bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating the Effects of Cancer Mutations on Mre11 Function and the DNA Damage Response
Authors: Harris Caitlin , Savas Jessica , Shanle Erin Karina 
Published: June 2021

When DNA is damaged, a DNA damage response (DDR) pathway is initiated to prevent mutations from being passed on to daughter cells. The MRN complex in humans (MRX in yeast) functions to start the DDR pathway by detecting DNA damage and is comprised of Mre11, Rad50, and Nbs1 in humans. Although mutations in members of the MRN complex pre‐dispose individuals to cancer, the functional effects of Mre11 cancer mutations have not been well characterized. The goal of this project was to investigate the effects of Mre11 cancer mutations on the DDR pathway using yeast‐based assays because the complex is highly conserved in eukaryotes. Using a database of human tumor mutations, we identified 135 cancer mutations in human Mre11, 46 of which occurred at residues that are conserved between yeast and human. A subset of these mutations were screened for functional effects in yeast by creating the cancer mutations at the conserved residues in yeast Mre11. Yeast expressing wild‐type or mutant Mre11 were then assessed for DNA damage sensitivity using ultraviolet light and methyl methanesulfonate. Results indicate that N‐terminal point mutations at in yeast Mre11 do not confer sensitivity to DNA damage in yeast cells suggesting that these regions may have conserved sequence without conserved function. Using yeast‐based assays, cancer mutations can rapidly be screened for functional effects on DDR pathways.Support or Funding InformationLongwood University Faculty Research and Development GrantsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for the Accumulation of 3‐Hydroxypropionate and β‐Alanine in Arabidopsis thaliana Seedlings via Isoleucine Degradation
Authors: Goldfarb Margo H , Rouhier Kerry A 
Published: June 2021

Amino acids serve several cellular processes outside of proteinogenesis that influence the growth and development of the plant, as well as the generation of metabolic energy. Here, we examine the contribution of branched‐chain amino acid degradation to the production of β‐alanine in the model organism, Arabidopsis thaliana. β‐alanine is a key intermediate in the synthesis of the essential molecules vitamin B5 and coenzyme A (CoA). Despite the importance, the current knowledge surrounding β‐alanine lacks strong evidence for the primary biosynthetic pathways in higher plants. Previous studies found that polyamines, uracil, and propionate can all serve as sources for β‐alanine. Given that the catabolism of branched‐chain amino acids (BCAAs) valine and isoleucine result in the production of propionyl‐CoA (a derivative of propionate), we hypothesized that these BCAAs could also serve as a source of β‐alanine. Propionyl‐CoA is typically converted to acetyl‐CoA via β‐oxidation, however, one of the intermediates, malonate semialdehyde, can react to form β‐alanine. To trace the production of β‐alanine from propionyl‐CoA and the BCAA isoleucine, we treated wild‐type and transgenic seedlings with isotopically‐labeled precursors and analyzed extracts using 13C‐NMR and GC‐MS. We observed the production of both β‐alanine and a key intermediate, 3‐hydroxypropionate (3HP) in samples treated with propionate and isoleucine. Our data shows that the fed 13C‐isotopes were efficiently incorporated into and recycled through propionyl‐CoA metabolism, resulting in the production of 13C‐labeled β‐alanine. These findings suggest that while there is evidence for the production of β‐alanine via polyamines, uracil, and propionate in other plants; isoleucine, and possibly valine, can also serve as a precursor to this metabolically important molecule.Support or Funding InformationClare Boothe Luce Undergraduate Scholars program funded by the Henry Luce Foundation (Grant #9601)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effects of Uncaria tomentosa on acetylsalicylic acid and paclitaxel
Authors: Contreras Jessica Irene , Larios Kelsy N. , Nogaj Luiza A. 
Published: June 2021

