bluuebunny/crossref_metadata_2025_split huggingface dataset:
The lower base of corrinoid small molecules regulates reductive dehalogenase enzyme function in Dehalococcoides species
Authors: Jiang Nannan , Yan Jun , Löffler Frank E. 
Published: June 2021

Key interactions between proteins and small molecules regulate numerous environmental reactions. Such is the case with reductive dehalogenases that dechlorinate toxic chlorinated solvents only in the presence of corrinoid small molecules. Previous studies have demonstrated that the lower bases attached to these small molecule cofactors affect the activity of reductive dehalogenases differently in corrinoid auxotrophic Dehalococcoides mccartyi strains, which prefer the 5,6‐dimethylbenzimidazole (DMB) lower base. In contrast, Geobacter sulfurreducens strain PCA produces a small molecule corrinoid with 5‐hydroxybenzimidazole (5‐OHBza) as the lower base, which fails to support dechlorination catalyzed by reductive dehalogenases. Here, we cloned and heterologously expressed a unique Geobacter lovleyi O‐methyltransferase gene implicated in the methylation of 5‐OHBza to 5‐methoxybenzimidazole (5‐OMeBza). Unlike 5‐OHBza, 5‐OMeBza does support D. mccartyi reductive dechlorination activity. We demonstrated using HPLC and LC‐MS that the G. sulfurreducens mutant expressed the O‐methyltransferase, as evidenced by the native 5‐OHBza‐cobamide being converted into the 5‐OMeBza‐cobamide. Next, we co‐cultured D. mccartyi and the recombinant G. sulfurreducens in the absence of exogenous vitamin B12, which supported D. mccartyi growth and the complete dechlorination of cis‐1,2‐dichloroethene to ethene. These studies demonstrated that the O‐methyltransferase catalyzes a critical step in lower base biosynthesis and provides a genetic basis for the observation that D. mccartyi reductive dechlorination activity occurs in co‐cultures with G. lovleyi but not with G. sulfurreducens. Our results emphasize the critical role of small molecule interactions directing protein function in the case of dechlorinating toxic chlorinated solvents.Support or Funding InformationThe authors acknowledge funding support from the NIH and SERDP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improving Ligand Geometry in Protein Data Bank Structures Computationally
Authors: Miller Beck , Hudson Brian P , Shao Chenghua , Wang Lu , Zardecki Christine , Burley Stephen K. 
Published: June 2021

Information about the three‐dimensional structures of complex biological molecules is an indispensable research tool for many fields of study, including biology, chemistry, pharmacology, and computer science. The Protein Data Bank (PDB) is an public digital archive providing access to structures of nucleic acids, proteins, and large macromolecular machines determined experimentally by X‐ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and 3‐dimensional electron microscopy (3DEM). PDB structures undergo validation for structural plausibility upon their deposition into the PDB using the program Mogul, which cross references each deposited structure against similar data in the Cambridge Structural Database (CSD). Ligands associated with PDB structures are a common source of structural inaccuracy. In this study, a programmatic pipeline was developed to determine if ligand geometric accuracy could be improved using known computational tools. Ligands with dubious geometry were identified through a Python mining code. A sample set of the identified structures was remodeled into minimum energy conformations using the molecular dynamics program AMBER and then fit to the original experimental density maps using the molecular modeling program Coot. The refined models were compared to the original models in the PDB as well as to similar structures in the CSD using the deposition statistical validation server. Preliminary results suggest that the pipeline effectively identifies and improves ligand structures that possess inaccurate geometry. This method can be used to evaluate and improve the accuracy of specific ligands in structures both archived and pending deposition.Support or Funding InformationThis work was supported by an NSF REU and the RCSB PDB [NSF (DBI‐1338415), NIH, and DOE.]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomaterials with Functionally Graded Stiffness
Authors: Siemers Bram , Stempien Alana , Craven Rachel , Napiwocki Brett , Crone Wendy 
Published: June 2021

