bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Vitamin D and Serotonin Pathway Variations in Patients with Irritable Bowel Syndrome
Authors: Dussik Christopher M , Grozić Aleksandra , Hockley Maryam , Zhang Lin , Park Jin , Wang Jie , Nickerson Cheryl , Yale Steven H , Foxx‐Orenstein Amy , Sandrin Todd , Jurutka Peter 
Published: November 2023

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with chronic abdominal pain along with recurrent diarrhea, constipation, or both that afflicts millions of people worldwide. Currently, the etiological foundation of the disorder remains enigmatic. However, there is mounting evidence suggesting that serotonin may play a critical role in the pathophysiology of IBS. DNA microarrays analyzing the transcriptomes of 11 IBS patients versus 13 asymptomatic control patients have identified a series of 858 differentially‐expressed genes that may characterize the IBS pathological state. Gene ontology (GO) analysis of this subset of genes revealed that nodes featuring serotonin‐related pathways displayed the highest number of functions relative to other GO groups. Quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis of IBS patient‐derived RNA displayed reduced expression of the rate‐limiting enzyme of the serotonin biosynthetic pathway, tryptophan hydroxylase 1 (TPH1), relative to a pool of RNA comprised of eight control patients. Further, this analysis revealed potential compensatory regulation of several other genes integral to the maintenance of serotonin metabolism. In addition, concentrations of serum 25‐hydroxyvitamin D in both the IBS and control sampled populations indicated a trend towards decreased vitamin D levels within the IBS cohort, regardless of IBS subtype. Finally, vitamin D was shown to be effective in regulating the expression of several key serotonin metabolism genes, alluding to a potential link between vitamin D and serotonin pathways modulating IBS. These findings suggest that the pathogenesis of IBS may be associated with abnormal serotonin metabolism and/or vitamin D deficiency, and provide novel insight into possible treatment regimes.Support or Funding InformationWisconsin Network for Health Research
Large scale structural rearrangement provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase
Authors: Johnson R. Jeremy , Smith Mackenzie , Hart Whitney 
Published: November 2023

The serine hydrolase FTT258 from the highly pathogenic bacterium Francisella tularensis exists in distinct open and closed conformations. Based on comparison to the homologous human acyl protein thioesterase, this large structural rearrangement was proposed to provide connected control over the catalytic and membrane binding activity of FTT258. Using comprehensive mutagenesis and differential kinetic and membrane‐binding measurements, we determined the contribution of a key flexible loop to controlling the structural rearrangement and biological activity of FTT258. For the catalytic activity, a centrally located tryptophan residue (Trp66) was deemed essential, with the resulting alanine variant showing complete ablation of enzyme activity. Other amino acids localized near this essential tryptophan residue, including Met63, Arg64, Tyr67, and Asp68, also significantly decreased the hydrolase activity, indicating a critical target area for controlling the enzyme activity and structural rearrangement of FTT258. Combinatorial variants containing the Trp66 substitution with other important residues showed enzymatic activity similar to the Trp66 variant alone, suggesting a controlling role for this residue. Liposome‐binding experiments with the tryptophan variants did not however show significant changes in membrane binding activity. Instead, removal of a proximal positively‐charged arginine residue (Arg64) significantly decreased the membrane binding activity of FTT258. Together, the conformational change in FTT258 dually controlled the catalytic and membrane binding activity of FTT258, but through distinct subsections of a key flexible loop.Support or Funding InformationFunding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.
Dynamic Equilibria Studies of FTT258, a Serine Hydrolase from Francisella tularensis
Authors: Rabin Perry , Johnson R. Jeremy 
Published: November 2023

