bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determining the Mechanism of Hemolytic Function and Type Vb Secretion by a Gram‐Negative Pathogen
Authors: Wade Gina , Grilley Dan , Weaver Todd 
Published: June 2021

Proteus mirabilis is a pathogenic opportunistic Gram‐negative bacterium that produces and secretes a toxic protein called hemolysin A (HpmA). P. mirabilis utilizes a type Vb, or two‐partner (TPS), secretion system to simultaneously transport and activate HpmA across its outer membrane and release it into the blood of a host where it destroys red blood cells (hemolysis). Like all other TPS proteins, HpmA contains an N‐terminal TPS domain (≈300 residues) essential for secretion and a separate C‐terminal functional domain (>1200 residues) responsible for virulent functions. Our goals are to determine how secretion of HpmA is prompted by its folding and identify regions of HpmA critical to its hemolytic function. In the absence of secretion, the unfolded full‐length HpmA requires a folded TPS domain to act as a template protein to achieve its hemolytically active form. We term these Template Assisted Hemolytic Activity (TAHA) assays. Performing TAHA assays with genetically altered HpmA, we have quantitated the importance of different regions on HpmA folding and function. We have determined the borders of the hemolytic functional domain, marking the first identification of a functional region on a TPS hemolysin. Other hemolytic proteins are known to adopt a modular method of function. To determine if the function of HpmA is dictated by similar modules, we have created small deletions and amino acid randomizations within the hemolytic functional region. Separately, by rationally designing deletions and variants within the TPS domain of full‐length HpmA, we have determined that the folded template recognizes specific sequences within the unfolded TPS domain to initiate folding. This supports our N‐terminal first secretion model and suggests a continuity of structure and linkage of folding between the TPS domain and the remainder of HpmA. A continuous β‐helical structure starting from the TPS domain could explain this link between domains of secretion and function. To test this link and characterize formation of secondary structure during templated folding, we have performed structural studies using CD spectroscopy. By identifying regions necessary for hemolysis and characterizing how TPS domain folding is required to induce hemolytic activation, our results have expanded our working model for HpmA secretion and function.Support or Funding InformationThis research was supported by an Undergraduate Research and Creativity grant and a Dean's Distinguished Summer Fellowship to GW.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unraveling the Biosynthesis of the Essential Lipoyl Cofactor in Staphylococcus aureus
Authors: Dill Zerick Robert , McCarthy Erin L , Booker Squire J 
Published: June 2021

Staphylococcus aureus (S. aureus) is the leading cause of infections acquired in a hospital setting. In recent years, the rise and spread of methicillin‐resistant S. aureus (MRSA) that has evolved resistance to antibiotics previously successful at eradicating the infection has become a major global health concern. Thus, the search for novel antibiotic targets has been the focus of many research efforts. One potential new target is the biosynthesis of the lipoyl cofactor, which plays an essential role in the oxidative decarboxylation of various α‐keto acids and breakdown of glycine. The second step of the de novo pathway for the biosynthesis of the lipoyl cofactor, the attachment of two sulfur atoms to C6 and C8 of an n‐octanoyl chain connected to a target lysyl residue on a lipoyl carrier protein, is catalyzed by lipoyl synthase (LipA), a member of the radical S‐adenosylmethionine (SAM) superfamily. In addition to its [4Fe–4S] radical SAM cluster, LipA contains a second [4Fe–4S] ‘auxiliary cluster’ which is sacrificed during catalysis to supply the requisite sulfur atoms. The destruction of its auxiliary cluster renders LipA inactive in the absence of a system to restore it. Further studies have identified that Escherichia coli (E. coli) has an additional protein, NfuA, an iron‐sulfur cluster carrier protein, that can target LipA and regenerate its auxiliary cluster to reactivate it for catalysis. However, while E. coli LipA has been extensively studied, the S. aureus LipA homolog has not been fully characterized. Interestingly, in vitro studies have shown that the activity of S. aureus LipA is unaffected by S. aureus Nfu alone, indicating that a second factor may be involved. Recent in vivo studies have reported that an S. aureus protein, SufT, is involved in iron‐sulfur cluster assembly and is implicated in lipoic acid biosynthesis; however, the role of SufT is unknown. We will, therefore, use a combination of genetic, biochemical, and spectroscopic approaches to functionally characterize Staphylococcus aureus LipA and understand the potential roles of Staphylococcus aureus Nfu and SufT in the regeneration of LipA's auxiliary cluster. Ultimately, this information could be used to design antibiotics that target this pathway and obstruct the essential biosynthesis of lipoic acid, inducing death to pathogenic bacteria.Support or Funding InformationI acknowledge support from the HHMI EXROP program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulating Activity of ABC Transporters in Blood Brain Barrier Cells in Culture
Authors: Holcomb‐Webb Kasi L. , Nanayakkara Amila K. , Aljowni Maha , Lippert Alexander R. , Vogel Pia D. , Wise John G. 
Published: June 2021

