bluuebunny/crossref_metadata_2025_split huggingface dataset:
A role for underlying glycan structure in influenza binding: extending the species specificity paradigm
Authors: Woods Robert J , Grant Oliver C , Hadden Jodi A , Smith Hannah M.K. , Peng Wenjie , De Vries Robert , McBride Ryan , Paulson James C. 
Published: November 2023

Recently, a new source for carbohydrate specificity data has emerged through the extensive development of glycan microarrays. Glycan array screening provides rapid insight into binding specificities, limited only by the number of elements in the array, the largest of which currently contain on the order of 600 members. Although rich in information, array data do not provide direct insight into the 3D structural properties of the glycan‐protein complexes responsible for the observed specificities.Here we employ a new technology [1,2], Computational Carbohydrate Grafting (CCG), to generate 3D models of glycan‐hemagglutinin complexes that provide a structural rationalization for the sometimes perplexing data from glycan array screening. Given a structure for the protein in complex with a minimal binding determinant, CCG can be employed to generate a 3D model of any related glycan bound to the complex, by splicing the additional branches of the glycan into the bound fragment. This virtual approach overcomes many of the experimental challenges associated with generating structures for these complexes. CCG is a high‐throughput screening method that can be readily validated by comparison with specificity data from glycan array screening.We show that CCG predictions explain differences in specificity for a range of sialylated N‐linked glycans interacting with human and avian influenza hemagglutinins. The analysis suggests that glycans with poly‐LacNAc extensions can bind in a bidentate fashion to the trimeric HA, consistent with affinity enhancements seen for some hemagglutinins in glycan array data. CGG analysis may prove valuable in elucidating the preferred receptors for influenza adhesion.Support or Funding InformationSupported by the National Institutes of Health GM094919 (RJW), and AI050143 & AI099141 (JCP), and the Science Foundation of Ireland 08/IN.1/B2070 (RJW).
Investigating the use of Chk1 Inhibitors for Triple‐Negative Breast Cancer
Authors: Bindeman Wendy , Redwood Abena , Piwnica‐Worms Helen 
Published: November 2023

Triple‐negative breast cancer (TNBC) accounts for 15–20% of all diagnosed breast cancers. It is characterized by lack of expression of estrogen receptor and progesterone receptor and an absence of HER2 amplification. TNBC is aggressive, highly metastatic, and ~50% of patients do not respond to first line chemotherapy. TNBC frequently harbors mutations in TP53, suggesting that therapies that exploit this vulnerability may be an effective treatment option. In particular, p53‐deficient cells are hypothesized to be highly sensitive to inhibitors of checkpoint kinase 1 (Chk1) due to checkpoint abrogation. We evaluated the efficacy of two clinically relevant Chk1 inhibitors, LY2606368 (LY) and SCH900776 (SCH), to elucidate the reasons for differing cellular response to the two drugs and evaluate the potential for synergy of each drug in combination with different DNA damaging agents. We hypothesized that both agents would synergize with gemcitabine regardless of p53 status, but would only synergize in combination with cisplatin in p53‐deficient cells. We tested the effects of LY and SCH alone and in combination with cisplatin or gemcitabine on cell viability, cell proliferation, and relevant signaling pathways in a TNBC cell line. Our data suggest that the differential cellular response to the two drugs is mainly due to a difference in potency. LY, but not SCH, preferentially sensitizes p53‐deficient cells to cisplatin. Notably, both LY and SCH synergize with gemcitabine regardless of p53 status, suggesting that Chk1 inhibitors + gemcitabine might be effective in both p53‐proficient and p53‐deficient backgrounds. Further studies are necessary to more fully evaluate the synergistic potential between cisplatin and LY or SCH, to characterize the sensitivity of cells carrying mutant p53, and to verify these results in vivo.Support or Funding InformationKomen Foundation; Cancer Prevention & Research Institute of Texas
ProKinO: A unified resource for mining the cancer kinome
Authors: Kannan Natarajan 
Published: November 2023

