bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of Novel Cycloheptatrienylidene Fluorophores for Detection of Metals
Authors: Kaur Kirandeep , Hyatt Ivan D. 
Published: June 2021

Fluorophores are fluorescent molecules with various biochemical applications such as indicators for polarity, biomarkers for antibodies, or stains for analytical methods, like imaging or spectroscopy. The purpose of this research is to synthesize asymmetrical cycloheptatrienylidene fluorophores and to react them with a metal to monitor the aggregation induced emission. Previous synthetic scheme involved the development of the target molecule intermolecularly, but the current scheme focuses on intramolecular mechanism via Catellani reaction. This palladium catalyzed reaction will produce the precursor, which will intramolecularly react to form the ring motif. It is predicted that aromatization of the cycloheptatrienylidene will cause aggregation and turn on the light emitting fluorescence as the molecule undergoes a restriction of intramolecular rotation. While the synthesis of a novel CHT was in progress, reproduction of a model system via a synthesis of fluorene and cycloheptatrienyl tetrafluoroborate reactants was performed.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering a Color Palette of FRET‐BRET ATP Sensors
Authors: Min Sehong , Tantama Mathew 
Published: June 2021

ATP, a key player in energy metabolism, is involved in various cellular processes and is shown to be an indicator of cellular health. There are several methods to measure ATP levels, and genetically‐encoded sensors are excellent tools for visualizing energy metabolism in live cells. Currently, however, there has been a lack of color options. By building new variants of hybrid FRET‐BRET (Förster or bioluminescence resonance energy transfer) ATP sensors, our goal is to increase the flexibility and utility of these tools with different experimental formats. For example, differently colored sensors could be used to simultaneously monitor ATP levels in different individual cells or cell types as well as in different compartments within a single cell. Here, we have developed ATP sensors which employ different fluorescent protein FRET pairs in conjunction with the NanoLuc luciferase. We characterized the both the fluorescence and bioluminescence purified sensor proteins in response to ATP. Furthermore, we carried out live‐cell imaging experiments to validate that our sensors can be used for ratiometric quantification of ATP levels. These new color variants of hybrid FRET‐BRET ATP sensors will be an effective method for studying energy metabolism both in vitro and in vivo.Support or Funding InformationNIH/NINDS R21 NS092010This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kd determination and characterization of zinc complexes by fluorescence titration
Authors: Weiland Hunter S , Wolyniak Michael J , Mueller Paul H 
Published: June 2021

Resent research has focused on the properties of metallo‐coenzymes as a potential potent antioxidant for cancer prevention. Specifically, zinc holds particular promise for these clinical applications. Fluorescent probes, in this case ligands that form fluorescent complexes with zinc ions, act in research as a standard due to their versatility, sensitivity, and quantitative capabilities. Of particular interest to us is the ability of these probes to be coupled with microscopy to determine zinc concentrations in a variety of living and non‐living samples. Fluorescence for metals such as zinc require metal chelating or binding moieties and one fluorophore capable of absorbing and emitting light. Our laboratory has attempted to synthesize a novel zinc complex designed to register fluorescence in the 500–581 nm range to maximize a detectable fluorescent signal. While previous researchers in our group have worked to construct this probe, we have, as of yet, not been able to verify its proper synthesis. Thus, this work focuses on establishing the Kd of our novel zinc complex in the hopes of proving that we have developed an efficient probe for use in zinc biology‐related projects. Development of a PIPES zinc chloride solution has been made to test the initial fluorescence using spectroscopy that standardizes the zinc metal complex. A DMF and zinc chloride solution was made to more fully characterize this complex. Current research is focused on the characterization of the complex via spectroscopy on a Shimadzu rf6000 spectro‐fluorophotometer. This is being applied towards conjugation of the probe to cell cultures with an eye towards several biochemical applications ranging from kinetics, detection of viral and bacterial pathogens, to ligand‐membrane receptor interactions.Support or Funding InformationHampden‐Sydney College Office of Undergraduate ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxytocin Analysis from Human Serum by LCMS after Derivatization
Authors: Franke Adrian A , Dabalos Chester , Custer Laurie J , Li Xingnan , Lai Jennifer F 
Published: June 2021

