bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation, Purification, Modeling, and binding site analysis of the Salmonella Bacteriophage ɛ34 repressor for subsequent crystallization and structure‐function studies
Authors: Jackson Doba 
Published: June 2021

The bacteriophage ɛ34 is one of many phages that infect the bacteria Salmonella which is the main cause of salmonellosis (food poisoning). The bacteriophage ɛ34 encodes a protein called the cI repressor that is like other phage repressors. It has a C‐terminal protease domain which is about 80% identical to the repressors of phage λ (which infects E. coli), but its DNA binding N‐terminal portion is only about 50% identical to its closest known relatives in phages λ, and Lex A. This means that the ɛ34 repressor may have the same pathway for inactivation as phage λ, but probably has a different operator binding specificity due to lower sequence homology of its DNA‐binding domain. The N‐terminal DNA‐binding domain is homologous to the Helix‐Turn‐Helix family of DNA‐binding domains.The main goal of this project was to initiate a structure‐function study on the repressor protein of phage ɛ34. The ɛ34 Bacteriophage DNA was first isolated and purified. The gene encoding the bacteriophage ɛ34 repressor was amplified by PCR and cloned into a pET11a vector. The cI repressor, was overexpressed and purified by immobilized metal affinity chromatography and ion exchange chromatography. We also generated a homology model of this protein using the bacteriophage λ as a template in the SWISS‐MODEL homology modelling server. Our models predict a unique structure for the N‐terminal Helix‐Turn‐Helix domain. Finally, we have identified a potential binding site upstream of the cI gene on the Bacteriophage λ DNA. Crystallization trials are currently in progress. This project was done as a collaborative venture between two laboratories, Huntingdon College (Dr. Doba Jackson) and Alabama State University (laboratory of Dr. Robert Villafane). This project is in line with Dr. Robert Villafane's research into the Genomic analysis of bacteriophage ɛ34 of Salmonella enteric serovar Anatum.Support or Funding Information1Department of Chemistry & Biochemistry, Huntingdon College2Department of Biology, Alabama State UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Amino Acid Residues in The Active Site of Dibenzothiophene Monooxygenase (DszC)
Authors: Johnson Kellee , Mendez Gilberto , Gonzalez‐Osorio Liliana , Jirde Samatar , Palfey Bruce , Vey Jessica 
Published: June 2021

Fossil fuel combustion releases sulfur dioxide into the atmosphere, which is a harmful air pollutant and precursor of sulfate aerosols that contribute to acid rain. It is necessary to reduce atmospheric sulfur levels to mitigate these effects. A current method, hydrodesulfurization, is costly and inefficient at removing sulfur from complex aromatic compounds. Biodesulfurization is a cost‐effective alternative that utilizes bacteria to remove sulfur. Rhodococcus erythropolis uses a four enzyme pathway to remove sulfur from dibenzothiophene. The first enzyme of the pathway, dibenzothiophene monooxygenase (DszC), requires molecular oxygen, reduced flavin, and the formation of an intermediate, C4a‐hydroperoxyflavin, to sequentially oxidize dibenzothiophene to dibenzothiophene sulfone. This study aims to elucidate the roles of DszC active site residues in catalysis and binding and to enable rational engineering of DszC for future application in fuel refinement. Active site residue mutants were created using polymerase chain reaction, followed by plasmid DNA isolation and sequence confirmation. Steady state activity assays of DszC, quantitated by reverse‐phase HPLC, compared the amount of product formed by the mutants relative to wild type‐DszC. The ability of the mutants to form the intermediate was studied using stopped‐flow spectrophotometry. In this study, seven active site residue mutants were created and the percent of product formation by each mutant relative to wild type‐DszC was: His92Asn (12%), His92Gln (20%), Tyr96Phe (12%), His388Ala (38%), His388Asn (39%), His391Val (0%) and Asp392Ala (3%). These results indicate that all of the residues analyzed are necessary for proper product formation. Additionally, the intermediate formation rate constant for Tyr96Phe (1.03 × 104 M−1 s−1) was similar to wild type‐DszC (1.69 × 104 M−1 s−1). These results indicate that Tyr96 is not critical for proper intermediate formation, but has another role in catalysis as evidenced by the low (12%) product formation. His92Asn and Asp392Ala were also analyzed using stopped‐flow spectrophotometry but showed no intermediate formation signifying that these residues are essential to proper intermediate formation. Overall, these results show the necessity of all the residues presented in product formation and the fundamental roles of residues His92 and Asp392 in intermediate formation.Support or Funding InformationNational Institute of Health (NIH SCORE 5SC2AI109500)Research Corporation (CCSA 22672)MBRS/RISE Program/National Institute of Health (Grant 2R25GM063787‐10)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Teaching the Use of Automated Pipettes to Undergrads through Direct Visualization of Data: A Quantitative Chemical Analysis Lab Focusing on Technique, Skill, and Accuracy in Solution Chemistry
Authors: Sostarecz Audra Goach , Sturgeon Bradley E. , Distin Steven P. 
Published: June 2021

