bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phage Display Selection and Identification of Novel Pancreatic Cancer Targeting Peptides
Authors: Asar Mallika , Soendergaard Mette 
Published: June 2021

Pancreatic ductal adenocarcinoma (PDA) is currently the fourth leading cause of cancer deaths in the U.S. It has been recently estimated that the disease causes approximately 30,000 deaths per year in the U.S., and that pancreatic cancer patients exhibit a 5‐year survival rate of only 3%. The high lethality of pancreatic cancer is due to the fact that most cases are diagnosed at advanced metastatic stages. While there are diagnostic methods in use clinically, most are inadequate, potentially leading to delayed diagnosis and further spreading of the cancer prior to treatment. Due to insufficient diagnostic techniques, it is imperative to develop new methods, which may facilitate early diagnosis. One such modality employs the use of peptides with high binding affinity and specificity to a pancreatic cancer biomarker.Bacteriophage (phage) display technology is a high throughput method for discovering peptides that confer specific binding properties to a single molecular target. The peptides are typically displayed on the distal end of coat protein III of the Fd filamentous phage. Here we report using phage display technology to select peptides with specific binding to human pancreatic cancer cells. To do so, a naïve fUSE5 15‐mer library (kind gift from Dr. George Smith) was negatively selected against normal pancreatic ductal cells (hTERT‐hPNE). Unbound cells were collected, amplified by infection of E. coli K91BK overnight, and isolated using polyethylene glycol (PEG), NaCl precipitation and centrifugation. Amplified phage were then subjected to four rounds of positive selection against pancreatic cancer cells (Mia‐Paca‐2) grown to 80–90% confluency. Between each round of selection, phage were collected, amplified in E. coli K91BK and isolated as described above. Stringency was increased in the fourth round of selection by performing the selection against cells grown to 60% confluency. After the final round of selection, phage DNA was extracted and polymerase chain reaction (PCR) amplicons were identified by paired‐end (2×150bp) next generation sequencing (MiSeq) followed by translation and sorting of the sequences (Genewiz, NJ). The “top” 10 peptides identified by this method will next be screened in cell binding assays to determine individual binding constants. Peptides that exhibit high binding affinity and specificity for Mia‐Paca‐2 cells may subsequently be employed as probes in the detection of pancreatic cancer.Support or Funding InformationWestern Illinois University Research Council GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolomic Responses to Burn Injury in a Rodent Model
Authors: Alkhalil Abdulnaser , Ball Robert , Day Anna , Kumar Raina , Hammamieh Rasha , Garg Guarav , Moffatt Lauren T , Shupp Jeffrey W 
Published: June 2021

Management of total body surface area (TBSA) burn injuries remains clinically challenging and is an understudied area. In order to explore the role of supplementary nutrient interventions and supportive tissue building component administration in the clinic to improve wound healing and patient survivability, we assessed metabolomic of a rodent model for burn injury of 30% TBSA. Nine mice were subjected to a 30% TBSA by submersion in 100 °C water. Mice sera or skin biopsies (4 mm) were taken on euthanasia at hour 2, 6, and 24 after injury (n=3 at each time point). An equal number of mice underwent sham procedures and biopsy collection for each time point with similar administration of anesthetic and fluid resuscitation. Samples were analyzed by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLCQ‐TOF‐MS). Multivariate analysis, the principal components analysis (PCA), was calculated using stats and pathway analysis using metaboanalyst R packages to analyze metabolic changes. Large changes in metabolite presence were detected in sham treated animals in both specimen types relative to baseline. These changes are caused by animal preparation steps and anesthesia were characterized by comparative analysis of results of specimens from thermally challenged animals relative to either baseline or to sham of the same time point across the study to identify burn associated changes. Metabolite responses in serum were more rapid than changes in skin, and while burn caused larger changes during early time points (1 and 2 hours post injury) in positively charged metabolites relative to negatively charged ones in serum samples, and to a lesser degree in sham treated skin samples, no difference was seen in burned skin samples. Pathway enrichment analysis of metabolites common to all time points from skin identified glycerophospholipid, ascorbate and aldarate, and phenylalanine metabolism pathways as the top three affected pathways. In serum, arachidoinc acid, starch and sucrose, and galactose metabolism were the top three pathways. These results underscore the differences in the kinetics and specimen‐dependent nature of metabolome responses.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.
Assessing Fucosyltransferase Activity via MSn
Authors: Ashline David , Reinhold Vernon 
Published: June 2021