There are many different tissues in the body, each possessing different mechanical properties and varying amounts of stiffness that are needed for cells to function properly and survive. A challenge to recreating in vitro tissues is to accurately recapitulate the mechanical properties of the native tissue. The focus of this research was to produce a substrate with a functionally graded stiffness for use with in vitro culture of cardiomyocytes, the cells of the heart responsible for contraction.Young's modulus, a measure of stiffness of materials, is a key characteristic parameter for tissues. Currently, there is a lack of substrate systems that span the range of Young's modulus needed to represent both healthy and diseased myocardium. This is problematic when trying to model cardiovascular disease. For example, after myocardial infarction, there are regions of soft and stiff underlying tissue that impact the behavior of cardiomyocytes differently.The goal of this research was to create a substrate system with a stiffness gradient spanning from 5 to 50 kPa upon which the cardiomyocytes derived from stem cell could be cultured. This in vitro substrate system was designed to mimic the range of physiological environments in which cardiomyocytes exist in vivo. This substrate model is specifically being designed to aid in better understanding and reducing cardiovascular disease.The method investigated that showed the most promise was UV curing of polyacrylamide hydrogels. A gradient photomask was designed to allow different amounts of UV light to penetrate over the span of the substrate. This mask was then placed over the prepolymer gel during UV exposure. This method produced a stiffness gradient of 55 to 75 kPa over a 1 cm distance. Further work is needed to optimize the photomasks and gel reagents to produce the lower end of modulus representative of healthy myocardium.Support or Funding InformationSupported with funding from the Karen Thompson Medhi Professor at the University of Wisconsin‐Madison and the National Science Foundation through the University of Wisconsin‐Madison Materials Research Science and Engineering Center (DMR‐0520527) and Nanoscale Science and Engineering Center (DMR‐0425880). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Olfactory Receptor Family 7 Subfamily C Member 1 Expression in T‐cell Acute Lymphoblastic leukemia Identifies a Potential Stem Cell Sub‐population
Authors: Jeon Dayoung , Silva‐Espinoza Juan Carlos , Ahmed Haidar , Vines Charlotte , Bill Colin 
Published: June 2021

Cancer stem cells (CSCs) are thought to be a self‐renewing population that is difficult to kill during standard chemotherapy due to the capacity of the cells for self‐renewal and multidrug resistance. Our lab studies T‐cell acute lymphoblastic leukemia (T‐ALL), which is an aggressive form of leukemia. T‐ALL cells, which can hide from chemotherapeutic treatment regimens within the central nervous system ( CNS), use different chemokine and adhesion receptors to invade the CNS; entry into the CNS contributes to the aggressiveness of the disease. Therefore, migration into the CNS necessitates the use of aggressive treatments such as cranial irradiation and intensified intrathecal chemotherapy, which causes neurocognitive deficits, endocrine disorders, and growth impairment. A recent study showed that the ectopic expression of Olfactory Receptor Family 7 Subfamily C Member 1 (OR7C1) correlates with expression of stem cell markers including SOX2, POU5F1, and LGR5. These stem‐cell markers correlate with decreased survival rates, implicating the expression of these stem cells in chemo‐resistant cancers. Therefore, we hypothesized that the expression of these stem cell markers in T‐ALL cell lines would contribute to the aggressiveness of the disease. Using flow cytometry, we found approximately 1% of HuT78, adult human T‐ALL cells, express OR7C1. Furthermore, while we were able to detect OR7C1 protein expression in HuT78 cells through both Western blot and RT‐PCR, we found that peripheral blood mononuclear cells (PBMCs) lack OR7C1. Currently, we are determining if the expression of OR7C1 leads to the expression of stem cell transcription factors in HuT78, CCRF‐CEM pediatric T cell lines, and PBMCs. These findings will allow us to determine the prognostic significance of OR7C1 in the progression of T‐ALL.Support or Funding InformationNSF award DUE‐1565063This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ferric Reductase Knockdown in Drosophila S2 Cells
Authors: Arismendiz Omar Alexis Rodriguez , Holst Jessica , Ochoa Alma , Gorman Maureen J. , Ragan Emily J. 
Published: June 2021

