical characterization of R2lox and illuminates the potential reactivity of this novel cofactor. As electrons are a form of chemical currency, the reduction potentials reported here provide a handle on the accessible energy available for catalysis.Support or Funding InformationOhio State University Graduate School Presidential Fellowship; NIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein‐Polymer Bioconjugation Effects on Activity and Stability of Cellulase (FnCel5a) with Varying Molecular Weights of Polymer Chains
Authors: Ram Natasha 
Published: June 2021

An alternative method for energy production which decreases dependency on nonrenewable resources and utilizes a greater amount of the available plant biomass is cellulosic ethanol production. However, there is a need for a more cost effective and energy efficient method to produce a stable form of cellulase that can withstand the harsh chemical and physical industrial processes. One method to enhance protein stability has been demonstrated through protein‐polymer bioconjugation. Thermophilic cellulase, FnCel5a, has been shown to have increased activity when conjugated to polymers via amidation reactions onto lysine residues of the enzyme. However, due to numerous lysine residues there was a lack of site‐specific binding of the polymers. Therefore, a novel site‐specific approach was taken in this study to conjugate polymers onto cellulase via a maleimide method using thiolene click chemistry onto a cysteine residue. In addition, polymer chain length was manipulated to investigate its effects on enzymatic stability and activity using the amidation method. Polymerization methods of RAFT and ATRP were utilized to synthesize N, N‐dimethylacrylamide (DMAm) polymers and OEOA polymers of varying degrees of polymerization. These polymers were conjugated using a “graft to” method onto lysine residues. SDS‐PAGE electrophoresis confirmed conjugation of the polymers. And an activity assay using carboxymethylcellulose substrate indicated the presence of a maximum percent enzymatic activity with increased polymer chain length. For site‐specific conjugation, a mutagenesis at C296S and C102S was done for selective polymer binding. SDS‐PAGE electrophoresis confirmed DMam and OEOA polymer binding at the C296 position of FnCel5a. Overall this study confirmed the effective use of click chemistry method as a technique for site‐specific polymer binding of RAFT and ATRP at a cysteine residue. This method can also be utilized to better investigate how polymer chain length can affect the stability and activity of FnCel5a with controlled site‐specific reactions.Support or Funding InformationREU Site: Summer Research in Chemistry and Biochemistry at Miami University at Miami University, Award No. 1460862This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
INTERACTION OF DEXTRIN CAPPED CADMIUM SULFIDE QUANTUM DOTS WITH BLOOD COMPONENTS: COAGULATION AND HEMOLYSIS STUDIES
Authors: Rodríguez‐López Anahí , Rodríguez‐Fragoso Patricia , De la Cruz Gerardo González , Rodriguez‐Fragoso Lourdes , Reyes‐Esparza Jorge 
Published: June 2021

Evaluate at interaction of dextrin capped cadmium sulfide quantum dots with blood components. The intravenous administration of quantum dots (QDs) has been widely reported as a promising alternative for delivery of drugs to specific cells. Therefore, previous studies have pointed out the importance of studying of, at least, factors like coagulation effect; and their hemolytic character when nanoparticles are intended to be administered for intravenous injection. The hemolytic activity and platelets aggregation of the nanoparticles was tested on isolated rat erythrocytes and platelets. Atomic force microscopy (AFM) facilitates the real‐time studies of blood cells and the morphological, structural, optical properties of the CdS‐dex QDs. In this work a methodology for erythrocytes and platelets study by platelet aggregation, hemolysis and AFM was developed. The results show that CdS‐Dex/QDs caused platelet aggregation at concentration‐dependent manner, this effect being greater at 1000 ug/mL, where the increase was up to 57% (p<0.05). The AFM measurements establish a more direct correlation between the surface topography of platelets with the surface nanostructure and reveal that modified the morphology of the platelets from the concentration of 0.01 ug/mL. The CdS‐Dex/QDs do not cause lysis of erythrocytes significantly, although they modified their morphology, suggesting that they can cause eriptosis. The CdS‐Dex/QDs, caused effects in oval and biconcave morphology of the erythrocytes, making it round and of edges not defined, semi‐flat and with cracks in its surface and lacking central depression in a concentration‐dependent manner and observed in its surface some artifacts that may correspond to agglomerated NPs. We found that CdS‐dex/QDs caused aggregation of rats platelets in vitro; however; there were both quantitative and qualitative differences depends to various concentrations.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The assembly and function of poxvirus proteins
Authors: Jafari Nafiseh Jandaghi , Szymczyna Blair 
Published: June 2021

