bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A
Authors: Stuttgen Gage Michael , Weaver Todd Michael , Grilley Daniel Patrick 
Published: June 2021

The protein structure and function paradigm is a foundational tenet of biomolecular science that underlies many infectious diseases. Hemolysin A (HpmA), a hemolytic protein produced by Proteus mirabilis, was used as a model to investigate the protein structure‐function paradigm. HpmA is a member of the two‐partner secretion (TPS) pathway, which is used by gram‐negative bacteria to export predominantly virulent proteins outside of the cell. Through this mechanism, the A‐component (HpmA) is translocated, folded, and activated by its cognate B‐component in the absence of high‐energy bond and electrochemical gradient dependency. All TpsA components are relatively large and can be further divided into two domains, the two‐partner secretion domain and the functional domain. Universally, all known TPS domains harbor a right‐handed, parallel b‐helix architecture, which has been shown critical for cognate TpsB‐dependent recognition and secretion. Conversely, functional domains provide TpsA diversification including, cytolysis, host cell adhesion, contact‐growth inhibition, and iron sequestration. A truncated version of hemolysin A (HpmA265) was implemented to define the contributions of the TPS domain toward TpsA structure and function. Recently, our group has further dissected HpmA265 into three sequentially folded structural units termed the polar core, non‐polar core, and carboxy‐terminal subdomains. This research project aims to expand upon our recent results and structurally map the role of the nonpolar core during TPS domain dependent secretion, folding, and function. Specifically, residues within the non‐polar core subdomain have been selectively targeted and modified. The structural and functional effects of these site‐selective modifications have been evaluated via chemical denaturation, protease sensitivity, and template‐assisted hemolytic activity. Site‐selective alterations within the non‐polar core subdomain shift four‐state b‐helix unfolding to a three‐state model, increased protease sensitivity, and decrease template‐assisted hemolysis. In contrast, site‐selective alterations on the exterior of the non‐polar core subdomain maintain four‐state unfolding and protease sensitivity, and increase template‐assisted hemolysis. Collectively, these results point toward TPS b‐helix stability as a requisite for robust template‐assisted hemolysis. Conservation of the TPS domain across gram‐negative bacteria makes development of antibiotics targeting b‐helix structure and function attractive.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic Allosteric Biomolecular Design Using Artificial Intelligence
Authors: Thayer Kelly , Beveridge David , Langley David 
Published: June 2021

Protein‐ligand binding mediated by an allosteric effector can be considered from the vantage of dynamic macromolecular structure. Recently, we have developed a methodology, MD‐based Markov State Models (MD‐MSMs) making use of this view. We envision gaining insight into the workings of allosteric systems, and applying those principles to allosterically engineer biomolecules as a new class of therapeutics. Such allosteric regulators hold promise for modulating currently “undruggable” targets. In our approach, Molecular dynamics simulations (MD) generate the Boltzmann ensemble of conformations of a system. Using machine learning to process trajectories, the number of conformational substates can be learned from the conformations of the system (Figure 1A). Network theory captures the dynamic interchange between conformations (Figure 1B). Our recent findings from the application of MD‐MSMs to allosteric proteins (Figure 1C) and principles for engineering allosteric effectors will be presented.Support or Funding InformationNIH R15 GM128102 to KMTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does SpNox directly produce hydrogen peroxide?
Authors: Scott James , Notice Brittany , Gonzalez Victoria , Thomas Sarah , Vermot Annelise , Petit‐Hartlein Isabelle , Fieschi Frank , Smith Susan ME 
Published: June 2021