For thousands of years, the indigenous women of Peru have relied on traditional medicine as a method for treating illnesses. While conventional medicine exists in Peru today, women continuously turn to the remedies they know and trust when modern medicine is inaccessible. The vast amounts of ethnopharmacological plants are utilized for a large span of conditions such as digestion, headaches, and inflammation. In particular, Uncaria tomentosa commonly known as Cat's claw, a bark like plant native to the Amazonian regions of Peru has been investigated for its historical anti‐inflammatory uses and was found to contain many antioxidant properties. However, not much is known about the effects of U. tomentosa on the molecular mechanism of drugs such as paclitaxel (taxol) and acetylsalicylic acid (aspirin). To address this question, a randomized survey was conducted on women and health care providers within the region of Cusco, Peru. The survey revealed that U. tomentosa remains a prominent healing aid within the Peruvian culture. In addition the survey showed that women overwhelmingly chose natural remedies over professional medical care. The following investigation was conducted to test the in vitro effects of U. tomentosa on the molecular mechanism of drugs paclitaxel and acetylsalicylic acid on MCF‐7 cells. An optimal concentration of paclitaxel and acetylsalicylic acid was established for further use in viability assays, MTT and Cytotoxicity. Preliminary data suggests that a 0.6 uM concentration of paclitaxel decreases cell viability by 55% and a 0.4 mM concentration of aspirin decreases cell viability by 20%. In addition, the preliminary data from molecular viability assay shows that the addition of U. tomentosa to acetylsalicylic acid and paclitaxel has an additive effect. Further analysis is necessary to determine the molecular mechanism of U. tomentosa on the activity of acetylsalicylic acid and paclitaxel.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Factors contained in the expansion medium regulate the expression stem cell markers CD146 and SUSD2 on human placenta‐derived mesenchymal stromal cells and modulate their differentiation capacity in vitro
Authors: Abruzzese Tanja , Aicher Wilhelm K 
Published: June 2021

Human mesenchymal stromal cells (MSCs) are investigated in vitro and in vivo manifold to serve as cellular therapy in different clinical situations including the regeneration of muscular tissues eventually. For production of MSCs for experimental purposes expansion media often include fetal bovine serum (FBS) as source of growth factors. In clinical situations xenogenic components such as FBS must be avoided in many countries. Therefore, different xeno‐free MSC expansion media were developed and FBS was replaced e.g. by human serum (hS), platelet lysate (hPL) or hPL plus human plasma (hPLP). However, differences in these medium complements could influence the phenotype and characteristics of MSCs. We therefore compared the expression of cell surface markers, the differentiation capacity and the expression of key cytokines involved in muscular regeneration in human placenta‐derived MSC‐like cells (pMSCs) expanded in different xeno‐free and GMP‐compliant expansion media. We report that addition of 5% hPL to either commercial MSC expansion medium (medium with FBS + 5% hPL) or to GMP‐compliant MSC expansion medium (DMEM + 5% hS + 5% hPL) significantly increased the MSC proliferation (p<0.05), and reduced the expression of CD146 (p<0.05), whereas CD73 and CD90 and other markers remained unchanged, on both transcript and protein levels. Expression of stem cell marker SUSD2 was not significantly regulated by hPL. The osteogenic differentiation capacity was reduced in pMSCs expanded in medium complemented with 5% hP + 5% hPL when compared to pMSCs incubated in medium containing FBS without hPL, but not in DMEM + hS compared to DMEM+ hS + hPL. Significant changes in the expression of neuro‐ or myo‐regenerative cytokines including BDNF, bFGF, GDNF, HGF, IGF‐1, IGF‐2, TGFβ, or VEGF in MSCs was not influenced by the composition of the GMP‐compliant expansion media. However, some interindividual differences between pMSCs from individual donors and between batches of hPL were noted. In contrast, the composition of expansion media did not influence osteogenesis or expression of stem cell markers CD146 and SUSDA on human bone marrow‐derived MSCs. We conclude that the expression of CD146 and the osteogenic differentiation potential of human pMSCs is influenced by the expansion medium. The expression of the regenerative cytokines investigated was not biased by hPL in the expansion medium. Expansion of pMSCs in GMP‐compliant medium enriched by hP + hPL could therefore be an advantage when bone formation by the MSCs applied should be avoided in clinical situations.Support or Funding InformationThis work was supported in part by grants from the DFG, BMBF and by institutional funding.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying Lysine Acetylation of Aminoacyl‐tRNA Synthetases in Escherichia coli
Authors: Fan Chenguang , Gan Qinglei 
Published: June 2021

