bluuebunny/crossref_metadata_2025_split huggingface dataset:
Further Objections
Authors: Coady C. A. J. 
Published: June 2021

Abstract
Chapter 2 discusses objections to the definition of terrorism from Chapter 1 that criticize it for being either too narrow or too broad. The narrowness criticisms object that (a) there are terrorist acts that target combatants, (b) there are terrorist acts that do not involve a political motive, such as certain criminal or religious acts, and (c) some non-intentional violence afflicting non-combatants should be encompassed by the definition. The “too broad” category of objections argues that (a) the tactical definition should include an ingredient of intentionally provoking fear, (b) the definition’s encompassing terrorist acts by states is mistaken, (c) including non-combatant property in the definition is mistaken, and (d) this approach simply defines terrorist acts as murder and loses what is distinctive of such acts. These objections are criticized and rejected for the most part, though some elicit concessional comments about their possible ancillary benefits in relation to the preferred tactical definition.
Counter-Terrorism and Its Ethical Hazards
Authors: Coady C. A. J. 
Published: June 2021

Abstract
Chapter 7 discusses ethical problems faced by contemporary counter-terrorism measures. The discussion is primarily focused on reactions of states to sub-state terrorism broadly understood. There is an initial discussion of whether, and in what circumstances, terrorists should be treated as combatants or criminals and the bearing of this upon military and non-military forms of counter-terrorism. Problems with military responses connected with the inflammatory slogan “the war on terror,” including “targeted killing,” are also discussed. Thereafter, the chapter deals mostly with non-military responses and their moral and political hazards. These are examined under the three categories of: (1) legal and regulatory measures; (2) diplomatic measures; and (3) measures to remove the grievance. Under (1), difficulties connected with legal definitions of terrorism, and their tendency to promote abuses of civil rights, are explored; under (2) and (3) the path of political diplomacy is supported, but obstacles to its success in practice are discussed.
Effects of Oleate and Inflammatory Cytokines on Dermal Fibroblasts in Type 1 Diabetics
Authors: Jones Albert Richard , Deeney Jude T , Corkey Barbara 
Published: June 2021

BackgroundDermal fibroblasts are crucial to wound healing which can be impaired in type 1 diabetics. We set out to determine if exposure to inflammatory cytokines and oleate changed the metabolism and ROS production of fibroblasts.MethodsCells were acutely exposed to 25 or 50 μM of oleate and chronically exposed to 100 μM of oleate. Cells were exposed to 10 ng/mL of TNF‐ α. Oxygen Consumption was measured using the SeaHorse XF24 Flux Analyzer, ROS was measured using DCF dye. Lipid accumulation was measured using Nile Red staining. β‐oxidation was measured using radioactive oleate.ResultsThe data shows type 1 diabetics sequester more lipid droplets than controls. As a result, less fatty acid is available for β‐oxidation and the stimulation of respiration, which explains the decreases in oxygen consumption, proton leak, β‐oxidation and ROS generation in diabetic fibroblasts. We believe that diabetics express an inflammatory phenotype that reduces β‐oxidation in the cell causing a decrease in oxygen consumption and ROS production.Future ResearchWe plan to measure the mRNA and protein expression of key components of β‐oxidation and inflammation in fibroblasts.Funded by NIH
High fat feeding enhances the ability of mesenchymal stem cell therapy to modulate mitochondria in the infarcted heart
Authors: Hughey Curtis , Ma Lianli , James Freyja D , Bracy Deanna P , Wasserman David H , Rottman Jeffrey N , Shearer Jane 
Published: June 2021

