bluuebunny/crossref_metadata_2025_split huggingface dataset:
Highly Sensitive and Selective Determination of Redox States of Coenzymes Q9 and Q10 in Mice Tissues: Application of HR/AM Orbitrap Mass Spectrometry
Authors: Tiziani Stefano 
Published: June 2021

Coenzyme Q (CoQ) is a redox active molecule that plays a fundamental role in mitochondrial energy generation and functions as a potent endogenous antioxidant. Redox ratio of CoQ has been suggested as a good marker of mitochondrial dysfunction and oxidative stress. Nevertheless, simultaneous measurement of redox states of CoQ is challenging owing to its hydrophobicity and instability of the reduced form. In order to improve the analytical methodology, paying special attention to this instability, we developed a highly sensitive and selective high‐resolution/accurate‐mass (HR/AM) UPLC‐MS/MS method for the rapid determination of redox states of CoQ9 and CoQ10 by ultra‐performance liquid chromatography‐hybrid quadrupole‐Orbitrap mass spectrometry. CoQs were extracted using hexane with the addition of butylated hydroxytoluene to limit oxidation during sample preparation. Chromatographic separation of the analytes was achieved on a Kinetex C18 column with the isocratic elution of 5 mM ammonium formate in 2‐propanol/methanol (60:40) within 4 min. A full MS/all ion fragmentation (AIF) acquisition mode with mass accuracy < 5ppm was used for detection and determination of redox states of CoQ9 and CoQ10 in healthy mice tissues using reduced and oxidized CoQ4 as internal standards. The validated method showed good linearity (r2≥ 0.9991), intraday, inter‐day precision (CVs ≤ 11.9%) and accuracy (≤ ±15.2%). In contrast to existing methods, the current method offers enhanced sensitivity (up to 52 fold) with LOD and LOQ ranging from 0.01–0.49 ng mL−1 and 0.04–1.48 ng mL−1, respectively. Moreover, we evaluated various diluents to investigate bench top stability (at 4°C) of targeted analytes in tissue samples during LC‐MS assay up to 24 h. Ethanol was determined to be the optimum diluent without any significant oxidation of reduced CoQ up to 24 h. The developed method offers a rapid, highly sensitive and selective strategy for the measurement of redox states of CoQs in clinical studies.Support or Funding InformationUniversity of Texas System (ST: STAR Award), Hyundai Hope on Wheels research program, NIH R01 CA206210‐01, and Cancer Prevention & Research Institute of Texas (CPRIT, DP150061).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the secondary structure and protein binding affinity of an intramolecular DNA four‐way junction.
Authors: Smith Andrew , Kebede Yonatan , Moorhead Gaston , Huang Raymond , Bell Anthony Jerome 
Published: June 2021

Oligodeoxynucleotides (ODN) offer great promise as antigene and antisense reagents due to their high binding specificity, straight‐forward synthesis and low toxicity. Research shows that ODNs can also target proteins such as the DNA‐binding cytokine, High Mobility Group B1 (HMGB1). HMGB1 was initially classified as a highly abundant architectural nuclear protein. It is now clear that HMGB1 also functions as a redox sensitive damage associated molecular pattern (DAMP) molecule. And several studies link deleterious HMGB1 inflammatory signaling with an array of diseases and immune disorders such as: atherosclerosis, cystic fibrosis, lupus and sepsis. Hence, HMGB1 is gathering a great deal of attention as a biomarker and therapeutic target. We hypothesize that ODNs based on DNA four‐way junctions (4WJs) can be used to target HMGB1. In order to enhance the stability of 4WJs for ex vivo studies, the helical termini of a DNA junction are modified to generate the intramolecular construct, i‐J1. The secondary structure, thermostability and protein binding affinity of i‐J1 are investigated vs. a control 4WJ (J1) and the intramolecular 4WJ, J4. Non‐denaturing gel electrophoresis assays show that i‐J1 possesses a global structural conformation that is analogous to the control 4WJ. Circular dichroism (CD) studies show that the secondary structure of i‐J1 is composed of B‐ and A‐form DNA helices. Moreover, the melting temperature (Tm) of i‐J1 is significantly higher than J1. The Tm of i‐J1 is ~ 30°C higher than J1. Finally, electrophoretic mobility shift assays (EMSAs) show that the HMG protein, HMGB1b, binds i‐J1 with nearly identical affinity to J1. The data clearly shows that the junction lattice of a DNA four‐way junction can be altered to enhance conformational stability and retain protein binding affinity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA‐binding peptide dendrimer for efficient and selective intracellular delivery
Authors: Kam Antony , Wong Clarence Tsun Ting , Loo Shining , Tam James P. 
Published: June 2021