Aminoacyl‐tRNA synthetases (AARSs) generate aminoacylated tRNAs for ribosomal protein synthesis, and play key roles in maintaining high fidelity of translation. A series of acetylome studies have shown that the family of AARSs is one of the protein families which have both the most acetylation sites and the highest acetylation levels. However, those putative acetylation targets have been less studied, mostly because of the challenge to produce homogeneously acetylated proteins at specific positions which needs the knowledge of specific acetyltransferases for target lysine residues. To overcome this issue, we applied the genetic code expansion strategy to co‐translationally incorporate acetyllysine at the target positions directly. We totally generated 25 site‐specifically acetylated variants of four AARSs. Kinetic analyses were performed to biochemically characterize the impact of site‐specific acetylation on AARS functions, including both tRNA aminoacylation and editing activities. The results showed that impacts of acetylation were different for class I and class II AARSs, and also varied among the same class of AARSs. Combined with quantitative proteomic studies, we proposed that the impact of acetylation on AARS editing activities could be more significant than that on tRNA aminoacylation activities. To the best of our knowledge, this is the first work to show the association of protein acetylation with the AARS editing function. Furthermore, using threonyl‐tRNA synthetase (ThrRS) as a representative, both in vivo and in vitro studies were performed to characterize the acetylation and deacetylation processes of AARSs. Although nonenzymatic acetylation and CobB‐dependent deacetylation were concluded, our results also indicated existence of additional acetyltransferases and deacetylases for AARSs.Support or Funding InformationThis work was supported by Oak Ridge Associated Universities, Arkansas Biosciences Institute, and the University of Arkansas.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational determination of the binding kinetics of the pH‐Low Insertion Peptide
Authors: Freshwater Kasey , Flint Bethann , Mertz Blake 
Published: June 2021

Membrane active peptides (MAPs) have the potential to be utilized in a number of biomedical applications due to their ability to alter the membrane through surface interactions. MAPs with pH‐dependent activity can be harnessed for use in medical imaging and targeted drug delivery to cells afflicted with diseases characterized by acidosis, such as cancer, arthritis, and heart disease. An example of this type of MAP is the pH‐Low Insertion Peptide (pHLIP), which interacts with the membrane in three states: in State I, pHLIP is randomly coiled in solution; upon addition of lipid vesicles, pHLIP binds to the membrane surface and gains partial helicity (State II), and upon acidification, pHLIP fully folds, inserting unidirectionally into the membrane (State III). Currently, the peptide‐membrane interactions leading to formation of stable complexes (i.e., state II) are poorly understood. To better understand this mechanism, we employed coarse‐grained molecular dynamics (CGMD) simulations of pHLIP interacting with either 1‐palmitoyl‐2‐oleoyl‐sn‐3‐phosphocholine (POPC) or mixed POPC:1‐palmitoyl‐2‐oleoyl‐3‐sn‐phosphoserine (POPS) lipid bilayers to determine the effect of headgroup charge on the ability of pHLIP to bind to a membrane surface. Our goal was to rigorously determine the kinetics of binding between pHLIP and the bilayer and to identify the key biophysical interactions that lead to a stable binding event, as well as characterizing the effect of headgroup composition on these behaviors. Our preliminary results show that the interaction of pHLIP with a POPC bilayer initiates at the N‐terminus and induces partial helicity. As the concentration of the anionic POPS lipids increases, the interactions between the anionic residues of pHLIP in the C‐terminus and the charged lipid head groups will be less favorable due electrostatic repulsions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Substrate Selectivity of a P4‐ATPase Flippase
Authors: Dudek David , Daleke David 
Published: June 2021

