bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analyzing Protein Kinase C Interactions With Rho‐type GTPases in the Filamentous Fungus Aspergillus nidulans
Authors: Carroll Maleiah , Craft Alexis , Freyaldenhoven W. Toler , Betton Brianna , Campbell Lynsey , Hameed Muhammad Y. , Vanderwall David , Hill Terry , Jackson‐Hayes Loretta 
Published: June 2021

Cell division in filamentous fungi involves coalescence and subsequent constriction of a contractile actomyosin ring (CAR), as in other fungi and metazoans. In fungi, constriction of the CAR is followed by deposition of cell wall material resulting in a septum. In the model organism, Aspergillus nidulans, many proteins are involved in septation including protein kinase C (PkcA), the formin SepA, and the GTPase Rho4. The current study focuses on the relationships among PkcA, SepA, and Rho‐type GTPases during septation. PkcA contains two putative N‐terminal Rho binding domains designated HR1A and HR1B. The formin SepA belongs to the Diaphanous‐related family of formins that contain an N‐terminal Rho binding domain that upon binding of the appropriate Rho GTPase results in formin activation. Reports from another group suggest that in A. nidulans Rho4 is likely the GTPase required for SepA activation. In previous work we showed that PkcA both physically and functionally interacts with SepA via one of its three formin homology domains (FH2), and in the sepA1 temperature sensitive mutant (FH2 domain‐L1369S) which is aseptate at elevated temperature, PkcA fails to localize to septation sites at elevated temperature. Here we report via yeast two‐hybrid assay that PkcA interacts with SepA via one of its putative Rho binding domains (HR1A), and that expressing extra copies of pkcA in a rho4‐null mutant increased colony diameter and improved sporulation. PkcA localization to septation sites is reduced in a rhoA mutant while septation is unaffected. The spatiotemporal relationship among PkcA, SepA, Rho4, and RhoA will be further investigated via yeast two‐hybrid, bimolecular complementation assays, and localization of fluorescence labeled proteins in mutant strains.Support or Funding InformationSupport for this work was provided by the National Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI).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 The Effect of O‐GlcNAc Modification and Other Stabilizers on Nod1 and Nod2
Authors: Drake Walter Richard , Hou Ching‐Wen , Ukaegbu Ophelia , Zachara Natasha , Grimes Catherine Leimkuhler 
Published: June 2021

In order to distinguish infectious pathogens from the trillions of commensal bacteria on the body, humans utilize their innate immune systems, which deploy pattern recognition receptors (PRRs) to detect microbe associated molecular patterns (MAMPs) specific to certain microorganisms. The PRRs Nod1 and Nod2 detect MAMPs found in the peptidoglycan (PG) of bacterial cell walls, and upon binding initiate an immune response via the NF‐kB and other pathways, leading to the expression of pro‐inflammatory cytokines and chemokines. Misregulation of Nod1 signaling is associated with various diseases, including gastric ulcers, stomach cancer, and lung cancer. Improper Nod2 signaling has been linked to Crohn's disease, and three different Crohn's associated variants of Nod2 lead to increased susceptibility to the disease and are unstable relative to the wild type. The enzyme O‐GlcNAc Transferase (OGT), which adds a GlcNAc monosaccharide to certain serines/threonines, post‐translationally modifies both Nod1 and Nod2. This post‐translational modification (PTM) is common among thousands of proteins in the nucleus, membrane, and cytosol, and is mediated by only two enzymes: OGT, which adds GlcNAc, and O‐GlcNAcase (OGA), which removes it. The effect of this PTM is diverse among the numerous client proteins. Targets include transcription factors, including oncogenes and tumor suppressors, as well as insulin receptors, kinases, chaperones, and others. We investigate the role of O‐GlcNAcylation on Nod1 and Nod2. Modification increases the stability of both PRRs according to multiple assays, including a cellular thermal shift assay (CETSA) that measures thermal stability and a cycloheximide‐chase assay that measures cellular proteosomal degradation rates. O‐GlcNAc modification also leads to increased PG‐induced NF‐kB activity. The Crohn's associated variants of Nod2 are modified by OGT as well, and the modification rescues the particularly unstable 1007fs variant to near‐wild type. Additionally, we measure the stability effects gained from PG fragment binding and chaperone proteins.Support or Funding InformationMITZUTANI GLYCOSCIENCE FOUNDATIONNATIONAL SCIENCE FOUNDATION (CAREER CHE 1554967)NIH NATIONAL HEART, LUNG, AND BLOOD INSTITUTE (P01HL107153 to NEZ)NIH NIGMS COBRE P20GM104316‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase (RibB) of riboflavin biosynthesis has a mononuclear magnesium active site
Authors: Kenjic Nikola , Moran Graham R , Lamb Audrey L 
Published: June 2021

