bluuebunny/crossref_metadata_2025 huggingface dataset:
Bioanalytical Method Development for the Detection of the Rac/Cdc42 Inhibitor MBQ‐167 in Mouse Tissue
Authors: Rosado‐González Gabriela Tatiana , Del Mar Maldonado María , Bloom Joseph , Hernández‐O'Farril Eliud , Rodríguez‐Orengo José F , Dharmawardhane Suranganie
Published: June 2021

Metastatic breast cancer is the second most common cancer in US women. The mortality rate has decreased approximately 40% in the last 25 years due to advancement in treatment and diagnostics. However, it is estimated that 20–30% of all breast cancer patients will develop metastatic breast cancer. Therefore, there is a need to develop targeted therapeutics to reduce cancer cell migration and subsequent metastasis. The novel small molecule MBQ‐167 inhibits Rac and Cdc‐42, proteins involved in cancer cell growth, migration and invasion. In vivo studies have shown that MBQ‐167 inhibits mammary tumor growth and metastasis in immunocompromised mice by ~90% (Humphries‐Bickley, et al., 2017). However, to continue validating this drug for FDA approval, it is necessary to study the pharmacokinetic profile of the drug, which includes elucidating the tissue distribution of the compound in the body. Hence, it is necessary to develop a rapid and sensitive method to quantify MBQ‐167 in tissues. The purpose of this study was to develop and validate a method for the quantification of MBQ‐167 in mouse tissues using Supercritical fluid chromatography coupled with electrospray ionization tandem mass spectrometry (SFC‐MS/MS). Mouse livers were homogenized, spiked with MBQ‐167, and liquid‐liquid extractions were performed with different organic solvents (heptane, ethyl acetate, 2‐propanol, toluene, and acetonitrile), in order to determine the optimal sample preparation method to selectively separate the drug MBQ‐167 from endogenous material in mouse tissue. Separation was performed on an ACQUITY UPC2BEH (3.0 × 100 mm, 1.7 μm) column at 40 °C with a mobile phase consisting of CO2 and methanol 0.1 % formic acid (95:5, v/v) at a flow rate of 1 mL/min. A tandem triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode. High recovery of MBQ‐167 (> 65%) and lower matrix effects were observed upon extraction with heptane:ethyl acetate (1:1). Consistent recovery was observed in other tissues such as kidney, lungs and heart. The method was sensitive with a lower limit of quantification (LLOQ) of 10 ng/mL. Linearity was observed over the concentration range of 10–1000 ng/mL. The data obtained supports the use of SFC‐MS/MS as a highly specific method to evaluate the tissue distribution of MBQ‐167 in mice.

Support or Funding Information

This study is supported by MARC grant NIH/NIGMS 5T34GM007821‐39 (to GRG) and NIGMS‐RISE 25 GM061838 (to MM).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Accuracy of Canine Scent Detection of Lung Cancer in Blood Serum
Authors: Junqueira Heather , Quinn Thomas , Biringer Roger , Hussein Mohamed , Smeriglio Courtney , Barrueto Luisa , Finizio Jordan , Huang Michelle
Published: June 2021

Canines are able to differentiate between serum samples taken from cancer patients and samples taken from normal controls. This study further supports the use of dogs as biomedical research tools for detection of cancer biomarkers. In particular, this study was designed to determine the accuracy of canines' ability to detect, by scent alone, lung cancer biomarkers in blood serum.

Operant conditioning was used in the form of clicker training to train four beagles to distinguish, by scent alone, blood serum from malignant lung cancer patients when presented along with healthy controls in a double‐blind format. Non‐small cell lung cancer and healthy control blood serum samples were presented to 2‐year‐old beagles.

Three dogs were able to correctly identify the cancer samples with a sensitivity of 96.7%, specificity of 97.5%, positive predictive value (PPV) of 90.6% and negative predictive value (NPV) of 99.2%. One of the dogs, Snuggles, was unmotivated to perform during training and tested with 80% specificity and 60% sensitivity. This study paves the way for a larger scale research project designed to explore the use of canine scent detection as a tool for detecting cancer biomarkers, ultimately leading to their identification.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

NIH/NINDS R21 NS092010

This 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 Information

Hampden‐Sydney College Office of Undergraduate Research

This 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

BACKGROUND

Oxytocin (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.

