bluuebunny/crossref_metadata_2025 huggingface dataset:
Are heme proteins major targets for homocysteine‐induced modifications?
Authors: Sharma Gurumayum Suraj , Singh Laishram Rajendrakumar
Published: June 2021

The prime cause of homocysteine (Hcy) toxicity towards proteins has been attributed to the modification of protein by homocysteine thiolactone (HTL). But the effects of Hcy (the parent molecule) on protein structure and function are still scarce. Hcy also has the ability to modify proteins, which could in turn affect the functionality and proper folding of the modified protein. Hcy is a highly pro‐oxidizing agent, thus Hcy‐induced oxidative stress has been commonly used to explain the toxicity of Hcy on endothelial cells and other tissues. Moreover, Hcy is known to modify cysteine residues in proteins in a process termed as S‐homocysteinylation. However, the structural and functional consequences of such modification are not yet clearly understood. The present study aims at investigating possible effects of Hcy on proteins structure and function. Using RNase‐A, Lyz and horseradish peroxidise (HRP) as models, we studied the effects of Hcy on the enzyme function and conformational status of these proteins. Enzyme activity measurements after overnight incubation with Hcy led to gradual decrease in enzyme function of HRP in a concentration dependent manner but not in Lyz and RNase‐A. Conformational analyses revealed no gross structural alterations in all proteins studied. However, certain alteration in the heme‐Trp distance was observed in case of HRP as evident from Trp fluorescence measurements. The loss of function in HRP was due to disruption of heme redox state in the presence of Hcy. Similar findings were obtained for three other common heme metalloprotein (Cytochrome c, Cyt c; Hemoglobin, Hb; and Myoglobin, Myo), wherein Hcy induced certain alterations in the heme environment rendering it reduced with further disruption of the heme‐Trp distance. In addition, Hcy was found to alter the heme redox states without direct interaction with the proteins (in case of Cyt c and Myo). The findings suggest that heme and heme containing proteins could be major targets for Hcy‐induced modifications and involved in Hcy‐induced cytotoxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analyzing the Three Dimensional Structure of Cortactin Protein
Authors: Gurumani Margaret , Kruchten Anne
Published: June 2021

Cortactin is a cytoskeletal protein which increases the metastasis of cancer cells. Cortactin is commonly located and over‐expressed in cancer cells. Because of its role in cancer, we sought to better understand the structure of cortactin. Our analysis involved expression of cortactin from E. coli, purification using His‐tag and ion exchange column purification, dialysis, and analysis by SDS‐PAGE. Previous work in the laboratory suggested that ion exchange purification resulted in the isolation of folding variants of cortactin. To understand the structures of these folding variants, we used the apoptotic enzymes calpain and caspase to probe solvent exposed regions of the protein and analyze the resulting protein fragments by SDS‐PAGE. There are three putative caspase sites on cortactin (at the N‐terminus, middle, and C‐terminus) and several putative calpain sites distributed throughout the structure. In the future, we plan on further analyzing cortactin by digging deeper into its conformation through other chemical analysis.

Support or Funding Information

This work is supported by the College of Saint Scholastica SSC Summer Research Fellowships.

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 Study of Arc and Its Interactions with Endocytic Binding Partners
Authors: Boldridge Melissa , Wang Lei
Published: June 2021

Alzheimer's disease is a devastating neurodegenerative disease that causes great hardship to afflicted individuals as well as their caregivers. Currently available treatments seek to slow the disease's progression, but there is no known cure. Recent studies have shown that the activity‐regulated cytoskeleton‐associated protein Arc could act as a potential new drug target for the treatment of Alzheimer's disease. The Arc gene is an immediate early gene whose expression is induced by neuronal activation. Its expression is essential for memory consolidation. Behavioral studies of Arc−/− mice in implicit and explicit learning tasks demonstrate deficits in long‐term memory formation, though short‐term memory is unaffected. Still, the mechanism of Arc's role in memory formation is unknown. Previous studies have identified the endocytic proteins dynamin and endophilin as binding partners of the Arc protein. Dynamin is a large GTPase that plays a role in pinching off and fissioning the membrane during endocytosis as well as recruitment other proteins involved in endosome formation. Endophilin is implicated in vesicle formation and function in the brain. Arc interacts with both dynamin and endophilin to enhance AMPA receptor endocytosis and decrease surface expression. In this study, we have produced fragment proteins of dynamin, endophilin, and Arc using the E. coli expression system, and we have collected structural data on these proteins and aim to identify their structures and interactions.