The massive and exponential growth of genomic, structural, biochemical and clinical data on the human kinome poses major challenges in using existing data for cancer and clinical research. Here we describe an ontological framework for integrating and conceptualizing diverse forms of protein kinase data in a machine readable, human understandable form. We demonstrate the utility of this framework in mining the cancer kinome and in generating testable hypotheses for experimental studies. Through the iterative process of aggregate ontology querying, hypothesis generation and experimental validation, we identify novel mutational hotspots in key regulatory regions of the kinase domain, and demonstrate the impact of identified mutations on kinase activity and drug sensitivity. We find that mutations mapping to the regulatory spine activate the kinase domain through distinct mechanisms and contribute to drug resistance. User‐friendly tools to mine cancer mutations in the context of naturally occurring variants, pathways, and drug sensitivity profiles will be discussed.Support or Funding InformationNIH RO1 GM 11409‐01
Ceramide is A Key Factor That Regulates The Crosstalk Between TGF‐β and Sonic Hedgehog Signaling at The Basal Cilia To Control Cell Migration and Tumor Metastasis
Authors: Gencer Salih , Oleinik Natalia , Dany Mohammed , Ogretmen Besim 
Published: November 2023

Bioactive sphingolipid ceramide is a second messenger in cell membrane in response to inflammation and stress. Recent studies indicate that ceramide species with different fatty‐acid chain lengths play diverse biological functions in various cellular processes, highlighting the importance of ceramide synthases (CerS) in these processes. Migration and cell mobility, a part of these processes, also are effected by ceramide metabolism. However, the molecular mechanism of CerS/ceramide involved is unknown. Here, we investigated the effect of CerS/and ceramide on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4/ceramide is involved to cell migration and tumor metastasis. Here, we also have generated CerS4−/− mice for in vivo studies. Interestingly, we observed that genetically loss of CerS4 resulted in severe irreversible alopecia, which was associated with hyper‐proliferation and migration of keratinocytes. Mechanistically, we show here that genetic loss or shRNA‐mediated knockdown of CerS4 enhances cell migration by which ligand‐independent signaling of TGF‐beta receptors I and II in various cell types, including keratinocytes, mouse embryonic fibroblasts and cancer cells. Moreover, we found that ceramide directly interact with Smad7 and this interaction was decreased by shRNA‐mediated knockdown of CerS4. Thus, ceramide‐Smad7 binding modulates plasma membrane association of TGF‐βR1 at primary basal cilia, and inhibits its signaling through Sonic‐Hedgehog (Shh) for migration. Furthermore, Ceramide accumulation at the primary basal cilia was decreased by knockdown of CerS4, and this was associated with direct interaction of TGF‐βR1 and SMO receptors in cilia. In fact, inhibition of TGF‐βR /Shh signaling or cilia formation using molecular or pharmacologic inhibitors almost completely prevented cell migration in response to CerS4 knockdown. These data revealed that CerS4/ceramide signaling plays key roles in the regulation of cell migration and metastasis via controlling the TGF‐βR and Shh axis at primary basal cilia.Support or Funding Information1R01 DE016572
ER‐Dependent Ca++‐Mediated Production of Cytosolic ROS as an Effector for Induction of Mitochondrial Apoptotic Signal and ATM in Gallic Acid‐treated Human Oral Cancer Cell
Authors: Lu Yao‐Cheng , Lin Meng‐Liang , Su Hong‐Lin , Chen Shih‐Shun 
Published: November 2023

Release of calcium (Ca++) from the endoplasmic reticulum (ER) has been proposed to be involved in induction of apoptosis by oxidative stress. Using ER Ca++ release inhibitor dantrolene and mitochondrial Ca++ uptake inhibitor Ru‐360, we demonstrated that Ca++ release from the ER was associated with loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS), and apoptosis of human oral cancer (OC) cells induced by gallic acid (GA). Chemical chaperone 4‐phenylbutyrate inhibits tunicamycin‐induced induction of 78 kDa glucose‐regulated protein, C/EBP homologous protein and pro‐caspase‐12 cleavage, cytosolic Ca++ increase, and apoptosis, but did not attenuate the increase in cytosolic Ca++ level and apoptosis induced by GA. Ataxia telangiectasia mutated (ATM)‐mediated c‐Jun N‐terminal kinase (JNK) phosphorylation and apoptosis in response to GA was blocked by dantrolene. The specificity of ROS‐mediated ATM activation was confirmed by the treatment of N‐acetylcysteine, an ROS scavenger. Blockade of ATM activation by a specific inhibitor KU55933, short hairpin RNA, or kinase‐dead ATM overexpression suppressed JNK phosphorylation but did not completely inhibit cytosolic ROS production, mitochondrial cytochrome c release, pro‐caspase‐3 cleavage, and apoptosis induced by GA. When taken together, these results indicate that GA induces OC cell apoptosis by inducing the activation of mitochondrial apoptotic signal and ATM, likely through ER Ca++‐mediated ROS production.Support or Funding Information
Single‐Molecule Studies of Eukaryotic DNA Replication
Authors: Bell Stephen P , Ticau Simina , Friedman Larry J , Gelles Jeff 
Published: November 2023