Riboflavin (vitamin B2) is a precursor for the synthesis of the cofactors FAD and FMN. Like other vitamins, riboflavin cannot be synthesized by mammals and must be obtained from the diet. Bacteria and plants synthesize riboflavin in five steps using the enzymes RibA‐E. The focus of this study is the 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase, otherwise known as RibB. RibB is a magnesium dependent enzyme that catalyzes the conversion of the sugar ribulose‐5‐phosphate (Ru5P), a product of the pentose phosphate pathway, into 3,4‐dihydroxy‐2‐butanone‐4‐phosphate (DHBP). The reaction catalyzed by RibB is an unusual deformylation reaction in which the fourth carbon of the five carbon sugar is removed as formate. Bacher and colleagues1 have proposed that the mechanism is dependent on a skeletal rearrangement linking C3 and C5. We propose a fragmentation mechanism, in which the bond between carbons C3 and C4 is broken allowing formation of the new link between carbons C3 and C5 followed by release of formate. To test these competing hypotheses, we are using NMR and X‐ray crystallography to observe the intermediates formed during catalysis. We have produced 13C labeled Ru5P for use in NMR experiments and shown enzymatic formation of DHBP and formate. We also show the 1.1 Å structure of apo RibB. Previously reported product bound structures of RibB have been modeled with two metal ions in the active site2. However, we report here intrinsic tyrosine fluorescence and activity assay profiles that indicate that RibB requires a single magnesium for full activity.Support or Funding InformationWeaver Graduate Student Fellowship, the National Science Foundation CHE‐1403293, The University of Kansas 2018 General Research FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Salt dependence of a self‐splicing enzyme from an extreme halophile
Authors: Cawood Alexandra , Mills Kenneth V 
Published: June 2021

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an internal protein, or intein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitantly with the ligation of the exteins. The extremophile Haloferax volcanii exists in a high salt environment. We are interested in the halophile because we want to learn how efficiently the intein that has invaded its PolB gene splices as a function of salt concentration. We have shown that we can induce splicing of the intein in vitro as a function of salt concentration and time. Others (http://www.ncbi.nlm.nih.gov/pubmed/27462108) have demonstrated that the intein has an active homing endonuclease domain in vivo, and that the presence of the intein has a high fitness cost for the organism. We are interested in studying the endonuclease activity of the intein in vitro, given that a salt‐dependent conditionally‐active nuclease may have utility in biotechnology applications.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Under Pressure: Pressure and Temperature Dependent Nuclease Activity
Authors: Wibben Amanda C , Comeau Hannah Y , Mills Kenneth V 
Published: June 2021

An intein is an intervening protein segment that can be found in proteins from extremophiles and is typically self‐excised to allow for complete flanking protein activity. We are interested in studying specific inteins that contain a homing endonuclease (HE), an enzyme that cleaves double stranded DNA in order to initiate insertion of an intein gene by homologous recombination. We are focused on the homing endonuclease activity of inteins from two hyperthermophilic archaea: Thermococcus barophilus (Tba), isolated from a deep sea hydrothermal vent, and Thermococcus kodakarensis (Tko), isolated from a volcanic area on the shore of a Japanese island. Because these intein‐containing extremophiles have been isolated from very high temperatures but from different depths, we are interested in the differential homing endonuclease activity of these extremophiles at varied temperatures and pressures. We have shown that Tba HE is active at pressures up to 1000 atm. Given that Tko is a surfaced‐welling organism, we suspect that the Tko HE will be pressure sensitive while Tba, which lives naturally at a pressure near 400 atm, is not. Additionally, we have found that both Tba HE and Tko HE show temperature dependence, which we attribute to the extreme temperatures that both organisms reside in.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus 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.
Structural Analysis of Halophilic and Thermophilic Inteins
Authors: Conroy Owen Ryan , Chavez Angel , Mills Kenneth Vincent 
Published: June 2021