METHODS

200 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.

RESULTS

Reduction 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.

DISCUSSION

Alkylation 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.

CONCLUSION

Improving 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 Information

None

This 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 Information

Research 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 Information

This 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 Information

Funding provided by Department of Chemistry, Slippery Rock University

This 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.
Comparison study of exosomes molecules driven from (NCI1975) NSCLC cell culture supernatant isolation and characterization techniques
Authors: Mahgoub Elham Omer , Haik Yosef , Qadri Shahnaz
Published: June 2021

Background

Lung cancer consider as one of leading cause of death around the world, normally the disease detection is difficult in early stages, Anew methods for diagnostic using non‐invasive way are developed using Nano‐molecules called exosomes. Exosomes are cell derived vesicles carrying different kind of molecules (proteins, DNA, RNA, microRNA) and also, displaying many proteins on their membrane surfaces. Those exosomes molecules are identical of parental cancer cells that induce them.

Methods

In this study, exosomes were isolated and characterized using multiple centrifugations, ultrafiltration, and chemical precipitation techniques. The resulted products were captured by two electro‐Microscopes (EM) methods for characterizing the morphology of exosomes as to differentiate exosomes from other extracellular vesicles Besides, the resulted products also further characterized by blotting the proteins extracted from exosomes against different monoclonal antibodies (CD81, CD63, CD9) that confirmed the existence of ligands attached to exosomes surfaces in western blot test.

Results and Discussion

the exosomes isolated products were screened under Transmission Electron Microscopy (TEM). The size distributions of extracellular vesicles were significantly different from exosomes 160 nm P < 0.001). The size distributions of apoptotic vesicles (APV) 450nm and necrotic bodies, (NCB) 280 nm were significantly different from exosomes (P < 0.001). The same size band was observed in PVDF membranes of western blot test in all three methods using each monoclonal antibody. However, the filtration devices give higher protein yield and greater purity.

In Conclusions

The current results confirmed the accessible use of exosomes as biomarker to detect lung cancer cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antiproliferative Properties of Lichens Parmelia vagans and Parmelia sulcata
Authors: Bondarenko Vladimir , Raymond Ky
Published: June 2021

The purpose of this project was to analyze antiproliferative properties of the secondary metabolites extracted from lichens Parmelia vagans and Parmelia sulcata collected from the semi‐deserts of Russia. An isolation and characterization of novel natural compounds that can selectively slow down or stop proliferation of cancer cells can enhance development of anticancer therapeutics.

Various lichen extracts were prepared using either water or organic solvents. We assessed the antiproliferative activity against several human cancer cell lines; human hepatocellular carcinoma (Hep G2), human non‐small cell lung cancer (NCI‐H1299), and human mammary gland ductal carcinoma (T‐47D). The same number of cells of different cell lines were plated into a 96‐well plate and allowed to adhere overnight. Next day, different dilutions of the lichen extracts were added to the adhered cells and the plate was placed in CO2 incubator for 6–72 hours. By the end of the incubation time a picture of each well was taken to assess cell confluency and the cells were subjected to Resazurin assay to analyze the effect of the lichen extracts on cell growth and proliferation. After Resazurin assay, the cells in each well were treated with trypsin, suspended in growth medium and counted using Bio‐Rad cell counter to evaluate a percent of live cells.

Our experimental data from cell viability assay and live cells count indicate that bottled water and ethyl acetate extracts of P. vagans and P. sulcata significantly suppress (30%–60%) the growth of human hepatocellular carcinoma and human non‐small cell lung carcinoma. We also found that cancer cells treated by the lichen extracts results in their morphological changes that resemble an activation of apoptosis.