The earliest step in eukaryotic DNA replication is the loading of the Mcm2–7 replicative DNA helicase onto origin DNA. This event marks all potential origins of replication and establishes the head‐to‐head configuration required for bidirectional replication initiation. Helicase loading requires the coordinate action of the Mcm2–7 helicase, and three helicase‐loading proteins: the origin‐recognition complex (ORC), Cdc6 and Cdt1. In an ATP‐hydrolysis‐dependent process, these proteins open the Mcm2–7 ring, insert the double stranded DNA into the central channel, and close the Mcm2–7 ring. The final outcome of this process is a head‐to‐head Mcm2–7 double hexamer.We have developed a reconstituted single‐molecule helicase‐loading assay using Colocalization Single Molecule Spectroscopy (CoSMoS). We use co‐localization of the fluorescently‐labeled DNA and proteins as a real‐time assay for protein‐DNA binding and helicase loading. This assay recapitulates all the know requirements of Mcm2–7‐loading. Using this assay, we found that initial association and loading of the two Mcm2–7 complexes at the origin DNA occurs sequentially. Furthermore, each of the two Mcm2–7 loading events is driven by distinct Cdc6 and Cdt1 molecules that are sequentially recruited and released. In contrast to Cdc6 and Cdt1, a single ORC complex is sufficient to load both of the oppositely‐oriented helicases of the double hexamer. We have extended this assay by developing a FRET‐based method to monitor opening and closing of the Mcm2–7 ring during helicase loading. We are using this assay to determine when these events occurs during the loading reaction and what proteins mediate these changes. We will discuss the implications of our findings for the mechanism of Mcm2–7 loading.Support or Funding InformationThis work was supported by NIH grants GM52339 (S.P.B.) and R01 GM81648 (J.G.) and a grant from the G. Har‐ old and Leila Y. Mathers Foundation (J.G.). S.T. was supported in part by an NIH Pre‐Doctoral Training Grant (GM007287). S.P.B. is an investigator with the Howard Hughes Medical Institute.
Structural Mechanisms for Initiating DNA Replication
Authors: Berger James 
Published: November 2023

RecA and AAA+ family ATPases play key roles in cellular events ranging from vesicle trafficking and proteolysis to chromatin remodeling and DNA replication/repair. A large subset of these enzymes form multimeric rings or spirals that respond to cycles of ATP binding and hydrolysis by undergoing changes in oligomeric and/or conformational state, and then transducing these physical rearrangements onto target macromolecules for controlling key biological processes. New structural and biochemical findings pertaining to how protein and DNA targets are moved or remodeled by RecA/AAA+ ATPases during the initiation of DNA replication will be discussed.Support or Funding InformationNIGMS RO1 GM‐071747
Effects of Divalent Metal Ions on the Structure and Stability of SIKE
Authors: Kim Seung‐Hwan , Bell Ellis , Bell Jessica 
Published: November 2023

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure of SIKE. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. A Three dimensional model of SIKE was made using the Phyre2 server using a variety of template molecules and ab initio modelling. The optimal model (with 97% of the sequence modelled at >90% confidence) was submitted to the 3DLigandSite server to predict potential binding sites. The model was predicted to contain a divalent metal ion binding site comprised of Aspartate 143, Leucine 173 and Glutamate 176 residues. His tagged SIKE was expressed, extracted using a Guanidine Hydrochloride solubilization procedure and purified using Nickel NTA affinity chromatography after renaturation on the resin. The purified protein was used in Fluorescence based Thermal Shift assays to in the presence and absence of a variety of divalent metal ions to screen for binding. Preliminary analysis of the results suggest that divalent metal ions including zinc do in fact affect the thermal stability of SIKE suggesting the presence of a metal binding site consistent with the optimal model.Support or Funding InformationThis work was supported by NIH grant R21A1 107447 to JKB
1Arg188 is Indispensible for RhoC Membrane Localization
Authors: Williams‐Perez Sophia , Shirar Sarah , Feller Gillian , Feller Samantha , Rooney Patrick , Ellerbroek Shawn Michael 
Published: November 2023