Fucosyltransferases add fucose residues to oligosaccharides. Resulting fucosylated epitopes include the common core fucosylation of N‐glycans, as well as important ligands such as Lewis structures and H antigens. Many of these structural motifs can be found on N‐linked glycans, O‐linked glycans, and glycosphingolipids. In complex biological samples, multiple fucosyltransferases, as well as multiple substrate specificities of individual fucosyltransferases, can result in multiple isomeric forms within individual glycan compositions. Intact mass analysis alone will not distinguish these isomers. Chromatographic separation may or may not resolve these structures, and, will not provide direct structure identification.Typically, MS/MS analysis of fucosylated N‐linked glycans can distinguish core fucosylation from antennal fucosylation. Finer details such as distinguishing Lewis structures from H antigens is a more difficult problem, as fucosylated lactosamine antennae fragments are produced from both structures. Disassembling these structural fragments to a further stage, MS3, in an ion trap, can easily distinguish Lewis from H antigen structures, providing direct empirical evidence of structure.We demonstrate MSn data from a variety of samples. Fucosyltransferase activity was assessed through transfection of fucosyltransferase (3,4,5,6,7, and 9) genes into mesenchymal stem cells, with comparison to non‐transfected cells as a control. MCF‐7 and MDA‐MB‐231 cultured cells were also interrogated for fucosylation isomers. Glycophorin C was also interrogated, with fucosylation on polylactosamine structures.For cultured cells, glycolipids were extracted via solvent extraction. Proteins were enzymatically digested, followed by enzymatic release of N‐linked glycans. Released N‐glycans were separated from O‐glycopeptides; O‐glycans were released by reductive b‐elimination. Purified glycan and glycosphingolipid fractions were permethylated. Glycan samples were directly infused into the mass spectrometer via nano‐ESI. Precursor ion selection was made manually.Fucosyltransferase‐transfected mesenchymal stem cells were interrogated for synthesis of Lewis X structures, with data shown to indicate that different fucosyltransferases show differing preferences for synthesizing Lewis X vs. sialylated Lewis X.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examining the Accuracy of Monosaccharide Structures Calculated with Density Functional Theory by Experimental Comparisons
Authors: Ziperman Emily D. , Calixte Emvia I. , McCutcheon Meg , Pulipaka Srinivas , Gallagher Elyssia S. 
Published: June 2021