Protein splicing is a post‐translational modification by which a segment of protein, known as an intein, self‐catalyzes its excision from the flanking polypeptides while simultaneously ligating the exteins. Inteins can be found in many types of archaebacteria, including halophiles, which thrive in environments with high salt concentrations. Two halophilic inteins that interrupt the Cdc21 gene, CdcA and CdcD, have been shown to splice at different salt concentrations. In addition, the PolII intein from the extreme thermophile Pyrococcus horikoshii requires incubation at high temperature in order to splice. We are interested in solving the structure of all three inteins by NMR to better probe the relationship between a protein's structure and its requirements of extreme conditions for activity. To obtain the proteins at sufficiently high concentrations for structural analysis, we sub‐cloned the inteins of interest into expression vectors with an N‐terminal GB1 tag and optimized expression conditions.Support or Funding InformationThis work was supported by the National Science Foundation (MCB‐1517138, KVM) and the Dreyfus Foundation (KVM).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enzymology of Columbamide Biosynthesis
Authors: D'Agostino Gabriel , Manley Olivia M. , Makris Thomas M. 
Published: June 2021

Marine cyanobacteria represent a unique, underexplored reservoir of bioactive natural products. Recent genomic analysis and mass spectrometry studies have revealed a novel chlorinated compound known as columbamide from Moorea bouillonii PNG isolated off the coast of New Guinea. Mass spectral shows columbamide to be a dihalogenated compound with chlorine atoms appended to the C6 and C12 positions of a hydrocarbon chain that is thought to be constructed from dodecanoic acid. Genomic studies have proposed a pathway for columbamide biosynthesis but it has yet to be supported by in vitro studies. Of particular interest, the enzymes proposed to catalyze the halogenation reactions, ColD and ColE, are most homologous to dinuclear iron‐dependent enzymes. Although diiron enzymes have been shown to have a great deal of functional diversity, halogenation is not amongst these reported activities. Our study seeks to characterize and reconstruct the early stages of the biosynthetic pathway by generating protein‐tethered substrates for the halogenases ColD and ColE. ColC is believed to be an acyl‐carrier protein for dodecanoic acid as well as a substrate of ColD/ColE. The first enzyme encoded by the gene cluster, ColA, is believed to attach dodecanoic acid to ColC. To investigate the biosynthetic pathway leading to halogenation, the role of ColA will be confirmed, and its enzymology characterized. Additionally, the well‐characterized enzyme acyl‐acyl carrier protein synthetase from the organism Vibrio harveyi will be used to efficiently generate large amounts of acylated‐ColC with non‐native fatty acids. Our study will seek to use the generated substrate‐library in concert with various spectroscopic techniques—such as optical spectra analysis, rapid‐mix stopped‐flow kinetics, and electron paramagnetic resonance (EPR)—to characterize and elucidate the mechanistic details of enzymes ColD/ColE.Support or Funding InformationNational Science Foundation; South Carolina Honors College‐‐Undergraduate Research FundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Fitness Cost of Two Inteins in Halobacterium salinarum”
Authors: Exconde Patrick M. , Janton Christopher J. , Makkay Andrea M. , Papke R. Thane , Gomez Alvin V. , Mills Kenneth V. 
Published: June 2021