Our findings indicate that these lichen species produce secondary metabolites that slow cancer cells growth and proliferation. Future experiments will be focused on isolation and characterization of the compounds from P. vagans and P. sulcata crude extracts that are responsible for the inhibition of cancer cells growth. Another important direction of the future research is to identify the cellular target(s) of the antiproliferative compound(s) and its molecular mechanism of action. I summary, the lichen's secondary metabolites may hold a vast medicinal potential and could be a viable source of a novel of anticancer drugs.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
UPLC‐MS Analysis of Characterization of Bioactive Compounds Produced by Endophytic Bacillus tequilensis ALR‐2 from Aloe vera Plant
Authors: Akinsanya Mushafau Adewale , Goh Joo Kheng , Ting Adeline Su Yien
Published: June 2021

Endophytic Bacillus tequilensis ALR‐2 isolated from Aloe vera plant, was found to have antimicrobial activities attributed to a series of lipopeptide antibiotics and biosurfactants produced. Partial‐purification of its ethylacetate extract by column chromatography gave SF5 (5mg mL−1) which effectively inhibited Bacillus cereus and Staphylococcus aureus (28.6±0.5 and 30.3±0.5 mm zone of inhibition, respectively), and significantly more effective than the standard antibiotics Ciprofloxacin (5μg) (23.6 ±0.5 and 18.6 ±0.5 mm, respectively) and Kanamycin (30μg) (20.3 ±0.5 and 13.6 ±0.5 mm, respectively) against the two pathogens. UPLC‐MS analysis of SF5 showed three series of ion peaks. The quadrupole‐orthogonal ionization time‐of‐flight mass spectrometer revealed [M+H]+ of 678.4601; 679.4690 and 680.5076 were shown to correspond to the oligopeptide antibiotics, monamycin (mol. wt. 677.84) and [M+H]+ of 1119.8870; 1120.8992 and 1121.9119 correspond to antibiotic complex 61–26 (mol. wt. 1120.348) a lipopeptide. However, [M+H]+ of 1356.0151 and 1358.0664 could not be classified to what is known in the literatures.

Support or Funding Information

This work was supported by Higher Degree for Research (HDR) School of Science, Monash University Malaysia and Sponsorship from Tertiary Education Trust Fund of Federal Republic of Nigeria. We wish to acknowledge the assistance provided by Ms Amreeta Sarjit of Microbiology Teaching Laboratory of Monash University Malaysia for the provision of bacteria pathogens used for this project.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Investigation of Milly, a K2 Mycobacteriophage
Authors: Conte Amy , Ganey John , Cornely Kathleen
Published: June 2021

Mycobacteriophages are viruses that infect bacteria of the genus Mycobacterium. These phages are capable of undergoing two life cycles: the lytic cycle and the lysogenic cycle. In the lytic cycle, the phage inserts its DNA into a bacterial host, and in response, the host produces all of the proteins and other materials necessary to create more phage. The phage then multiply exponentially within the bacterial host until the bacterium lyses, releasing thousands of new phage to infect more bacteria. On the other hand, during lysogeny, the bacterium incorporates the phage DNA into its own genome, allowing it to replicate without producing new phage or lysing. Due to the increasing prevalence of antibiotic resistance, we are interested in taking advantage of the lytic cycle in order to treat bacterial infections, such as tuberculosis, with mycobacteriophage therapies. Using the Bacteriophage Recombineering of Electroporated DNA (BRED) method, we hypothesized that we could delete the gene coding for the repressor protein on Milly's genome, which would prevent Milly from undergoing lysogeny and make it lytic‐only. Here we report a characterization and manipulation of the K2 cluster mycobacteriophage Milly, isolated at Georgia College and State University, which we have determined to be capable of both the lytic and lysogenic phage life cycles. We are in the process of demonstrating the deletion of Milly's repressor gene so that it loses its ability to undergo lysogeny. This way, we can utilize Milly's lytic cycle as a way to infect and kill disease‐causing bacteria.