Glycans are prominent in biological processes. Glycan structural characterizations can have significant implications for disease progression, e.g. cancer metastasis.1 Glycans are challenging to analyze with traditional chemical techniques due to a lack in diversity of functional groups; thus, computational modeling of glycans can help bridge this gap in knowledge regarding glycan structures. However, it is necessary that the computational models adequately represent carbohydrate structures and align with experimental results. Experimentally, mass spectrometry (MS) is one technique that is used for glycan characterization. In MS analyses, glycans are often analyzed as ion adducts. A group of computational methods and basis sets have been used to compare the energies of a subset of monosaccharides2, but this work has not been expanded to the ion adducts detected with MS, nor has it included experimental comparison. Herein, we optimized the geometry of both anomers of fucose and glucose in neutral, protonated, ammoniated, and sodiated forms using Density Functional Theory with the B3LYP or B3PW91 methods and 15 basis sets. Three basis sets (cc‐pVQZ, GEN, and GENECP) were unsuccessful in running calculations to completion. For the remaining computations, we examined values from the output files for variations between methods and basis sets. If computational methods yield accurate structures, the calculated values for molecular parameters, e.g. dipole moment, should be similar. Yet, if there are incorrect approximations in the basis set, the calculated electron density around the molecule will be incorrect and alter the values of the parameters using that specific functional. Greater variation occurred in the average dipole moments calculated for all methods and basis sets of fucose (standard deviation (SD) = 1.5) compared to glucose (SD = 0.68). The addition of ion adducts increased error in dipole moment calculations compared to the neutral molecules. The variation in these results suggests that some methods and basis sets are not reliable when modelling carbohydrates. Currently, molecular dynamics runs using GROMACS are being done to obtain over 50 different coordinate sets of each system for input into MOBCAL.3 MOBCAL will be used to obtain theoretical collisional cross sections (CCS) which will be compared with experimentally measured CCS by ion mobility MS. These results will illustrate which computational methods yield accurate carbohydrate structures, allowing future application of this work to address the biological utility of glycans.Support or Funding InformationBaylor University Office of the Vice Provost for Research and the Undergraduate Research and Scholarly Achievement Small Grant Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unveiling the Mechanotransduction Mechanism of Substrate Stiffness‐modulated Cancer Cell Motility via ROCK1 and ROCK2 Differentially Regulated Manner
Authors: Peng Yueting , Chen Yu , Qin Xiang , Li Shun , Liu Yiyao 
Published: June 2021

In addition to soluble factors and stromal cells, the mechanical properties of extracellular matrix (ECM) are considered to be an important component of tumor microenvironment. In particular, substrate stiffness is a mechanical characteristic of the ECM by which certain anchorage cells sense and respond to a variety of cellular behaviors. At the same time, clinical studies have observed that substrate stiffness was increased during tumorigenesis, which could promote tumor malignant transformation and metastasis. However, the biomechanical behavior of tumor cells and the underlying mechanotransduction pathways remain unclear. Here, using stiffness‐regulable polyacrylamide (PAA) substrates to simulate the tissue stiffness at different progress stages of breast cancer in vitro, we found that moderate substrate stiffness induced the emergence of malignant phenotypes and further unregulated breast cancer cell motility. Subcellular structure observation showed that moderate substrate stiffness promote the formation of leading‐edge protrusion by accelerating focal adhesion maturation and polarizing intracellular traction force distribution. Moreover, the substrate stiffness directly activated integrin β1 and focal adhesion kinase (FAK), which accelerate focal adhesion (FA) maturation and induce the downstream cascades of intracellular signals of the RhoA/ROCK pathway. Interestingly, the differential regulatory mechanism between two ROCK isoforms (ROCK1 and ROCK2) in cell motility and mechanotransduction was clearly identified. ROCK1 phosphorylated the myosin‐regulatory light chain (MRLC) and facilitated the generation of traction force, while ROCK2 phosphorylated cofilin and regulated the cytoskeletal remodeling by suppressing F‐actin depolymerization. The ROCK isoforms differentially regulated the pathways of RhoA/ROCK1/p‐MLC and RhoA/ROCK2/p‐cofilin in a coordinate fashion to modulate breast cancer cell motility in a substrate stiffness‐dependent manner via integrin β1‐activated FAK signaling. Our findings provide new insights into the mechanisms of matrix mechanical property‐induced cancer cell migration and malignant behaviors. Taming these physical forces by implanting scaffolds with specific stiffness or by inhibiting specific ROCK isoforms could have potential implications for improving therapeutic outcomes in cancer.Support or Funding InformationThis work was supported, in part or in whole, by the National Natural Science Foundation of China (11772088, 31470906, 31700811, 11802056, and 31800780).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Function of Yeast Bax Inhibitor BXI1 in Redox and Calcium Homeostasis of the Endoplasmic Reticulum and Programmed Cell Death in Saccharomyces cerevisiae
Authors: Jacob Walter , Alisch Joseph , McDonough Liam , Abaherah Ibrahim , Andrews Nick , Bittner Michael , Raffa Amanda , Kalhorn Jack , Austriaco Nicanor 
Published: June 2021