OBJECTIVEEvaluate the effect of mesenchymal stem cell (MSC) therapy on cardiac mitochondria following a myocardial infarction (MI).METHODSC57BL6 mice were high fat‐fed (HFF) for eight weeks and separated into three groups: HFF‐SHAM, HFF‐MI+PBS mice given a MI followed by intramyocardial injection of saline and HFF‐MI+MSC mice receiving MSCs. HFF groups were age‐matched with chow‐fed mice.RESULTSMitochondrial function was evaluated via high‐resolution respirometry in permeabilized, peri‐infarct fibers 5 weeks post‐MI. In chow‐fed mice, maximal oxygen flux (155.30 vs 122.63 vs 135.61 pmol/s/mgww/CS; SHAM vs MI+PBS vs MI+MSC; n=9–10) and oxidative phosphorylation (OXPHOS) efficiency (RCR = 3.17 vs 2.34 vs 2.84) was preserved by MSC therapy. Also, MSC treatment prevented a decline in mitochondrial content as indicated by citrate synthase activity (1.28 vs 0.90 vs 1.34 mmol/min/mg pro). In HFF mice, the MSC treatment maintained mitochondrial content (1.21 vs 0.99 vs 1.24) and OXPHOS efficiency (RCR = 2.18 vs 1.88 vs 2.23). Also, HFF‐MI+MSC hearts exhibited an exaggerated decline in futile oxygen flux (61.39 vs 50.69 vs 38.85; n=8–9). Immunoblots show the HFF‐MI+MSC hearts have lower mitochondrial complex I and II, UCP3 and TFAM protein levels (n=6).CONCLUSIONSMSC therapy minimizes mitochondrial dysfunction post‐MI and HFF enhances the therapeutic potential of MSCs. Funded by CIHR and Killam Trusts.
Association of Genetic Polymorphism of Peroxisome Proliferator‐Activated Receptor‐Gamma Gene and Polycystic Ovary Syndrome
Authors: Hemimi Neveen Salah Eldine , Shaafie Ishtiyaq Ahmad , Alshawa Haya Husni A 
Published: June 2021

BackgroundPeroxisome proliferative‐ activated receptor gamma (PPARs) is a nuclear receptor which controls transcription of genes involved in free fatty acid uptake and lipogenesis, and is essential for differention and survival of adipocytes.AimTo investigate the association of Pro12Ala polymorphism in PPAR gamma gene with polycystic ovary syndrome (PCOS) and its related metabolic disorders.Material and MethodThe study consisted of 50 women diagnosed with PCOS and 96 healthy women as a control group. Individuals were genotyped for Pro12Ala genetic polymorphism of PPAR gamma by using polymerase chain reaction‐ restriction fragment length polymorphism. Statistical analysis was done by SPSS program.ResultsFrequency of Pro12/Pro12 and Pro12/Ala12 genotypes were 86% and 14% in PCOS group compared to 77.1% and 22.9% in control group. The carriers of Pro12/Pro12 genotype exhibited a significant greater risk for PCOS compared to carriers of Pro12/Ala12 genotype (OR= 1.8, 95%CI= 0.7–4.6). Furthermore they were at higher risk of different metabolic disorders such as obesity, insulin resistance and diabetes mellitus.ConclusionPro12/Pro12 genotype is a risk factor for PCOS and its related metabolic disorders.Source of Fund: Dubai Medical College for girls.
Characterization of a novel interaction between BLIP‐II and Staphylococcus aureus PBP2a
Authors: Adamski Carolyn J , Chow Dar‐Chone , Brown Nicholas , Palzkill Timothy 
Published: June 2021

