bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of an Adenosine Receptor Bitopic Ligand on The Cardiovascular System
Authors: Cooper Samantha Louise , Wragg Edward , March Julie , Hill Stephen , Woolard Jeanette 
Published: April 2020

Adenosine regulates physiological and pathophysiological processes via the activation of four G‐protein coupled receptors identified as the A1, A2A, A2B and A3 receptors. All of these receptors are known to be involved in regulation of the cardiovascular system
[1]. Recently, we described the effects of A1‐receptor selective agonists and the novel A1‐receptor bitopic ligand VCP746 on the rat cardiovascular system
[2]. Here, we assess the haemodynamic responses of VCP746 in the presence and absence of selective A2A and A2B‐receptor antagonists (SCH58261 and PSB1115, respectively).As described previously
[3], under anaesthetic, rats were instrumented with miniature pulsed Doppler flow probes for the measurement of vascular conductance at the renal, mesenteric and hindquarters vascular beds. Intra‐arterial and intra‐venous catheters were implanted for measurement of heart rate (HR) and mean arterial pressure (MAP), and drug administration, respectively. All procedures were carried out with approval of the University of Nottingham Animal Welfare Ethical Review Board, under Home Office Project and Personal License Authority.Experimental studies were conducted in conscious, freely‐moving rats. All compounds were prepared in a propylene glycol (PEG) buffer (5% PEG, 2% Tween, 0.9% saline). A 0.1ml bolus of SCH58261 (1.0 mg/kg), PSB1115 (10mg/kg) or vehicle was administered 10 minutes prior to increasing doses of VCP746 (6, 20, 60 μg/kg/min). Doses of VCP746 were administered as 3 minute infusions over a total of 9 minutes at an infusion rate of 0.1 ml/min.VCP746 produced tachycardic effects, which were associated with renal and mesenteric vasodilatations (Figure). There was little effect in the hindquarter vascular bed and MAP was not significantly affected (Figure). The haemodynamic effects of VCP746 were partially antagonised by SCH58261 and PSB1115 fully attenuated haemodynamic responses to VCP746.This study suggests that VCP746, the A1‐receptor bitopic ligand, mediates haemodynamic responses through the activation of the A2‐receptors, particularly the A2B‐receptor, rather than the A1‐receptor. This has implications for the design of bitopic ligands in the future.Support or Funding InformationWe would like to thank the BHF and COMPARE for funding.Cardiovascular responses to VCP746. Rats were dosed with either PSB1115 (0.1 ml bolus dose of 10 mg/kg i.v., n=8) or vehicle (0.1 ml bolus PEG buffer, n=8). Approximately 10 min later, all animals received an infusion of VCP746 (6, 20, and 60 μg/kg/min; each dose infused over 3 minutes). Data presented as mean ± SEM. *P < 0.05 versus baseline (Friedman test). A Wilcoxon test for integrated area under or above curve analysis was used for comparisons between groups (♯ P<0.05).Figure 1
SPARC: SODA, an interactive software for curating SPARC datasets
Authors: Patel Bhavesh , Srivastava Harshit , Aghasafari Parya , Helmer Karl 
Published: April 2020

