bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Bacterial Arginine Kinases in Species from the Order Myxococcales
Authors: Fannin Lindsay , Aryal Manish , Stock Katie , Snider Mark , Fraga Dean 
Published: June 2021

Phosphagen kinases are found in many eukaryotes and function in energy buffering within the cell through the exchange of a phosphate group between ATP and one of eight substrates (Ellington, 2001). Arginine kinases (AKs) have been identified in a few bacteria and phylogenetic analysis suggest they arrived through horizontal gene transfer (Andrews et al., 2008; Bragg et al., 2012; Suzuki et al., 2013). Recently, the bacterial AK in the well‐studied soil bacteria, Myxococcus xanthus, was knocked out and the deletion strain characterized. The results were impairment in both recovery from some forms of stress and completing a starvation induced development pathway that leads to spore formation. To update our understanding of the bacterial AKs, we conducted a BLAST search of bacterial genomes and found a total of 37 bacterial species with AKs. The majority of the bacterial AKs were deltaproteobacteria (20) with 5 in the genus Myxococcus. Interestingly, the AKs seen in the Myxococcus species seems to have arrived through two independent HGT events and thus present an opportunity to better understand how two similar evolutionary events (HGT of an AK into a soil bacterial genome) have subsequently evolved. Will the outcomes be the same or different? We will describe the biochemistry and phylogenetics of the AKs found within the deltaproteobacteria order Myxococcales.Support or Funding InformationFunding provided through 2014 NSF‐PGRP 1444539 and the Whitmore‐Williams Science Scholarship at the College of Wooster.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A New Membrane Potential (ΔΨ)‐Independent Iron Indicator Selectively Detects Mitochondrial Chelatable Iron but Not Calcium in Living Cells
Authors: Hu Jiangting , Nieminen Anna‐Liisa , Kholmukhamedov Andaleb , Lindsey Christopher C. , Beeson Craig C. , Lemasters John J. 
Published: June 2021

Mitochondrial chelatable iron contributes to several injury processes, including ischemia/reperfusion injury, oxidative stress and the hepatotoxicity of acetaminophen. To measure mitochondrial chelatable iron in living cells and tissues, we synthesized a new fluorescent Fe2+ indicator, mitoferrofluor (MFeF), which has a reactive group that forms covalent adducts with mitochondrial proteins to allow retention of MFeF after subsequent mitochondrial depolarization (Hepatology 60 [Suppl], 812A). MFeF selectively accumulates in the polarized mitochondria of cultured rat hepatocytes. Here we assessed whether MFeF specifically reports mitochondrial iron independent of calcium. After loading cultured rat hepatocytes with 1 uM MFeF and 500 nM rhodamine 123 (Rh123) or 1 μM calcein‐AM for 30 min, confocal microscopy revealed red MFeF fluorescence that co‐localized with green mitochondrial Rh123. Membrane‐permeant Fe3+/8‐hydroxyquinoline(8‐HQ) (10–50 μM) progressively quenched mitochondrial MFeF fluorescence to ~80% within 15 min, but mitochondrial Rhodamine 123 was unchanged. Fe2+ specific calcein fluorescence in the cytosol was also quenched, confirming that Fe3+/8‐HQ undergoes rapid intracellular reduction to Fe2+. MFeF fluorescence increased when the lipophilic transition metal chelator pyridoxal isonicotinoyl hydrazone (PIH) was added to competitively bind mitochondrial chelatable iron. Importantly, MFeF was retained in mitochondria after mitochondrial de‐energization with CCCP (10 μM), oligomycin (10 μg/ml) and myxothiazol (10 μM), confirming mitochondrial retention of the covalently adducted MFeF. In hepatocytes loaded with MFeF, red fluorescence did not decrease after adding 2 μM of Ca2+‐mobilizing thapsigargin, whereas parallel experiments showed that Ca2+‐indicating mitochondrial Rhod‐2 fluorescence did increase, showing that MFeF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In acetaminophen‐induced hepatotoxicity, progressive quenching of MFeF began at ~4 h after overdose acetaminophen (10 mM) in mouse hepatocytes, signifying increased mitochondrial chelatable Fe2+. Mitochondria then depolarized after ~10 h. Dipyridyl, a membrane‐permeable iron chelator, partially restored MFeF fluorescence. In conclusion, MFeF is a ΔΨ‐independent iron indicator that selectively detects mitochondrial chelatable Fe2+ but not Ca2+.Support or Funding InformationNIH grants: DK073336, AA021191 and T32 DK083262This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Planar Enteroids Reveal an Autonomous WNT And BMP Circuit Controlling Intestinal Epithelial Growth and Organization
Authors: Thorne Curtis A. , Chen Ina W. , Sanman Laura E. , Cobb Melanie H. , Wu Lani F. , Altschuler Steven J. 
Published: June 2021