Halobacterium salinarum is from the domain Archaea and an obligate inhabitant of extremely saline conditions, including saturated brines. Its PolII and Cdc21a genes are both interrupted by an intein. Inteins are genetic elements within an organism's genome, which, at the protein level, self‐catalyze their own excision to concomitantly form a functional protein. We previously showed that protein splicing of these inteins depends on high salt concentrations, requiring a minimum of 2.0 M NaCl to be active. This suggests that the intein may be active in the native cellular environment of H. salinarum, allowing for production of functional protein, and that its presence might not be detrimental to its host. Conversely, a study by Naor et al. (PNAS (2016) 113(32), E4654) showed that in another halophilic archaeon, Haloferax volcanii, there is a > 7% fitness cost for maintaining the polB‐c intein. We are interested in whether the inteins in H. salinarum exert a similar fitness cost, and will test this using a growth‐competition assay between wild type and intein‐less strains of H. salinarum.Support or Funding InformationThis work was supported by NASA (grant NNX15AM09G to RTP), the National Science Foundation (grants MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Personalized mechanisms of Lafora disease using Differential Scanning Fluorimetry”
Authors: Donohue Kit , Brewer M. Katherine , Vander Kooi Craig W , Gentry Matthew S 
Published: June 2021

Lafora disease is an autosomal recessive neurodegenerative disease. Patients grow up normally for their first decade of life, then develop severe epilepsy, and die in their 20s or 30s from massive neurodegeneration. The field has demonstrated that the pathogenic agent of the disease are aberrant glycogen molecules.Glycogen phosphorylation impacts the branch point and chain length distribution within the glycogen molecule, determining both the granule shape and its accessibility to regulating enzymes. Laforin is the only known mammalian phosphatase capable of modifying the phosphorylation status of glycogen. When laforin is mutated, aberrant glycogen forms inclusion bodies and drives Lafora disease progression. Therefore, it is important to understand how different mutations in laforin affect its ability to bind and dephosphorylate glycogen. However, defining protein‐carbohydrate interactions can be challenging because many binding techniques cannot accurately measure low binding affinities.We utilized differential scanning calorimetry (DSC) in combination with differential scanning fluorimetry (DSF) and a unique algorithm to consistently predict a true Kd using DSF data. Through the use of short polysaccharides that approximate glycogen micro‐structures, we show that the DSF method can be used in a high‐throughput assay to assess laforin‐carbohydrate interactions. With this data, we compare how different mutations impact the binding affinity of laforin to glycogen and how changes to glycogen structure alter the ability of laforin to bind and dephosphorylate glycogen. These results provide unique insights into personalized mechanisms of Lafora disease.Support or Funding InformationThis work was supported by the National Science Foundation through NSF MCB1817417 and NSF OIA 1355438This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microbial VOC Fingerprints: Rapid Detection of Antimicrobial Resistance in Pathogenic Bacteria
Authors: Dailey Allyson , Saha Jessica , Zaidi Fatima , Couch Robin 
Published: June 2021

The US Centers for Disease Control and Prevention (CDC) estimates that at minimum, 2 million people in the United States are infected with antimicrobial resistant (AMR) bacteria, resulting in 23,000 deaths each year. With no projected decrease in the number of deaths and the evergrowing threat of multi‐drug resistant bacteria, the development of a rapid and accurate diagnostic test is crucial, not only to reduce the burden on the public healthcare system, but to save countless lives.Microbial volatile organic compounds (mVOCs) are a structurally diverse group of microbial‐derived metabolites, generally related by their volatility at ambient temperature. There is great diagnostic potential for mVOCs, as numerous studies have identified unique mVOC‐fingerprints indicative of specific bacterial pathogens. In addition, specialized headspace sampling methods, such as solid‐phase microextraction (hSPME), coupled to gas chromatography, have enhanced the isolation, preconcentration, and analysis of mVOCs from biological specimens. Herein, we describe the development and assessment of our newly developed and patented metabolomics device and technique known as “simultaneous multifiber headspace solid‐phase microextraction (simulti‐hSPME),” for the rapid and minimally invasive preparation of mVOC fingerprints. This method is evaluated against antimicrobial susceptible and resistant strains of Yersinia pestis and Francisella tularensis.Support or Funding InformationFunding for this project is provided by DTRA (Grant Number: HDTRA 1‐16‐1‐0040).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Longitudinal Analysis of DNA Methylation Status Linked to Post‐Traumatic Stress Disorder in Deployed Service Members
Authors: Gautam Aarti , Yang Ruoting , Miller Stacy , Chakraborty Nabarun , Abu‐Amara Duna , Blessing Esther , Hammamieh Rasha , Marmar Charles , Jett Marti 
Published: June 2021