The prediction and manipulation of protein‐protein interactions remains a difficult task. Model systems such as the β‐lactamase inhibitory protein II (BLIP‐II) and β‐lactamases have been used to investigate the principles of protein‐protein interactions. Previous studies focused on the determinants of binding affinity and specificity between BLIP‐II and class A β‐lactamases. However, interactions between BLIP‐II and other proteins have yet to be explored. In this study, we characterized the novel interaction between BLIP‐II and penicillin binding protein 2a (PBP2a) from methicillin resistant Staphylococcus aureus. Mutagenesis and surface plasmon resonance experiments were used to determine how specificity is achieved between BLIP‐II and its binding partners. The results suggest that an outer ring of residues on the BLIP‐II interface plays a critical role in binding PBP2a while an inner ring of residues on the binding surface is primarily responsible for the binding of β‐lactamases. Changes in BLIP‐II binding affinity for PBP2a and β‐lactamases were found to be largely due to changes in kon and koff, respectively. In summary, the results of the study indicate BLIP‐II binds PBP2a in addition to β‐lactamases and provide insights into how BLIP‐II binds a wide range of target proteins.Research support provided by grants R01 A132956 and R21 A1092063 to T.P.
Monitoring the Structural Dynamics of LRRK2 using Split‐ Luciferase Protein‐Fragment‐Assisted Complementation
Authors: Nardozzi Jonathan , Baker Olivia , Wong Salinda , LaVoie Matthew J. 
Published: June 2021

Missense mutation of the PARK8 gene, which encodes the protein Leucine‐Rich Repeat Kinase 2 (LRRK2), is the most common genetic cause of Parkinson's Disease (PD), yet the physiological role of this protein is largely unknown. LRRK2 dimerization has been linked to its activity and localization in the cell, as dimers have higher kinase activity and are preferentially membrane‐associated. In this study we utilized a Split‐Luciferase Protein‐ Fragment‐Assisted Complementation assay (PCA) to assess dimerization of LRRK2. PCA has numerous advantages over other techniques used to study protein‐protein interactions, as the luciferase reporter displays specificity, reversibility, and signal intensity. We have developed a quantitative bioluminescent assay of LRRK2 dimerization as a means to understanding the regulators of LRRK2 structure and function. Numerous split‐luciferase fragment pairs were explored from both the Renilla and Firefly luciferase reporters, and one fragment pair was optimized for investigating LRRK2 dimerization. Interestingly, we observed that fusion and orientation of luciferase fragments to their respective LRRK2 monomers were important factors for reporter efficiency. Efforts to characterize small molecule regulators of LRRK2 dimerization are ongoing.NIH grant #NS072604
Sensitization of NCI‐H460 Cells to Anticancer Drugs with Aminoglycosides
Authors: Cuccarese Michael 
Published: June 2021

Aminoglycosides, characterized by their toxic interaction with the bacterial ribosome, are also known for their effects on eukaryotic cells. While aminoglycosides are routinely used in the clinic as antibiotic agents, they are currently being evaluated therapeutically for their ability to rescue protein synthesis that is suppressed by nonsense‐mutation (e.g., in the case of cystic fibrosis). In this current study we aim to determine if the aminoglycoside read‐though effect on eukaryotic cells can be leveraged for use in cancer chemotherapy.Herein we describe our efforts to find the aminoglycoside read‐though effect on non‐small cell lung carcinoma cells (NCI‐H460) and it application to cancer cell chemotherapy. Specifically, these studies involve the search for sensitization effects of sub‐toxic doses of known read‐though inducing aminoglycosides in cancer cell cytotoxicity assays with known anticancer agents. For NCI‐H460 cancer cells, a dose dependent sensitization effect was found for known read‐though inducing aminoglycosides for some, but not all anticancer drugs. Studies aimed at the mode of action for sensitization due to aminoglycoside exposure is ongoing.
Structure activity relationship study of the Cleistriosides and Cleistetrosides for antibacterial/anticancer activity
Authors: Shi Pei 
Published: June 2021