NADPH oxidases (NOXs) are a family of transmembrane enzymes that generate reactive oxygen species (ROS), which in humans acts both as a cytotoxic agent in autoimmunity and as a signaling molecule in many physiological processes. Human NOXes can produce different ROS species; for example, NOX2 isoform generates superoxide whereas NOX4 isoform produces mainly hydrogen peroxide. The ROS species produced affects downstream signaling, and misregulation produces disease states. A recently discovered prokaryotic NOX, SpNOX, provides an ideal model system to investigate controls on ROS product. SpNOX was previously shown to produce superoxide. Because superoxide spontaneously dismutes at a fast rate, assays that detect hydrogen peroxide cannot be used to assess direct hydrogen peroxide formation. Instead, as in the case of xanthine oxidase, we estimate total electron flux and also superoxide production; we assume that any difference between these values reflects direct hydrogen peroxide production by the enzyme. We estimate electron flux using three assays: i/ monitoring NADPH consumption using absorbance (340 nm); ii/ monitoring NADPH consumption using fluorescence (ex 345/em 454); iii/ monitoring cytochrome c reduction by the FAD of a dehydrogenase‐domain only construct of SpNOX. Superoxide generation from full‐length SpNOX is estimated by the superoxide‐dismutase‐inhibitable portion of cytochrome c reduction, monitored by absorbance at 550 nm. Here we present our findings regarding relative production of superoxide and hydrogen peroxide by SpNOX, as a first step toward manipulating ROS product in this model system.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Hydrogen Peroxide on Collagen
Authors: Valdes Diana , Tellez Jorge , Keenan Kelly 
Published: June 2021

Americans spend billions of dollars every year on teeth whitening products including whitening strips; the active ingredient is hydrogen peroxide (H2O2). Collagen accounts for 95% of the protein found in the dentin layer of teeth, which lies beneath the enamel. Previous research done in our laboratory has indicated that treatment of teeth with over‐the‐counter whitening strips results in reduced amounts of collagen and hydroxyproline. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used on collagen extracted from teeth. For the untreated teeth, the extracted collagen showed the expected molecular weights (MW) on SDS PAGE but these bands were absent for the collagen extracted from treated teeth. For this reason, pure Type I collagen was used and treated either with water or with H2O2 for an hour before pepsin was added. Our results indicate that pre‐treatment of Type I collagen with low concentrations of H2O2 produced a lack of bands at the expected MW for collagen and these bands were present in collagen treated with water. This effect was seen as soon as 10 minutes after H2O2 was added. The impact of adding the protease pepsin was further assessed. These results suggest that hydrogen peroxide, even at the concentration used in the whitening strip, can damage the collagen in the dentin provided that the H2O2 can penetrate to the dentin in the teeth. This work was supported by Research and Professional Development Grant from Stockton University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of hydrogen sulphide in regulating invasion in trophoblast cells
Authors: Saxena Shobhit , Gupta Sunil Kumar , Arora Pallavi , Chetlapalli Pallavi , Sirohi Neerja , Sharma Arundhati , Dwivedi Sadananad , Luthra Kalpna , Bhatla Neerja , Dhingra Renu 
Published: June 2021

IntroductionHydrogen sulphide is recently being considered as endogenous gaseous mediator which has shown to be involved in regulating many physiological processes like cell proliferation migration, invasion and phenomena like oxidative stress, angiogenesis, vasoregulation and neuromodulation. However, its role in modulating invasion in trophoblast cells, the key component of placenta is not known. Hence, in the present study we sought to explore the effects of hydrogen sulphide (H2S) on invasion capacity of trophoblast cells using HTR‐8/SVneo cell lineMaterial and methodsThe invasion potential of HTR‐8/SVneo cells was assessed by transwell invasion assay. The Matrigel invasion assay was performed at 37°C using 24 well transwell inserts (corning Greiner Bio‐One) coated with semi solid Matrigel 10% (Becton and Dickinson). The HTR‐8/SVneo cells received various treatments and then were transferred to the transwell(Boyden's) chamber. The cells were exposed to NaHS (sodium hydrosulphide:Hydrogen sulphide chemical mimic), its inhibitor AOAA (amino‐oxiacetate) in different doses for various time periods and hypoxia /reoxygenation. Cells (2500) suspended in 200ul of serum free medium were seeded onto the upper chamber. However,400ul of 10% FBS was added to lower chamber. Matrigel was removed from upper surface. Cells that migrated and invaded through the membrane to the under surface were methanol fixed and stained by 0.5% crystal violet. The cells were counted under Nikon Ti‐S Eclipse microscope using NIS‐AR software. The percentage invasion was calculated by the formula: mean number of cells invading the Matrigel membrane (test group) / mean number of cells invading through the insert membrane (control group) X100ResultsThe Percentage invasion was much higher when the cells received hydrogen sulphide mimic i.e. NaHS 100mM for 10 minutes (220%) as compared to cells which did not receive any treatment (100%). The trophoblast cells which were subjected to hypoxia reoxygenation (two cycles) showed reduction in the percentage invasion (20%). However, the cells of hypoxia reoxygenation treatment group regained their invasion capacity (46%) after they were exposed to NaHS 100 μM. On the other hand, the treatment of cells using AOAA [Cystathionine beta synthase (H2S producing enzyme) inhibitor] treatment decreased the cell invasion capacity (32%)ConclusionThe results from present study point towards the role of H2S in invasion of trophoblast cells, thereby predicting its role if any in placentation. These findings may also indicate that decrease in H2S may contribute to the pathogenesis of preeclampsia and also the other clinical outcomes associated with it like Fetal growth restriction (FGR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional Control of Fibroblast Plasticity
Authors: Small Eric 
Published: June 2021