The correct use of automated pipettes in undergraduate labs has been demonstrated with an activity primarily for second semester chemistry and biology majors. This lab involves the use of automated pipettes for transferring a specific amount of dye mixtures to a cotton bandanna. The data from this lab comprise a color wheel on a bandanna that displays the amount of dye transferred based on the volume set on the automated pipette by the student. This allows for the students to see their mistakes and does not allow them to restart. Student learning outcomes include the ability to choose the correct automated pipette for the volume needed; to set the correct volume on an automated pipette; to properly draw up the solution; and to use proper dispensing technique. Pre‐surveys assessing students' confidences in accurately using automated pipettes show that the average freshman student (60% of those surveyed) has not used automated pipettes in high school but has used them in their first semester chemistry course. The majority of those students also have confidence in their ability to choose the correct automated pipette; to draw up the liquid, and to dispense it properly. There was less confidence in setting the correct volume on the automated pipette. The results show that some students still have difficulties in setting volumes on automated pipettes correctly and in dispensing the liquid. However, the post survey suggests that setting the correct volume on the automated pipette was the most learned skill from performing the lab (26% to 88% increase in confidence by the students). After performing the lab, 96% of the class was confident in their ability to use automated pipettes. The learning outcomes were met along with the unintentional learning outcome of following directions. Automated pipettes are increasingly being utilized in our labs at our institution for both classroom lab activities and research lab activities. The ability to use automated pipettes accurately and precisely is a vital skill for chemistry, biochemistry, and biology majors with the increased use of this instrument in scientific jobs and in research. The use of these pipettes is a learned skill and it should not be assumed that students know how to correctly utilize them. Feedback from students supports this conclusion.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the structural basis of the Cross‐Talk between GlcNAcylation and Phosphorylation Using Physical Models
Authors: Shania Sharon , Schwabe Michael , Garcia Nina Marie , Sonnenschein Halie , Bell Ellis 
Published: June 2021

The focus of this project was to explore Structure Function relationships in the enzyme UDP‐GlcNAc O transferase protein and design and build a physical model that illustrates key functional features of the protein. In particular our focus is on the Cross‐Talk between O‐GlcNAcylation and Phosphorylation that plays a critical role in overall regulation. Using the pdb file 4gyy.pdb from the paper “Crystal structure of human O‐GlcNAc Transferase with UDP‐5SGlcNAc and a peptide substrate” The structure illustrates the binding sites for both UDPGlcNAc and the peptide YPGGSTPVSSANMM with the requisite PV prior to the GlcNAcylatable S. Recent work by Leney et al (Proc Natl Acad Sci U S A. 2017 Aug 29;114(35):E7255–E7261) has shown that the preceding T can be phosphorylated and that the sequence TPVS is common in proteins thought to undergo GlcNAc‐Phosphorylation cross talk. We have also used computational models to create and built models of the peptide phosphorylated at the N‐3 Threonine. These models clearly illustrate that the phosphate group clashes with the U in UDP‐GlcNAC. Since the kinetic mechanism of OGT is ordered Bi‐Bi with UDPGlcNAc as the obligate first substrate, the phosphorylated peptide can no longer bind in the peptide substrate pocket explaining why excludes GlcNAcylation. The models described here can be used to illustrate three different aspects contained in the ASBMB 8 Core Concepts of Macromolecular Structure and Function: #3. Structure and function are related, #4. Macromolecular interactions, and #6. The biological activity of macromolecules is often regulated. In addition construction and use of the models illustrates the use of core concept #8, A variety of experimental and computational approaches can be used to observe and quantitatively measure the structure, dynamics and function of biological macromolecule.Support or Funding InformationFunded in part by NSF‐DUE 1725940 for the CREST Project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circulating Caspase‐3 p17 Fragment as a Novel Marker for Cardiac Apoptosis During Cardioplegia
Authors: Lorinsky Michael K. , Kim MJ , Gold Courtney , Lahey Stephen , Fusco Daniel , Rosinski David , Pawlak Dorota , Liang Bruce 
Published: June 2021