The de novo asymmetric syntheses of several partially acylated dodecanyl tri‐ and tetra‐rhamnoside oligosaccharides have been achieved by using the divergent route. The desired sugar absolute stereochemistry is achieved by the use of highly enantioselective Noyori reduction and a diastereoselective palladium‐catalyzed glycosylation.1In addition; all remaining triol functionalities of the sugar moieties were established via corresponding post‐glycosylation transformations with the use of only three or less protecting groups.As one of the primary components of bacterial cell‐walls,2 oligosaccharides and the rhamnan in particular, play important biological roles, such as cell‐cell interaction, cell adhesion, and immunogenic recognition.3,4 Since the bacterial cell wall oligo‐rhamnose‐containing structurally motifs are not present in mammalian cells,5 they are potentially interesting targets for the development of anti‐bacterial agents.With these ten oligosaccharides in hand, biological activities were measured against several bacteria strains and NCI‐H460 human lung cancer epithelial cell line. This study for the first time reports anticancer activity and comprehensively the antibacterial activity for this class of natural products. In addition, seven of these oligosaccharides were found to have potent activities against the NCI panel of 60 cell lines. Interestingly, the observed varieties of antibacterial and anticancer activities suggest that the degree of acylation and location of the specific acetate groups might play an important role on the underlying modes of action.6
Expression and Characterization of Recombinant Neutral Sphingomyelinase 2 in Escherichia coli: Evidence of Sensitivity to Redox State
Authors: Dotson P. Patrick , Nikolova‐Karakashian Mariana 
Published: June 2021

Neutral sphingomyelinases are a family of lipid‐metabolizing enzymes that hydrolyze sphingomyelin to ceramide. Neutral sphingomyelinase 2 (nSMase‐2), in particular, was identified as the main cytokine‐inducible sphingomyelinase. Several recent studies have correlated nSMase‐2 activity with the state of oxidative stress. Depletion of cellular GSH, NADH oxidase, or exposure to agents that generate reactive oxygen species have been linked to upregulation of nSMase‐2 activity in various mammalian cells. The mechanisms for activation of nSMase‐2 during oxidative stress conditions, however, remain not completely understood.In this study, we investigate whether nSMase‐2 is a redox sensitive enzyme. A recombinant mouse nSMase‐2 construct was overexpressed in an Escherichia coli strain whereby recombinant proteins were produced that, based on in vitro activity assays, were functionally active. Interestingly, we demonstrate that nSMase‐2 exists in multimeric forms that are sensitive to reducing agents, which could correspond to cysteine‐mediated oligomerization between nSMase‐2 molecules. To directly test this, 17 of the 22‐ cysteine residues within nSMase‐2 were gradually mutated to serine. Gradual mutagenesis resulted in a loss of nSMase‐2 multimers with cysteine residues at position C561 and C617, within the catalytic domain, being identified as being involved in nSMase‐2 oligomerization. Furthermore, we show that the cysteine‐mediated nSMase‐2 oligomerization may be modulated through the thiol‐reducing enzyme thioredoxin. Together, these results suggest that nSMase‐2 is a redox‐sensitive enzyme and the redox state of specific cysteine residues may regulate nSMase‐2 activity.
The iron transport inhibitor ferristatin II induces degradation of transferrin receptor‐1
Authors: Byrne Shaina L , Buckett Peter D , Sanford Jack , Chen Juxing , Enns Caroline , Wessling‐Resnick Marianne 
Published: June 2021

Ferristatin II, a polyaromatic compound previously proposed to be an iron uptake inhibitor, induces degradation of transferrin (Tf) receptor‐1 (TfR1) in HeLa cells within 4 h of treatment. Dose response experiments established an IC50 of 12 μM for inhibition of 55Fe‐Tf uptake, consistent with receptor degradation. Ferristatin II‐induced TfR1 degradation was blocked by nystatin, a cholesterol‐depleting agent that disrupts lipid raft membrane microdomains. siRNA knockdown of flotillin, a protein component of lipid rafts, also blocked TfR1 degradation by ferristatin II. Constitutive trafficking of TfR1 through clathrin‐mediated endocytosis occurs with or without ligand. In contrast, Tf antagonized TfR1 degradation by ferristatin II, an effect that was abrogated for the Tf binding mutant G647A TfR1. Combined, these data indicate that ferristatin II‐induced trafficking and degradation of TfR1 occurs through a lipid raft pathway gated in a ligand dependent manner.This work is supported by NIH grant DK064750 to M.W‐R., DK054488 to C.E., S.L.B is supported by NHGRI/NIEHS Training Grant T32ES016645.
Artemisia princeps var. orientalis induces cell cycle arrest accompanied by upregulation of p21CIP1 and caspase‐dependent apoptosis in human breast carcinoma cells
Authors: Choi Eun‐Jeong , Kim Gun‐Hee 
Published: June 2021

