bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Mechanisms of Enzyme Catalyzed Protein Unfolding and Translocation by Class 1 AAA+ Motors
Authors: Lucius Aaron L.
Published: June 2021

AAA+ molecular motors involved in protein quality control are at the heart of many biological functions. Here I will discuss our efforts on two model AAA+ driven motors, prokaryotic ClpA and ClpB. ClpA is a hexameric ring motor that uses the energy from ATP binding/hydrolysis to processively translocate a polypeptide substrate through its axial channel for one of two purposes: protein remodeling or ATP dependent proteolysis. When catalyzing proteolysis, ClpA associates with the tetradecameric serine protease, ClpP, to form the ATP dependent protease ClpAP, which is architecturally identical to the 26 S proteasome in humans. ClpB, on the other hand, has the unique ability to disrupt protein aggregates in vivo and does not associate with any known protease. Due to the structural similarity between ClpA and ClpB it has long been proposed that ClpB processively translocates a polypeptide through the axial channel of its hexameric ring structure; as do ClpA and ClpX. However, for ClpB and related enzymes, this has been difficult to show because ClpB does not covalently modify the substrate on which it translocates. We have developed a transient state polypeptide translocation strategy that is sensitive to processive translocation catalyzed by ClpA in the absence of proteolytic degradation catalyzed by ClpP. Using this approach, we have shown that ClpA employs a different molecular mechanism when translocating polypeptide in the absence of the protease vs. when it is associated with the protease. Applying this approach to ClpB, we have shown that ClpB takes, at most, two translocation steps on the polypeptide substrate before rapid dissociation. These results reveal ClpB to be a non‐processive translocase that is not likely to fully translocate a polypeptide through its axial channel during protein disaggregation. These findings and the application of these approaches have broad impact on the examination of many AAA+ molecular motors that do not covalently modify the substrate on which they operate.

Support or Funding Information

NSF MCB‐1412624

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in Sympathetic Control of Vascular Tone: Influences of Aging
Authors: Charkoudian Nisha
Published: June 2021

Reproductive hormones have important non‐reproductive influences on cardiovascular structure and function. In women, these include important effects on the autonomic control of the circulation. Women under the age of 40 tend to have less tonic activity of sympathetic vasoconstrictor nerves in the periphery (usually measured as muscle sympathetic nerve activity), as well as smaller hearts and lower cardiac output compared to men of similar age and health status. Together, these result in a tendency for lower blood pressures in young women, including decreased risk for hypertension, and increased risk of hypotensive disorders such as orthostatic intolerance. Female reproductive hormones (particularly estrogen) appear to contribute to increased functional activity of vascular beta‐2 adrenergic receptors, which offset alpha‐adrenergic vasoconstriction and result in less vasoconstriction for a given amount of sympathetic neural activity (or local norepinephrine), also contributing to the lower blood pressure in young women compared to young men. At menopause, the influences of the hormones decreases, and sex differences are minimized or reversed: sympathetic nerve activity and blood pressure increase to levels similar to, or higher than, those seen in older men. The vasodilator influence of beta‐2 receptors decreases, and sympathetic nerve activity becomes more tightly linked to vasoconstriction and blood pressure. Sympathetic neural activity thus becomes a greater contributor to blood pressure with aging in both men and women, and is a factor in the increased risk of hypertension in older age in both sexes.

Support or Funding Information

Funded by USAMRMC; author views not official US Army or DOD policy

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dietary Adaptations of the Feeding Apparatus in Bats
Authors: Santana Sharlene
Published: June 2021

Diet evolution is considered a major driver of morphological differences and species diversification in mammals, but quantitative tests of morphological adaptation and diversification are scarce. Bats are an ideal system to investigate dietary adaptations; they represent 20% of all mammals, encompass nearly the full spectrum of mammal diets, and have highly diverse cranial morphologies and feeding behaviors. Here, we present research aimed at identifying whether and how the bat feeding apparatus has diversified in conjunction with diet evolution. We combine modern laboratory and field approaches to quantify and map the evolution of skull shape, muscle morphology, and feeding performance across major bat lineages. These methods include: Computed Tomography (CT) scanning and Diffusible Iodine Contrast Enhanced CT scanning, three‐dimensional biomechanical modeling, in vivo bite force measurements, and statistical tests of evolutionary adaptive regimes. With these detailed anatomical and performance datasets, we have been able to identify potential drivers and mechanisms of morphological and functional diversification of the bat feeding apparatus. Analyses of selective regimes indicate that the current diversity of bat skull shapes is best explained by adaptive evolution for both dietary and sensory demands, with striking differences among bats that use different forms of echolocation, or none at all, and among diet specialists. Species that specialize on different diets differ in skull size and shape and in the morphology of their jaw muscles, including physiological cross‐sectional areas, origin and insertion areas, and fiber architecture. Importantly, our biomechanical models indicate that these morphological differences translate into differences in feeding performance metrics that are relevant to food processing, including maximum bite force and gape. For example, for their size, species with a foreshortened rostrum and large temporalis muscles are able to generate high bite forces at the expense of a wide gape, and this allows them to consume tough but relatively small food items. Altogether, these results illuminate the potential drivers and mechanisms of morphological and ecological diversification in bats, and possibly other mammals.