Dendrimeric peptides or peptide dendrimers are branched peptides with multiple peptidyl units. Multiple antigen peptides (MAPs) are examples of dendrimeric peptides first introduced in 1988 by our laboratory as immunogens for producing monoclonal and polyclonal antibodies. We also designed antimicrobial dendrimeric peptides, such as D4R, using a tetrapeptide RLYR which contains a ChC‐motif (cationic‐hydrophobic‐cationic) found in the naturally‐occurring antimicrobial peptides such as tachyplesins and protegrins. Advantages of D4R as a short‐chain, tetra‐branched peptide dendrimer include its short preparation time (a few hours), its polycationic, low membranolytic activity to human red blood cells, and high proteolytic stability. Here, we show that D4R is also a cell‐penetrating and an intracellular DNA‐binding peptide dendrimer. Using solid‐phase synthesis, we exploited different chemistries to attach tags or small molecules to the α‐ or ɛ‐amino groups of lysine to form a tetra‐branched dendrimer with four chains of RLYR. An example is D4R‐f, a C‐terminal 5‐FAM‐labeled D4R which we showed that it can enter living cells through endocytosis‐independent mechanisms as determined by using epi‐fluorescence microscopy. Furthermore, we showed that D4R‐f is distributed in different intracellular organelles during different stages of the cell cycles and colocalizes with nuclear DNA in cells undergoing mitosis. Using agarose gel shift assay, we showed that D4R binds to double‐stranded DNA. Co‐incubation of D4R with DNA showed resistance against DNase‐mediated digestion. The control linear 16‐residue peptide with four tandemly repeating RLYR units was not cell‐penetrating like D4R. Modifying the ChC motifs of the short‐chain peptide dendrimers produced different results, suggesting that the RLYR motif is tunable. Taken together, this study highlights D4R as a prototypic DNA‐binding peptide dendrimer which penetrates into living cells through endocytosis‐independent uptake mechanisms. Our work also suggests the potentials of short‐chain DNA‐binding peptide dendrimers as a lead to design anti‐mitotic agents and a convenient new tool for non‐viral gene delivery.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐Synzyme and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utility of AMP Detection System for Monitoring the Activities of Diverse Enzyme Reactions
Authors: Goueli Said , Hsiao Kevin , Mondal Subhanjan 
Published: June 2021

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are disregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP‐Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono‐ and poly‐ADP‐ribosyltransferases. Sulfotransferases use 3′‐Phosphoadenosine‐5′‐phosphosulfate (PAPS) as the universal sulfo‐group donor and Phosphoadenosine‐5′‐phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgiresident PAP‐specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP‐Glo system we can measure the activities of sulfotransferases. Thus by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP‐Glo assay.Support or Funding InformationThe research was supported by Promega Corp.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of tetradentate metal Schiff base complexes towards the synthesis of novel antimicrobials
Authors: Guimaraes Guilherme J. , Wolyniak Michael J. , Deifel Nicholas P. 
Published: June 2021

Schiff bases are molecules that carry a characteristic imine bond that are derived from carbonyl compounds and are widely studied due to the versatility of their steric and electronic properties. Schiff bases are significant because of their biologically active properties that can improve and develop agrochemicals, anti‐inflammatories, antivirals, and even radiopharmaceuticals for cancer targeting. This study is focused on synthesizing a family of novel metal Schiff base complexes that have potential to act as antimicrobials over a wide range of pathogenic species.In a previous study, a family of metal Schiff base complexes with an NN'NN coordination sphere was synthesized. This study found that antimicrobial activity using Schiff base complexes with chromium, iron, or manganese as their center showed greater inhibition of Aspergillus sp. growth than a variety of known antimicrobial drugs (Kumar et al., 2012). In an attempt to expand the antimicrobial potential of these compounds, we changed the structure of these Schiff base ligands to utilize an NN'OS coordination sphere with the same set of metal centers. We hypothesized that the chemical activity of the sulfur atom would provide an improved range of antimicrobial function.Our synthesis of NN'OS coordination sphere‐based antimicrobials began with the compound cyclopentanone. Cyclopentanone was condensed with carbon disulfide, recrystallized, protonated, and methylated with dimethyl sulfate. The resultant methylated product was aminated with two different diamines to form a set of novel primary amines. These primary amines were reacted with salicylaldehydes with different substitutions to form a family of Schiff base ligands. The last step in the synthesis process was to add a transition metal to the Schiff base ligands (chromium, iron, or manganese) through a reflux reaction. After synthesis, each metal Schiff base complex was characterized through 1H and 13C nuclear magnetic resonance, infrared, and gas chromatography‐mass spectroscopy.With synthesis complete, current work focuses on the examination of these compounds as effective antimicrobials. The compounds are being tested for these properties against Aspergillus furmigatus as a representative of a broad range of Aspergillus species. Characterized Schiff base complexes are being compared for their level of antimicrobial activity against that shown by Miconazole, an established antimicrobial used to treat an array of fungal infections. The results of these tests may establish Schiff base ligands with NN'OS coordination spheres as a novel effective means of treating an array of dangerous microbial infections.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 a High Resolution Melt Curve Assay to Examine the Single Nucleotide Polymorphisms of the HFE Gene
Authors: Ehrke Mariah Kay , Zwiesler‐Vollick Julie 
Published: June 2021