The aim of the present study was to investigate the anticancer activity and mechanism of action of methanol extract from Artemisia princeps var. orientalis (APME). First, the antiproliferative activity of APME against T47D cells was at various concentrations (5–200 μg/mL) and treatment times (24–72 h) determined by MTT assay. APME significantly inhibited cell proliferation in a dose‐dependent manner, regardless of treatment times. Under the same condition, cytotoxicity (LDH release) of APME was only appeared at 72 h. In addition, treatment with the IC50 concentration of APME for 24 h caused cell cycle arrest and apoptosis. APME arrested cells in both G1 and G2/M phases of the cell cycle, a response that was related to the induction of CDK1, cyclin B, p21Cip1 expression, and repression of CDK2 and cyclin A. The induction of p21Cip1 by APME caused activation of p53 and modulation of its downstream targets Bcl‐2 and Bax. Subsequently, caspase‐8 and ‐9 were cleaved in cells treated with APME. Activation of these caspases in turn led to increased activation of caspase‐3. The results of this study suggest that APME produce anticancer effects in T47D cells through cell cycle arrest associated with the induction of p21Cip1 and apoptosis mediated by p53 and caspase‐depedent pathways.This research was supported by Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012–0006811)
The conformation of active receptor‐bound arrestin
Authors: Gurevich Vsevolod , Kim Miyeon , Vishnivetskiy Sergey A , Van Eps Ned , Alexander Nathan S , Cleghorn Whitney M , Zhan Xuanzhi , Hanson Susan M , Morizumi Takefumi , Ernst Oliver P , Meiler Jens , Hubbell Wayne L 
Published: June 2021

Visual arrestin‐1 binds light‐activated phosphorylated rhodopsin (P‐Rh*) to terminate G‐protein signaling. To map conformational changes upon binding to the receptor, pairs of spin labels were introduced in arrestin‐1 and double electron–electron resonance was used to monitor inter‐spin distance changes upon P‐Rh* binding. We found that the relative position of the N‐and C‐domains remains largely unchanged, contrary to expectations of a “clam‐shell” model. A loop implicated in P‐Rh* binding that connects b‐strands V and VI (the “finger” loop,” residues 67–79) moves toward the P‐Rh* in the complex, but does not assume a fully extended conformation. A striking and unexpected movement of a loop containing residue 139 away from the adjacent finger loop was observed, which appears to facilitate P‐Rh* binding. We detected smaller movements of distal loops containing residues 157 and 344 at the tips of the N‐and C‐domains, which correspond to “plastic” regions of arrestin‐1 that have distinct conformations in monomers of the crystal tetramer. Remarkably, the loops containing residues 139, 157, and 344 have high flexibility in both free and P‐Rh*‐bound arrestin‐1.Support: NIH Grants EY011500, GM077561, GM081756 (VVG), GM080403, MH090192 (JM), EY005216, EY00331, and Jules Stein Professorship Endowment (WLH), Anne and Max Tanenbaum Chair in Neuroscience and Canada Excellence Research Chair program (OPE).
AKAP79 Interacts with the Axon Guidance Receptor Robo2
Authors: Samelson Bret , Colledge Marcie , Scott John 
Published: June 2021