Support or Funding Information

This project is sponsored by the grants from the National Institute of Health (NIGMS‐INBRE III, award number: P20GM103466; NIGMS‐AREA, award number: 1R15GM126499‐01) and the Hawaii Community Foundation (Alan M. Krassner Fund, award number: 16ADVC‐78888). The content is solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or the Hawaii Community Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical characterization of Suppressor of IKKepsilon structure, stability, and metal binding properties
Authors: Shikwana Flowreen , Bell J. Ellis , Bell Jessica K.
Published: June 2021

Suppressor of IKKepsilon (SIKE) is phosphorylated in the signaling cascade that initiates the anti‐viral innate immune response mediated by Toll‐like receptor 3. Although shown to be associated with cytoskeletal proteins, the function of SIKE in association with the cytoskeleton or as part of the innate immune response is not yet known. To gain insight into SIKE's function, structural and biophysical characterization were undertaken. Fluorescence‐based thermal shift assays (FTSA) indicated that the SIKE structure displayed hydrophobic residues prior to thermal denaturation. With increasing temperature, additional hydrophobic character was displayed giving rise to unique FTS curves showing three transitions, initial binding of fluor, unfolding, and quenching of fluorescence, presumably by protein aggregation. From modeling software, a divalent cation binding site was predicted. Using FTSA, the addition of zinc and calcium appear to alter the thermal stability of SIKE. Using ANS as a reporter group or direct tryptophan fluorescence of SIKE, the binding affinity of SIKE to various divalent cations, zinc, magnesium, manganese and calcium, was assessed. Fluorescence quenching experiments with neutral and charged quenchers were used to probe the local environment of the lone Trp residue of SIKE. The quenching coefficient for the Trp in SIKE determined from acrylamide studies was significantly lower than that of free Trp in solution indicating that the residue is fairly protected. Together, these studies provide biophysical parameters with which to interrogate and refine our predicted SIKE structure that is being employed in interpreting how SIKE interacts with cytoskeletal proteins to mediate host defense mechanisms.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and purification of human Brain and Muscle Arnt‐like 1 protein (hBMAL1)
Authors: Moreno Brenda , Li Diyang , Sarabia‐Gonzalez Alejandro , Rodriguez Jose , Choi Jennie , Ray Supriyo , Xiao Chuan
Published: June 2021

The circadian rhythm is an intrinsic biological clock in living organisms that oscillates every 24‐hours. It is influenced by external environmental cues to regulate many behavioral and physiological processes. Prolonged disruptions to circadian rhythms in humans have been associated to sleep disorders, cardiovascular and metabolic diseases, as well as high risks of cancer. Within every individual cell at a molecular level, the circadian rhythm is maintained by a negative feedback loop made of several core proteins' accumulation and degradation in a daily cycle. One of the core proteins involved in this cycle is Brain and Muscle ARNT‐Like 1 (BMAL1). Crystal structures of the N‐terminal DNA binding part of BMAL1 has provided some understanding of its DNA‐binding function, however, the lack of C‐terminal structural information of BMAL1 hinders our understanding of its regulatory role in the circadian rhythm. With the purpose of obtaining the atomic structure of full length circadian protein BMAL1, the complete human BMAL1 (hBMAL1) gene was cloned into bacterial systems for protein expression. However, the recombinant hBMAL1 is in its mis‐folded and aggregated form. Various methods in expression and purification of hBMAL1 are being developed to obtain high homogenous soluble hBMAL1. After achieving that objective, the full length hBMAL1 protein will be used in in vitro studies using various structural and functional analysis tools. The results will deepen our understanding of the regulatory mechanism of the circadian rhythm and build the foundation to develop therapeutic agents for circadian rhythm related diseases. In addition, the research project has been chosen for the Research‐Teaching‐Integration program. A teaching module will be developed and delivered to senior biochemistry students to raise their interests of scientific research and apply the biochemistry principles that they learn during the semester to analyze real experimental data.

Support or Funding Information

Research reported has used instruments sponsored by the BUILDing SCHOLARS program funded by National Institute of General Medical Sciences of NIH under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970.

Research reported has used the core facility at the BBRC supported by National Institute of Minority Health and Health Disparities under award number 5G12MD007592.

Research reported was also supported by the Campus Office of Undergraduate Research Initiatives (COURI), Summer Undergraduate Research Mentoring Experience (SURME), BUILD Research Teaching Integration (RTI), and Research Initiative for Scientific Enhancement (RISE) program awards.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using TTR50‐127 fragments as probes to detect amyloidogenic seeding
Authors: Nelson Luke Thomas , Schonhoft J.D. , Kelly Jeffrey , Duerr Jeffrey
Published: June 2021