Francisella tularensis is a highly infectious bacterium and the causative agent of tularemia. In this project, we investigated two different structural forms of FTT258, a serine hydrolase from F. tularensis and a potential novel drug target. Specifically, the goal of my project was to determine the role of the active site of FTT258 in controlling the equilibria between its inactive and active forms. Substitutional analysis combined with size exclusion chromatography was used to analyze the importance of amino acids in the active site and in a flexible loop region of FTT258 to controlling this equilibria. Although amino acids within the flexible loop were found to not control the structural equilibria of FTT258, deletion of the catalytic serine active site disrupted the transition between the inactive and active forms of FTT258. Furthermore, the structural composition of FTT258 was found to be highly concentration dependent. These experiments confirmed the essential role of the active site and catalytic amino acids in controlling the structural shifts in FTT258. Understanding this regulation will allow for new methodology in purifying the different forms of FTT258 and analyzing the potential of FTT258 as a novel drug target.Support or Funding InformationFunding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.
DISSOCIATION CONSTANTS FOR REVERSIBLE HOMODIMERS OF RECOMBINANT MONOMERIC HUMAN ACETYLCHOLINESTERASE
Authors: RADIC ZORAN 
Published: November 2023

Size exclusion chromatography profiles of highly purified monomeric recombinant human acetylcholinesterase (hAChE; EC 3.1.1.7) at micro molar concentration range reveal elution volumes smaller than expected for a monomeric globular protein comparable in size to the bovine serum albumin. Dilution of hAChE to the nano molar concentration range results in the elution peak shifts towards larger volumes indicating formation of hAChE monomers. This observation is consistent with small angle X‐ray scattering (SAXS) experiments on hAChE that indicate formation of reversible homodimers of the micro molar monomeric recombinant enzyme in solution (Blumenthal et al., EB2016 abstract).FPLC chromatography of a series of concentrations of recombinant monomeric hAChE in the range of 0.00001 – 1 mg/ml (150 pM‐15uM) was performed to assess concentration dependent elution shifts and determine dimer dissociation constants in 50 mM Tris/HCl, 100 mM NaCl, pH 7.4 at 4 ºC. Peaks of enzyme activity in FPLC elution fractions were detected using Ellman spectrophotometric activity assay. Wild type hAChE constructs with and without N‐terminal FLAG tag sequence as well as Tyr337Ala/Phe338Ala double mutant hAChE not containing FLAG tag were assayed. Size of the hAChE active center gorge in the choline binding domain was increased in this double mutant. Corresponding dimer dissociation constants were determined to be in the range of 1 – 2 uM for both wild type and mutant hAChE, consistent with hAChE oligomerisation interface residing outside of the active center gorge opening as indicated by crystallographic dimers of hAChE.Support or Funding InformationThis work was supported by the Counter ACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.
OVERLAY INDEPENDENT COMPARISON OF ACETYLCHOLINESTERASE 3D STRUCTURES REVEALS LIGAND SPECIFIC BACKBONE CHANGES UPON REVERSIBLE AND COVALENT BINDING
Authors: RADIC ZORAN , Rohrer Jacqueline , Sidhom Morgana , Han Jingting 
Published: November 2023

We developed a simple overlay independent comparison of protein 3D structure alpha carbon backbones and applied it to PDB deposited structures of acetylcholinesterase (AChE; EC 3.1.1.7) in order to identify backbone domains amenable to ligand binding associated conformational flexibility. Distance of each alpha carbon in the structure to a frame of reference point was determined first. We then calculated differences in corresponding distances between two different PDB structures and plotted as a function of amino acid number, or color coded and mapped onto ribbon or surface representation of an AChE structure. To reduce the influence of crystal packing we averaged corresponding distances determined in individual chains of crystallographic dimers before comparing different structures. We were able to observe systematic, previously undetected, conformational changes upon binding of tight and weak reversible ligands as well as covalent conjugates, phosphonylated and aged. Analogous structures of AChE from different species, human mouse and fish were compared revealing consistent conformational changes. Finally, our simplified comparative 3D structure analysis was contrasted to the sophisticated error‐scaled difference distance matrix analysis.Modest to moderate fluctuations of AChE backbone conformation upon complex or adduct formation may remain undetected in classical overlay analysis. Small, but systematic conformational changes in protein structures determined at low, 100 K temperature conditions, may prove as an important indicator of larger conformational changes at physiological conditions relevant for structure based design of modern therapeutics.
Structural, Biochemical Characterization, and Evolutionary Origin of Alcohol/Aldehyde Dehydrogenases (ADHE) in Entamoeba
Authors: Leitao Joshua , Espinosa Avelina 
Published: November 2023