Support or Funding Information

National Science Foundation award 1557125

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering Plant Signal Transduction for Water Smart Crops
Authors: Cutler Sean R.
Published: June 2021

Plant hormones are a structurally diverse collection of small molecules that control plant growth, development, and environmental responses. Work over the past two decades has established that many plant hormones directly stabilize protein‐protein complexes and act analogously to chemical dimerization agents, which were first described for the immunosuppressants rapamycin, FK506, and cyclosporin. For example, abscisic acid (ABA), which regulates plant transpiration, stabilizes a complex between soluble ABA receptors and downstream phosphatases; the ABA‐induced complex inhibits phosphatase activity, which in turn derepresses downstream kinases and activates signaling. I will describe my lab's work in discovering this intriguing sensing module and our efforts to design both synthetic ABA receptor agonists and engineer receptors with altered ligand binding properties. An unusually large family of receptors encodes ABA receptors and, until recently, it was unclear if selective or pan‐agonists would be required to effectively control plant water use and drought tolerance. Our discovery and characterization of multiple selective agonists resolved this issue and defined the ABA receptor Pyrabactin Resistance 1 (PYR1) and its close relatives as key targets that can be leveraged to control transpiration. I will also describe a new fully orthogonal signaling module that is insulated from endogenous signaling components and controlled by a non‐native agrochemical. This engineered dimerization module provides a simple platform technology for programming crops with agrochemically controlled traits.

Support or Funding Information

Supported by NSF (IOS1258175 and 1656890) and Syngenta Crop Protection AG.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beyond the Central Dogma: The tRNA epitranscriptome and an alternative genetic code tune translation during stress in eukaryotes, prokaryotes and viral infections
Authors: Dedon Peter
Published: June 2021

The Central Dogma defines the “what” of biology: genes are transcribed into messenger RNAs that are translated into proteins. But it says nothing about the “when” or “how much” of gene expression. Application of new convergent and data‐driven technologies to the already‐known >150 chemical modifications of DNA, RNA and histone proteins is revealing information‐rich systems for scheduling gene expression ‐‐ the epigenome, the epitranscriptome and the epiproteome. Here we explore one of the newest of these ‐‐ a universal “system of systems” in which the transfer RNA epitranscriptome interacts with an alternative genetic code to fast‐track production of survival proteins during stress in eukaryotes, prokaryotes and viruses. The tRNA molecules themselves are further repurposed as small regulatory RNAs in a system that is only now emerging with the development of new analytical and data mining tools. We will explore examples of the epitranscriptome and small regulatory RNAs in model bacterial and viral pathogens, and in human cells. The components of these systems are now being recognized as drivers of many human diseases and are emerging as new tools for academic and industrial research and development.

Support or Funding Information

This work has been funded by the US National Institutes of Health, the US National Science Foundation, the National Research Foundation of Singapore through the Singapore‐MIT Alliance for Research and Technology, the United States‐Israel Binational Science Foundation, Agilent Technologies and NTUitive.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Triacylglycerol synthesis, energy metabolism, and glucose homeostasis: A gut reaction?
Authors: Yen C.‐L. Eric , Nelson David , Yen Mei‐I , Amador‐Noguez Daniel , Veldkamp Kelsey
Published: June 2021