The aggregation and amyloid deposition of soluble transthyretin (TTR) has been linked to the development sporadic and familial forms of cardiac amyloidosis. TTR associated cardiomyopathies are often diagnosed when the disease state has reached advanced stages (if at all), precluding effective clinical intervention. This diagnostic problem can largely be attributed to a lack of fundamental understanding of the mechanism of disease pathogenesis and the clinical similarities of TTR cardiomyopathies to other heart failure related conditions. In the human prion diseases and tau protein related neurodegenerative diseases, the amyloidogenic protein conformations have the ability to “seed” and transform non‐amyloidogenic states into pathogenic amyloid‐like states. Therefore, we hypothesized that TTR related cardiomyopathies may undergo similar seed based aggregation mechanisms. To test this hypothesis, we synthesized C‐terminal fragments of Transthyretin that are fluorescently labeled and upon assembly, the fluorescent signal is quenched and provides a convenient method to measure aggregation. We specifically chose the C‐terminal peptide fragment (TTR50–127) because at autopsy it has been shown that the C‐terminal region is preferentially deposited in heart tissue compared to the full length sequence. First we used these fluorescent probes to analyze the kinetics involved in the transition from monomeric TTR to the stable polymeric form of TTR and thus demonstrated proof‐of‐principle. As C‐terminally cleaved TTR is primarily found in biopsies of cardiomyopathy patients, introduction of rhodamine labeled TTR50–127 to cardiomyopathy and polyneuropathy patient plasma revealed distinct aggregation profiles in Native PAGE gels. While preliminary in nature, these results suggest that plasma of symptomatic patients may accelerate the aggregation of Transthyretin.

Support or Funding Information

Special thanks to staff of the Kelly Lab at TSRI as well as mentors James Smart Ph.D. and Jeff Duerr Ph.D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards the Evaluation of Quorum Sensing Signal Synthase Inhibitors
Authors: Higgins Erin Leigh , Payne Sterling , Ulrich Scott
Published: June 2021

Quorum sensing is a system through which bacteria sense and respond to their cell density by altering their gene expression. This behavior is executed through the synthesis, secretion, and detection of a class of small molecules called autoinducers. Some bacteria that are clinically relevant human pathogens contain a quorum sensing system that controls genes related to virulence and biofilm formation. As such, quorum sensing represents a reasonable antibacterial drug target for the control of pathogenic behavior without targeting bacterial growth or survival. Acyl‐homoserine lactones, the autoinducer for gram‐negative bacteria, is catalyzed by the LuxI family of autoinducer synthases. This project involves the expression, purification, and kinetic characterization of the LuxI family of autoinducer synthases and design of synthase inhibitors to block quorum‐sensing induced gene expression.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screemomg, expression, and characterization of Baeyer‐Villiger Monooxygenase for the biotransformation of ricinoleic acid
Authors: Yun Joohyun , Choi Kwon‐Young
Published: June 2021

Here, the biotransformation of ricinoleic acid into of 9‐(nonanoyloxy)nonanoic acidusing Baeyer‐Villiger monooxygenases (BVMOs) was investigated. The whole cell biotransformation of oleic acid includes OhyA (hydratase), ADH (alcohol dehydrogenase), and BVMO Enzymes consecutively. BVMOs are known to catalyze regiospecific Baeyer‐Villiger oxygenation of a variety of cyclic and linear ketones to generate the corresponding lactones and esters, respectively. However, the enzymes are difficult to overexpress in a soluble form in microorganisms. Thereby, this study has focused on screening and functional expression of the BVMOs in Escherichia coli. Initially BVMOs were selected by protein sequence analysis and were examined for their ability to express in soluble and active form to generate 9‐(nonanoyloxy)nonanoic acid from oleic acid. Secondly various optimization strategies of inducer concentrations, co‐expression with molecular chaperones, and different media conditions were investigated. Among the 9 BVMOs screened, three BVMOs were found to produce the target product and among these, Di_BVMO3 isolated from Dietzia sp. D5 was found to be best. Further, the soluble expression of Di_BVMO3 was enhanced by adding phosphoglycerate kinase as N‐terminal fusion tag. The whole cell biotransformation with this fusion enzyme resulted in 3 to 5‐fold enhancement in product formation compared with the non‐fusion counterpart. Final productivity up to 105.3 mgL−1 was achieved. Besides Di_BVMO3, other two new BVMOs of Rh_BVMO4 from Rhodococcus sp. RHA1 and AFL838 from Aspergillus flavus NRRL3357, were screened for production of 9‐(nonanoyloxy)nonanoic acid and could be used for whole cell biotransformation reaction of other long chain ketones.

Support or Funding Information

This work was supported by the Industrial Strategic Technology Development program (No.10044604), funded by the Ministry of Trade, Industry & Energy (MI, Korea)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Snapshots of the catalytic cycle of an O2, pyridoxal phosphate‐dependent hydroxylase
Authors: Hedges Jason B. , Kuatsjah Eugene , Du Yi‐Ling , Eltis Lindsay D. , Ryan Katherine S.
Published: June 2021