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are essential for anaerobic respiration in protists and bacteria (i.e. group‐A‐Streptococcus, Entamoeba histolytica, Pasteurella multocida, Escherichia coli, Clostridium perfingens, Clostridium difficile). Structural and biochemical analysis of ADHE enzymes would contribute to the understanding of their evolution in prokaryotic and eukaryotic lineages. We have previously characterized the bifunctional Entamoeba histolytica alcohol/aldehyde dehydrogenase (EhADH2). Since EhADH2 is required for amoebic survival, it is an ideal target for the search of antiamoebic alternatives. Here we elucidate the ADHE structure and function of six glycolytic proteins from six Entamoeba varieties (isolated, cloned and expressed from E. dispar, E. invadens‐IP‐1, E. invadens VK, E. moshkovskii Snake, E. moshkovskii Laredo, and E. terrapinae). Alcohol and aldehyde dehydrogenase activities from each ADHE enzyme were analyzed. Considering that the evolutionary origin of ADHE enzymes appears to be from diverse bacterial lineages, the purpose of this study is to examine the kinetic properties and efficiency of Entamoeba ADHEs. Concurrently, inhibitory studies using pyrazoline derivatives on Entamoeba growth by targeting its EhADH2 enzyme are being pursued in our laboratory as a novel anti‐amoebic strategy.Support or Funding InformationAmerican Society for Microbiology (ASM) Undergraduate Research Fellowship
Biodiesel Production via Genetically Altered Lipase from Proteus mirabilis Transesterification in Methyl Acetate
Authors: McDonnell Alaina Nicole , Altier Jake , Carey Demetrius , Witherow D. Scott 
Published: November 2023

Lipase catalyzed biodiesel production presents a potential methodology that exceeds the traditionally utilized acid/base protocols. Advantages include economic favorability and convenience due to more efficient catalysis, which leads to less time, starting material, and energy required for production. A lipase has been previously genetically engineered for methanol tolerance and was shown to be an effective catalysis for transesterification reactions utilizing methanol to produce fatty acid methyl esters, or biodiesel, and glycerol. An alternate acyl acceptor to methanol, methyl acetate, produces a fuel stabilizer, triacetin, as opposed to glycerol when used in the transesterification. This methodology has not been previously utilized with the characterization of these genetically engineered lipases, which are also described as dieselzymes. Dieselzyme mutations have been expressed in E.coli using a pET28 bacterial expression vector and purified utilizing immobilized metal ion affinity chromatography. Kinetic optimization was performed for three mutants using p‐nitrophenyl palmitate as substrate at varying pHs and in methyl acetate versus methanol reactions. Further investigation of the dieselzymes includes use of synthetic triglycerides as substrate to continue kinetic optimization.Support or Funding InformationUniversity of Tampa Department of Biochemistry, Chemistry and Physics Summer Fellowship; University of Tampa Honors Program Fellowship; University of Tampa David Delo Research Professor Grant; University of Tampa Dana Foundation Grant
Antiseptic mouthwash use and effect on resting metabolic rate and blood pressure among healthy females: a randomized, double‐blind, crossover study
Authors: Sundqvist Michaela Linn , Lundberg Jon , Weitzberg Eddie 
Published: November 2023