RhoA and RhoC GTPases are 92% identical and possess unique cellular functions. Phosphorylation of Ser188 has widely been reported to inhibit RhoA activity. RhoC retains a canonical PKA recognition sequence (KRR) at positions 185–187, but possesses Arg188 in place of Ser188. We report here that transiently expressed RhoC was more membrane associated than RhoA. Further, RhoC‐R188S was a PKA substrate in vitro and exhibited less GTP loading compared to wild‐type RhoC when expressed in cells. RhoC‐R188S was less membrane associated than RhoC regardless of GTP‐binding status (Q63L). Surprisingly, RhoC‐R188A shared a similar membrane fractionation profile as RhoC‐R188S. In accordance, RhoA‐S188R was more membrane associated than either RhoA or RhoA‐S188A regardless of GTP‐binding status (Q63L). Altogether, these data suggest that expansion of the polybasic carboxyl tract through divergent appearance of Arg188 contributes more to increased RhoC membrane stabilization than loss of phospho‐regulation.Support or Funding InformationThis work was supported by a research grant from the McElroy Trust Foundation
Lipid Raft Coalescence Links HIV‐1 gp120 Induced Oxidative Stress to Neurodegeneration
Authors: Smith Lisa K , Walsh Keifer P , Whittington Lindsey G , Shaw Alisa E , Minamide Laurie S , Bamburg James R , Kuhn Thomas B 
Published: November 2023

Host response to infection is a key determinant to disease pathogenesis. Hence, molecular understanding of host‐pathogen interactions provides a means of identifying novel therapeutic strategies in disease intervention. A number of pathogens, including the human immunodeficiency virus (HIV), utilize lipid rafts as their principal host entry portal. HIV1‐lipid raft interactions in the CNS are intimately linked to progressive neurodegeneration in a subset of infected individuals. Lipid rafts also play a critical role in other neurodegenerative diseases including Alzheimer disease. A signaling pathway common to amyloid β, cytokines, as well as HIV viral surface glycoprotein gp120 links oxidative stress and the formation of cofilin‐actin bundles (rods) to defects in synaptic function. Enhanced lipid raft coalescence mediated by the cellular prion protein (PrPC) and associated activation of NADPH oxidase are key to this pathway. Application of gp120 to SH‐SY5Y human neuroblastoma cells induced a time and dose‐dependent increase in lipid raft density reflected by a coalescence of lipid rafts into large macrodomains. Primary rat hippocampal neurons exposed to gp120 responded with a dose‐dependent formation of rods and increased lipid raft density. A rapid gp120‐induced production of reactive oxygen species (ROS) in hippocampal neurons was observed with the ROS‐sensitive fluorescence indicator DCF. NADPH oxidase activation in response to gp120 was causal to the formation of rods in hippocampal neurons. Blocking NADPH oxidase using either pharmacological inhibitors or by overexpressing dominant negative p22phox (a small membrane subunit of NOX) negated gp120‐mediated rod formation. More recent studies have identified a number of natural products that potently reduce viral infectivity. We demonstrate that a non‐polar fraction obtain from crude extracts of Alaskan wild bog blueberries (Vaccinium uliginosum) disrupt lipid raft coalescence in neurons exposed to neurotoxic stresses including gp120 and significantly decrease rod formation. In this scenario, lipid rafts serve as a signaling platform to progressive neurodegeneration associated with viral (AIDS dementia) as well as chronic degenerative CNS disorders. Natural products with the capability of modulating neurotoxic lipid raft signaling pathways could provide a novel approach to blunt progression of neurodegenerative processes.Support or Funding InformationSupported in part by NIH grants AG044812, AG049668, Camille & Henry Dreyfus Sr. Scientist Mentor Award, and CSU Core Infrastructure Grant Microscope Imaging (JRB), and P20GM103395 and the Alzheimer's Resources of Alaska (TBK)
Sex‐Specific Differences of Transforming Growth Factor β Expression in Cardiac Hypertrophy and Heart Failure
Authors: James Maya 
Published: November 2023