The organic cofactor pyridoxal phosphate (PLP) is used by enzymes to catalyze numerous modifications of amino acid substrates. Recently, the diversity of PLP‐dependent enzymes has been expanded with the discovery of several enzymes that use O2 as a co‐substrate to perform chemically challenging oxidative reactions. The interesting chemistry catalyzed by such O2‐, PLP‐dependent oxidases prompted us to undertake a bioinformatic search for new O2‐, PLP‐dependent enzymes. We identified one widely distributed enzyme, which we named RohP. Through in vitro biochemical studies we characterized the activity of RohP and found that it uses PLP and O2 to catalyze the hydroxylation of an sp3‐hybridized carbon in l‐arginine, giving 4‐hydroxy‐2‐ketoarginine, along with stoichiometric conversion of O2 to H2O2. Surprisingly, we found that the hydroxyl group in the 4‐hydroxy‐2‐ketoarginine product is derived from water, raising questions about the mechanism of RohP. To provide insight into the mechanism behind this hydroxylation, we used X‐ray crystallography to obtain four ~1.5 Å resolution snapshots interpreted to represent RohP at different stages of its catalytic cycle: the holo enzyme, two different PLP‐bound intermediates, and the enzyme in complex with (S)‐4‐hydroxy‐2‐ketoarginine. These structures reveal that the dynamic N‐terminus of RohP, which is required for catalysis, becomes ordered upon substrate binding, sealing off the active site from the bulk solvent. Concurrently, several conformational changes of residues in the active site help to properly orient the substrate for catalysis. Through our structural work we also identify a conserved histidine, which we demonstrate is essential for the later steps in catalysis, and we suggest that this residue may be key to catalyzing a stereospecific alkene hydration. Collectively, our biochemical and X‐ray crystallographic investigation provides insights into how O2‐, PLP‐dependent oxidases can catalyze the challenging hydroxylation of an unactivated sp3‐hybridized carbon in l‐arginine and sets the stage for further mechanistic studies on this exciting group of enzymes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Monoamine Oxidases (MAOs) by Green Tea Extracts
Authors: Topaz Gemma Rose , Rodriguez Joyce , Mahmood Farah , Depeiza Damien , Lorenzo Loren , Destine Rodely , Stieglitz Kimberly
Published: June 2021

Monoamine oxidase (MAO) performs deamination of amines and is found bound to the outer mitochondrial membrane at high‐concentration in neuronal cells. There are two isoforms of MAO: MAO_A which oxidizes serotonin, noradrenaline and adrenaline, and MAO_B which oxidizes dopamine, b‐phenylethylamine (PEA), and benzylamine. Alterations in MAO activity can occur in some central and peripheral nervous system diseases. More specifically, heightened MAO_B activity in the brain occurs in Alzheimer's disease, Huntington's disease, Parkinson's disease and normal aging. Abnormal MAO_A activity has found to be associated with depression, anxiety and psychiatric disorders. Drugs have been developed and continue to be developed for both MAO_A and MAO_B as targets. MAO_A is inhibited by clorgyline and MAO_B is potently inhibited by both deprenyl and pargyline. Using these inhibitors as controls, a fluorescent activity assay was performed with commercially available catechins (green tea extracts), serotonin, and benzylamine substrates for MAO_A and MAO_B respectively, to investigate and confirm recent studies suggesting that green tea catechins (polyphenols) may be preventative for certain degenerative diseases and emotional illnesses utilizing MAOs as a target. Using a fluorescent assay, the Km value of serotonin to MAO_A was 1.75 μM and the Km value of benzylamine to MAO_B was 0.75 μM. The IC50 of clorgyline to MAO_A was 3.25 nM and that of deprenyl to MAO_B was 7.25 nM, in close agreement with the literature. The commercial catechins tested were found to have IC50s in the low‐to‐mid μM range (~50–750 μM). Efforts to purify catechins are underway to repeat these studies. Molecular docking of specific catechins into the MAO_A and MAO_B active sites resulted in binding constants in the low μM range (in agreement with experimentally determined Km values for natural substrates). Crystallization studies of MAO/catechin complexes are in progress.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ATP as an Allosteric Modulator and Chelator of Fe3+ from Fe3+‐transferrin. Is ATP a Major Fe3+ Carrier
Authors: Cowart Richard E. , Shaffer Tyler B. , O'Hara Ryan D. , Campbell Jacquelyn A. , Schneider‐Tugan Lori L. , Binz Sara K. , Pope Mitchell A. , Gregory Robert B.
Published: June 2021