Proteins play essential roles in biological processes, and atomic details of their structure and function provide an insight into their biological roles and how they can be manipulated for therapeutic purposes. One class of proteins that are still poorly characterized at the structural level are those involved in pox virus replication. This work describes the structural and functional studies of several proteins involved in Tanapox virus replication replication. The Tanapox virus is a poxvirus that is related to the medially important vaccinia, variola and Monkeypox viruses. The variola virus is the causative agent of smallpox, which is estimated to have caused 300–500 million deaths in the 19th century alone and is a potential bioterrorism threat. Structural and functional insights into the proteins and biomolecular complexes that regulate the lifecycle of the virus will aid in the development of therapeutic agents against poxviruses and the development of poxviruses as therapeutic tools against cancer. The main goals of the study were to assess the structure and function of Tanapox proteins using biochemical and biophysical methods. Many Tanapox virus protein construct samples were generated and studied. Nuclear magnetic resonance (NMR) spectroscopy was performed on several samples to assess their structural and conformationally dynamic properties. Circular dichroism spectroscopy, size exclusion chromatography and electrophoretic mobility shift assays were used to determine the roles of these proteins molecular function. The results reveal that Tanapox virus proteins are generally insoluble when expressed alone. While biophysical studies of insoluble proteins are difficult, the samples generated are ideal for generating antibodies for in vivo functional studies.Support or Funding InformationThank you to Dr. Karim Essani for providing Tanapox genome.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐Term Consumption of High Protein Disrupts Dog Gut Microbiome and Metabolites
Authors: Gebreselassie Eden Ephraim Ephraim , Jewell Dennis E. 
Published: June 2021

There are controversial views on the optimum level of protein required by adult dogs. According to the guidelines by the Association of American Feed Control Officials, adult maintenance dog foods must contain a minimum of 18% protein based on dry matter. However, there are varying levels of protein in commercial dog foods ranging from 18% to over 60%. We conducted a study to evaluate the impact of a long‐term consumption of low (18.99%), medium (25.34%) and high (45.77%) protein foods on the body composition, metabolites and gut microbial composition of 30 adult dogs. All dogs were maintained on a prefeed food for 14 days and were randomly assigned to one of the 6 different groups of 5 dogs. Each group consumed the low, medium and high protein foods, each for 90 days, in a distinct sequence following the William's Latin Square Design. Body composition and levels of metabolites and microbial composition were assessed at the end of each 90 days feeding period. The body composition of the dogs was not affected by the varying levels of protein consumption. However, dogs had increased circulating levels of urea and indole sulfates after the consumption of the high protein food compared to the lower protein levels. High protein consumption also led to increased fecal levels of indole and branched‐chain fatty acids and reduced levels of the short‐chain fatty acids butyrate, propionate and lactic acid. Further, the consumption of high protein food increased the proportions of the genera Clostridium and Streptococcus that are implicated to have negative health associations. This was accompanied by a reduction in the beneficial bacteria belonging to the genera Prevotella and Bifidobacterium. The results suggest that long‐term consumption of high protein foods is associated with negative microbial and metabolic profiles.Support or Funding InformationThe study was funded by Hill's Pet Nutrition, Inc. Topeka, Kansas, United States of AmericaThis 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 the CRISPR‐Cas13b systems in Porphyromonas gingivalis
Authors: Zhou Xia , Zhu Jiang , Du Zhihua , Huang Xiaolan 
Published: June 2021

CRISPR‐based technologies have revolutionized biochemical and biomedical research. In the past few years, many CRISPR systems have been discovered and characterized. These CRISPR systems greatly expand the CRISPR toolkit. Cas13b is the most recently identified CRISPR system. The usefulness of the Cas13b system as a gene editing tool has been demonstrated in human cells. Cas13b system can be exploited to achieve not only RNA knockdown, but also highly specific and efficient RNA editing. The RNA editing ability of the Cas13b‐based technology has been used for the correction of human disease‐relevant mutations. Despite the rapid progress of Cas13b research, little is known about the structures and catalytic mechanisms of this important family of CRISPR enzymes. A cross‐disciplinary research approach is employed in our study to characterize the two CRISPR‐Cas13b systems in Porphyromonas gingivalis (Pgi5 and Pgi8 Cas13b systems). Both Cas13b enzymes possess dual RNase activities for pre‐crRNA processing (to produce the mature crRNAs) and target RNA cleavage. The systems are capable of mRNA knockdown in human cells.Support or Funding Information1. SIUC Seed Grant2. NIH FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Celecoxib Inhibits Proliferation, Mitochondrial Respiratory Rate, and Membrane Potential in Myoblasts
Authors: Kolb Alexander , Reynoso Marinaliz , Geddis Alyssa , Matheny Ronald 
Published: June 2021