Phospholipids, the primary structural components of cell membranes, are distributed unequally across the membrane. The sphingolipids, including sphingomyelin (SM) and glycosphingolipids, are enriched on the exofacial surface of the plasma membrane or the luminal surfaces of internal organelles, while the cytofacial leaflet is enriched in the aminophospholipids, phophatidylserine (PS) and phosphatidylethanolamine (PE). Contributing to maintenance of this asymmetry is a combination of slow passive transbilayer phospholipid movement, trapping by cytofacial proteins and the activity of a number of energy‐dependent transporters. The substrate specificity and directionality of these pumps defines the transbilayer asymmetry of phospholipids. Two transporter superfamilies, ABC transporters and P‐type ATPases, account for these activities and move lipids in either the exofacial (floppase) or cytofacial (flippase) directions, respectively. In previous studies we have characterized the biochemical requirements and substrate specificity for a PS specific flippase activity in human red blood cells. Transport is ATP‐dependent and sensitive to vanadate and sulfhydryl‐modification. This flippase is selective for the sn‐1,2‐isomer of PS. Transport is diminished by modification of the PS headgroup amine (with the exception of mono‐methylation), carboxyl and phosphate groups, while a variety of fatty acyl groups and the D‐serine analog are accommodated. Red blood cells express two members of the P4‐type ATPase subfamily, ATP8A1 and ATP11C, that are likely responsible for this activity. Here, we report on the heterologous expression, purification, and functional assaying of hexahistidine‐tagged constructs of Atp8a1 with and without its cognate accessory subunit, Cdc50a. Purified Atp8a1 is vanadate‐sensitive and dependent upon Cdc50a co‐expression and PS for robust ATPase activity, although lipid specificity is not dependent on the presence of Cdc50a. In addition to reconstituting the flippase activity of Atp8a1 and ATP11c in vitro, our efforts are focused on rational mutagenesis of Atp8a1 to identify the lipid transport path and the determinants of substrate specificity, directed by homology modeling and the lipid‐binding characteristics of the closest members of the P‐type superfamily. X‐ray crystal structures of the SERCA pump in conformation E2 (PDB 2AGV) display a PE glycerol backbone and head group tightly pinched by helices M2 and M4 with PE's acyl chains extending between helices M2 and M6. This PE‐binding site implies a mechanism for how the P4lipid‐binding site and transport pathway evolved from ancestor P‐types designed to move simple cations. With these clues, we aim to identify the PS binding site and deduce the origin of the sn‐1,2‐stereospecificity of Atp8a1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating regulation of the PI 4‐kinase Pik1 at the Saccharomyces cerevisiae trans‐golgi network
Authors: Diefenderfer Carolyn M , Fromme J. Christopher 
Published: June 2021

Vesicle‐mediated protein transport among eukaryotic cellular compartments is dynamic and tightly‐regulated. At the Saccharomyces cerevisiae trans‐Golgi Network (TGN), the conserved small GTPases Ypt31 (Rab11 homologue) and Arf1 collaborate with an essential pool of phosphatidylinositol 4‐phosphate (PI4P) to recruit vesicle coats, vesicle transport motors, and tethering proteins to nascent outbound vesicles. The conserved lipid kinase Pik1 (PI4KIIIβ homologue) generates PI4P at the TGN, and work in several model systems suggests that it is regulated by close ties to the activities of Ypt31, Arf1, and the ArfGEF (guanine nucleotide exchange factor) Sec7 (BIG1/2 homologue). However, the mechanisms underlying this regulatory network are unclear. Using a combination of imaging and in vitro protein‐protein interaction studies, we have found that Pik1 relies on Sec7‐activated Arf1 for proper localization to the TGN and may, once at the TGN, influence the activity of Ypt31. Together, these data provide a clearer picture of how cooperation among Ypt31, Arf1, and Pik1/PI4P choreographs trafficking from the TGN.Support or Funding InformationNSF GRFP DGE‐1650441; NIH/NIGMSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Papuamine prevents the fusion between autophagosomes and lysosomes by inhibiting the maturation of autophagosomes
Authors: Fujimoto Keiko , Tadakuma Ryo , Tanaka Chiaki , Hirota Yuko , Miyauchi Yuu , Ishii Yuji , Tanaka Yoshitaka 
Published: June 2021