The epithelial lining of the human intestine is a prime example of tight homeostatic control of cell proliferation, organization and fate determination. As a mechanism for dealing with constant mechanical, chemical and pathogen‐derived insults, the epithelium is in constant turnover, completely renewing every four days. Amazingly, this active process produces multiple cell types at just the right ratios and locations throughout our life span. Failure of this exquisite control is the basis for diseases including Crohn's disease, ulcerative colitis, and cancers of the esophagus, stomach, small and large intestine.A central challenge is to elucidate mechanisms required for robust control of tissue renewal. Opposing WNT and BMP signaling is essential in establishing epithelial homeostasis. However, it has been difficult to disentangle contributions from multiple sources of morphogenic signals in the tissue. Here, to dissect epithelial‐autonomous morphogenic signaling circuits, we developed a planar enteroid intestinal culture system that recapitulates key properties of the intestinal epithelium, specifically the ability to: maintain proliferative and differentiated zones, self‐renew, polarize and generate major intestinal cell types. We systematically perturb intrinsic and extrinsic sources of WNT and BMP signals to reveal a core morphogenic circuit that controls proliferation, tissue organization and cell fate. Our work demonstrates the ability of intestinal epithelium, even in the absence of 3D tissue architecture, to control its own growth and organization through morphogen‐mediated feedback.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DETERMINATION OF THE THREE‐DIMENSIONAL STRUCTURE OF FULL‐LENGTH HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR BY CRYO‐ELECTRON TOMOGRAPHY
Authors: Purba Endang R. , Akhouri Reeresh R. , Ofverstedt Lars‐goran , Skoglund Ulf , Maruyama Ichiro 
Published: June 2021

Human epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility and differentiation. Mice lacking EGFR die shortly after birth due to multi‐organ failure. Aberrant activation of EGFR is implicated in many human cancers. EGFR is synthesized as a single‐pass transmembrane protein, which consists of an extracellular ligand‐binding domain and an intracellular kinase domain separated by a single transmembrane domain. While crystal structures of EGFR domains are available, three‐dimensional structural determination of the full‐length EGFR has not been successful so far. In the conference, we will present cryo‐electron tomography structures of the intact human EGFR in the presence of bound EGF ligand. We successfully purified the full‐length EGFR with autophosphorylation activity upon EGF stimulation. We then prepared vitrified specimens and collected 30 tilt series projections spanning −60 to +60 degrees, with tilt increments of 0.5 degrees, using a FEI Titan Krios at 39,000× magnification and □1–2 μm underfocus. Tilt series data was analyzed with COMET software. These three‐dimensional structures of the full‐length EGFR are invaluable for understanding of mechanisms underlying activation of EGFR by ligand binding.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Soluble Activin Type IIB Receptor Decoy changes Gene Expression Profiles of Bone Cells in the OIM and not the G610C Mouse Model of Osteogenesis Imperfecta
Authors: OMOSULE CATHERINE LAARIPUOH , Jeong Youngjae , Phillips Charlotte , Pfeiffer Ferris 
Published: June 2021