Myocardial cellular necrosis occurs with acute myocardial infarction and can be identified through the measurement of serum biomarkers, such as troponin I and the MB isozyme of creatine kinase (CK‐MB). However, both ischemia and reperfusion of cardiac myocytes in mouse models has been shown to result in cell death via apoptosis, in addition to necrosis. Apoptosis of cardiac myocytes during myocardial ischemia in humans has less, but nonetheless supporting evidence. Caspase‐3 is an end‐effecter of cardiac myocyte apoptosis, which yields a measurable p17 fragment in the serum after activation of the apoptotic cascade. Here we show the presence of elevated p17 levels in patients that undergo cardioplegia for cardiac surgery, which is a clinically controlled ischemia‐reperfusion condition. Serum levels of p17 were measured along with the traditional cardiac necrosis biomarkers CK‐MB and cardiac troponin I. Blood was drawn before, during, and after cardioplegia in three locations—peripheral venous, aorta, and coronary sinus. Compared to biomarker levels in healthy subjects, the levels of p17 were statistically significantly elevated before, during, and after cardioplegia, in conjunction with similar elevations of the traditional necrotic biomarkers. In addition, the ratio of caspase‐3 in the coronary sinus to that in aorta after reperfusion was greater than 1.0—a finding that was also found with the traditional cardiac biomarkers, indicating a cardiac origin of these biomarkers. These findings support that apoptosis of cardiac myocytes is the primary source of the elevated p17 after reperfusion of hypoxic cardiac myocytes during cardioplegia and that, in addition to necrosis, cardiac apoptosis occurs during cardioplegia and likely contributes to cardiac injury in this commonly performed operation.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 Regulation of mTORC1 Activity in The Plasma Membrane
Authors: Zhong Yanghao , Zhou Xin , Zhang Jin 
Published: June 2021

The mechanistic/mammalian target of rapamycin (mTOR) complex 1 (mTORC1) plays a central role in cells by sensing a variety of signal molecules and integrating those signals to control pivotal cellular functions, including protein synthesis, cell growth and autophagy. Dysregulation of mTORC1 signaling pathway can cause many severe diseases such as cancer and type‐II diabetes. Emerging studies have shown that mTORC1 is regulated in a location‐ and signal‐specific manner, however, the mechanism underlying mTORC1 activities in different cellular compartments is poorly understood. By using a novel mTORC1 kinase activity reporter (TORCAR) based on fluorescence resonance energy transfer (FRET), our study aims to elucidate how mTORC1 activity is regulated in the plasma membrane compartment in response to different stimulations. We have shown that the growth factor signal but not the amino acid signal can induce mTORC1 activity in the plasma membrane compartment. Interestingly, we found that platelet derived growth factor (PDGF) induced mTORC1 activity showed a two‐phase pattern. Depleting calcium removed the fast responding component while keeping the slow one intact. Our preliminary results indicate calcium‐dependent regulation of mTORC1 activity at the plasma membrane. This result suggests signaling pathways including G‐protein coupled receptor (GPCR), especially Gq subclass, might contribute to the regulation of mTORC1 activity in the plasma membrane.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LOW‐LEVEL LASER THERAPY REDUCED BLOOD PRESSURE RESPONSE TO MAXIMUM EXERCISE IN POLICE OFFICERS
Authors: De Moura Jose Roberto , Alves Cleber Rene , Lemos Jose Ribeiro , Jordão Camila Paixão , Vicente Wanda Rafaela , Dalboni Maria Aparecida , Trombetta Ivani Credidio 
Published: June 2021