BACKGROUNDOxytocin (OT) needs to be measured accurately to research its associations with social behaviors, conditions, and diseases in humans. Available enzyme‐linked immunosorbent assays lack the sensitivity to measure the very low (pg/mL) circulating levels in non‐pregnant/‐lactating humans (NPLH) reliably while radioimmunoassays are accurate but involve hazardous radioactive materials. One‐dimensional liquid chromatography mass spectrometry (LCMS) methods are currently not sensitive enough. Our recently developed LCMS method (Franke, 2018 PMID 30091853) can quantitate serum OT down to 10 pg/mL but precludes quantification in NPLH, which is known to be < 5 pg/mL. Thus, we sought to improve the lower limits of detection (LLOD) of OT measurements by forming OT products with higher lipophilicity thereby increasing the MS sensitivity as well as achieving better liquid‐liquid extraction (LLE) or solid‐phase extraction (SPE) outcomes.METHODS200 uL aq. OT (0.01–10 ng/mL) was reduced using 1,4‐dithiothreitol(DTT) or tris(2‐carboxyethyl)phosphine (TCEP) to cleave the dithio ether followed by alkylation with N‐alkyl iodoacetamide using alkyl chains (C10 to C18) according to Williams et al. 2009 (PMID 19734056). A second strategy involved derivatization of the phenolic tyrosine hydroxyl with biphenyl sulfonyl or dansyl chloride. The resulting products were then concentrated using LLE or SPE.RESULTSReduction using DTT or TCEP resulted in >95% cleavage to OT‐dithiol. Subsequent alkylation products derived from N‐alkyl iodoacetamide alkyl chains C10 and C12 were chosen over those with C14 or C16 for further analysis due to better product sensitivity. However, these products produced low yields and lower LCMS sensitivity than the underivatized OT. Enriching these products by LLE or SPE were also unsuccessful. Derivatization using biphenyl sulfonyl or dansyl chloride yielded mono‐tagged adducts of the reduced and unreduced OT but had lower LCMS sensitivity than the underivatized OT. Enriching these adducts with LLE or SPE did not improve LLOD via concentration.DISCUSSIONAlkylation of the thiol groups of the reduced OT with N‐alkyl iodoacetamide or sulfonylation of native OT with an array of sulfonyl chlorides led to products less sensitive than underivatized OT. Attempts for enrichment of these products by SPE and LLE were unsuccessful.CONCLUSIONImproving LCMS LLODs of OT by derivatization approaches were unsuccessful as was enrichment of these products by LLE or SPE. Current attempts to enrich native OT from human serum for the improvements of LLOD during LCMS analyses are presented.Support or Funding InformationNoneThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An open source bioinformatic pipeline to decipher how the human milk metabolome protects infants from pediatric obesity
Authors: Lemas Dominick J , Kirpich Alex , Francois Magda , Manfio Luran , Hentschel Austen , Cacho Nicole J , Thompson Lindsay J , Parker Leslie , Hogan William , Neu Joe , Jobin Christian , Garrett Timothy 
Published: June 2021