The intestine plays a pivotal role in regulating systemic metabolism. It is the portal and sensor for incoming nutrients and the origin of neural and endocrine signals that determine responses to diet. Importantly, the intestine is the location where host, diet, and gut microbiota interact. We have discovered that acyl CoA:monoacylglycerol acyltransferase 2 (MGAT2) catalyzes triacylglycerol synthesis in enterocytes, mediates fat absorption in the intestine, and regulates whole body energy balance. Mice without a functional gene encoding the enzyme (Mogat2−/−) absorb a normal amount of dietary fat. However, they exhibit delayed fat absorption, altered gut hormone secretion, and increased energy expenditure. As a result, they are resistant to obesity and related metabolic disorders induced by a high‐fat diet. Using tissue‐specific gain‐ and loss‐of‐function models, we showed that intestinal MGAT2 enhances metabolic efficiency and promotes weight gain. When fed a fat‐free diet, Mogat2−/− mice still exhibit increased energy expenditure, suggesting the effects are not limited to calories from fat. Accordingly, MGAT2 deficiency protects the genetically hyperphagic Agouti mouse fed a regular chow from excessive weight gain, hepatic steatosis, and glucose intolerance. We also found that inactivation of MGAT2 in adult mice decreases weight gain and enhances glucose regulation, even in already obese mice. Surprisingly, Mogat2−/− mice are also protected from both chemical and genetic insults to the pancreatic beta‐cells. We hypothesize that loss of MGAT2 may act on beta‐cells through its effect on bile acid‐modifying gut microbiota. Some of these findings and approaches we are taking to test this hypothesis will be discussed in this presentation.

Support or Funding Information

We gratefully acknowledge our funding support from the U.S. National Institutes of Health (DK088210) and U.S. Department of Agriculture (WIS01442).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
First detailed anatomical study of bonobos exposes bonobos as best model for human‐chimp ancestor and just‐so stories of human evolution, bipedalism and tool use
Authors: Diogo Rui
Published: June 2021

Just‐so stories are prominent in human evolution literature because of our tendency to create simple progressionist narratives about our ‘special’ place in nature, despite the fact that these stories are almost exclusively based on hard tissue data. How can we be so certain about the evolution of human facial communication, bipedalism, tool use, or speech without detailed knowledge of the internal anatomy of, for instance, one of the two extant species more closely related to us, the bonobos? Here I show how many of these stories now become obsolete, after such a comprehensive knowledge on the anatomy of bonobos and other primates is finally put together. Specifically, each and every muscle that has been long accepted to be ‘uniquely human’ and to provide ‘crucial singular functional adaptations' for our bipedalism, tool use and/or vocal/facial communication, is actually present as a variant or even as normal phenotype in bonobos and/or other apes. Importantly, chimps and in particular bonobos also provide a remarkable case of evolutionary stasis: within >120 head‐neck (HN) and forelimb (FL) muscles, since the chimp‐human split c.8Mya there were only four minor changes in the chimp clade, and all were reversions to the ancestral condition. Outstandingly, since the common chimp‐bonobo split c.2 Mya not even a single change occurred in bonobos, i.e. regarding the HN‐FL musculature bonobos are the best living model for the common chimp‐bonobo LCA and the chimp‐human LCA. The extremely static Pan evolution becomes even clearer when hindlimb data are added: only two muscle absence/presence differences occur between common chimps‐bonobos, and none of these species has muscles not found in other primates. Puzzlingly, there is an evolutionary mosaicism between each of these species and humans. I discuss these data in the context of available genomic information and debates on whether the common chimp‐bonobo divergence is linked to heterochrony, and put these data together with that available for humans and other other primates in order to provide a more comprehensive, and less biased, understanding of primate and human anatomical evolution.

Support or Funding Information

This project has received funding from NSF grants 1516557 and 1440624, as well as an American Association of Anatomists Innovation grant to RD

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Biochemical Investigation of Plant‐Nematode Interactions
Authors: De Santiago Perez Alejandro , Holland Cynthia , Jez Joseph
Published: June 2021

Chorismate, the branch point metabolite in aromatic amino acid biosynthesis, is synthesized by plants, bacteria, and fungi. In plants, the three aromatic amino acids, phenylalanine, tyrosine, and tryptophan produce several compounds such as plant growth hormone indole‐3‐acetic‐acid (IAA) and the defense compound salicylic acid (SA). Several plant parasitic nematodes, such as Meloidogyne javanica and Heterodera schachtii secrete chorismate mutase (MjCM and HsCM, respectively), an enzyme that converts chorismate to prephenate, during early stages of infection. The secretion of chorismate mutase competes with the plant for chorismate, reducing IAA and SA, and inhibiting the vascularization and formation of roots. A recently discovered Arabidopsis thaliana (thale cress) protein, GH3.7, is capable of conjugating chorismate to cysteine, and may play an important role during nematode infections. Here, we found that wild‐type and gh3.7 plants infected with the pathogenic bacterium Pseudomonas syringae pv. tomato do not show a significant difference in pathogenic susceptibility. Steady‐state kinetic assays determined that the turnover rate and catalytic efficiency of MjCM and HsCM is significantly reduced compared to the chorismate mutases in Arabidopsis thaliana. MjCM and HsCM structural models were generated through computation modeling and most closely resembled Mycobacterium tuberculosis chorismate mutase. The kinetic and structural characterization of nematode chorismate mutase will provide an insight into the molecular machinery used by plants to ward off parasitic infections.