Support or Funding Information

This research is funded by the Providence College Undergraduate Research Committee. We would also like to thank the Providence College Department of Chemistry and Biochemistry for their support.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Step Past Phage Hunters, Modification of the Mycobacteriophage ZoeJ Genome for Phage Therapies
Authors: Scano Colin J , Jancevski Alicia , Scola Shelby , Ferrante Aaron
Published: June 2021

Mycobacteriophages are viruses that infect host mycobacterial cells like the mycobacterium tuberculosis. When infected, the host bacterial cell will either enter a lytic or lysogenic phase. In the lytic cycle, the host cell is transformed into a phage DNA producing factory. This host cell then lyses, releasing numerous new phages that infect other bacterial cells, carrying on the cycle. During lysogeny, however, the phage genome is integrated into the bacterial cell genome and the host cell does not lyse, remaining dormant for periods of time. The use of phages for the treatment of bacterial infections known as phage therapy is an alternative to the conventional use of antibiotics and could potentially replace it entirely. Since only the lytic cycle offers antimicrobial treatment, problems arise in phage therapy due to the inability to control the lifecycle of phages. A solution to this problem involves the creation of a lytic‐only phage through alterations of the phage genome. The phage genes responsible for the lysogenic cycle and genome integrations are the repressor and integrase genes. The goal of this project is to alter the phage genome in order to construct lytic‐only phage that lacks the repressor or integrase genes. This project focuses on the K2 cluster phage ZoeJ that was discovered in the SEA‐PHAGES/Phage Hunters program at Providence College. We have previously demonstrated that the repressor gene can be knocked out in the ZoeJ phage, and now aim to knock out the integrase gene in the wild‐type ZoeJ phage, as well as in the Δgp46 ZoeJ phage. Using the BRED method developed by the Hatfull laboratory, we aim to take advantage of the homologous recombination of bacteria DNA with phage DNA to delete the integrase gene from the ZoeJ phage genome.

Support or Funding Information

Funding for the project and trip has been supplied by the Providence College Biochemistry department through the PC small grants for undergraduate research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Demonstration of Photodynamic Molecules as Larvicides towards the Yellow Fever Mosquito Aedes aegypti
Authors: Meier Cole , Rouhier Matthew
Published: June 2021

Each year, mosquitoes serve as a vector to transmit diseases that kill upwards of 750,000 people. Although the need for vector control of these organisms is clear, the misuse of modern pesticides has caused significant ecological damage as well as the development of pesticide‐resistant mosquitoes. Photosensitive insecticide technology has shown potential to minimize ecological damage by targeting classically resistant mosquitoes in new ways. These photoactive molecules, specifically safranin‐O (Saf‐O) and methylene blue (MB), transfer their energy to molecular oxygen to form reactive oxygen species (ROS). These species damage biological systems in the mosquito that differ from the systems excessively targeted by modern pesticides. By targeting new and less resistant biological systems, photosensitive insecticides present a novel strategy for vector control. Currently the literature lacks the in vivo pathway describing ROS generation by photodynamic insecticides. Here we show the generation of ROS in vivo using Saf‐O and MB along with a model of the photodynamic interaction with the Aedes aegypti larva.

Support or Funding Information

The authors would like to thank the Kenyon College Summer Scholars Program for the funding received to pursue this project.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the Chemistry and Biological Action of Midwestern Medicinal Plants
Authors: Nguyen Nguyen‐Anh Thi , Shima Rafaella Tami , Abelkis Alexandra , Carter Jasmine , Cornwell Claire , Scerbak Courtney , Deibel Michael
Published: June 2021

Literature has demonstrated Caenorhabditis elegans possess similar cellular mechanisms when responding to toxic stress and increasing lifespan. In addition, a plant‐based diet with antioxidant activity and high phenolic content are associated with increased health. We tested the hypothesis that medicinal plant extracts would improve C. elegans survival rates when exposed to toxic (hot and cold temperature) stresses. The chemical content and biological activity of mulberry, goldenrod root, goldenrod leaf, blue violet, black raspberry, and red root plant extracts were measured and their compound class fractions (anthocyanin, flavonol, tannin, and phenolic acid). We found that with increased phenolic content, extracts had higher antioxidant activity; this correlation is largely attributed to anthocyanin content. Additionally, phytochemicals not isolated in our fractions and/or synergistic interactions contributed to the overall antioxidant capacity of extracts. We did not find a correlation between antioxidant capacities and C. elegans survival rate under toxic stresses. However, mulberry (50 μg/mL) significantly increased the survival rate in response to cold stress and blue violet ( 400 μg/mL) extract treatment trended towards increased survival rate in heat stress. This research is a preliminary step for further study and discussion to understand the comparison between medicinal plant activity and C. elegans survival and longevity.