In this era of data‐intensive research, data sharing and reuse are becoming increasingly important for rapid discovery and innovation. While sharing can be as easy as a few clicks thanks to numerous data sharing platforms, data reuse is still an issue as standards for data curation and annotation are not rigorously or consistently followed by all researchers. As a result, it is often difficult, if not impossible, for researchers to understand and work with data they were not involved with during collection or analysis. To address this concern, the NIH SPARC program has imposed strict guidelines for curating and sharing data resulting from SPARC‐funded research. Complying with the guidelines requires additional time investment from the researchers (as with any data curation and submission standards), and the data curation process could thus become overwhelming as more and more data is generated. The objective of this project was to develop computer software to improve the curation workflow for SPARC researchers.To achieve that, we are developing a software named SODA (Software for Organizing Data Automatically) to assist SPARC investigators in curating and annotating their datasets. Distributed as an open‐source desktop application (Windows, MAC, Linux), the goal of SODA is to bridge a long‐standing and overlooked gap between comprehensive data standards and their convenient application by researchers. SODA provides an interactive interface that, without requiring any coding knowledge, walks SPARC investigators step‐by‐step through the data organization and sharing requirements, while automating repetitive, complex and/or time‐consuming tasks (Fig
1). An introduction to the software and the algorithms implemented will be given. Quantitative results from ongoing performance evaluation (time to curate a SPARC dataset with and without SODA) by beta testers, all of whom receive funding from the SPARC program, will be presented as well.Overall, the beta testers for early versions have acknowledged that SODA has the potential to improve their curation workflow. Data curation is becoming a critical issue, even beyond the SPARC consortium, and SODA could impact the broader research community by providing an exemplar and foundational tool for convenient and time‐efficient curation of data, which could be adopted by other projects. Potential applications, beyond the SPARC program, will be thus discussed as well.Support or Funding InformationSPARC OT2OD025308‐01S2Overview of the major features included in SODA during the on‐going development phaseFigure 1
Molecular Basis of 14‐3‐3 Protein Dependent Regulation of Caspase‐2
Authors: Obsilova Veronika , Kalabova Dana , Filandr Frantisek , Alblova Miroslava , Petrvalska Olivia , Horvath Matej , Man Petr , Obsil Tomas 
Published: April 2020

Among all species, caspase‐2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164, within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14‐3‐3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14‐3‐3‐dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14‐3‐3 by hydrogen/deuterium mass spectrometry (HDX‐MS) and protein crystallography to determine the molecular basis for 14‐3‐3‐mediated inhibition of C2 activation. Our data reveal that the 14‐3‐3 dimer interacts with proC2 not only through ligand‐binding grooves but also through other regions outside the central channel, thus explaining the isoform‐dependent specificity of 14‐3‐3 protein binding to proC2 and the substantially higher binding affinity of 14‐3‐3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14‐3‐3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14‐3‐3 protein interacts with and masks both the nuclear localization sequence (NLS) and the C‐terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14‐3‐3. This masked region of p12 domain is involved in caspase‐2 dimerization. Therefore, 14‐3‐3 protein likely inhibits proC2 activation by blocking its dimerization surface.Support or Funding InformationFundingThis study was funded by the Czech Science Foundation (V.O., grant number 19‐00121S), the Czech Academy of Sciences (RVO:67985823 of the Institute of Physiology), project BIOCEV (CZ.1.05/1.1.00.02.0109 and LQ1604) and MEYS CR (LM2015043 CIISB, Biocev, Biophysical methods, Structural Mass Spectrometry).Crystal structure of the caspase‐2 peptide pS139+pS164 bound to the 14‐3‐3 dimer.Figure 1
Ca2+ signalling is important for loss of keratinocyte adhesion in pemphigus vulgaris
Authors: Schmitt Thomas , Waschke Jens 
Published: April 2020

Pemphigus vulgaris (PV) is a severe blistering skin disease caused by autoantibodies (PV‐IgG) against desmogleins (Dsg)1 and 3. Ca2+ release in response to PV‐IgG has been reported. However, its contribution to loss of keratinocyte adhesion is largely unknown. Phosphatidylinositol‐4‐kinase‐a (PI4K) and phospholipase‐C‐γ1 (PLC) release inositol‐3‐phos‐phate (IP3) activating the IP3‐receptor (IP3R) and thereby causing Ca2+ flux from the endoplasmic reticulum (ER) into the cytoplasm. Low Ca2+ in the ER activates the Ca2+ release‐activated‐channel (CRAC) to replenish the Ca2+ store. Inhibitors against PI4K, PLC, IP3R or CRAC were applied under conditions under which they effectively blocked Ca2+ influx. All inhibitors ameliorated PV‐IgG‐induced alterations of Dsg1 and Dsg3 localization, keratin filament retraction and loss of cell adhesion in dissociation assays. Inhibition of PLC completely abolished PV‐IgG‐induced and also ameliorated AK23‐mediated loss of adhesion. These data demonstrate that Ca2+ signalling is important for loss of cell adhesion in PV but independent from antibodies against Dsg3 supporting the hypothesis that autoantibodies engage different signalling mechanisms to reduce cell adhesion.Support or Funding InformationFunded by the DFGCa‐dependendent interacellular signalling in pemphigus vulgaris. Black arrows indicate calcium‐flux.Figure 1
3D Kinetics, an Upgrade to the Classical Michaelis‐Menten Method
Authors: Abbasi Armina , Jones Jeff 
Published: April 2020