Nonsteroidal anti‐inflammatory drugs (NSAIDs) are commonly prescribed for their analgesic and anti‐inflammatory properties, and their use is widespread in the general, athletic, and military populations. It is relatively well‐documented that long‐term consumption of NSAIDs is associated with increased risk of heart attack, stroke, gastrointestinal bleeding, and kidney disease; however, much less is known regarding the positive or negative effects of NSAIDs in skeletal muscle. Given this, we performed a series of experiments to determine the degree and mechanisms through which the NSAID celecoxib perturbs cultured skeletal muscle cell proliferation, intracellular signaling, and mitochondrial function. Myoblasts were incubated with celecoxib or NS‐398 (a selective COX2 inhibitor) for 48‐h. Cell viability was tested using 3‐(4,5‐Dimethylthiazol‐2‐Yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay, anabolic signaling pathways were evaluated by Western blot, mitochondrial metabolism was determined via cytochrome c oxidase activity assay, and mitochondrial membrane potential was determined by membrane‐sensitive JC‐1 dye. First, cells were treated with celecoxib at concentrations ranging from 200 μM to 0.1 μM. Cell viability was significantly depressed (100% to 19%; P<0.01) by celecoxib in a dose‐dependent fashion at concentrations from 200 μM to 3.125 μM. Myoblasts treated with NS‐398 showed no decrease in viability. Phosphorylation of ribosomal protein S6 (S240/244) was decreased by 77% in myoblasts treated with 50 μM celecoxib (P<0.05), whereas treatment with NS‐398 had no effect on S6 phosphorylation. Celecoxib inhibited the mitochondrial respiratory rate of the following Kreb's cycle intermediates (percent reduction in parenthesis): cis‐aconitic acid (25%; P<0.01), alpha‐keto‐glutaric acid (50%; P<0.05), succinic acid (45%; P<0.05), and L‐malic acid (37%; P<0.05). Fifty and 25 μM celecoxib reduced mitochondrial membrane potential by 40% and 25%, respectively (P<0.001 for both) Together, these data demonstrate that decreased myoblast viability following celecoxib treatment was associated with reductions in proliferative signaling, mitochondrial enzyme activity, and membrane potential. The mechanism(s) and degree to which celexoxib‐induced perturbations of mitochondrial enzyme activity influence myoblast viability remain to be determined.The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This abstract has been approved for public release with unlimited distribution.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 MAP kinase docking specificity with yeast genetic screens
Authors: Shi Guangda , Turk Benjamin 
Published: June 2021

Mitogen‐activated protein kinase (MAPK) pathways play pivotal roles in making vital cellular decisions. In order to carry out their unique functions, each MAPK recognizes its own repertoire of substrates. Aside from a rather degenerate phosphosite motif (S/T‐P), MAPKs utilize a non‐catalytic site docking interaction to specifically recognize their substrates as well as their regulators. Docking interactions occur between a linear docking site peptide (D‐site) and a conserved docking groove on the MAPK. While a general D‐sites sequence motif has been described, unique features of sequences that specifically target individual MAPKs is not completely understood. Investigations into these interactions is difficult due to the weak and transient nature of the interaction.Here I present screening methods using S.cerevisiae to identify functional docking site sequences that direct to specific MAPKs. I found that co‐expressing a mammalian MAPK and its MKK activator in yeast leads to growth suppression. I have used this phenomenon as the basis of a pooled yeast genetic screen, where cells expressing MKK variants compete in one pooled culture. The change in representation of every single D‐site in the culture over time is monitored by next‐generation sequencing. Variants having more functional D‐sites activate the pathway better, grow slower, and are depleted from the population. I generated and screened a library of ~12,000 candidate D‐sites found in the human proteome against the MAPKs p38α and JNK1.These screens revealed sequence motifs that allow discrimination between the two MAPKs, and also defined a D‐site dependent MAPK interactome which contains many new targets that could broaden our understanding of MAPK biology. Our screens serve as guides to more completely understand weak interactions that play an important factor in the dynamic nature of MAPK signaling networks.Support or Funding InformationThis research was supported by National Institutes of Health grant R01 GM104047.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein phosphatase 2A‐B56 controls mitosis by interacting with LS/TPI/V motif containing protein interactors
Authors: Chaudhuri Sibapriya , Moorhead Gregory 
Published: June 2021

Mitosis, like many other cellular processes, is tightly regulated by reversible protein phosphorylation which is achieved by coordinated activities of mitotic protein kinases and mitotic protein phosphatases. PP2A‐B56 is an important mitotic protein phosphatase which contributes to the timely execution of several mitotic events. Recent proteomic studies show that PP2A‐B56 recognizes a short linear motif, LXXIXE, on its interactors. We hypothesize that LS/TPI/V is a consensus motif for the recruitment of PP2A‐B56 in an isoform‐dependent manner, particularly during mitosis. We also speculate that this motif is phosphorylated during mitosis and dephosphorylated at mitotic exit. Recruitment of PP2A‐B56 is regulated by the phosphorylation status of this motif and this, in turn, plays an important role in controlling the progression of the cell cycle. Our findings show that LS/TPI/V motif is present in more than 100 mitotic regulators and several of them interact with PP2A‐B56 in a phosphorylation‐dependent and isoform‐dependent manner. Further understanding of how interactions between the target proteins and PP2A‐B56 affect mitotic progression and/or mitotic exit will provide valuable insight into the mechanisms of specific recruitment and mitotic functions of PP2A‐B56.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.