Transforming Growth Factor β (TGFβ) ligands and receptors are known to be pro‐hypertrophic and pro‐fibrotic factors in the heart, which could contribute to the development of cardiac hypertrophy and heart failure. It is well established that premenopausal females possess a lower incidence of these pathologies. To better understand the sex‐specific roles of TGFb family members in cardiac hypertrophy and heart failure, we analyzed gene expression in a rodent model of hypertrophy/heart failure and in human heart specimens. Gene expression analysis of sex‐specific rat neonatal cardiomyocyte hypertrophy model demonstrated that TGFβ ligands and receptors are expressed in a sex‐specific manner both in the presence and absence of pro‐hypertrophy agent phenylephrine. Sex differential expression of TGFb was also observed in a transaortic constriction model of cardiac hypertrophy in which males develop more severe hypertrophy. In contradiction, an angiotensin II (AngII) eluding pump mouse model did not result in sex differences and expression of TGFβ family members were more similar. This suggests that AngII, a known activator of TGFβ, may circumvent the cardioprotection afforded to females potentially through the direct activation of TGFβ. Preliminary analysis of human hearts reveals some sex differential gene regulation in both non‐failing and failing hearts. For example, TGFβ1 ligand is expressed at a lower level in non‐failing females hearts as compared to non‐failing males with levels increasing only in the failing hearts of females. Taken together these results provide evidence that sex specific differences in the development of cardiac hypertrophy and heart failure are mediated in part by TGFβ signaling.Support or Funding InformationEastern Kentucky University Department of Biological Sciences NIH 1R15HL 108192‐01NIH 1 R25 GM102776‐01
Developmental Factors Create a Niche for Cardiac Regeneration
Authors: Perrera Alec Bennett , Hoffman Kristen D , Camenisch Todd D 
Published: November 2023

Heart disease is the number one cause of death in the United States. The adult mammalian heart has limited ability to repair itself, which is not sufficient to regenerate myocardial tissue injured by myocardial infarction (MI). Previous research has established that cardiac fibroblasts can be reprogrammed to adult cardiomyocyte‐like cells in vitro by administration of Gata4, Mef2c, and Tbx5 (GMT) as well as thymosin β4 (Tβ4). We have reported previously that hyaluronan (HA) supports a hematopoietic niche in the bone marrow as well as serving as a vital biomolecule for endocardial cushion and epicardial development during mouse embryogenesis. Epicardial cells are also implicated in myocardial regeneration, however the mechanisms of how this occurs in mammalian cells is not clear. TGFβ2 and HA are important for the induction of epicardial cell differentiation and invasion through regulation of epithelial to mesenchymal transformation (EMT). Thus, we hypothesize that TGFβ2 and HA alongside Tβ4 create a regenerative niche potentiating effective reprogramming of cells into cardiomyocytes. These factors should promote cellular differentiation without driving proliferation. We therefore tested whether alone or together HA, TGFβ2 and Tβ4 induces epicardial cell proliferation. Additional experiments are defining the differentiation pathway triggered by the regenerative cocktail of HA, TGFβ2 and Tβ4. These data are vital for understanding how upstream factors can promote GMT‐induced cardiac regeneration.Support or Funding InformationNIEHS R25ES025494
Induction of Fatty Acid Synthase is Required for Profibrotic TGFβ Signaling
Authors: Jung Mi‐Yeon , Kang Jeong‐Han , Wang Youli , Hernandez Danielle , Lupu Ruth , Leof Edward B. 
Published: November 2023

Alterations in cellular metabolism are known to play critical roles in a variety of normal as well as pathological processes, including (but not limited to) cancer, hepatic steatosis, and cell differentiation. While TGFβ is routinely considered a pleotropic cytokine due to its ability to impact numerous diseases and biological phenotypes, the role of metabolism in profibrotic TGFβ signaling is relatively unexplored/unknown. Fatty Acid Synthase (FASN) is the primary anabolic enzyme responsible for the de novo synthesis of fatty acids. Renewed interest in FASN has been sparked by the recognition of the critical inter‐relation of metabolic dysregulation and tumor progression with the findings that (i) FASN levels are significantly increased in numerous human tumors including breast, prostate, colorectal, and ovarian; and (ii) the efficacy of ErbB receptor inhibitors is enhanced by inhibiting FASN activity. In the current study we show that FASN expression is regulated by TGFβ in multiple murine and human fibroblasts, required for profibrotic TGFβ signaling, and, most importantly, treatment with C75 ((trans‐4‐carboxy‐5‐octyl‐3‐methylenebutyrolactone, a novel cerulenin‐derived semi‐synthetic FASN inhibitor) abrogates pathologic and physiologic changes in a murine model of lung fibrosis.Support or Funding InformationThis work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.