Most of the kinetic data published nowadays are derived from different extensions of what Michaelis and Menten introduced to this field more than hundred years ago. The key assumption to Michaelis‐Menten kinetics is the steady state approximation where the change in the concentration of any enzyme‐substrate intermediate complex is assumed to be zero. This assumption has two underlying requirements with the first one being [S]≫[E] and the second being the limitation of the reaction time to the short initial linear region of the progress curve. The historical need for the steady state assumption was to simplify data treatment to such a degree that the analytical mathematics could be done without need for an abundance of computing power. However, with the computation power at our disposal today, none of the limiting assumptions are necessary anymore especially considering that many of these assumptions rarely hold true. The time course of aldehyde oxidase with one of it substrates, O6‐benzylguanine, is provided here as an example of the statement above. In addition to the obvious instant curvature in the time course plot which makes it difficult to identify the precise linear region, the simulation done on the ES complex based on the data further proves how the steady state approximation is a poor assumption to make especially during the shorter “linear” region of the progress curve as this region is the one in which the concentration of ES is changing the most (Figure
1). The easiest way to approach kinetic data today is to parameterize realistically complex kinetic reaction mechanisms through 3D kinetics. 3D numerical modeling uses the data derived from experiments that monitor the product formation as a function of both incubation time and substrate concentration. With this approach, scientists would be able to derive both the traditional Km and Vmax values as well as the micro‐rate constants related to each step of the enzymatic reaction (Figure
2).Support or Funding InformationThis work was supported by the National Institutes of Health grant: GM100874A‐1) Numerical fitting of product formation with two different models (A‐2 and A‐3) shows that Michaelis Menten (dashed line) does not fit the data well over longer period of time. B) Simulation of the ES complex through the A‐3 model shows a significant change in this species’s concentration over the initial period of the time course where it is assumed to be a constant in deriving the Michaelis‐Menten equation.Figure 1A) Numerical fitting of the product formation over time of different initial substrate concentrations with the kinetic model provided on the right side of the graph to solve for the micro‐rate constants k3‐k5. B) Classical kinetic parameters such as Km and Vmax can be obtained from the same data set by plotting the rate versus substrate concentration for each time point.Figure 2
Inhibition of Major Cytochrome P450 Enzyme Activities in Human Liver Microsomes by 9‐a and WES‐1, two Novel Carbonic Anhydrase Inhibitors.
Authors: Nguyn Van , Espiritu Michael , Eldehna Wagdy , Abdelrahman Mohamed , Abdel-Aziz Hatem , Abou-Serid Sahar , Elbarbry Fawzy 
Published: April 2020