The objectives of this study were to examine two chelators in the removal of Fe3+ from Fe3+‐transferrin. One was pyrophosphate (PPi) as it is a reasonably effective chelator in this reaction, and the other was ATP in that it consists of PPi attached to adenosine monophosphate. We reasoned that ATP might bind to a nucleotide binding site if present and in close proximity to the metal center, to enhance Fe3+ removal and give insight into the mechanism of Fe3+ release. In this study we used uv/vis spectroscopy to examine the removal of Fe3+ from Fe3+‐transferrin by time‐course and Vi reactions. At acidic pH we determined association constants of Fe3+‐transferrin alone, and Fe3+‐transferrin in the presence of ATP. We also employed molecular modeling to identify and characterize a putative ATP binding site. Our results show that sigmoidal kinetics were obtained as a function of pH when ATP was the chelator. At pH 7.5 the kinetic data showed there was no removal of Fe3+ by ATP. ATP was found to exert positive heterotropic effects at acidic pH and resulted in the increase in the association constant of Fe3+‐transferrin. These effects were not seen with PPi. We concluded that there were two destabilizations of Fe3+‐transferrin necessary for Fe3+ release; the first due to a low pH induced conformational change in the protein and the second due to ATP, also at low pH, where ATP served as an allosteric modulator. Essentially 100% of Fe3+ was released from both the N‐ and C‐lobes at pH 5.0 in the presence of ATP in ~ 3 minutes as determined by time‐course reaction. Molecular modeling studies identified a putative 25 amino acid ATP binding channel in the N‐lobe of human serum transferrin. These conserved amino acids were identified on each open face of the NI/NII subdomains of apo‐transferrin with the channel forming upon NI/NII subdomain closure as Fe3+ is acquired. At neutral pH ATP bound approximately 9 Å from Fe3+ in the metal center. At acidic pH the protein conformational change positioned ATP immediately adjacent to Fe3+ for rapid chelation. In that ATP is an extracellular, allosteric signaling molecule a mechanism of Fe3+ release from Fe3+‐transferrin is proposed. Here, extracellular ATP is acquired by transferrin in the serum at neutral pH where chelation does not occur. Upon binding to the transferrin receptor and internalization into an acidic endosome, a pH induced conformational change along with positive heterotropic effects occur to allow ATP to efficiently chelate Fe3+. We conclude that transferrin is an allosteric protein, ATP is an allosteric modulator and effective chelator, and that ATP is likely a major Fe3+ carrier in higher organisms.

Support or Funding Information

State of Iowa grant GIVF

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying Substandard Medications in Developing Nations
Authors: Omanovich Esad , Donels Jordan , Trier Justin , Thomsen Alli , Dollen Julia , Miller Austin , Memic Nejla , Zea Corbin
Published: June 2021

Access to adequate and quality health care is a worldwide concern. Without proper access to medicine individuals will often suffer diseases that could be treated or prevented with the proper medicine. Proper dosage is also necessary so that patients can recover from the disease as well as stop the production of drug resistant bacteria and parasites. In collaboration with Norte Dame University, a protocol, using High Performance Liquid Chromatography (HPLC), was developed to quantify the amount of Albendazole, a deworming medication, in tablets from locations in Africa to ensure proper dosage. A Spike and Recovery, a secondary verification method, was developed to verify the efficiency of the column and protocol used. Optimization of the Spike and Recovery method was completed and showed that the efficiency of the column was 97 ± 1 %. Using this protocol, multiple samples have been quantified with one sample falling below the advertised dosage.

Support or Funding Information

Grow Iowa Values Fund

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Light Controlled Intracellular Protein Release: Tracking Ras Interactions With Superresolution Fluorescence Microscopy
Authors: Yun Jason , Phelps Carey , Morales Demosthenes , Nan Xiaolin , Reich Norbert
Published: June 2021

Although fusion proteins have been extremely helpful in the study of intracellular protein dynamics, this approach has significant drawbacks. We circumvent such problems by delivering dye‐conjugated proteins using our Nano‐optogenetic platform, which relies on hollow gold nanoshells to release proteins with NIR laser exposure. Herein, we report the delivery of a NS1 monobody, known to prevent dimerization of RAS GTPases that inhibits downstream signal transduction for cell division. Our modular nanoplatform is appended with thiol linked scaffolds containing a streptavidin‐biotin cell‐penetrating peptide for cell internalization and a nitriloacetic acid moiety to bind the polyhistidine dye‐labeled NS1 in the presence of copper. Near‐infrared laser activation allows for controlled spatio‐temporal endosomal release of the dye‐labeled NS1 to colocalize with RAS GTPase. Our approach can deliver any dye‐label His‐tagged protein of interest with controlled spatio‐temporal release inside cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Creation and Characterization of Gold‐Nanoparticle Containing Conductive Scaffolds for Culturing Cardiomyocytes
Authors: Maldonado Marcos , Castellanos Brisa Pena , Bonham Andrew J. , Park Dae Won
Published: June 2021