Exclusive breastfeeding is associated with protection against pediatric obesity independent of maternal obesity; however, the mechanisms that account for these observations are poorly characterized. Recent advances in untargeted metabolomics provides a novel opportunity to comprehensively investigate how the human milk metabolome is associated with infant adiposity during the first year of life. The goal of this project is to develop an open source bioinformatics pipeline using Nexflow that leverages high‐resolution mass spectrometry to interrogate the human milk metabolome in a longitudinal maternal‐infant cohort. Nextflow is a scalable and reproducible scientific workflow that can implement metabolomics tools using containers on high‐performance computers. Data for this study will be collected as part of the Breastfeeding and Early Child Health (BEACH) study. The BEACH study investigates the effects of breastfeeding on numerous postpartum outcomes through questionnaires and longitudinal collection of milk, stool, and blood samples. More specifically, BEACH study will follow normoglycemic pregnant mothers and full‐term babies through the first year of life. Mothers will be recruited between 36–38 weeks of pregnancy that plan to deliver in Gainesville, FL and exclusively breastfeed their infant for at least 2‐months. Non‐invasive biological specimens (i.e. stool, urine), body composition, self‐reported questionnaires and clinical variables will be collected at the Clinical Research Center from mothers during 3rd trimester and mom‐baby pairs at 2‐weeks, 2‐months and 1‐year. Human milk metabolomics will be completed by the Southeast Center for Integrated Metabolomics (SECIM) and high‐throughput data analysis will be implemented using Nextflow on the HiperGator. Our preliminary analysis demonstrates human milk metabolites differ across milk fractions (whole, skim, lipid) and metabolite identification is sensitive to background‐subtraction techniques. Collectively, these results highlight rigorous and reproducible data processing procedures as critical to elucidation of human milk metabolites that may reduce the risk of pediatric obesity.Support or Funding InformationResearch was supported in part by the University of Florida Clinical and Translational Science Institute and Southeast Center for Integrated Metabolomics, which is supported in part by the NIH National Center for Advancing Translational Sciences under award number UL1TR001427 and the NIH Common Fund, respectively.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabase: A New Programming Framework for Analyzing, Visualizing, and Integrating Multi‐Omics Data for Nutritional Intervention Studies
Authors: Zhu Chenghao , Zhang Ruihan , Zivkovic Angela M. 
Published: June 2021

With the rapid development of sequencing technology and mass spectrometry, high through‐put experimental methods are being adopted into the field of nutritional intervention studies. –Omics methods such as metabolomics, lipidomics, proteomics, and microbiome methods generate large datasets, with high sensitivity, and are powerful to allow researchers to discover subtle metabolic alterations. All –omics methods have their own communities and data analysis workflows that make the integration multi‐omics datasets a challenge for nutritional/clinical researchers. ‐‐R is an open‐source programming language with a large community in the field of data science and bioinformatics. Here we developed a data analysis framework based on the R language, that aims to analyze, visualize, and integrate tabular datasets from different –omics experiments. The Metabase package uses an S4 object‐oriented design. It allows researchers to easily store, manipulate, summarize, and transform –omics data, followed by statistical analysis and visualization. Researchers can adjust their hypothesis for testing and statistical model easily. The Metabase package is inspired by data analysis software from different –omics communities, and is capable of analyzing metabolomic, lipidomic, proteomic, and microbiome data. It is designed with large extensibility and can be extended and customized to handle other experimental data such as anthropometric and dietary data. The Metabase package allows nutritional researchers to analyze and integrate their multi‐omics data and improve reproducibility and data presentation quality.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutations L163P and R190C in NKX2‐5 Confer More Stability to the Protein's Binding to DNA
Authors: Tizon Lech Havel Orquin , Bangalan Marielle Alyanna T. , Billones Junie B. 
Published: June 2021