Papuamine, an alkaloid isolated from a marine sponge Haliclona sp., has been shown to have antifungal and antimycobacterial activities. Recent studies have reported that papuamine exhibits cytotoxicity and induces apoptosis in MCF7 breast cancer cells, which are attributable to the induction of autophagy. However, the effect of papuamine on autophagic flux remains uncertain. Therefore, the aim of this study was to investigate the effect of papuamine on autophagy.Western blot analyses showed that treatment of HeLa cells with papuamine causes a markedly increase in LC3‐II, an autophagosome‐specific marker. Likewise, confocal immunofluorescence microscopy revealed that not only the number of, but also the size of, LC3‐positive vesicles increased in papuamine‐treated cells. However, in cells treated with papuamine LC3‐positive vesicles were not significantly colocalized with a late endosome/lysosome marker LAMP‐1. In addition, papuamine did not affect lysosomal properties including acidic pH, activities and distribution of lysosomal enzymes. These results demonstrate that papuamine inhibits the fusion between autophagosomes and lysosomes, thereby leading to the accumulation of autophagosomes. Furthermore, our data suggest that papuamine‐induced impairment of both the autophagosomes‐lysosomes and the enlarged autophagosomes formation is due to a blockade of Rab5‐to‐Rab7 conversion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MiR‐590‐3p Restores Autophagy in Cultured Human Glomerular Endothelial Cells Exposed to High Glucose
Authors: Liu Wenjie , Shi Yi 
Published: June 2021

BackgroundAutophagy is an important mechanism for homeostasis. Previous data mining indicated that miR‐590‐3p was involved in diabetes‐induced endothelial dysfunction. The present study was designed to investigate the role of microRNA‐590‐3p (miR‐590‐3p) in the regulation of autophagy in cultured endothelial cells incubated with high glucose.MethodsCultured human aortic endothelial cells (HAEC) and glomerular endothelial cells (HGEC) were subjected to high glucose (44.4mM). To overexpress miRNA‐590‐3p, lipofectamine RNAiMAX was used for transfection. Cells were collected for quantitative real‐time polymerase chain reaction and western blotting.ResultsAfter six hours stimulation with high glucose, expressions of miR‐590‐3p were significantly reduced in HGEC, but not in HAEC, while expressions of miR‐590‐5p were not changed in both cells. After 48 hours, high glucose treatment blunted autophagy in HGEC, which was shown as reduced protein expressions of microtubule‐associated proteins 1A/1B light chain 3B‐II (LC3B‐II), autophagy‐related genes (ATG) 5, and beclin‐1, as well as increased protein expressions of SQSTM1/p62. Overexpressing miR‐590‐3p restored the impairment of autophagy in HGEC. Incubation of HGEC with high glucose reduced phosphorylation of 5′ AMP‐activated protein kinase (AMPK) and increased phosphorylation of mammalian target of rapamycin (mTOR), two upstream signaling of autophagy. Overexpressing miR‐590‐3p increased the phosphorylation of AMPK and reduced the phosphorylation of mTOR, suggesting that miR‐590‐3p restored autophagy in HGEC through AMPK/mTOR pathway. High glucose stimulation did not alter apoptosic protein expressions in HGEC or HAEC, including bcl‐2, pBAD and caspase‐3.ConclusionHigh glucose incubation reduces autophagy in HGEC which is prevented by overexpressing MiR‐590‐3p through AMPK/mTOR signaling pathways.Support or Funding InformationThe present study was supported by the National Nature Science Foundation of China (81573418) and ZSYXRC‐014This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.