The blood‐brain barrier (BBB) is a physical and dynamic barrier that separates the central nervous system from the circulatory system in order to protect the brain from harmful endogenous and exogenous chemicals. The capillary endothelial cells that form the BBB are firmly sealed via tight junctions and express relatively high amounts of ATP‐binding cassette efflux transporters like P‐glycoprotein (P‐gp) and the breast cancer resistant protein (BCRP) to protect the brain from entry of potentially harmful chemicals. While protective to the brain under normal circumstances, this high level of expression of these proteins at the BBB are problematic when attempting to treat diseases of the brain like neurodegenerative diseases or brain cancers since P‐gp and BCRP impede entry of therapeutic drugs into the brain. Previously, we have computationally identified several drug‐like molecules that inhibit or modulate P‐gp and/or BCRP activities. The cell line, hCMEC/d3, was used in our studies as a model for the BBB. Using qPCR, we confirmed a higher level of expression of both P‐gp and BCRP in hCMEC/d3 cells when compared to non‐cancerous HFL‐1 cells. When hCMEC/d3 cells were treated with the P‐gp and BCRP inhibitors, accumulation of the P‐gp substrate rhodamine‐123 as well as the P‐gp and BCRP substrates, mitoxantrone and daunorubicin, was observed. We conclude that our newly discovered P‐gp and BCRP inhibitors may make good leads for the development of co‐therapeutics to inhibit and/or modulate the activity of P‐gp and BCRP in the BBB and facilitate therapeutic drug uptake through the BBB and into the brain.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas as well as the Hamilton Undergraduate Research Scholars Program, the SMU Summer Research and Undergraduate Research Assistantship Programs as well as the SMU Engaged Learning Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pitavastatin Selectively Kills PTEN Knock Out Cells and Cancer Organoids in Mouse Model via the Mevalonate Pathway
Authors: Jiao Zhihua , Cai Huaqing , Long Yu , Sirka Orit Katarina , Ewald Andrew , Devreotes Peter N 
Published: June 2021

PTEN is one of the most frequently mutated tumor suppressors in human cancer. Loss of PTEN leads to hyperactivation of the PI3K–AKT signaling pathway, cell migration, and cell proliferation. Aiming to identify specific drugs that could target cancer cells, we screened 2650 FDA approved compounds and discovered that certain statins selectively kill Dictyostelium pten‐cells. This result carries over to mammalian cells: MCF10A cells lacking PTEN are more sensitive to statins than MCF10A wild type (WT) cells. By testing the differential sensitivity between MCF10A WT and pten−/− cells with multiple statins, we found that pitavastatin showed the best performance. Mevalonic acid and geranylgeranyl pyrophosphate (GGPP), but not farnesyl phyrophosphate or squalene, rescued pitavastatin treated MCF10A pten−/− cells. Pitavastatin induced an increase in RhoA expression suggesting that the requirement for geranylgeranylation of this GTPase may mediate the sensitivity of MCF10A pten−/− cells. The different sensitivity between MCF10A WT and pten−/− cells also carried over to three‐dimensional culture model (3D). Moreover, we found pitavastatin significantly decreased the growth and Twist1‐induced cell dissemination in 3D culture of murine mammary gland organoids. Finally, we tested the effects of pitavastatin on growth and survival of WT(FVB) and tumor organoids (MMTV‐PyMT (luminal breast cancer model), C31Tag (basal breast cancer model)) in 3D culture. Both MMTV‐PyMT and C31Tag were killed by 100 nM pitavastatin, while WT organoids survived. These findings implicate pitavastatin as a potential therapeutic candidate for tumors.Support or Funding InformationThis work was supported by NIH Grant R35 GM118177.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical evaluation of drug inhibition of human MDR1 P‐glycoprotein
Authors: Oliveira Maisa C. , Okwuone Dakota D.D. , Farokhnia Roxana , Jensen Madeline K. , Edgar Jennifer M. , Wise John G. , Vogel Pia D. 
Published: June 2021