Pharmacogenetics is the study of how inherited genetic variations correspond to an individual's specific reaction to medication. Pharmacogenetic testing determines an individual's genotype. This is beneficial in the medical field because it informs doctors and patients about drug efficacy, drug dosage recommendations, and the risk of adverse side effects for their genotype. The purpose of this project is to determine how to use high resolution melting analysis to characterize the different alleles caused by single nucleotide polymorphisms (SNPs). The SNP being tested is in the HFE gene which codes for an iron‐sensing protein which also regulates iron uptake. This SNP has been shown to predict the effectiveness of treatment with Humira® (adalimumab) in patients with Crohn's disease. We are presently developing a protocol for high resolution melting analysis of this SNP using the Roche LightCycler 480. The high resolution melting analysis should be specific enough to differentiate between the different SNPs. We will verify our SNPs results with DNA sequencing. The results of this study will be presented.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monomethyl Branched Chain Fatty Acids link Mitochondrial Amino Acid Metabolism and Adipose Tissue Lipogenesis to Fatty Acid Diversity
Authors: wallace martina , Green Courtney , Roberts Lindsay , Lee Michelle , Cabrales Pedro , Ayres Janelle , Nomura Daniel , Loomba Rohit , Metallo Christian 
Published: June 2021

BCAA's are important substrates for protein synthesis and bioenergetics in mammals, and this metabolic pathway is dysregulated in various disease states. Fatty acid biosynthesis occurs in many tissues, but the balance of lipogenesis in liver and adipose tissue is disturbed in the context of obesity and metabolic syndrome. Here, using in vitro and in vivo stable isotope tracing we demonstrate that these pathways are linked within adipose tissue, where mammalian FAS produces monomethyl branched chain fatty acids (mmBCFAs). BCAA catabolic flux and short chain fatty acids from the gut microbiome initiate fatty acid synthesis in brown and beige adipocytes to influence the diversity of fatty acids present in the body. Diet‐induced obesity decreases BCAA catabolism and adipose tissue de novo lipogenesis such that mmBCFAs are the most reduced fatty acid species detected and mmBCFA abundance correlates negatively with markers of non‐alcoholic steatohepatitis. These results highlight the origin and regulation of mmBCFAs in mammals, which may play functional roles in metabolic homeostasis, nutrient sensing, and membrane function in human disease.Support or Funding InformationAjinomoto Innovation Alliance Program Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antioxidants Protect Diaphragm Function subjected to Ionizing Irradiation
Authors: Zuo Li , Lu Lanchun , Wu Shiyong , Zhou Tingyang 
Published: June 2021