BACKGROUNDInorganic nitrate, from dietary or endogenous sources can be metabolized in vivo to generate bio‐active nitrogen oxides including nitric oxide (NO) with robust NO‐like cardiovascular and metabolic effects. The bio‐activation of nitrate involves its active accumulation in saliva followed by a sequential reduction to nitrite, nitric oxide, and other reactive nitrogen species. Commensal oral bacteria have a key role in nitrate bio‐activation since they reduce salivary nitrate to the more reactive nitrite anion.A recent study demonstrates that resting metabolic rate (RMR) decreases significantly after dietary supplementation with nitrate. This effect is likely caused by bio‐active nitrogen oxides directly interacting with mitochondria to affect proton leak and other parameters. It is unclear however if endogenous nitrate, derived mainly from the oxidation of NO in blood and tissues, also has this effect. We recently showed that blocking oral bacterial conversion of nitrate to nitrite with an antiseptic mouthwash, leads to a reduction in salivary and plasma nitrite levels together with a concomitant increase in blood pressure. Such effect indicates that endogenous nitrate is sufficient to have physiological effects after reduction to nitrite and then NO.OBJECTIVEHere we studied the effects of an antiseptic mouthwash on nitrate and nitrite kinetics and RMR in humans.DESIGNIn a randomized, double‐blind, crossover study, we measured the RMR using indirect calorimetry in 17 healthy young females after a 3‐d intervention with a chlorhexidine‐containing antiseptic mouthwash. The subjects were instructed to use antiseptic mouthwash or placebo 3 times a day after meals. We measured 24h ambulatory blood pressure (ABP) and collected saliva‐ and blood samples in the end of the intervention period.RESULTSSaliva nitrate levels were significantly increased after mouthwash compared to placebo (mouthwash: 1443 ± 262 μmol/L; placebo: 652 ± 199 μmol/L, p = 0.002), but nitrate and nitrite levels in plasma showed no significant changes. There was no significant increase in RMR after mouthwash compared to placebo (p = 0.982) and saliva nitrate did not correlate with RMR. The 3‐d use of antiseptic mouthwash did not significantly change ABP.CONCLUSIONSDifferently from a recent study, the use of an antiseptic mouthwash did not affect circulating nitrite levels in this group of healthy young females, despite markedly reducing salivary conversion of nitrate to nitrite. Similarly, RMR was not affected by this intervention. Further studies are needed to clarify the role of endogenous nitrate, derived from NOS activity, in regulation of NO homeostasis in blood and tissues.
Anthocyanins Improved Mitochondrial Dysfunction in Mice of Non‐alcoholic Fatty Liver Disease Induced by High Fat Diet
Authors: Ling Wenhua , Shen Tianran , Tang Xilan , Jiang Xinwei 
Published: November 2023

Recent studies indicate that mitochondrial dysfunction plays a critical role in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD. The goal of this study is to investigate whether anthocyanins (ACNs) from bilberries and blackcurrants possess a protective effect on mitochondrial dysfunction and eventually contribute to attenuation of NAFLD in mice induced by feeding a high fat (HF) diet. Twenty‐four male C57BL6/J mice were randomly divided into three groups with different diets respectively for sixteen weeks: the control group was fed a low‐fat diet and the other two groups were fed a HF diet with ACNs or not. Our results illustrated that ACNs administration at a dosage of 100 mg in 1 kg diet significantly inhibited HF diet‐induced hepatic steatosis and inflammatory cell infiltration. In addition, increase of mitochondrial swelling, decrease of mitochondrial content and damage of mitochondrial biogenesis, oxidative phosphorylation and β‐oxidation were observed in mice fed with a HF diet, which were significantly normalized through ACNs supplementation. This appears to be attributed to activation of phosphorylated AMP‐activated protein kinase (p‐AMPK) and its downstream peroxisome proliferator‐activated receptor‐gamma coactivator‐1α (PGC‐1α). Our work presents the beneficial effects of ACNs on mitochondrial dysfunction in HF‐induced NAFLD mice by promoting activation of AMPK/PGC1α signaling axis, signifying that ACNs may be a useful nutrient for the treatment of NAFLD.All animal procedures were approved by the Animal Care and Protection Committee of Sun Yat‐Sen University (2013–10).Support or Funding InformationThis research was funded by grants from the National Basic Research Program (973 Program, 2012CB517506) and the National Nature Science Foundation (81172655, 81372994).
Whole Ethanolic Moringa Oleifera Leaf Extract Generates Kinetic Growth Curves of Escherichia coli Comparable to Pharmaceuticals
Authors: Smith Brandon , Orders Tanner , Tallapaneni Jetha , Nair Srijith , Ravi Vivek , Emrani Jahangir , Idassi Joshua , King James , Zwetsloot Kevin A. , Mowa Chishimba 
Published: November 2023