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer. To search for P‐gp inhibitors as co‐therapeutics to combat MDR, we had previously used high‐throughput in silico ligand docking studies to identify drug‐like compounds and their effects on P‐gp ATPase hydrolysis activity, followed by assays of MDR‐reversal of a prostate and ovarian cancer cell lines. More recently, additional computationally identified compounds were shown to reverse MDR in cancer cells. Biochemical assays using purified P‐gp provide important information regarding the mechanism of inhibition and have been used as primary screens in our drug finding process. Some of these newly found compounds were observed to be inhibitors of ATP hydrolysis activity of a cysteine‐less mouse MDR3 homolog of the human MDR1 P‐glycoprotein. Even though human and mouse P‐gp share about 87% identity in amino acid sequence, there may still be sufficient differences between the two proteins that may prevent MDR3 from faithfully reporting on inhibitor action on the human protein. High expression of human P‐gp in Pichia pastoris in a biologically active form was previously reported and the purified P‐gp was used on our recent assays. Lipid activation of ABC transporters, as shown by others, is a requirement to achieve optimal enzyme activity, but in our hands has proven to be more challenging than expected. The best P‐gp activation was achieved using L‐α‐phosphatidyl choline. The resulting lipid‐activated P‐gp showed low basal activity (~8.5 nmol NADH/min/mg P‐gp), but more than 30‐fold activation by the P‐gp transport substrate, verapamil (~306 nmol NADH/min/mg P‐gp). Here we report our optimized strategy to lipid‐activate wild‐type human P‐gp as well as the evaluation of the potential of our previously discovered drug‐like compounds to serve as inhibitors of human P‐gp ATP hydrolysis activity.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Inhibitors of Glutaminyl‐tRNA synthetase from Pseudomonas aeruginosa
Authors: Escamilla Yaritza , Hughes Casey A , Bullard James M 
Published: June 2021

IntroductionAntibiotic resistance is a growing global threat and infections caused by resistant bacteria have become increasingly difficult to treat with most current antibiotics. Pseudomonas aeruginosa is a Gram‐negative opportunistic pathogen and is the leading cause of mortality in cystic fibrosis patients. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. The glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was over‐expressed, enzymatically characterized and developed into a screening platform for the discovery of chemical compounds that inhibit protein synthesis.MethodsThe P. aeruginosa GlnRS was developed, using scintillation proximity assay (SPA) technology, as a platform to screen chemical compounds for detection of compounds that inhibited function. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with inhibitory activity.ResultsFrom this screening campaign three compounds (BM02E04, BM04B05, and BM04H03) were identified and confirmed as inhibitors of the activity of P. aeruginosa GlnRS. BM02E04, BM04B05, and BM04H03 were observed to inhibit the activity of GlnRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. The minimum inhibitory concentration (MICs) was determined against a panel of bacteria including; E. coli, E. coli tolC mutant, E. faecalis, H. influenzae, M. cattarrhalis, P. aeruginosa, P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain, S. aureus, and S. pneumoniae. There was no activity observed against the wild‐type strain of E. coli or P. aeruginosa, but broad spectrum activity was observed against other Gram‐negative and Gram‐positive bacteria. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus and M. catarrhalis. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, there was no toxicity observed by either BM02E04 or BM04B05 at any concentration tested up to 400 μg/ml.ConclusionP. aeruginosa GlnRS was cloned, over‐expressed, characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms. Three compounds were identified as having inhibitory activity against the aminoacylation activity of GlnRS. Two of the compounds were further characterized for inhibition of enzymatic activity, bacterial growth and toxic effects in human cell cultures.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)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 effect of caffeine on actin filament assembly
Authors: Zhai Arianna , Kang Hyeran 
Published: June 2021