Iron is an essential micronutrient that is potentially harmful due to its redox properties, which is why its transport between cells must be a highly regulated process. As compared to mammals, very little is known about iron uptake in insects, because receptors for ferritin or transferrin have not been identified in insects. The goal of the proposed research is to understand the physiological mechanisms of iron transport in insects. We are testing the hypothesis that the reduction of iron ions in the hemolymph leads to iron transport into cells. Hemolymph contains iron in the ferric form, but iron transporters in animal cells are specific to ferrous iron; therefore iron uptake via an influx transporter is likely to require a reduction step. We silenced a putative ferric reductase CG8399 by RNA interference (RNAi) to determine if it contributes to ferric reductase activity and impacts the iron content of the cells. We analyzed the efficiency of knockdown of CG8399 by real time, quantitative PCR (qPCR).Support or Funding InformationNSF IOS‐1656407This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pilot Studies of Two Possible Iron Uptake Mechanisms in Insect Cells
Authors: Coca Michelle , Najera Diana G , Gorman Maureen J 
Published: June 2021

Iron plays an important role in energy metabolism and other essential physiological processes; however, because iron can also be toxic, its uptake by cells must be strictly regulated. In humans, there is a well‐understood pathway of iron uptake and multiple poorly understood pathways. How iron is transported into insect cells is unknown. The goal of this study was to test two models of iron uptake by cultured insect cells. The first model involves endocytic uptake of protein‐bound iron. The second model involves uptake of iron through a ferrous transporter. For our first set of experiments, we used the well‐studied Drosophila melanogaster S2 cell line. We found that S2 cells express rab5 (an essential component of endosomes), secreted ferritin, membrane‐bound transferrin, two ferric reductases, and a ferrous transporter. Treating cells with endocytosis inhibitors had no effect on iron content; therefore, endocytosis does not seem to be required for iron uptake by S2 cells. In contrast, sequestering iron with a ferrous chelator decreased the iron content of treated cells, suggesting that the ferrous form of iron is involved in the uptake process. We wanted to take advantage of a newly developed chemically defined medium (ZB) to learn more about the mechanism of iron uptake, but we were unable to grow S2 cells in ZB medium. We found that Kc167 and Cl.8 cells, which do grow in ZB medium, express the same set of iron‐related genes as S2 cells. To test the hypothesis that endocytosis is involved in iron uptake by D. melanogaster cells, we are in the process of evaluating the effect of RNAi‐mediated knockdown of rab5 on cellular iron content. Toward this goal, we have synthesized dsRNA that targets rab5 and optimized a method to measure cellular iron content. To test the hypothesis that a ferrous transporter is involved in iron uptake, we will measure the effect of RNAi‐mediated knockdown of divalent metal transporter 1 (DMT1) on cellular iron content. Our tentative conclusion is that insect cells take up iron via a mechanism that involves a ferrous transporter. A better understanding of iron uptake in insects could lead to better insect control strategies and also provide insight into the less understood iron uptake mechanisms of humans.Support or Funding InformationNSF IOS 1656388This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biological Synthesis of Short Glycogen Polymers
Authors: Beacham Branden , Cuellar Itzayana G. , Snyder Nicole L. , Campbell A. Malcolm 
Published: June 2021

Although humans naturally synthesize full‐length glycogen, chemically producing short glycogen polymers in the laboratory is complex and takes months. The goal of our research is to efficiently synthesize short glucose polymersusing the human primer enzyme glycogenin. Glycogen is large polymer of glucose that is produced by many organisms, including humans. Inside cells, glycogen production requires two protein enzymes. The first enzyme, glycogenin, assembles up to 10 glucose molecules to form a glycogen “primer” molecule. Glycogen synthase then uses the short glucose polymer as aprimer to synthesize the much larger glycogen molecule. In our study, we cloned the human glycogenin cDNA and used it to produce the primer enzyme inside E. coli cells. We then assessed the ability of therecombinant human protein to produce short glucose polymers in vitroand analyzed the results to confirm glucose primer production. Here we present our preliminary results demonstrating the potential for our method to produce glycogen primers that can serve as a scaffoldsto produce derivatized glucose‐basedpolymers. The resulting glycopolymers are potential candidates for therapeutic applications.Support or Funding InformationFunding provided by the Davidson Research Initiative, NSF grants to Davidson College (RUI‐1613203 and RUI‐1708546) and the James G. Martin Genomics 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.
The resilience of cancer‐specific protein galectin‐3: Implications in biological functions
Authors: Dam Tarun , Kadav Priyanka , Welch Christina , Edwards Jared , Bandyopadhyay Purnima 
Published: June 2021