Support or Funding Information

This work was funded by the National Science Foundation and supported by the Howard Hughes Medical Institute.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide
Authors: Mohammad Adeba , Ramos Adriana Bon , Lee Bobby W.K , Cohen Spencer W. , Kiani Maryam K. , Iwata‐Reuyl Dirk , Stec Boguslaw , Swairjo Manal A.
Published: June 2021

QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent reduction of the nitrile group of 7‐cyano‐7‐deazaguanine (preQ0) to 7‐aminomethyl‐7‐deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF‐catalyzed reaction involves the formation of a covalent thioimide intermediate with a conserved active site cysteine (Cys55) that is prone to oxidation in vivo under different conditions. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide bond formed between the catalytically active Cys55 and a conserved Cys99 located near the active site. When compared to the substrate‐bound structure, this structure is more symmetric and exhibits major rearrangement of the loops responsible for substrate binding. It has been demonstrated that the disulfide does not play a catalytic role because mutation of Cys99 to Ala/Ser does not compromise enzyme activity. Inactivation of the wild‐type enzyme via peroxidation is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible. This is consistent with protection of Cys55 from irreversible oxidation by disulfide formation with Cys99. Conservation of the cysteine pair, and the reported in vivo interaction of QueF with the thioredoxin‐like hydroperoxide reductase AhpC in Escherichia coli suggest that regulation by the thioredoxin disulfide‐thiol exchange system may constitute a general protective mechanism of QueF from oxidative stress in vivo. Docking of QueF onto this enzyme complex sheds structural light on the mechanism of redox control and may inform future mutagenesis experiments.

Support or Funding Information

This project was supported by National Science Foundation grant CHE‐1309323 to D. Iwata‐Reuyl and M.A. Swairjo, National Institutes of Health (NIH) grant GM110588 to M.A. Swairjo and D. Iwata‐Reuyl, and NIH grant GM70641 to D. Iwata‐Reuyl. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), Stanford Linear Accelerator Center National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE‐AC02‐76SF00515. The SSRL Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research, by the NIH, and the National Institute of General Medical Sciences (NIGMS) (including grant P41GM103393).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Direct Route for Asparaginyl‐tRNA Formation in B. subtilis
Authors: Wales Rebecca , Sheppard Kelly
Published: June 2021

Attaching an amino acid to its cognate tRNA is an essential step for the fidelity of protein synthesis. Two distinct routes have evolved for ligating asparagine to tRNAAsn. In the direct route, asparagine is first synthesized by an asparagine synthetase. Asparagine is then attached to tRNAAsn by asparaginyl‐tRNA synthetase. For the indirect path, asparagine is synthesized on the tRNA. First a non‐discriminating aspartyl‐tRNA synthetase ligates aspartate to tRNAAsn and the amidotransferase GatCAB amidates the tRNA‐bound aspartate to asparagine. Bacillus subtilis encodes both routes for asparaginyl‐tRNAAsn formation. Why it acquired an asparaginyl‐tRNA synthetase is not clear. We hypothesize that it enables the organism to adapt to different environmental conditions. To test this hypothesis, we have purified the B. subtilis asparaginyl‐tRNA synthetase and asparagine synthetase B. We will report on characterizing in vitro the two enzymes for the direct route for asparaginyl‐tRNA synthesis under different physiologically relevant conditions as well as knocking out asks in the B. subtilis genome that encodes asparaginyl‐tRNA synthetase. The work will provide insight into how bacteria have adapted amino acid metabolism and use in protein synthesis for different environmental niches.