Heart disease is associated with the accumulation of resident cardiac fibroblasts that secrete extracellular matrix (ECM) and drive the development of pathological fibrosis and heart failure. However, the mechanisms underlying fibroblast proliferation and activation remain poorly defined. To address this gap in understanding we have utilized transcriptional profiling to characterize the cardiac fibroblast phenotype in health and disease. Cardiac fibroblasts exhibit dynamic changes in genes encoding cell cycle regulators and ECM proteins. Here, we report that the cardiac fibroblast cell cycle is in part controlled by Sprr2b, a small proline‐rich protein that is induced in heart disease and extinguished during exercise. Sprr2b is a novel regulatory component of the USP7/MDM2‐containing ubiquitination complex. Sprr2b stimulates MDM2‐dependent p53 degradation, thus facilitating the proliferation of pathological cardiac fibroblasts. These findings establish a paradigm of transcriptional regulation that governs cardiac fibroblast phenotypic plasticity and the development of pathological cardiac fibrosis.Support or Funding InformationNIH / NHLBI R01‐HL133761 and R01‐HL120919This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mast Cells Augment Neuroinflammation and Neurodegeneration
Authors: Duraisamy Kempuraj , Premkumar Keerthivass , Selvakumar Govindhasamy Pushpavathi , Thangavel Ramasamy , Ahmed Mohammad Ejaz , Zaheer Smita A , Raikwar Sudhanshu P , Dubova Luliia , Iyer Shankar S , Zaheer Asgar 
Published: June 2021

Activated glial cells, and neurons express several proinflammatory mediators that are implicated in neuroinflammation and neurodegeneration in neurodegenerative diseases including Parkinson's disease (PD), Alzheimer's disease (AD) and Multiple sclerosis (MS). Mast cells are immune and inflammatory cells that are implicated in neuroinflammation by releasing several prestored and newly synthesized multifunctional cytokines, chemokines and neurotoxic mediators. Protease activated receptor‐2 (PAR‐2) expression is increased in neuroinflammatory conditions. PD is characterized by neurodegeneration of dopaminergic neurons in the brain. Here, we have investigated how mast cells can contribute to neuroinflammation, and to the pathogenesis of PD. We demonstrate that inflammatory mediators such as interleukin‐1beta (IL‐1β), tumor necrosis factor‐alpha (TNF‐α) and the chemokine (C‐C motif) ligand 2 (CCL2) were lower in the brains of mast cell deficient (MC‐KO) mice and glia maturation factor‐deficient (GMF‐KO) mice when compared to wild type mice brains after acute 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) administration. Further, we found that reconstitution of mast cells in MC‐KO mice increases neuroinflammation in the MPTP‐injected mice brains as compared to the MC‐KO brains without mast cell reconstitution. Our immunofluorescence staining revealed that the PAR‐2 expression is increased in MPTP‐injected mice brains as compared to control wild type mice brains without MPTP administration. We also show significantly increased PAR‐2 expression in the cultured primary mouse astrocytes and neurons incubated with 1‐methyl‐4‐phenylpyridinium (MPP+), inflammatory protein GMF, mouse mast cell protease‐6 (MMCP‐6), and MMCP‐7. We conclude that mast cells augment neuroinflammation and neurodegeneration in neurodegenerative diseases including PD and inhibition of mast cell activation could be a therapeutic option in neuroinflammatory and neurodegenerative disorders.Support or Funding InformationThis work was supported by Veteran Affairs Merit Award I01BX002477 and National Institutes of Health Grants AG048205 & NS073670 to Prof. Asgar ZaheerThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interferon Induces Expression of the Immune Checkpoint PD‐L1 via Bcl3 Upregulation in Ovarian Cancer Cells
Authors: Zou Yue , Uddin Mohammad , Padmanabhan Sveta , Gaire Bijaya , Vancurova Ivana 
Published: June 2021