The NKX2‐5 is a gene that encodes for a transcription factor containing the homeobox domain which functions mainly in heart formation and development. Mutations in this gene have been known to cause heart malformation diseases and congenital hypothyroidism. The objective of this study is to determine, through methods in computational biology, the effect of mutations on the binding affinity of NKX2‐5 to DNA and thus predict their phenotypic consequences. The mutations used were determined using UCSC Genome Browser based on regions of highest conservation. Structure‐function prediction was performed for these mutations using Polyphen‐2, Mutation Assessor, HOPE, and Mutation Taster. Molecular docking experiments for the binding of NKX2‐5 to DNA were then performed using the ZDock module of Discovery Studio (Biovia). Results include the mutations rs757605578 (p. L163P) and rs104893906 (p. R190C), which were identified from regions of high conservation (across metazoans). The functional predictions for the mutant L163P using the tools Polyphen‐2, Mutation Assessor, HOPE, and Mutation Taster are “Probably Damaging” (Score: 1.000), “Low” (Score: 1.45), “Probably Not Damaging”, and “Could Not Retrieve a Sequence”, respectively. For mutant R190C, the predictions are “Probably Damaging” (Score: 1.000), “High” (Score: 4.015), “Severity: Disease”, and “Disease Causing” (Score: 180), in the same order as the previous. Results of the docking experiments show that the best poses for the wild‐type, L163P, and R190C had structural energies of −80.2641 kJ/mol, −82.1326 kJ/mol, and −81.4704 kJ/mol, respectively. These show that the mutant NKX2‐5 proteins had more stable binding to DNA. Examination of the non‐covalent interactions formed by mutated residues compared to wild‐type reveals that there are fewer interactions in the mutated residues compared to the wild‐type. These suggest that it may be the nature of the interactions (charge interactions, H‐bonds, polar interactions, or hydrophobic interactions) and/or steric effects that are responsible for the increased stability of binding by NKX2‐5 to DNA. It is concluded that mutations L163P and R190C are functionally highly important and are predicted to be, in general, probably disease‐causing. The increased stability of binding to DNA by the mutants, therefore increased transcription factor activity, may be the mechanism by which this predicted phenotype is achieved.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanisms of exercise protein FNDC5/Irisin in vasculature during zebrafish development
Authors: HUANG YI‐SHAN , Wu Chang‐Yi 
Published: June 2021

Vascular development plays an important role in vertebrates, and the molecular mechanism how formation of intersegmental vessels (ISV) and caudal vein plexus (CVP) in zebrafish is still not complete understood. FNDC5 (Fibronectin type III domain containing protein 5) gene encodes a type I transmembrane protein that is processed proteolytically cleavage and released into the blood, termed as exercise protein, Irisin. Irisin converts white adipocytes into brown adipocytes consumed energy to produce heat, involved in increase total body energy expenditure, reduced body weight. Previous studies have pointed out that adding Irisin to cell culture medium can activate the VEGF/VEGFR2/Akt signaling pathway. Irisin promote angiogenesis of endothelial cells by activating ERK signaling pathway, increase vascular migration and angiogenesis. However, the mechanism and fuction of FNDC5/Irisin in vasculature during embryo development is still unknown.Our in‐situ hybridization results showed fndc5a and fndc5b mRNA are expressed on vasculature from 18 somite to 30 hpf stages, suggesting its role in vascular development. Knockdown of fndc5a or fndc5b by morpholino injection caused vascular defects and abnormality of vascular circulation, indicating the role of fndc5a and fndc5b in ISV, CVP growth and tube formation. Our AO staining and TUNEL assay results showed that vascular defects do not caused by cell death, but due to the impairment of migration and/or proliferation by examining ISV cell numbers and integrity in Tg(kdrl:mCherry; fli:nEGFP)y7 fish. We found that fndc5a and fndc5b morphants showed a decreased expression of vascular markers ephrinb2, flt4, mrc1, flk and stabilin, indicating molecular mechanisms of vascular defects by fndc5a and fndc5b morphants. We further use Human Irisin to enhance vascular growth of ISV. The results indicate human Irisin can rescue vascular defects by knockdown of fndc5a and fndc5b, indicating Irisin has a highly conservation between human and zebrafish.In summary, our findings provided evidence that fndc5a and fndc5b may play an important role in vasculature during zebrafish development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA miR‐145 Modulates p38 MAP Kinase Pathway in Cardiac Fibroblasts to Suppress Cardiac Fibrosis
Authors: Marosis Sam , Sawant Dwitiya , McCallinhart Patricia , Trask Aaron , Thomas Shelby , Lilly Brenda 
Published: June 2021