Yeast Bax Inhibitor‐1 (BXI1/YBH3) encodes a protein (Bxi1p) that belongs to the Bax Inhibitor family of proteins. In mammals, this family of proteins has cytoprotective properties that are most evident in paradigms of endoplasmic reticulum (ER) stress and have been linked to several human cancers. Both the crystal structure of a prokaryotic member of the family (BsYetJ) and our own published studies suggest that yeast Bxi1p is a pH‐sensitive calcium leak channel localized to the ER membrane. It is involved in the unfolded protein response (UPR). Bxi1p is thought to act via a mechanism involving altered calcium dynamics. We now show that cells lacking BXI1 have an altered redox microenvironment in the ER. We also report that cells lacking BXI1 accumulate more calcium in the ER than wild type cells. Together, our data suggest that Bxi1p is involved in regulating the ER microenvironment and, consequently, programmed cell death.Support or Funding InformationOur laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interplay of reactive oxygen species, apoptosis and necroptosis in atypical cell death of Tak1‐deficient macrophages and mouse mortality
Authors: Lopez‐Perez Wilfred , Sakamachi Yosuke , Sai Kazuhito , Mihaly September , Takaesu Giichi , Ninomiya‐Tsuji Jun 
Published: June 2021

TAK1 is an intracellular signaling intermediate of TNF signaling pathway, in which it mediates inflammatory responses and plays a critical role in blocking TNF‐induced cell death. Tak1‐deficiency makes most cell types hyper‐sensitive to TNF‐induced apoptosis, which is mediated by caspase 8 with typical apoptotic features, e.g. caspase 3 activation and membrane blebbing. On the contrary, Tak1‐deficienct macrophages die with features closer to necrosis accompanied with marginal activation of caspases. TNF‐induced necrosis, known as necroptosis, is mediated by a protein kinase complex within which receptor interacted protein kinase 3 (RIPK3) plays a critical role. However, Tak1‐deficient macrophages only marginally activates RIPK3 pathway, and their cell death was not rescued by deletion of Ripk3. In the current study, we attempted to define the mechanism of this atypical Tak1‐deficient macrophage death. We found that mitochondria‐derived reactive oxygen species (ROS) were highly upregulated by ablation of TAK1 in macrophages. Unexpectedly, even though they lack typical apoptosis and necroptosis features, we found that double but not any single deletion of caspase 8 and RIPK3 largely abolished elevation of ROS and cell death in Tak1‐deficient macrophages. Interestingly, blockade of ROS diminished Tak1 deficiency‐induced marginal activation of the caspase and RIPK3 pathways. These demonstrate that ablation of TAK1 elicits a feedforward loop involving caspase 8, RIPK3 and mitochondrial ROS leading to atypical macrophage death. We also characterized mice having Casp8, Ripk3 and Tak1 gene deletion in the in vivo settings. Tak1 deletion caused animal mortality within several days with tissue injuries in the liver and the intestine. Compound double deletion of Casp8 and Ripk3 prolonged Tak1‐deficient animal life for an additional several days, but was insufficient for complete rescue. Thus, caspase 8 and RIPK3‐independent pathway(s) are also involved in Tak1‐deficient animal mortality.Support or Funding InformationThis work was supported by National Institutes of Health Grant GM068812 and GM112986 (J.N‐T). Y.S. and W.L‐P were partly supported by National Institutes of Health Training Grant T32 ES007046.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Growth and Transglutaminase 2 Expression within the Human Erythroleukemia Cell Line (HEL)
Authors: Schultz Shannon L , Birckbichler Paul J 
Published: June 2021