Support or Funding Information

This work was funded by the Earlham College Center for Global Health and a Senior Research Grant from the Indiana Academy of Sciences (to Courtney Scerbak).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of oxidized mitochondrial mRNA in human neurons under oxidative stress
Authors: Rana Joram , Kharel Prakash , Basu Soumitra
Published: June 2021

Mitochondrial dysfunction and the overproduction of reactive oxygen species (ROS) are key features of many neurological disorders. Electrons leaking from the electron transport chain (ETC) in the mitochondria reduces molecular oxygen to superoxide, which through enzymatic and non‐enzymatic reactions generates ROS. Under normal physiological conditions ROS are used in cellular homeostasis and cell signaling, but during stress conditions, they are often overproduced. ROS are known to damage biomolecules DNA, RNA, proteins, and lipids. RNA damage has received attention in recent years. Studies in our lab demonstrated that the selective oxidative damage to RNA could lead to altered gene expression and differential production of protein, contributing to neurodegeneration in multiple sclerosis (MS). The mitochondria contain its own DNA, which transcribes into RNA and subsequently gets translated into mitochondrial proteins using its unique machinery. Selective mitochondrial RNA oxidation could disrupt this process, damaging the mitochondrial respiratory chain proteins, and eventually causing more mitochondrial dysfunction, contributing towards the progression of neurodegeneration in MS and other neurological disorders. The SH‐SY5Y human neuronal cells were treated with sodium nitroprusside (SNP, NO·/·OH) to induce stress and generate ROS to mimic the diseased condition. By quantitating cellular RNA, we have displayed the differential mRNA expression of a number of genes during oxidative stress, for example, high expression of superoxide dismutase 1 and carbonyl reductase. In this study, we focused on mRNA transcribed specifically inside the mitochondria to examine which sequences are most susceptible to oxidation by ROS. We found that 9 of the 13 mRNAs transcribed by mitochondria are highly oxidized. The oxidation of these mitochondrial mRNAs might lead to complication in protein synthesis and might play a role in mitochondrial dysfunction.

Support or Funding Information

Division of Research & Sponsored Programs (SURE program), Kent State University Graduate Student Senate, Kent State University to PK.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergistic Chemopreventive Effect of Medicinal Plants against Azoxymethane‐induced Oxidative Stress and Colon Carcinogenesis
Authors: Waly Mostafa I
Published: June 2021

Background

Oxidative stress plays an essential role in the development of different types of cancer malignancy, including colon cancer. The protective effect of different medicinal plants against oxidative stress‐mediated colon carcinogenesis has been reported, yet their synergistic effect was not elucidated.

Objective

This study investigated the antioxidant potential properties of a mixture of five medicinal plants (Rumex vesicarius, Oxalis corniculate, Pteropyrum scoparium, Moringa peregrine, and Caralluma arabica) in a rat model of colon cancer.

Methods

Azoxymethane (AOM), a colon selective carcinogenic agent, was used to induce colon cancer in Sprague Dawley rats which were divided into 4 groups, n=10, (Control, AOM‐injected group and the other two groups were fed ad‐libitum for 12 weeks a diet supplemented with a mixture of the five different medicinal plants in the presence or absence of AOM injection). By the end of the experiment, all rats were sacrificed; colon tissues were excised and homogenized for biochemical measurements of: oxidative DNA damage, reduced glutathione, and total antioxidant capacity. Histopathological examination for colon tissues was also examined for aberrant crypt foci (ACF) development.

Results

AOM induced oxidative stress in the colonic milieu as evidenced by high level of oxidizing DNA, glutathione depletion, and impairment of cellular total antioxidant capacity. In addition, AOM increased the formation of ACF. Meanwhile rats fed with the five medicinal plants mixture showed a protective effect against the AOM‐induced oxidative insult and attenuated the colonic ACF formation.

Conclusion

This study revealed that the examined medicinal plants provided a colon specific protection and might be used as a functional dietary bioactive agent in the context of primary prevention of colon cancer.

Support or Funding Information

Sultan Qaboos university Funding, Internal grant (IG/AGR/FOOD/01/17)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.