Structural degradation is detrimental for protein activity and functions. We recently found that the tumor‐associated protein galectin‐3 (Gal‐3) is apparently an exception to this rule. Gal‐3 plays important roles in cancer but its functions are not fully understood. We have found that the affinity purified Gal‐3, when stored with lactose at 4°C for more than six weeks, undergoes complete degradation even in the absence of collagenases. Full‐length Gal‐3 after full degradation produced a single protein band of 17 kDa. This molecular weight is similar to that of the CRD (carbohydrate recognition domain) portion of Gal‐3. Conventionally, the CRD is separated from intact Gal‐3 by enzymatic digestion using collagenases. However, in the present case, Gal‐3 apparently self‐degraded to produce the CRD without the catalytic action of collagenases. Our data indicate that degradation of Gal‐3 significantly alters the protein but the CRD remains intact and functionally active. Like the full‐length Gal‐3, the CRDs interacted with thyroglobulin, chondroitin sulfate A and C. However, the CRDs could not cross‐link these multivalent ligands as revealed by our spectroscopic analysis. Our current observations suggest that Gal‐3 retains it ligand recognition properties even when it is substantially degraded in a challenging cellular environment. Thus the degraded Gal‐3 can still carry out some of the functions of intact Gal‐3. Although the CRDs retain the binding property of its intact precursor (Gal‐3), they lose the ability to cross‐link cellular receptors. As a result, the CRDs generated through degradation may influence cell signaling by competing with intact Gal‐3 for the same receptors in cellular environment.Support or Funding InformationNational Science FoundationThis 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 recurrent fusion genes across multiple cancer types
Authors: Luo Jianhua , Michalopoulos George , Yu Yanping 
Published: June 2021

Chromosome changes are one of the hallmarks of human malignancies. Chromosomal rearrangement is frequent in human cancers. One of the consequences of chromosomal rearrangement is gene fusions in the cancer genome. We have previously identified a panel of fusion genes in aggressive prostate cancers. In this study, we showed that 6 of these fusion genes are present in 7 different types of human malignancies with variable frequencies. Among them, the CCNH‐C5orf30 and TRMT11‐GRIK2 gene fusions were found in breast cancer, colon cancer, non‐small cell lung cancer, esophageal adenocarcinoma, glioblastoma multiforme, ovarian cancer and liver cancer, with frequencies ranging from 12.9% to 85%. In contrast, four other gene fusions (mTOR‐TP53BP1, TMEM135‐CCDC67, KDM4‐AC011523.2 and LRRC59‐FLJ60017) are less frequent. Both TRMT11‐GRIK2 and CCNH‐C5orf30 are also frequently present in lymph node metastatic cancer samples from the breast, colon and ovary. Thus, detecting these fusion transcripts may have significant biological and clinical implications in cancer patient management.Support or Funding InformationNCI, DOD and UPCIThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of STAT3 by Human Serotonin 2C Receptor
Authors: Maisha Musarrat , Curtis M.E. , Fears L.S , Lanaghan Zeljka Miletic , Ivy MT , Fentress Hugh 
Published: June 2021