Both 9‐a (sulphonamide derivative) and WES‐1 (coumarin derivative) were designed and synthesized as potential selective Carbonic Anhydrase Inhibitors, and are currently being tested for anti‐cancer activity. This study was undertaken to investigate their potential inhibitory effects on the major Cytochrome P450 (CYP) drug metabolizing enzymes in human liver microsomes using specific CYP probe substrates and liquid chromatography‐tandem mass spectrometry. 9a potently inhibited CYP2E1‐catalyzed chlorzoxazone‐6′‐hydrolylation and CYP2C9‐catalyzed tolbutamide‐4′‐hydrolylation with IC50 values of 0.05 and 0.7 μM, respectively. CYP2A6‐catalyzed coumarin‐7′‐hydroxylation and CYP3A4‐catalyzed testosterone‐6β‐hydroxylation were moderately inhibited by 9‐a with IC50 values of 2.7 and 12 μM, respectively. The rest of the tested CYP enzymes were weakly or negligibly inhibited by 9‐a. WES‐1, on the other hand, demonstrated weak inhibitory effects on CYP2C19‐mediated [S]‐mephenytoin‐4′‐hydrolylation, CYP2C9 activity, and CYP2D6‐catalyzed Dextromethorphan dealkylation with IC50 values of 39, 42.5, and 57.5 μM, respectively. This in‐vitro data indicates that more in vivo studies should be conducted to examine the potential pharmacokinetic drug‐drug interactions with 9‐a due to its potent and moderate inhibition of CYP2A6, CYP2C9, CYP2E1, and CYP3A4.Inhibitory effect of WES‐1 on the major CYP enzymes in human liver microsomesFigure 1Inhibitory effect of 9a on the major CYP enzymes in human liver microsomesFigure 2
Flos Magnoliae and Its Constituent Linoleic Acid suppress T Lymphocyte Activation via Store‐Operated Calcium Entry.
Authors: KIM HYUN JONG , Nam Joo Hyun 
Published: April 2020

Intracellular calcium signaling is crucial for type 2 helper T cell and mast cell activation, which is essential for allergic inflammation. It is initiated by antigen‐mediated receptor stimulation that triggers store‐operated calcium entry (SOCE) via ORAI1 calcium channel. Flos Magnoliae (FM) is widely used to treat allergic diseases such as allergic rhinitis and asthma. Although many studies have reported that FM regulates intracellular calcium signaling, research on the exact type of calcium channel modulated by FM is scarce. Therefore, we hypothesized that the anti‐allergic effects of FM might result from ORAI1 inhibition in T cells. We investigated whether a 70% ethanolic extract of FM (FMEtOH) and its constituents inhibit ORAI1 channel activity and subsequent T cell activation. We performed conventional whole‐cell patch clamp studies in STIM1 and ORAI1‐overexpressing HEK293T cells (HEKORAI1). Intracellular calcium concentration was determined using Fura‐2 dye and cytokine production measurement in Jurkat T lymphocytes. FMEtOH (0.03 mg/mL) and its fractions, especially hexane fraction (FMHex, 0.01 mg/mL), significantly inhibited SOCE and IL‐2 cytokine production in Jurkat T lymphocytes. GC/MS analysis showed linoleic acid (LA) as the major component of FMHex. FMHex at 0.01 mg/mL (equivalent to 10 μM LA) inhibited not only SOCE but also IL‐2 production, as well as CD3/CD28 receptor co‐stimulation induced calcium signaling in Jurkat T lymphocytes. FMEtOH and LA suppressed CD4+ T lymphocyte activation, at least in part, by inhibiting ISOCE. Thus, ISOCE inhibition may be a potential strategy to inhibit immune responses in inflammation.Linoleic acid (LA) inhibits ISOCE and IL‐2 production to similar levels.Figure 1FMEtOH and its fractions inhibit store‐operated Ca2+ entry mediated current (ISOCE) in ORAI1 and STIM1 co‐overexpressing HEK293T cells (HEKORAI1).Figure 2
Collaborating With Glycoscience Community To Improve Data Representation of Carbohydrates in the Protein Data Bank
Authors: Zardecki Christine , Shao Chenghua , Feng Zukang , Westbrook John D. , Persikova Irina , Burley Stephen K. , Young Jasmine Y 
Published: April 2020