Premature births are largely caused by preterm infections. Here, we report the anti‐bacterial activities of the medicinal plant Moringa oleifera (M. oleifera) against Escherichia coli (E. coli), one of the main causes implicated in infections associated with preterm labor. We screened different whole leaf extracts of M. oleifera for anti‐bacterial activities and determined its effects on the overall growth using in vitro kinetic curve liquid broth assays. In our assays, we compared the anti‐bacterial potency of the M. oleifera leaf extracts to commonly utilized antibiotics, such as tetracycline, ampicillin and streptomycin. The pure ethanol leaf extract showed the most anti‐bacterial activity at 20 μg against E. coli, with ethanol and ethanol/water extracts acting in a concentration‐dependent manner, and showed the greatest kinetic change at 6 μg against E. coli and completely inhibited growth, followed by tetracycline at 6 μg compared to control. This study confirms the anti‐bacterial properties of M. oleifera and its potential use as a natural anti‐bacterial agent for the management and prevention of infection‐induced preterm labor.Support or Funding InformationOffice of Student Research, Appalachian State University.
Structurally Diverse Positive Allosteric Modulators of the D1 Dopamine Receptor Potentiate G‐Protein and β‐Arrestin‐Mediated Signaling
Authors: Luderman Kathryn D. , Conroy Jennie L. , Free R Benjamin , Southall Noel T. , Ferrer Marc , Aubé Jeffrey , Frankowski Kevin J. , Sibley David R. 
Published: November 2023

The D1 dopamine receptor (D1R) is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition. Agents that enhance D1R signaling in the prefrontal cortex may be useful in the treatment of cognitive decline in schizophrenia, Alzheimer's disease and other disorders. Unfortunately, orthosteric D1R agonists have proved problematic as they frequently induce hypotension and exhibit a narrow therapeutic window. An alternative approach is to potentiate D1R activity using allosteric modulation. Hypothetically, a positive allosteric modulator of the D1R may exhibit highly selective actions and exhibit a larger therapeutic window. In order to identify allosteric ligands of the D1R, we implemented a high throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library. A D1R‐G15 calcium mobilization assay was used to identify library compounds that potentiated an EC20 concentration of dopamine. Initial hit compounds were characterized using endogenous G‐protein (cAMP stimulation) and ®‐arrestin (recruitment) signaling pathways, and parental cell lines were used as negative controls. A pool of compounds that exhibited positive allosteric modulation (PAM) of the D1R were identified and further counter‐screened against other dopamine receptor subtypes to establish subtype selectivity. Two structurally diverse compounds, MLS6585 and MLS1082, with PAM activity at the D1R were selected as lead compounds. These compounds potentiate both G‐protein and ®‐arrestin‐mediated D1R signaling, increasing the potency of dopamine by 5–10 fold for stimulating cAMP accumulation and 4–8 fold and for β‐arrestin recruitment. Both compounds also increased the maximum dopamine‐stimulated responses by 25–30%. Neither compound displayed any intrinsic agonist activity in either assay. Further, the two compounds displayed minimal to no ability to inhibit radioligand binding to the orthosteric‐binding site of the D1R. Both compounds potentiated the agonist activity of dopamine, as well as other full and partial agonists, in a dose‐dependent manner with EC50 values in the micromolar range. Importantly, they showed no ability to potentiate forskolin‐stimulated cAMP accumulation, or β‐arrestin recruitment by other receptor subtypes. MLS1082 and MLS6585 may serve as important scaffolds for the future development of optimized D1R PAMs for in vivo use and the discovery of therapeutic lead compounds.Support or Funding InformationNINDS Intramural Research Program