Interferon‐g (IFN) induces PD‐L1 expression in ovarian cancer (OC) cells, thus promoting OC growth. In addition to inhibiting anti‐tumor T responses, the tumor‐expressed PD‐L1 has tumor‐intrinsic effects that include increased cancer cell survival and proliferation. However, the mechanisms by which IFN induces PD‐L1 expression in OC cells are unknown. Here, we show that IFN increases gene and protein levels of the proto‐oncogene Bcl3 in ovarian cancer SKOV3 and OVCAR3 cells, resulting in their increased survival, proliferation, and migration. The IFN‐induced Bcl3 expression then promotes IFN‐mediated PD‐L1 transcription in OC cells. The mechanism consists of an IFN‐induced, Bcl3‐ and p300‐dependent PD‐L1 promoter occupancy by K314/315 acetylated p65 NFkB. Since blocking PD‐L1 by neutralizing antibody reduces proliferation of OC cells overexpressing Bcl3, these results indicate that the pro‐proliferative effect of Bcl3 in OC cells is mediated by PD‐L1. Together, our work demonstrates that IFN induces Bcl3 expression in OC cells, identifies PD‐L1 as a novel target of Bcl3, and links Bcl3 to IFN signaling and PD‐L1‐mediated immune escape.Support or Funding InformationSponsor: Dr. Ivana Vancurova (ASBMB member ID: 32743).Funding: NIH CA202775 grant (to Dr. Ivana Vancurova)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Semi‐Quantitative Analyses of Protein Expression in Polyphenon E‐Treated PC‐3 Cells
Authors: Murtagh Christopher Gerard , Carastro L. Michael 
Published: June 2021

In the United States, approximately 1 in 9 men will be diagnosed with prostate cancer (PCa) during their lifetime. Furthermore, PCa is the second leading cause of malignant‐related death in the United States. Polyphenon E (Poly E) is a standardized, caffeine‐free, green tea extract reported to inhibit the progression of PCa. The chemopreventive mechanisms of action for Poly E are not fully understood. In an attempt to further understand the role of Poly E in PCa, we sought to identify its potential molecular targets. The aim of this study was to detect changes in protein expression levels in Poly E‐treated PC‐3 cells. We previously obtained DNA microarray and qRT‐PCR data from PC‐3 cells treated with increasing concentrations of Poly E (100, 200, 300 mg/L) to identify potential molecular targets. We extracted total cellular protein from Poly‐E treated PC‐3 cells and performed immunoblot analysis for RGCC, ATM, RB1, and beta‐tubulin. Immunoblotting data was used in semi‐quantitation analyses of protein expression to determine fold changes in protein levels compared to the reference sample (untreated control). Immunoblotting data was normalized against beta‐actin protein loading control. Data obtained from semi‐quantification of protein levels for selected genes was compared to qRT‐PCR data obtained previously. RB1 protein expression levels for exhibited dose‐dependent decreases upon increasing Poly E treatment. At the highest concentration of Poly E treatment (300 mg/L), RB1 protein expression decreased by 44% compared to untreated cells, and was consistent with qRT‐PCR data. RGCC protein expression levels did not exhibit dose dependency, with 1.2 and 1.1‐fold increases in protein expression upon 200 and 300 mg/L Poly E treatment, respectively, which was consistent with observed trends for the corresponding qRT‐PCR data. A dose‐dependent decrease in ATM protein expression was observed for the Poly E‐treated PC‐3 cells. At the highest concentration of Poly E treatment (300 mg/L), ATM protein expression decreased 2‐fold. However, qRT‐PCR data obtained for ATM indicated marginal changes in gene expression in Poly E‐treated PC‐3 cells. Our semi‐quantitative immunoblotting data for RGCC and RB1 protein expression in Poly E‐treated PC‐3 cells was generally consistent with our qRT‐PCR data. However, the dose dependent decreased ATM protein expression observed did not comport with the lack of gene expression level changes observed by qRT‐PCR.Support or Funding InformationThis work was supported by a University of Tampa David Delo Research Professor Grant (to: L. M. C.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recombinant Immunotoxins against Latent Epstein Barr Virus Infections
Authors: Zhu Yuyi , Emmanuel Muhsinat , Weldon John E. 
Published: June 2021