Serotonin (5‐HT) is an indolamine neurotransmitter involved in a variety of functions including regulation of mood, appetite, sleep, learning and memory, and neuronal excitability. 5‐HT elicits its effects by binding to at least 14 different receptor subtypes that mediate both excitatory and inhibitory neurotransmission. The many classes of serotonin receptors have been subjected to significant studies in order to discern the functionality and produce effective therapeutic targets. One of the receptors, the 5‐HT2C receptor, has been studied and used as a target for antipsychotic medication. 5‐HT2C subtype receptor exists as a 7‐transmembrane spanning G protein‐coupled receptor (GPCR) found in the central nervous system, and is the only GPCR known to undergo RNA editing, creating 14 known isoforms in the human brain which affect its ability to couple with G proteins and other signaling proteins. The structurally similar 5‐HT2A receptor has been shown to activate the G protein independent JAK/STAT pathway. The purpose of this study was to determine if the human 5‐HT2C receptor also activates the JAK/STAT pathway. Human Embryonic Kidney (HEK) 293 cells were transfected with human 5‐HT2C receptors to produce stable cell lines. Cells were then treated with vehicle (control), 5‐HT, olanzapine (antagonist), and SB206553 (inverse agonist) for 30 minutes or 1 hour. Treated cells were lysed, proteins were separated by SDS‐PAGE, and levels of phosphorylated JAK2 and STAT3 were analyzed using western blots. In cells treated for 30 minutes with vehicle, phosphorylation of STAT3 was observed in cells transfected with the 5‐HT2C receptor but not in untransfected cells indicating constitutive phosphorylation of STAT3 by the 5‐HT2C receptor. Upon treatment with serotonin, phosphorylated levels of STAT3 were increased in cells expressing the 5‐HT2C receptor while STAT3 phosphorylation was completely blocked with cells treated with olanzapine or SB206553. Phosphorylated levels of JAK2 were unaffected across all treatments. Phosphorylated STAT3 levels showed the same patterns in cells expressing the 5‐HT2C receptor when treated for 1 hour. These findings indicate that 2C receptors are activating STAT3 independently of JAK2 and may be involved in cell growth, differentiation, transcription of other STAT genes, and immune response. Future studies will examine different edited isoforms of the 5‐HT2C receptor and whether STAT3 activation is dependent or independent of G‐protein activation to elucidate the phosphorylation mechanism of STAT3 by the human 5‐HT2C receptors.Support or Funding InformationSupported by NSF Grant HRD‐1238723 to HMFThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinase and Mitochondrial Targets for Reversing the Adverse Effects of Organophosphates on Axonal Transport
Authors: Naughton Sean X , Wei Zhe , Guangyu Wu , Terry Alvin V 
Published: June 2021

Organophosphates (OPs) represent a class of chemicals with a wide range of uses in industrial and agricultural settings as pesticides, but they have also been associated with a variety of adverse health effects in humans including deficits in cognition. While the acute toxicity of OPs is associated with the inhibition of cholinesterase enzymes, our laboratory has previously demonstrated that repeated exposures to levels of OPs below the threshold for acute toxicity can negatively affect non‐cholinesterase targets (i.e., effects that might contribute to the cognitive defects). These negative effects of OPs include deficits in the axonal transport of membrane bound organelles (MBOs) as well as alterations in both the transport and morphology of mitochondria. Postmortem analyses of brain tissues of rats treated with the OP diisopropylfluorophosphate (DFP) and in vitro phenotypic screening assays revealed that targeting specific signaling kinases that regulate axonal transport and approaches to improve mitochondrial function might represent rational therapeutic strategies to reverse the negative effects of OPs. These hypotheses were validated in live cell imaging studies in vitro, where DFP‐induced impairments in both anterograde and retrograde AXT were attenuated in a dose‐dependent manner with the glycogen synthase kinase (GSKIIIβ) inhibitor, lithium chloride. Similarly, the mitochondrial‐targeting antioxidant methylene blue also attenuated the negative effects of DFP on anterograde and retrograde axonal transport in a dose dependent manner. Studies are in progress to determine the effects of these potential therapeutic agents on specific components of the axonal transport process (e.g., number of stationary particles, directional changes, pauses, etc.).Support or Funding InformationCongressionally Directed Medical Research Programs (CDMRP), Medical College of Georgia Foundation, Augusta UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.