Cardiomyopathies, diseases of the heart, are one of the major causes of death in the United States, and thus there is great interest in preventing and treating these complications. However, due to the nature of limited availability of donors, many techniques and solutions are inadequate to meet the needs of the field. In particular, heart tissue transplantation, culturing human tissues/tissue‐derived cells and tissue engineering, and finding a suitable extracellular environment that closely resembles the natural environment of cardiomyocytes in vivo are difficult to attain. As such, a great deal of work has gone into efforts to produce polymers which mimic the natural cell environment in properties such as binding sites, stiffness, reactivity, and hydration. In our research, we are investigating the development and characterization of conductive polymer scaffolds providing cardiac tissue support, which will ideally aid in culturing cardiomyocytes for academic, research, and medical use. The core of our model is the incorporation of conductive gold nanorods into reverse thermal gel polymers. Synthesized gold nanorods utilized in this procedure are made with a high aspect ratio, at high purity and with defined surface functionalization. These conducting scaffolds should properly accommodate cardiac cells in building functional cardiac tissue constructs and further improving cell retention, spreading, cardiomyocytes long term survival and maturation, cell‐cell coupling and synchronized beating behavior at the tissue level. The nanoparticles have been synthesized, then covalently coupled into these scaffolds, resulting in order‐of‐magnitude increases in conductivity. The goal of this work aims to improve cardiac tissue engineering, so that it can be directed to ultimately repairing damaged heart muscle and improve overall cardiac function in cardiovascular diseases whether they have been acquired from past medical complications or developed through hereditary traits.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
iTraq‐based Proteomics analysis of colon mucosal proteins in a dextran sulfate sodium (DSS)‐induced colitis mouse model and the effects of dietary treatments with edible mushroom Pleurotus eryngii
Authors: YUAN BIAO , XU XIAO , HAN YANHUI , CAO XIAOQIONG , HU QIUHUI , HANG XIAO
Published: June 2021

Our previous studies have demonstrated the protective effects of an edible mushroom Pleurotus eryngii against the colitis in mice. Herein, an itraq‐based proteomics analysis has been utilized to establish the potential mechanism of actions of the mushroom. Three groups of mice (10 mice per group) were treated with DSS in drinking water (the colitic positive control group), DSS in drinking water plus Pleurotus eryngii (1.5% in diet, the treatment group), or regular drinking water (the negative control group), respectively, for 40 days as we described previously. The colon mucosa of mice were collected and subjected to an itraq‐based proteomic analysis. In total, 5794 proteins were identified by LC‐ESI‐MS/MS analysis in the colon mucosal of the mice. Among them, the expression levels of 626 proteins were different (392 up‐regulated proteins and 234 down‐regulated proteins) in the colitic positive control mice in comparison with the noncolitic negative control mice. There were 303 differentially expressed proteins (107 up‐regulated and 196 down‐regulated) in the treatment group compared to that of the positive control t group. The KEGG pathways that these differentially expressed proteins involved in were mainly: Steroid hormone, Arachidonic acid, Basal cell carcinoma, Linoleic acid metabolism, Phospholipase D signaling, viral myocarditis, chemical carcinogenesis, natural killer cell mediated, thyroid hormone signaling pathways. Twenty‐five proteins that were highly upregulated in the colitic positive control group (vs. the negative control group) were found significantly down‐regulated in the treatment group (vs. the colitic positive control group), whereas, twenty‐one proteins that were highly down‐regulated in the colitic positive control group (vs. the negative control group) were found significantly up‐regulated in the treatment group (vs. the colitic positive control group). Importantly, western blot analysis validated the changes in the protein expression described above. These results demonstrated that dietary mushroom treatment reversed, at least partially, the abnormal expression of proteins in the colon mucosa of colitic mice, which may be accounted for the protective effects of Pleurotus eryngii mushroom against pathogenesis of colitis in mice. Overall, our results provided preliminary but critical information on the potential mechanism of Pleurotus eryngii mushroom in inhibiting colitis.

Support or Funding Information

This research work was funded by the National Natural Science Foundation of China under Grant No. 31471927.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrated Therapeutic Dynamics by Applications of Multi‐Mathematical Functions in Multiple Degenerative Chronic Physiological Processes. A Case study
Authors: Einstein George P. , Tulp Orien L. , Konyk Carla M. , Kealoha Nicholas P.
Published: June 2021

Background

Most chronic diseases and conditions affect one or more organs. The degeneration process overlap in chronic diseases and is very difficult to treat. Nonetheless, 7 of every 10 Americans who die each year—more than 1.7 million people—succumb to a chronic disease. Health‐damaging behaviors, such as tobacco use, lack of physical activity, poor eating habits, and excessive alcohol use contribute to many chronic diseases, whereas others may represent the long‐term effect of early exposure to toxins and/or other environmental factors, especially in individuals with a higher genetic risk of disease. A shared aspect of many chronic diseases is chronic pain and other disease‐associated disability that interferes with quality of life. Both psychological factors and a general physiological medical condition are judged to have important roles in the onset, severity, exacerbation, or maintenance of the pain.