Epstein Barr virus (EBV), commonly associated with acute infectious mononucleosis, is an oncogenic virus implicated in thousands of cancer‐related deaths every year worldwide. Estimates suggest that 90–95% of the population worldwide are infected with EBV, but acute infections are controlled with minimal symptoms by a healthy immune system. Like all herpesviruses, however, EBV persists in a latent state even after the acute infection has cleared. Studies have shown a definitive link between latent EBV infections and the development of certain cancers such as nasopharyngeal cancer and Burkitt's lymphoma, but there are currently no treatments for latent infections. A potential treatment for both latent EBV infections and EBV‐induced malignancies is antibody therapy targeted to virus‐specific membrane proteins. We have identified two EBV‐specific membrane proteins expressed on infected cells, LMP1 and LMP2, and are developing HEK293 cell lines constitutively expressing each of these proteins. We plan to develop recombinant immunotoxins (RITs) targeted to each of these proteins. RITs are fusion proteins comprised of an antibody and protein toxin designed to identify and kill specific populations of cells. RITs using the bacterial toxin Pseudomonas exotoxin A (PE) have been extensively studied. Several RITs are in clinical trials, and one (moxetumomab pasudotox) has recently received FDA approval for the treatment of Hairy Cell Leukemia. Using a previously developed anti‐LMP1 antibody, we have begun work to clone, express, and purify an anti‐LMP1 single‐chain variable fragment antibody joined recombinantly to a cytotoxic fragment of PE. We plan to evaluate this and other RITs as potential therapeutics for EBV‐related malignancies.Support or Funding InformationThis project was supported by the Towson University's Fisher College of Science and Mathematics.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Energy and Cellular‐Defense Systems are Target for Anti‐Senescence Activity of Methylene Blue
Authors: Atamna Hani 
Published: June 2021

Methylene blue (MB) delays cellular senescence, induces complex‐IV, and activates Keap1/Nrf2. The molecular link of these effects to MB and cellular senescence is unclear. We investigated the effect of MB on specific factors that affect cell senescence. Since MB is redox‐active agent, we started by assaying its effect on NAD/NADH ratio. MB caused a transient increase in NAD/NADH. This increase triggered an investigation into the energy metabolism regulator AMPK. MB induced AMPK phosphorylation in a transient pattern, which was followed by the induction of PGC1α and SURF1: both are inducers of mitochondrial and complex‐IV biogenesis. As expected from previous results, MB‐treated cells doubled complex‐IV activity. In addition, a significant decline in cellular oxidants was measured in MB treated cells. The telomeres erosion rate was also significantly lower in MB‐treated cells. A previous research suggested that the pattern of AMPK activation (i.e., chronic vs. transient) determines AMPK's effect on cell senescence. We found that the anti‐senescence activity of MB (transient activator of AMPK) was 8‐times higher than that of AICAR (chronic activator of AMPK). We investigated the effect of MB on cell cycle. We found that MB lacked an effect on cell cycle, indicating that an MB‐dependent change to cell cycle is unlikely to contribute to the anti‐senescence activity of MB. The current findings in conjunction with the activation of Keap1/Nrf2 suggest a synchronized activation of the energy and cellular defense cellular systems as a possible key factor in MB's potent anti‐senescence activity.Support or Funding InformationNIHAFARAmesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.