A‐kinase anchoring proteins (AKAPs) are scaffolds that bring together complexes containing protein kinase A (PKA) and other signaling molecules. They localize these complexes to specific regions of the cell to regulate numerous signaling pathways. AKAP79 is a neuronal scaffold that coordinates PKA, protein kinase C (PKC), and protein phosphatase 2B (PP2B) signaling at the plasma membrane. The anchoring of these enzymes via AKAP79 is important for the function of numerous receptors.We have identified an interaction between AKAP79 and the axon guidance receptor Robo2 in a mass spectrometry screen. The Roundabout (Robo) family of receptors is involved in both the repulsion of axonal growth cones during pathfinding as well as the stimulation of dendritic branching. This interaction was confirmed by performing GST‐Robo2 pulldowns from mouse brain lysate as well as immunoprecipitations from HEK293 cells overexpressing Robo2 and AKAP79. Further studies demonstrated that the Robo2‐AKAP79 interaction is disrupted by calcium. In addition, we performed in vitro kinase assays to determine whether Robo2 was a substrate for any AKAP79 anchored kinases. We discovered that Robo2 can be phosphorylated by both PKA and PKC. Based on these findings, we hypothesize that the AKAP79 signaling complex may regulate the essential developmental functions of Robo2.This work was supported in part by NIH grant GM48231.
Phosphorylation‐dependent interaction of PRIP with Akt
Authors: Sugiyama Goro , Nagano Koki , Otani Takahito , Gao Jing , Takeuchi Hiroshi , Hirata Masato 
Published: June 2021

Phospholipase C (PLC)‐related but catalytically inactive protein (PRIP) is a protein with a domain organization similar to PLC‐δ1. We have reported that PRIP interacts with the catalytic subunits of protein phosphatases, PP1c and PP2Ac, depending on the phosphorylation level of PRIP. We also found that Akt was precipitated along with PRIP by anti‐PRIP antibody from neuronal cells. In this study, we investigated the interaction between PRIP and Akt.Co‐immunoprecipitation assay was performed using COS7 cells expressing glutathione S‐transferase (GST)‐fused PRIP. Both endogenous Akt and PP2Ac were co‐precipitated along with PRIP by GST antibody, and the precipitation was up‐regulated when the cells were stimulated with insulin. GST‐pull down experiments using recombinant GST‐Akt and his‐tagged (His‐) PRIP or the deletion mutant of PRIP (74–298) showed the direct binding of Akt to 74–298 residues in RPIP. Pretreatment of PRIP with Akt in the presence of ATP increased this interaction. Akt phosphorylated PRIP, but not the mutant PRIP whose Ser‐96 was replaced with Ala (S96A). Pre‐treatment of S96A mutant with Akt did not increase the binding to Akt, indicating that phosphorylation of PRIP on Ser‐96 increases the binding of Akt to PRIP. These results indicate that PRIP modulates Akt signaling by the regulation of complex formation between Akt and protein phosphatases.
Two unique phosphorylation‐driven signaling pathways crosstalk in Staphylococcus aureus: STK1 meets GraR
Authors: Fridman Michael , Golemi‐Kotra Dasantila 
Published: June 2021

The Stk1/Stp1 and GraSR signal transduction pathways are two distinct pathways in Staphylococcus aureus that rely on reversible phosphorylation processes in transducing external stimuli intracellularly. Our study shows for the first time that these two systems interact in S. aureus. GraR undergoes phosphorylation by Stk1 at three threonine residues in the DNA‐binding domain. Phosphorylation by Stk1 depends on the structural integrity of GraR and amino acid sequences flanking the phosphorylation sites. Its homolog in Bacillus subtilis, BceR, which shares 56% sequence identity with GraR, does not undergo phosphorylation. In addition, an unrelated response regulator of S. aureus, VraR, does not undergo phosphorylation by Stk1. These findings provide strong evidence that GraR is a target of Stk1. Stk1‐dependent phosphorylation of GraR increases its DNA‐binding affinity to one of the GraR‐target promoters. The crosstalk between these two otherwise independent pathways may facilitate S. aureus adaptation to diverse environmental cues and mediate its virulence.This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Ministry of Economic Development and Innovation (Ontario, Canada).