Police officers (PO) in Brazil are exposed to a stressor environment, being wide‐open to higher cardiovascular risk. Arterial stiffness and endothelial dysfunction and impairment blood pressure response to exercise are markers of cardiovascular risk. Previous experimental studies have shown that low‐level laser therapy (LLLT) promotes increase in flow‐mediated dilation (FMD). Thus, we hypotetized that LLLT would improves endothelial function and decrease blood pressure in response to maximum exercise in police officers. We studied PO, submitted to LLLT (n=11, 39.4±2 years; 91.9±5 kg) for 6 months, and a matched group for age and weight that remained without any therapy was studied as a control group (C, n=7, 38.2±3 years; 83.8±3 kg). LLLT was applied twice weekly in the sublingually, with a maximum of three days between one application and another. Endothelial function was evaluated by brachial artery FMD. Systolic and diastolic blood pressure (BP) were analyzed during cardiopulmonary exercise test. After intervention, LLLT increased FMD, whereas C did not change (Δ of increase= 19.5±2 and 1.5±5 %, respectively, P=0.002). Interestingly, BP response to maximum exercise decreased only in PO submitted to LLLT (pre vs. post, SBP=198±6 vs. 177±4, P=0.02; DBP= 106±2 vs. 90±1 mmHg; respectively, P<0.001) and did not change in C (p=0.19). In conclusion, our data demonstrated that LLLT could significantly increase the vascular reactivity also in humans, what seems to explain, at least in part, the improved BP response to exercise. In police officers, who are exposed to high levels of stress, LLLT would be an alternative therapeutic to prevent cardiovascular risk.Support or Funding InformationCAPES ‐ Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of the antioxidant enzyme‐mimetic activity and neuroprotective effects of cerium oxide nanoparticles stabilized with varying ratios of citric acid and EDTA
Authors: Estevez Ana Y. , Boateng Yvonne , Lipps Jennifer , Stadler Brad , Erlichman Joseph S. 
Published: June 2021

Cerium oxide (CeO2) nanoparticles (nanoceria or CeNPs) are potent antioxidants that are being explored as a potential therapeutic for diseases featuring oxidative stress. However, both beneficial and toxic effects of CeNPs have been reported. We hypothesize that the method of synthesis as well as the choice of stabilizers coating the particles can lead to disparate biological effects. In the present study we tested the antioxidant enzyme‐mimetic properties of a series of custom‐synthesized CeNPs that differed in the citric acid (CA) and EDTA ratio (CA:EDTA) used to stabilize them. We first spun each type of CeNP at 100,000 RPM (434,513 xg) in order to separate it into supernatant (SUP) and pellet (PEL) fractions. We then used commercially available enzymatic assay kits to assess the catalase‐ and superoxide dismutase (SOD)‐mimetic activity of each fraction. The CeNPs stabilized with only CA (CA:EDTA ratio of 100:0) exhibited significantly higher catalase‐mimetic activity in both the SUP and PEL fractions compared to the other samples. Varying the CA:EDTA ratio had opposing effects in the SOD‐mimetic activity of the SUP and PEL fractions. For example, CeNPs stabilized with only CA (CA:EDTA ratio of 100:0 ) displayed most of the SOD‐mimetic activity in the PEL. In contrast, CeNPs stabilized with only EDTA (CA:EDTA ratio of 0:100) showed highest level of SOD‐mimetic activity in the SUP. Intermediate ratios (CA:EDTA of 40:60) showed intermediate levels of SOD‐mimetic activity in the SUP and PEL fractions. We then tested the effects of these CeNPs in a mouse hippocampal brain slice model of ischemia where cell death was assessed using the fluorescence indicator SYTOX green. Interestingly, no significant neuroprotection was observed in brain slices treated with CeNPs stabilized with the extreme ratios of CA:EDTA (100:0 and 0:100). However, slices treated with CeNPs stabilized with intermediate ratios of CA:EDTA (50:50, 40:60, 30:70) showed significantly reduced ischemic cell death. When the SUP and PEL fractions of the CA:EDTA 50:50 sample were tested separately, only the slices treated with the pellet fraction showed significantly reduced cell death. These results demonstrate that CeNPs varying in the ratio of CA:EDTA used for stabilization display different enzyme‐mimetic activity. More importantly, our data suggest that CeNPs synthesized with intermediate CA:EDTA stabilization ratios demonstrate the best neuroprotection profile in ischemic brain slices and that the neuroprotective activity resides in the PEL fraction of the CeNPs.Support or Funding InformationThis work was supported by St. Lawrence University and Cerion Advanced Materials.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 D‐allulose transport is likely mediated by glucose transporter type 5 (GLUT5)
Authors: Kishida Kunihiro , Iida Tetsuo , Yamada Takako , Ferraris Ronaldo P. , Toyoda Yukiyasu 
Published: June 2021