The essential protein actin polymerizes into double‐stranded helical filaments that play critical roles in many cellular processes including cell shape change and movement. Actin is ubiquitous in eukaryotic cells and is particularly abundant in muscle cells. Caffeine is a chemical that is used to create mental alertness. A previous study shows that caffeine relaxes the smooth muscle of the body. We predicted that this effect of caffeine on muscle cells may be coupled to actin assembly/disassembly dynamics. To test the main hypothesis, we explored how caffeine affects actin polymerization with purified rabbit skeletal muscle actin. Solution containing actin filaments with varying concentrations of caffeine were sedimented down using ultracentrifuge. Supernatants and pellets were run through a Sodium Dodecyl Sulfate‐PAGE using gel electrophoresis. UV gel imaging analysis allowed us to determine the amount of polymerized actin in the presence of caffeine. Our preliminary results show that the higher concentrations of caffeine cause actin to polymerize to a greater degree, suggesting that caffeine aids in actin polymerization. This study suggests that caffeine promotes actin filament assembly and may have a stimulating effect on muscle cells.Support or Funding InformationThis study was supported by the UCF start‐up fund for Hyeran Kang.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tracking and Detection of Bactericidal Quantum Dots
Authors: Untracht Zachary , Ozcan Ali , Santra Swadeshmukul , Kang Hyeran 
Published: June 2021

Zinkicide is a bactericidal nanoparticle that combats Citrus Greening with exceptional field efficacy. Bactericidal nanoparticle detection is important for surpassing EPA criteria and bringing biocide products to market; however quantitative analysis on Zinkicide degradation in particular is currently lacking. Here, we have developed a protocol using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) to monitor and track bactericidal nanoparticles in varying solutions, including water and acidic buffer mimicking citrus pH. The nanoparticle detection was performed using UV fluorescent imaging of polyacrylamide gels. This assay is a cost‐effective and relatively simple approach for detecting and quantifying the intensity, concentration, and molecular weight change of nanoparticles. We observed intensity fluctuations of Zinkicide over time, indicating that the capping agent of the nanoparticle may be affected. Our results show that citric acid buffer induces the greatest degradation of Zinkicide evidenced by molecular weight changes. These findings are beneficial for general tracking and detection of bactericidal quantum dots.Support or Funding InformationThe authors would like thank the Kang Lab members for their continued support and assistance. This study was supported by the UCF start‐up fund (Kang). Authors acknowledge the UCF Materials Innovation and Sustainable Agriculture Center for facilities and technical support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spatiotemporal Control of Glycolysis Modulates ATP Generation and Enhances Restoration of Endothelial Barrier Function Following Inflammatory Injury
Authors: Gajwani Priyanka A , Wang Li , Ray Anne Marie A , Krantz Sarah A , Karginov Andrei A , Rehman Jalees 
Published: June 2021

BackgroundAcute inflammatory injury is characterized by dysfunction of the vascular endothelium, resulting in disruption of endothelial adherens junctions, increased vascular permeability and leukocyte infiltration. While the mechanisms of assembly and disassembly of endothelial adherens junctions have been extensively studied, little is known about the bioenergetics of the barrier repair process. The glycolysis regulatory enzyme PFKFB3 has recently been identified as a key mediator of endothelial metabolism and function and could thus serve as a potential mediator of barrier repair.Methods and ResultsTo investigate how the endothelial metabolic profile changes during inflammation, we performed a Seahorse bioenergetic flux assay on Human Lung Microvascular Endothelial cells (HLMVECs). Glycolysis, as measured by the extracellular acidification rate, increased by 36% following treatment with the inflammatory mediator TNFα, while oxidative phosphorylation was not affected. Pharmacological inhibition of PFKFB3 abolished TNFα mediated upregulation of glycolysis. PFKFB3 activity was also necessary for barrier restoration of HLMVECs following injury, as measured by a transendothelial electrical resistance (TER) assay (p<0.0001). Whole cell ATP levels, measured by live cell microscopy using the fluorescent ATP biosensor Perceval HR, increased 31% following TNFα treatment (p<0.0001), largely concentrated at the cell periphery. We hypothesized that this regional ATP generation is PFKFB3 dependent. To address this question, we engineered a novel construct in which PFKFB3 is targeted to the cell membrane in an inducible manner and thus regulating metabolism in a spatiotemporal manner. Importantly, PFKFB3 membrane targeting enhanced endothelial barrier restoration by increasing the extent and rate of recovery (p<0.0001) following barrier disruption.ConclusionsThis data suggests that spatial and temporal regulation of PFKFB3 mediated glycolysis is important for restoration of the endothelial barrier after inflammatory injury. A better understanding of the endothelial barrier bioenergetics could allow for the development of novel therapeutic approaches to treat diseases in which endothelial barrier function is acutely compromised.Support or Funding InformationAHA 18PRE34070092 National Institute of Health P01HL060678This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.