Bone is a highly dynamic tissue which is continually remodeled by osteoblasts and osteoclasts, with regulation by osteocytes. In Osteogenesis Imperfecta (OI), a clinically heterogeneous disorder characterized by bone fragility, the functions of osteoblasts and osteoclasts are thought to be impaired. As a mechanosensitive tissue, bone responds to mechanical loads, some of the strongest of which are from skeletal muscles, by changing its shape.The inhibition of myostatin, a negative regulator of muscle growth and a member of the TGF‐β superfamily, has been shown to induce muscle growth via hypertrophy. In this study, we investigated the effect of pharmacologically inhibiting myostatin through treatment with the soluble activin receptor type IIB decoy molecule (sActRIIB‐mFc), focusing on the gene expression patterns of bone cells derived from two molecularly distinct mouse models of OI (osteogenesis imperfecta murine [OIM] vs. G610C). Because the molecular defects that give rise to the G610C and OIM models differ and muscle function is differentially impacted, we hypothesize that the two models may respond differently to sActRIIB‐mFc treatment. Homozygous mice (oim/oim) in the OIM model have a single nucleotide deletion in the COL1A2 gene that leads to functional null proα2 (I) collagen chains and the generation of homotrimeric type I collagen, whereas the G610C mouse, heterozygous for a missense mutation in the COL1A2 gene, has a glycine to cysteine substitution at the 610 position of the triple helical domain.At two months of age, WT, +/G610C, and oim/oim male mice were treated bi‐weekly either with vehicle (Tris‐buffered saline) or sActRIIB‐mFc for 8 weeks. Quantitative RT‐PCR analyses demonstrated that tibias from sActRIIB‐mFc treated oim/oim mice exhibited increased gene expression levels of the markers of osteoblast, osteoclast, and osteocyte function and differentiation analyzed. This contrasts with the significant changes seen in the femoral cortical bone microarchitecture and biomechanical integrity of sActRIIB‐mFc‐treated WT and +/G610C mice, which was not observed in the oim/oim mice.In conclusion, our findings indicate that sActRIIB‐mFc treatment has differential effects on the G610C and OIM mice, which may in part reflect differences in severity (severe in oim/oim vs. moderately severe in +/G610C), and/or their unique molecular defects.Understanding the molecular impact of therapeutics is critical in order to develop more effective and targeted treatment for OI.Support or Funding InformationLeda J. Sears TrustKansas City Area life Sciences Institute Patton Trust Research, Inc. University of Missouri Research Board GrantMarch of DimesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Divergent C‐terminal Region of Gα12 and Gα13 Provides Unique Effector Binding and Growth Signaling Mechanisms
Authors: Quick Courtney R. , Stecky Rebecca C. , Mull Makenzy L. , Dover Ellen N. , Meigs Thomas E. 
Published: June 2021

Heterotrimeric G proteins of the G12/13 class play key signaling roles in cell growth, oncogenic transformation, migration and invasion, and various other cellular responses. This G protein class is represented by the closely related α subunits Gα12 and Gα13 in mammals, whereas invertebrates encode a single G12/13 homolog. We have sought to identify structural features that evolved in mammalian G12/13 proteins to allow their divergent mechanisms of growth signaling. Previously, we demonstrated that the Drosophila G12/13 homolog Concertina failed to drive growth signaling through serum response factor, and thus we generated a set of evolutionarily guided chimeras in which regions of mammalian Gα12 and Gα13 were replaced by the corresponding invertebrate sequence. These substitutions revealed a non‐conserved, 42‐residue region near the C‐terminus of Gα12 which is necessary for its growth signaling function, whereas the same invertebrate sequence replacement had no effect on Gα13 signaling. In addition, we utilized invertebrate amino acid substitutions to identify specific determinants in this region of Gα12 that are important for its interaction with specific effector proteins. Gα12 mutants uncoupled from signaling through serum response factor are currently being tested for ability to signal through more primitive pathways, including cytoskeletal rearrangements. To further evaluate the functional significance of this 42‐residue region, additional chimeras were constructed to place isolated C‐terminal regions of Gα12 into Gα13. Protein co‐precipitation experiments indicated the divergent 42‐residue region of Gα12 bestowed upon Gα13 the ability to interact with several Gα12‐specific binding partners. These findings indicate that Gα12 and Gα13 evolved to harbor different C‐terminal structural determinants of growth signaling and effector binding that are not present in invertebrate G12/13 homologs.Support or Funding InformationWe acknowledge support from the North Carolina GlaxoSmithKline Foundation, the C.D. Spangler Foundation, and UNC Lineberger Comprehensive Cancer Center.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of ANKDR49 in evaluating the prognosis of glioma
Authors: Duan Hubin , Hao Chunyan , Wang suping , Wang Huan , Gao Ling , Zheng Xiaojun , Duan Shengqiang , Gao Weiqin , Zhang Ce 
Published: June 2021