The Protein Data Bank (PDB) is the single global repository for experimentally‐determined 3D structures of biological macromolecules and their complexes with ligands. The Worldwide Protein Data Bank (wwPDB) is the international collaboration that manages the PDB archive according to the FAIR Principles: Findability, Accessibility, Interoperability, and Reusability. The PDB archive now holds more than 150,000 structures, which are all publicly accessible without restriction.A major focus of the wwPDB is maintaining consistency and accuracy across the archive. As the PDB grows, developments in structure determination methods and technologies can challenge how all structures are represented. The wwPDB addresses these challenges with “remediation” efforts to improve data representation in support of making PDB data FAIR.Understanding the structure and organization of carbohydrates is critical to comprehending their biological roles in health and disease. Approximately 10% of PDB structures contain carbohydrates. However, there is lack of uniform representation for carbohydrates due to complex nature of carbohydrates, which exhibit stereo‐isomers, anomeric configurations, and branched chains. As a result, glycoscientists and other experts are unable to fully utilize the rich structural information available for these structures.Working with the glycoscience community, carbohydrate‐appropriate annotation tools are being developed and implemented within the OneDep system for deposition, validation, and biocuration of PDB structures. These software tools will provide standard nomenclature and consistent oligosaccharide representation that can be easily translated to other representations commonly used by glycobiologists.wwPDB carbohydrate remediation efforts involve: (1) standardizing sugar nomenclature following IUPAC/IUBMB; (2) providing a uniform polymer representation for polysaccharides with appropriate descriptor(s); (3) adopting community software for reliable carbohydrate identification, assignment of standard nomenclature, and detection of intra/inter‐molecular connectivity between monosaccharides and other molecules/proteins; and (4) providing intra‐ and inter‐molecular connectivity at atom level explicitly.Support or Funding InformationThis carbohydrate remediation project is a collaborative wwPDB project carried out principally by the RCSB PDB with funding from NIGMS (U01 CA221216) in collaboration with the Complex Carbohydrate Research Center at the University of Georgia. RCSB PDB is funded by the NSF (DBI‐1832184), the US DOE (DE‐SC0019749), and the NCI, NIAID, and of the NIH under grant R01GM133198.N‐Glycosylation of cardosin A in PDB structure 1b5f (Frazao, C. et al. (1999) J Biol Chem 274: 27694–27701)Figure 1
Kinetic Characterization of Mutant Methylenetetrahydrofolate Reductase Enzymes His273Ala and His273Gln
Authors: Du Siyuan , Tetrick Maxwell G. , Li Richard , Chien Jason , Trimmer Elizabeth E. 
Published: April 2020

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism that binds flavin adenine dinucleotide (FAD) and uses NAD(P)H to catalyze the conversion of 5,10‐methylenetetrahydrofolate to 5‐methyltetrahydrofolate by a ping‐pong Bi‐Bi mechanism. The x‐ray crystal structure of the 5‐methyltetrahydrofolate and FAD
‐bound E. coli enzyme has revealed a hydrogen bonding network that involves amino acid residues Ser26, His273, Glu28, a structural water molecule, and the N10 of the folate. Based on previous studies that confirmed the importance of Glu28 in the catalysis of the folate half‐reaction, we hypothesized a proton relay mechanism that transports a proton from the aqueous solvent to the N10 of the folate through this hydrogen bonding network and catalyzes the formation of the putative 5‐iminium cation folate intermediate. In our study, we employed steady‐state oxidoreductase assays using menadione as an artificial electron receptor and measured kinetic parameters of the wild‐type E. coli enzyme and two mutants, His273Gln and His273Ala MTHFRs. We found that in the methyltetrahydrofolate‐menadione assay, where the folate half‐reaction is rate‐limiting, the kcat of the His273Ala mutant (0.047 ± 0.002 s−1) is more than 10‐fold lower than the wild‐type enzyme (kcat = 0.54 ± 0.01 s−1). His273Gln mutant showed similar activity to the wild‐type enzyme, but strong competitive substrate inhibition was observed. In the NADH‐menadione assay, both His273Ala and His273Gln showed insignificant change in kcat (1.2 – 1.3‐fold) and in Km (1.7 – 2‐fold). Together, these results suggest that the ability of the His273 residue to form hydrogen bonds is important in the catalysis of the folate half‐reaction and support the hypothetical proton relay mechanism. In addition, we report our use of Liquid Chromatography‐Mass Spectrometry (LC‐MS) to quantify the product in the folate‐menadione assay more accurately.Support or Funding InformationGrinnell College Department of ChemistryHydrogen‐bonding network in the X‐ray structure of E28Q MTHFRFigure 1v/E vs. [substrate] plots obtained from methyltetrahydrofolate‐menadione oxidoreductase assays at 4C.Figure 2