Diaphragm can be subjected to undesired radiotoxicity during radiotherapy when targeted tumors are located at the upper bodies such as in breast, stomach and lung cancers. This study tests the hypothesis that ionizing irradiation (IR) can induce reactive oxygen species (ROS) generation as well as functional decline in the diaphragm; while administration of antioxidants may attenuate muscle dysfunction associated with IR exposure. Male C57BL6 mice were irradiated with onetime IR of 10 Gy (6 MV energy, 600 cGy/min dose rate) on the diaphragmatic region. On days 1, 3, 5, 7 after IR, mice were sacrificed and the diaphragms were removed for experiments. Muscle force and extracellular ROS levels were measured during five‐min tetanic contraction. Control group followed the same protocol without IR exposure. Antioxidant‐treated mice received daily intraperitoneal injection of Tiron (200 mg/kg body wt) and N‐acetyl Cysteine (NAC, 100 mg/kg body wt) starting from one day before IR until sacrifice. These mice were sacrificed on days 5 and 7 after IR for muscle function and ROS assay. Our results show that IR exposure compromised diaphragmatic function compared to control on the 5th and 7th days after IR (n ≥ 6; p < 0.05). There was elevated ROS release associated with muscle contraction from days 3–7 after IR as compared to control muscles (n = 6; p < 0.05). In contrast, mice treated with antioxidants demonstrated no muscle functional decline on days 5 and 7 (n ≥ 5; p < 0.05). Further, antioxidants abolished IR‐induced oxidative stress during contraction on 5th and 7th days (n ≥ 10; p < 0.05). These data collectively suggest that 10‐Gy IR exposure can lead to decreased diaphragm function potentially via ROS formation. Antioxidant treatment may be regarded as a promising strategy for diaphragm protection among patients who receive radiotherapy on tissue/organs in proximity to diaphragm.Support or Funding InformationThis study was supported by American Physiology Society S&R Foundation Ryuji Ueno Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic Control of Two Dynamic Pools of Diacylglycerol in Budding Yeast
Authors: Ganesan Suriakarthiga , Tavassoli Marjan , Sosa Maria Laura , Wagner Kelsey , Terebiznik Mauricio , Zaremberg Vanina 
Published: June 2021

The location of lipids and their distributions across cellular membranes have critical biological consequences, particularly for lipids involved in cellular signaling. In this study, diacylglycerol pools were monitored in budding yeast under conditions where lipid homeostasis was altered. Two predominant pools of diacylglycerol were visualized using the C1 domain of mammalian PKCδ fused to GFP. One pool was associated with vacuolar membranes and the other localized to sites of polarized growth. Upon growth resumption, diacylglycerol pools appeared more dynamic than those of phosphatidic acid and phosphatidylserine. During this period, diacylglycerol enriched puncta and vacuolar rings experienced constant morphological changes with clusters of lipid droplets closely attached. Lack of conversion of diacylglycerol to phosphatidate by Dgk1 led to the accumulation of diacylglycerol in a vacuolar associated compartment. Diacylglycerol distribution was strongly affected in cells lacking the phosphatidylserine synthase Cho1/Pss1. Supplementation of lysophosphatidylserine to cho1 cells did not correct mislocalization of DAG, pointing to a role for phosphatidylserine synthesis and traffic in the establishment of cellular diacylglycerol pools.Support or Funding InformationThis work was supported by a National Science and Engineering Research Council of Canada to M.R. Terebiznik and V. Zaremberg.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uncovering Clinically Relevant Mutations in Membrane Transporters by Genetic Analysis Linked to the Determination of Erythrocyte Membrane Protein Expression
Authors: Sarkadi Balazs , Zambo Boglarka , Bartos Zsuzsanna , Mozner Orsolya , Szabo Edit , Varady Gyorgy , Homolya Laszlo 
Published: June 2021

The human red blood cell (RBC) membrane contains more than 300 integral membrane proteins, many with high relevance to disease conditions or pharmacological interventions. We have developed a flow cytometry method for the quantitative determination of the RBC membrane expression levels of selected membrane transporters, and examined their molecular genetic background, as well as their potential value in medical diagnostics as biomarkers. One example presented is the ABCG2 xeno‐ and endobiotic transporter, modulating the absorption and metabolism of pharmacological agents and causing multidrug resistance in cancer. ABCG2 is also involved in uric acid elimination and its impaired function is causative in gout. Analysis of ABCG2 expression in the erythrocyte membranes of healthy volunteers and gout patients showed an enrichment of lower expression levels in the gout patients, correlating with a genetic polymorphism, causing a Q141K variant protein. In addition, based on RBC expression levels, we found stop and frameshift mutations, as well as a relatively frequent, novel ABCG2 mutation (ABCG2‐M71V). This variant, according to cellular expression studies, causes reduced protein expression, although with preserved transporter capability. ABCG2‐M71V expression in vitro could be corrected by therapeutically relevant small molecules, with a potential in allele‐specific gout treatment. Another example is the plasma membrane calcium pump (PMCA4b) of the RBC membrane, which showed heterogeneous RBC expression levels in healthy volunteers. By genetic analysis we found a correlation of the low PMCA4b expression levels with a minor haplotype in the erythroid cell specific regulatory region of this protein. The respective regulatory regions in this minor haplotype have been characterized in detail. These results suggest that personalized medicine should consider the genetic analysis linked to RBC membrane protein expression, and these studies may provide a new tool to uncover clinically important variants of membrane proteins.Support or Funding InformationSupported by OTKA K115375, K111678 and FIEK 16‐1‐2016‐0005, NKTH, grants.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.