ObjectiveTo evaluate the role of ANKRD49 in the prognosis of glioma.MethodsTranscriptome expression data sets and the corresponding clinical information of ANKRD49 gene were downloaded from the website of The Cancer Genome Atlas (TCGA). A total of 558 samples, which contained transcriptional expression data of 548 tumor tissues and 10 normal tissues, were available for this analysis. Human malignant glioma cell lines U251 and U87 were cultured. Total RNA was isolated from indicated cells using TRIzol reagent. Quantitative real‐time PCR was used to analyze ANKRD49 gene expression. Total protein was isolated from indicated cells and West‐blot was used to analyze ANKRD49 protein expression. SPSS 16.0 software was used to analyze experimental data and clinical data.ResultsThe expression of ANKRD49 was predominantly higher in glioma samples compared with matched normal samples(fold change =2.11, P=2.99E‐09). The 5‐year overall survival of patients with highly expressed ANKDR49 was distinctively shorter than that of patients with lowly expressed ANKDR49(P<0.01).ConclusionANKRD49 is overexpressed in human glioma tissues, and high expression of ANKRD49 is significantly associated with poor survival. ANKDR49 might be a indicator for evaluating the prognosis of glioma.Support or Funding InformationThis study was supported by the National Natural Science Foundation Youth Fund (Fund No. 30600637), China Postdoctral Foundation(Fund No.2014M561207), the Shanxi Province Basic Research Program Youth Science and Technology Research Fund (Fund No. 2010021034‐4), the Shanxi Scholarship Council of China (Fund No. 2011‐096 and Key Projects No.4), The science and technology research projects of health department of Shanxi Province(Fund No.2016D011119), The Teaching Reform Program of Shanxi Higher Education (No. 2013‐033), The Education Reform Project of Postgraduate Students in Shanxi Province (No. 2017‐027). The Project of “131” Leading Talents of Shanxi Higher Education, The Education Reform Project of Shanxi Medical University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Implementing the CURE: Combining Wet‐Lab Protein Biochemistry with Computational Analysis to Provide Gains in Student Learning in the Biochemistry Teaching Lab
Authors: Pikaart Michael 
Published: June 2021

Most undergraduates studying biochemistry and molecular biology get their broadest exposure to wet‐lab techniques in protein and nucleic acid chemistry (and, increasingly, computer/visualization) in their upper‐level laboratory courses. These tend to be juniors and seniors with well‐defined career goals. Some of these students will have already have a research background in a traditional one‐to‐one (or one‐to‐few) research mentoring setting, for example a summer research program. This approach has proved effective at increasing student learning and persistence in the sciences. At the same time, extended full‐time PI‐directed research is limited in the number of students served, and can even present a barrier. To broaden the impact of teaching through research, many practitioners have adopted a CURE, or Course‐based Undergraduate Research Experience, approach.This presentation reports on “BASIL” (Biochemical Authentic Scientific Inquiry Laboratory), a team of faculty who have worked to bring computational and wet‐lab protein science to the biochemistry teaching lab. Together, we have developed a protein biochemistry CURE to determine enzymatic function of proteins of unknown activity. This work leverages the results of the Protein Structure Initiative, a fifteen‐year NIH‐funded effort which concluded in 2015 with the publication and distribution of more than 5000 previously uncharacterized proteins. The great majority of these are “orphans,” with high quality structures and pre‐cloned expression plasmids available, but no research on their enzymatic function or role in native organisms. The BASIL consortium of undergraduate biochemistry faculty and students seeks to identify functional properties of a subset of these uncharacterized proteins, seeking to unify structure and function relationships. Currently, implementable modules are available for faculty who wish to adopt them, and expected student results will be presented.Support or Funding InformationSupported by NSF IUSE 1709278This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gene Regulatory Pathways that Modulate Response to Dexamethasone and Daunorubicin in Breast Cancer Cell Lines
Authors: Macias Alejandro Rafael , Medh Rheem 
Published: June 2021