Pre‐deployment identification of risk and resilience factors is crucial in developing strategies to reduce or prevent post‐traumatic stress (PTSD) symptoms in military personnel. Combat and deployment itself can result in drastic changes in activity, diet, and stress, often with detectable molecular consequences. This longitudinal study was designed to assess the effects of deployment on DNA methylation in peripheral blood. Blood samples were collected pre‐deployment, immediately post‐deployment, and at a three month follow‐up. A battery of clinical tests and self‐reported psychological data were included in the study. DNA methylation assays, followed by repeated‐measures ANOVA, correlation analysis, and PTSD differential analysis were carried out to characterize methylation status alterations associated with significant PTSD Check List (PCL) changes (PCL difference >10). We identified 64 subjects with increases in PCL scores of 10 across the three phases of the sample collection. Repeated‐measures ANOVA testing, adjusted for cell composition and age, was conducted on 2415 methylation probes. 72 significantly differentially methylated probes were identified that were associated with Netrin, mTOR, hypoxia, and ubiquitination pathways. Correlation and differential analysis between PTSD and control samples identified 51 genes passing mean p‐value cutoffs across analyses. These genes are involved in the cAMP, mTOR, opioid, and CREB signaling pathways. Our findings will contribute to understanding the pathogenesis of PTSD co‐morbid conditions and provide mechanistic insights which may aid in averting the onset of chronic PTSD. Future work will integrate these epigenetic data with other molecular ‐omics and clinical findings to better understand PTSD co‐morbidities, as well as susceptibility and resiliency.Disclaimers:Research was conducted in compliance with all Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.Support or Funding InformationThis study was supported by USAMRMC grant No: 09284002.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
WWOX drives UV/cold shock‐induced bubbling cell death whereas without WWOX cells pop out
Authors: Chang Nan‐Shan , Tsai Cheng‐Chang , Sheu Hamm‐Ming 
Published: June 2021

Bubbling cell death (BCD) is known as release of an enlarging nuclear bubble from the surface of each cell at room temperature or lower post exposure of the cells to UV irradiation and cold shock. BCD concerns the severity of frostbite and UV. However, the molecular mechanism is largely unknown. Here, we determined that UV/cold shock induces tumor suppressor WWOX‐expressing L929S cells to undergo typical BCD. UV energy is rapidly taken up by the nuclear DNA to transmit signals and simultaneous rapid influx of calcium ion, followed by nuclear accumulation of numerous cytosolic and membrane proteins (e.g. p53, WWOX, TRAF2, NOS2, NF‐κB, C1q and Hyal‐2). Subsequently, release of nucleolar content leads to irreversible bubble formation and death without cell shrinkage. BCD is blocked at 37°C in L929S and other WWOX‐positive cells. Notably, transient overexpression of TIAF1 and SH3GLB2 restores BCD in TNF‐sensitive L929S cells at 37°C. In contrast, UV/cold shock‐treated WWOX‐deficient cells undergo shrinkage, then rapid nuclear bubble formation and final whole cell pop‐out, designated pop‐out death (POD). Antiapoptotic TRAF2 binds WWOX and the binding is abolished during BCD, suggesting that the dissociated WWOX is needed in inducing BCD.Support or Funding InformationResearch was supported by the Ministry of Science and Technology, Taiwan (MOST 105‐2320‐B‐006‐046, 105‐2320‐B‐006‐036,106‐2320‐B‐006‐017, and 106‐2320‐B‐006‐061), the Department of Defense, USA (W81XWH‐08–1‐0682), the National Health Research Institute, Taiwan (NHRI‐EX107‐10734NI) to NS Chang.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.