D‐allulose is found in a wide variety of food products in limited amounts, and therefore is referred to as a rare sugar. It is an epimer of fructose that is 70% as sweet as sucrose and has been demonstrated to have antihyperglycemic, antihyperlipidemic, and antiobesity effects by various studies including humans. The mechanism underlying intestinal absorption of D‐allulose is unknown, hence we tested the hypothesis that intestinal D‐allulose transport is mediated by glucose transporter type 5 (GLUT5). Adult rats were initially fed for a week a high‐fructose (control: high‐glucose) diet to induce GLUT5 expression and then gavaged with 1g/kg BW D‐allulose alone, D‐allulose with 1g/kg BW glucose or D‐allulose with 1g/kg BW fructose. Plasma D‐allulose concentrations were then compared to examine whether D‐allulose shares transporters with fructose and/or glucose and whether its absorption is affected by luminal glucose or fructose. When D‐allulose was gavaged alone, the fructose‐fed rats showed significantly higher plasma D‐allulose levels at 30, 60, 90 min and conversely lower (P = 0.05) level at 120 min after administration than those of the glucose‐fed rats. This suggests that more D‐allulose was rapidly absorbed via highly expressed GLUT5 in the fructose‐fed rats. When D‐allulose was gavaged with fructose, appearance of D‐allulose in the plasma was delayed compared to when D‐allulose alone was administrated, indicating that fructose inhibited D‐allulose transport into plasma. Moreover, the levels of plasma fructose peaked earlier than those of D‐allulose, indicating that luminal fructose is more rapidly absorbed than D‐allulose. When D‐allulose was gavaged with glucose, higher plasma D‐allulose concentrations at 30 min after administration were observed compared to when D‐allulose was gavaged with fructose in both glucose‐ and fructose‐fed rats. These results suggest that intestinal D‐allulose transport is likely mediated by GLUT5 and its affinity for GLUT5 is lower than that of fructose.Support or Funding InformationThis work was partly supported by Matsutani Chemical Industry Co., Ltd.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Cellular Mechanisms of DNA Replication Restart in Escherichia coli
Authors: McKenzie Aidan , Wessel Sarah , Keck James 
Published: June 2021

In E. coli, replisomes duplicate only a fraction of the chromosome before being ejected from the DNA due to collisions with damaged DNA or nucleoprotein complexes. Premature replisome ejection leaves an abandoned replication fork that must be reinitiated by DNA replication restart proteins (PriA, PriB, PriC, and DnaT in E. coli). PriC can mediate helicase loading without the help of other replication restart proteins in vitro and it directly interacts with the DnaB/DnaC (helicase/loader) complex. Interestingly, ΔpriC strains appear wild‐type but have synthetic lethal relationships with genes not implicated in DNA replication restart. Our poster describes recent structural efforts to probe the PriC/DnaB/DnaC complex and a transposon‐sequencing approach to identify genes linked to priC and other DNA replication restart genes in E. coli.Support or Funding InformationNIH R01GM098885.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.