Glucocorticoids (GCs) such as Dexamethasone (Dex) are anti‐cancer agents that induce apoptosis (programmed cell death) and counter side effects of chemotherapy through regulation of pro‐ and anti‐apoptotic genes such as E4BP4, FOXO3A, BIM and BIRC3. Anthracyclines, such as Daunorubicin (Dauno), are effective anti‐cancer agents for leukemias, breast cancer and ovarian cancer. Although several regimens combine both classes of chemotherapy agents, Dex has been reported to promote rather than retard breast cancer cell proliferation, and counter the pro‐apoptotic effects of other chemotherapeutic agents. E4BP4, a transcriptional regulator, is upregulated by GCs and is known to mediate Dex‐evoked apoptosis in leukemia cells, but has been reported to promote cell survival in other models of cancer, including breast cancer. The phosphorylation state of E4BP4 and its crosstalk with the FOXO3A and AKT pathways modulate its pro‐survival or pro‐apoptosis functions. We hypothesize that Dex‐evoked upregulation of E4BP4 and its subsequent modulation of proliferative signals may contribute to pro‐survival effects of Dex in breast cancer. In the present study we are investigating the response of breast cancer cell lines MCF‐7 (estrogen receptor positive) and MDA‐MB‐468 (triple negative) to Dex and Dauno (single agent and in combination) with respect to cell viability, apoptosis and regulation of pro‐ and anti‐apoptotic genes including E4BP4, FOXO3A, BIM and BIRC3. MTT cell viability assays determined that MDA‐MB‐468 and MCF‐7 cells are resistant to 1 μM Dex, with viable cells being about 92% and 140% compared to untreated controls, respectively. However, MDA‐MB‐468 cells were sensitive to 1 μM Dauno and in combination with 1 μM Dauno and 10 μM Dex with viability at 54% and 58% compared to control, respectively. To confirm that cell death occurs via apoptosis, cells are stained with Annexin V‐FITC followed by epifluorescence microscopy to detect apoptotic cells. Evaluation of Dex and Dauno‐mediated regulation of gene expression was done via reverse‐transcriptase and end‐point PCR analysis, followed by ImageJ quantitation, or by RT‐qPCR analysis. Our data suggest that Dauno upregulates while Dex downregulates E4BP4 in MDA‐MB‐468 cells. Combined treatment significantly enhances FOXO3 upregulation compared to either single agent, in correlation with increased cell death. Our data suggest that Dex and Dauno may cooperatively promote apoptosis in MDA‐MB‐468 cells.Support or Funding InformationThis project was funded by a NIH SCORE SC3 grant (GM081099) to Dr. Rheem D. Medh and NIGMS BUILD ( 8TL4GM118977) scholarship to Alejandro Macias.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of cancer stem cell sphere explosion by UV irradiation/cold shock or therapeutic chemicals: detection of autofluorescence using visible wavelength
Authors: Chang Nan‐Shan , Chang Katarina T. , Ho Pei‐Chuan 
Published: June 2021

We have recently identified a new type of cell death, called “bubbling cell death”. When cells are exposed to UV irradiation followed by incubation at 37°C, they undergo typical apoptosis. However, when the UV‐irradiated cells are incubated at room temperature or down to 4°C, they generate bubbles – one bubble per cell – from the nucleus. The bubbles contain nitric oxide. There is occurrence of no caspase activation, flip‐over of phosphatidylserine, and DNA fragmentation, indicating that bubbling death is a novel type of cell death (Oncotarget 2015;6:8007–18). Here, by time‐lapse microscopy, we demonstrated that when breast cancer 4T1 stem cell spheres were subjected to UV and a brief cold shock (5 min at 4°C), followed by imaging at room temperature. The spheres initially shrank, then slowly expanded and finally exploded, and the cells died. Similar results were observed when the spheres were exposed to an aliquot of cocktail of protease inhibitors and an ERK inhibitor U0126, or an anti‐lung cancer drug ceritinib. Notably, prior to sphere explosion, there was an emission of luminescent light under bright light stimulation. We developed a novel approach to measure visible wavelength‐induced autofluorescence up to energy for DAPI imaging. In addition, a time‐dependent initial increase and then reduction in redox activity and formation of stress fibers occurred. Loss of cellular lipids and nuclear uptake of DAPI first and then propidium iodide prior to death were also observed. The entire course lasts 24–48 hr at room temperature. However, sphere explosion is accelerated dramatically by blebbistatin, an inhibitor of myosin II, suggesting the participation of cytoskeletal proteins in the sphere explosion.Support or Funding InformationMOST, TaiwanThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.