Cardiac fibrosis is associated with many types of cardiovascular diseases and is characterized by the deposition of excess extracellular matrix by the cardiac fibroblasts. Previous studies demonstrated that microRNA miR‐145 acts as an inhibitor of fibrosis by targeting the profibrotic TGFβ pathway. Our preliminary data suggest that within the TGFβ pathway, miR‐145 specifically targets the p38 mitogen‐activated protein (MAP) kinase signaling, which is known to activate cardiac fibroblasts and drive the fibrotic response. Therefore, I hypothesize that miR‐145 targets the p38 MAP kinase pathway to regulate cardiac fibroblast activation and suppress fibrosis. Human cardiac fibroblasts (hCFs) were transfected with a miR‐145 mimic or a control mimic and TGFβ, a fibrotic agonist, was added to activate fibroblasts. Cells were processed for RNA and quantitative‐PCR was used to measure relative expression of genes within the p38 MAP kinase pathway. My findings show that MAP2K3 and MAP4K4 expression are inhibited by miR‐145 in activated hCFs. Further, TGFβ 3 and RGS2, both negative regulators of fibrosis, were induced in miR‐145 treated cells. These data indicate that miR‐145 modulates components of the p38 MAP kinase signaling pathway, which may contribute to its ability to regulate cardiac fibrosis. Ongoing studies will examine if overexpression of miR‐145 in a mouse model will affect the expression of these p38 MAP kinase mediators and contribute to a reduction in cardiac fibrosis.Support or Funding InformationThis project was supported by the American Physiological Society‐Undergraduate Research Excellency Fellowship, the American Heart Association Summer Undergraduate Research Fellowship, and the National Institute of Health grant R01‐HL‐135657 (to B. Lilly).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium Dependence of Transglutaminase and its Function in Apoptosis in Human Erythroleukemia Cell Lines
Authors: Lowther Warren J , Birckbichler Paul J 
Published: June 2021

The expression of transglutaminase (TG‐2) in human erythroleukemia cells is dependent on the intracellular calcium concentration. Human erythroleukemia cells (K562) arise from stem cells that have not fully differentiated to erythrocytes and maintain continuous, uncontrolled proliferation. The preferred apoptotic machinery reduces the degree of inflammation of the tumor. Previous studies have concluded that thapsigargin induces the rate of apoptosis in these cells and disrupts the expressivity of TG‐2. Expression of the TG‐2 protein can be visualized when complexed with CUB74, a monoclonal antibody to TG‐2, and GAM‐FITC. Immunofluorescent studies suggest that thapsigargin and an increase in available Ca2+ upregulates the expression of TG‐2 type proteins. Cells exposed to calcium shock also demonstrated an increase in the presence of TG‐2 on the periphery and intrarnuclear space of the cell. Calcium shocking cells may open the nuclear envelope to allow access of TG‐2, or stimulates the synthesis of the protein coding gene. EDTA, a popular chelating agent and DNase repressor, reduces the expression of TG‐2. The mechanism for this remains elusive. Investigations will be conducted to visualize any change in the amount of mRNA product as well as quantifying the difference in TG‐2 products.Support or Funding InformationFunding provided by Department of Chemistry, Slippery Rock UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing the fitness of HeLa cells expressing elongation factor 2 with mutations at the diphthamide histidine
Authors: Lowe Elliot Keith 
Published: June 2021

Elongation factor 2 (EF2), and its bacterial homolog EFG, are essential proteins in the process of translation. In Eukarya and Archaea, but not in Bacteria, a post‐translationally modified histidine, called diphthamide, is specific and unique to EF2. Diphthamide is a target of several bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A, and cholix toxin), but its role in translation remains elusive. Prior research in our laboratory investigated the viability and toxin resistance of HEK293 cells that express EF2 with mutations at the diphthamide histidine (His715 in mammals). These experiments indicated that all mutants except H715K were viable, but none displayed resistance to a diphthamide‐targeted toxin. In order to better understand the role that the diphthamide residue plays in translation we are generating and testing a library of EF2 mutations at His715 in the HeLa cell line. We plan to transfect this mutation library stably into HeLa cells, and evaluate the resulting clones that survive. We hypothesize that HeLa cells expressing these mutations containing are diphthamide‐deficient and, except for H715K, viable, yet not resistant to diphthamide‐targeted toxins. The frequency of mutants in the transfected cells will be assessed and compared to the mutant frequency of the original library to determine the overall fitness effect of each mutation. The end goal of this research is to independently assess the fitness of HeLa cells expressing EF2 mutants in order to evaluate the effect of different mutations at the diphthamide histidine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.