Materials and Methods

Patient suffering forty‐three years in chronic pain and countless medical diagnosis including COPD, Diabetes, MRSA, Irritable Bowel Syndrome, Fibromyalgia, Cronic Renal Failure, Ulcerative Cortices and Major Depressive Disorder. Patient was barely able to walk, with Multiple physio‐pathological processes of chronic diseases, and required a complex design to bring about a reversal therapeutic process. The Multi‐Mathematical Functions in Multiple Degenerative Chronic Physiological Processes (MMFPP) are represented by FN Specific Coherencies Therapeutic Functions and Specific Therapeutic (M} Matrices

Results

Brain activity was essential for Diagnostic Multilevel Physio Pathological processes. Integrative therapy by Specific Dynamic Matrixes of Sigma Quanta Energy by Biophysical‐Biochemical and Psycho‐neural Stimulation was effective in amelioration of symptoms.

Support or Funding Information

Supported by Institutional Resources of USAT Montserrat and the Einstein Medical Institute, NPB, FL

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Low pH Treatment on Cell Cycle and Cell Growth
Authors: Hu Yuli , Li Yang
Published: June 2021

Cancer growth affects the pH of microenvironment that becomes acidified and supports local invasion. Studies have reported that pH is a factor in cancer growth, division, and spread. Although cancer cells produce low extracellular pH (6.5–6.9), they are able to maintain their intracellular pH at favorable ranges (7.2–7.4). Our preliminary results showed that growth of cancer cells changes after low pH treatment. The objective of our research is to study how low pH affects cell morphology and behavior. In my preliminary experiments, HeLa cells were incubated with low pH solution for 10 min and harvested using trypsin into medium with normal pH. After 24 hours in culture, cell number was assessed using Trypan Blue Exclusion Assay. As a result, low pH treated group doubled 1.4 times; whereas control group doubled 2.3 times, suggesting a division latency in low pH treated group on Day 1. Cell morphology on Day 1 was recorded as images and was categorized into round, spindle, triangle, and irregular groups, among which spindle cells were likely to be at G1 phase. Percentage of the spindle cells gradually increased during 16–24 hours in experimental group; however, in control group, percentage of spindle cells decreased, suggesting that low pH had induced cell cycle arrest at G1 phase. In conclusion, low pH treatment affects cell growth potentially through G1 cell cycle arrest. Future work is to confirm cell cycle arrest at G1 phase after low pH treatment and to study the mechanism of low pH effect on cancer cell growth.

Support or Funding Information

Funding is provided partially by NIH (#NS081629) to YVL. Thanks to the Osteopathic Heritage Foundations, Graduate Assistantship Program at Ohio University Heritage College of Osteopathic Medicine for supporting graduate student YH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DMD‐10 and its Potential Effect on Regulation of the Glutamate Receptor GLR‐1 in C. elegans
Authors: Johnsky Lily Renee , McGehee Annette
Published: June 2021

An organism's nervous system processes decisions on how to respond to their environment. Studying how proteins of the nervous system are regulated enables us to identify how an organism responds to its environment and alters its behavior. In our lab, we study glutamate receptors, specifically GLR‐1. Glutamate is used for communication between neurons, and changes in glutamate receptors are believed to play an important role in learning and memory. The DAF‐7/TGF‐β signaling pathway regulates GLR‐1 in C. elegans and mutants in the DAF‐7/TGF‐β signaling pathway have increased GLR‐1. However, the mechanism through which GLR‐1 levels are increased in the daf‐7 mutant worms is unknown. My research addressed how GLR‐1 levels were increased in the daf‐7 worms. I explored the possibility that the transcription factor DMD‐10 is affected in the daf‐7 mutants, and then in turn affects GLR‐1. We tested this by measuring spontaneous reversals (a GLR‐1 dependent behavior) in both single mutant and double mutant worms. If DMD‐10 is affected in the daf‐7 mutants, we predict that the dmd‐10; daf‐7 double mutants would have fewer reversals than the daf‐7 mutant. The dmd‐10; daf‐7 double mutants did not have fewer spontaneous reversals than the single daf‐7 mutant, which suggests that the DAF‐7/TGF‐β signaling pathway does not use DMD‐10 to alter levels of GLR‐1. However, the dmd‐10 single mutant had fewer reversals than the wildtype, suggesting that DMD‐10 is required for normal reversals. The results of this experiment suggested that although DMD‐10 is not involved in DAF‐7/TGF‐β regulation of GLR‐1, it may have an independent effect on GLR‐1. My current research is aimed at determining if DMD‐10 is involved in the regulation of GLR‐1. I have generated a strain of dmd‐10 mutant worms that expresses GFP‐tagged GLR‐1 (GLR‐1::GFP). Imaging of GLR‐1::GFP in wild type and dmd‐10 mutant worms will allow us to test if DMD‐10 is involved in GLR‐1 regulation. This strain is being analyzed for total levels of GLR‐1::GFP using western blotting and will indicate if DMD‐10 is reducing total levels of GLR‐1. Synaptic levels of GLR‐1::GFP will be analyzed using fluorescence imaging that provides information on the location of GLR‐1 and whether it is localized in the cell body or synapses. Together, these results will indicate if DMD‐10 is affecting GLR‐1 and will give us insight into how regulation is occurring.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Expression of Piccolo Splice Isoforms During Cerebellar Development
Authors: Fenster Steven Daniel
Published: June 2021