The objective of the study was to explore the biological responses of retinoic acid and sodium butyrate on human erythroleukemia cells (HEL). The process of apoptosis is exemplary in that the cytoskeleton collapses and the DNA is broken into fragments. The cellular components of the dead cell after undergoing apoptosis are able to be recycled. Furthermore, a cell that has experienced apoptosis does not disturb its neighbors. By learning the basics of human cell experimentation, further analysis was conducted of the HEL cell lines, such as an apoptotic assay and indirect immunofluorescence. Cells were treated with either buffer (control), retinoic acid (20 uM), sodium butyrate (100 uM) or a combination of retinoic acid (20 uM) and sodium butyrate (100 uM). Cell growth rates declined when the cells were treated with the chemicals; the greatest difference being in the combination culture of retinoic acid and sodium butyrate. The enzyme transglutaminase (TG2) is an enzyme found in large amounts within some cancer cells, and is known to have multiple functions. When the cells were treated with a monoclonal antibody to TG2, transglutaminase enzyme fluorescence in the control cultures were observed to be cytoplasmic. The retinoic acid‐treated cultures appeared to have a strong presence of the enzyme in the cytoplasm. The amount of TG2 appeared to increase in all the cultures with time with the greatest increase occurring in the combination cultures. After 144 hours a portion of the combination cultures had been converted to apoptotic bodies. Future research will attempt to localize the actual location of the transglutaminase when treated with both chemicals, and if it promotes or suppresses the viability of the cancer cells. Studies are underway to determine the amount of transglutaminase that is present in each of the samples.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.
Embryonic Stem Cells Derived Exosomes Enhances Chemosensitivity of Doxorubicin in Breast Cancer Cells
Authors: Das Anindita , Hirsch Alexander , Samidurai Arun , Singla Ritu , Singla Dinender K , Kukreja Rakesh C 
Published: June 2021

Doxorubicin (DOX) is the most effective anti‐tumor agent to treat wide variety of solid tumors, including the breast cancer. However, it is not known whether combination treatment of embryonic stem cells derived exosomes (ES‐exos) with DOX influences the killing of breast cancer cells. To address this question, breast cancer cell lines, MDA‐MB231 and MCF7 were treated with DOX (1mM) ± ES‐exos (5 μg/ml, 10 μg/ml or 20 μg/ml) for 24 and 48 hrs. Cell death was assessed by trypan blue exclusion assay and cell proliferation was measured by MTS assay. DOX treatment of MDA‐MB‐231 cells caused significant increase in cell death at 48 hrs post treatment, from 11.4 ± 0.01 in non‐treated controls to 38.2 ±1.2 % (p<0.05, n=4; Figure 1A). However, combinatorial treatment with DOX and ES‐exos increased cell death to 49.2± 2.1% (p<0.05 vs DOX, n=4; Figure 1A). Identical results were obtained when MCF‐7 cells were treated with DOX ± ES‐exos (Figure 1B), although these cells were relatively less sensitive to cell death compared to the MDA‐MB231 cells. Similarly, co‐treatment with ES‐exos resulted in an additive effect on DOX‐induced reduction of proliferation in both cell lines. Treatment of cancer cell lines with ES‐exos alone (concentrations ranging from 5 μg/ml to 20 μg/ml) had no significant effect on cell death or proliferation. These results suggest that ES‐exos dramatically sensitize the cell killing effect of DOX in breast cancer cells which may translate to enhancing the anti‐tumor properties of DOX. We also identified 30 to 400 fold increase in miR‐96, miR‐124, miR‐200a, b, c, miR‐205, MiR‐211 in ES‐exos compared with MEF‐exos (exosome isolated from non‐ES cells) (Figure 1C). Further investigations are in progress to demonstrate the functional role of the miRs or their protein targets in enhancing the chemosensitivity of DOXSupport or Funding InformationRO1CA221813 Figure 1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.