Piccolo is a multi‐domain protein with restricted expression to the presynaptic active zone, the site of synaptic vesicle fusion and neurotransmitter release. Alternative splicing of the Piccolo gene (PCLO) results in two primary splice isoforms: isoform‐1 (I‐1) with two C2 domains (C2A and C2B) and isoform‐2 (I‐2) with only a single C2A motif. In addition, an exon‐skipping event generates Piccolo isoforms that alter the C2A domain resulting in exclusion of a 27‐nucleotide exon located in the C2A domain (e.g. short variant). The result is a lack of 9 amino acids in the domain resulting in increased calcium and phospholipid binding affinity versus the longer C2A domain (e.g. long variant) that includes the 9‐peptide region. Consistent with the importance of the Ca2+‐binding property of C2 domain‐containing proteins in synaptic function, genome‐wide association studies have identified variations in or near the C2A domain of human PCLO that predispose individuals to affective disorders. The most compelling evidence for the importance of the C2A domain comes from recent identification of the genetic cause of pontocerebellar hypoplasia type III (PCH3): a neurological disorder that results in severe underdevelopment of the cerebellum. Analysis of genomic DNA from two affected individuals with PCH3 identified a homozygous nonsense variant in PCLO that introduces a STOP codon in the 3′ coding region including the C2A domain. The purpose of this study was to examine the temporal expression of Piccolo splice variants during critical periods of mouse cerebellar and cortical development. We performed real‐time quantitative RT‐PCR gene expression analysis of Piccolo splice isoforms (e.g. I‐1, I‐2, long and short) from RNA isolated from cerebellum at key stages of cerebellar granule cell differentiation (P0, P5, P15, adult) and also from the cortex at the same ages. Our analysis demonstrated that expression levels of the I‐1 and I‐2 splice variants were relatively consistent between cerebellum and cortex at all examined developmental time points. In contrast, we showed that during initial stages of cerebellar development (P0 and P5), the more calcium‐sensitive short variant had significantly increased expression levels relative to the long variant. Our results indicate that increased expression of the short variant may play a role in neuronal proliferation during initial cerebellar development when considering the augmented calcium permeability of immature neurons during this time period.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Suppression and inhibition of Acetylcholinesterase (AChE) Gene Expression and Adenosine Deaminase (ADA) respectively in Cadmium Treated Rats by curcumin administration
Authors: Fadaka Adewale Oluwaseun , Akinyemi Ayodele J. , Adewale Olusola Bolaji , Olayide Israel Robinson , Onikanni Amos , Olaoye Oyindamola Adeniyi , Okoh Patrick Osemeyekeh
Published: June 2021

Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes have been reported to exert anticholinesterase and cognitive enhancing potentials with limited information on their scientific basis and how they regulate gene expression.

This study sought to evaluate the in vitro effect of curcumin on cerebral cortex acetylcholinesterase (AChE) activity, their mRNA gene expression level and adenosine deaminase (ADA) in cadmium (Cd)‐treated rats. Animals were divided into six groups (n=6): group 1 [(control) received saline/vehicle], group 2 [saline plus curcumin (25 mg/kg)], group 3 [saline plus curcumin (50 mg/kg)], group 4 [Cd (2.5 mg/kg) plus vehicle], group 5 [Cd (2.5 mg/kg) plus curcumin (25 mg/kg)] and group 6 [Cd (2.5 mg/kg) plus curcumin (50 mg/kg)] by oral gavage for 7 days. Acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities were determined and AChE expression was analyzed out using real time polymerase chain reaction (RT‐qPCR).

The results of this study revealed that ADA activity was increased in Cd‐poisoned rats, and curcumin co‐treatment reversed this effect comparable to the control. Also, it was observed that acute administration of Cd increased AChE activity and caused a significant (P<0.05) increase in AChE mRNA levels in whole cerebral cortex when compared to control group. However, co‐treatment with curcumin inhibited AChE activity and alters AChE mRNA levels when compared to Cd‐treated rats. Furthermore, Cd intoxication increased the level of lipid peroxidation in cerebral cortex with a concomitant decreased in functional sulfhydryl (−SH) group and nitric oxide (NO). However, the co‐treatment with curcumin at 25 and 50 mg/kg, respectively increased the non‐enzymatic antioxidant status and NO in cerebral cortex with a decreased in malondialdehyde (MDA) level.

In conclusion, inhibition of AChE and ADA activities, suppression of AChE mRNA gene expression level in Cd exposed rats, as well as increased antioxidant status by curcumin in Cd‐induced memory dysfunction could suggest some possible mechanism of action for their cognitive enhancing properties, thus providing some biochemical and molecular evidence on the therapeutic effect of this turmeric‐derived compound in treating neurological disorders including Alzheimer's disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.