Support or Funding Information

National Science Foundation (MCB‐1615770)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticodon‐Binding Domain Mutations of the Bacillus subtilis Non‐discriminating Aspartyl‐tRNA Synthetase to Increase tRNA Specificity
Authors: Shi Katherine C. , Rasmussen Karli C. , Sheppard Kelly
Published: June 2021

The fidelity of the genetic code is etablished by tRNA synthetases attaching an amino acid to the correct tRNA isoacceptor. The majority of prokaryotes lack an asparaginyl‐tRNA synthetase to ligate asparagine to tRNAAsn. Instead, these organisms possess a non‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS), an AspRS with relaxed substrate specificity, that can ligate aspartate to either tRNAAsn or tRNAAsp. An amidotransferase enzyme, GatCAB, then amidates Asp‐tRNAAsn to form Asn‐tRNAAsn. In many but not all organisms encoding an asparaginyl‐tRNA synthetase to directly ligate asparagine to tRNAAsn, the aspartyl‐tRNA synthetase has evolved to be specific for tRNAAsp, a discriminating aspartyl‐tRNA synthetase (D‐AspRS). How bacterial aspartyl‐tRNA synthetase evolved specificity for tRNAAsp is not clear. Bacillus subtilis encodes a ND‐AspRS while the related Bacillus anthracis codes for a D‐AspRS. The sequence similarity between the two enzymes provides an opportunity to alter the tRNA specificity of a ND‐AspRS. Using the sequence comparison along with available structures of aspartyl‐tRNA synthetases, we have mutated the anticodon binding domain of the B. subtilis ND‐AspRS and will report how the various mutations impacted specificity for tRNAAsp. The work expands our understanding of tRNA synthetase substrate specificity and how fidelity of protein synthesis is achieved.

Support or Funding Information

National Science Foundation (MCB‐1615770)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Dual Pathways for B. halodurans Asparaginyl‐tRNA Formation
Authors: Schroeder Caitlin M. , Sheppard Kelly
Published: June 2021

Protein synthesis requires the attachment of an amino acid to its cognate transfer RNA (tRNA). Two distinct pathways for attaching asparagine (Asn) to tRNAAsn are known: the direct pathway, in which Asn is directly attached to tRNAAsn by the enzyme AsnRS; and the indirect pathway, in which Asn is attached to tRNA by a non‐discriminating AspRS and GatCAB. Bacillus halodurans uses both routes for Asn‐tRNAAsn formation. To better understand why B. halodurans possesses both pathways, we have purified its AsnRS and AspRS in order to test and compare their activities under different physiologically relevant conditions such as in the presence of reactive oxygen species and changes in pH. This research will provide insight into how B. halodurans has adapted to survive in different environmental conditions.

Support or Funding Information

National Science Foundation (MCB‐1615770)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Functional Large Substrate Binding in Iterative Non‐ribosomal Peptide Synthesis Independent Synthesis (NIS) Enzymes
Authors: Hoffmann Katherine M.
Published: June 2021

Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation. One type of siderophore synthesis, increasingly associated with bacterial virulence, uses Non‐ribosomal peptide siderophore Independent Synthesis enzymes (NIS synthetases). Within a sub‐category in this family, an additional remarkable kinetic behavior exists whereby multiple bonds may be catalyzed on the same substrate (iterative.) Prior work in the field has identified a novel protein fold as well as the unique peptide bond chemistry, but no structures exist of an iterative protein. The iterative activity is correlated with broad substrate specificity, but this has never been delineated nor quantified. In fact, no iterative enzyme kinetic parameters have been satisfactorily described. We propose basic science to characterize the structure, function, and elasticity of the under‐described iterative proteins in this novel family of NIS sythetases. To do this, we will utilize the following key methodologies: (1) site‐directed mutagenesis to probe important residues; (2) isothermal titration calorimetry (ITC) to explore the thermodynamics of substrate and cofactor binding; (3) Kinetic assays to quantify and compare iterative vs. single bond formation; and (4) X‐ray crystallography to probe the structural changes of iterative substrate binding. The results of our work will be used as a foundation for future mechanism and inhibitor studies, an effort that could lead to a new class of antibiotics.

Support or Funding Information

This work was supported in part by a grant from the National Science Foundation (MCB‐1716986) and by California Lutheran 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.
Dynamics of Organophosphate‐Induced Structural Changes in Acetylcholinesterase Revealed by Time‐Resolved Small‐Angle X‐Ray Scattering and Inelastic Neutron Scattering
Authors: Blumenthal Donald K. , Kovalevsky Andrey , Gerlits Oksana , Fajer Mikolai , Cheng Xiaolin , Taylor Palmer , Radić Zoran
Published: June 2021

Organophosphates (OPs), the active compounds in nerve agents and insecticides such as paraoxon (POX), exert their toxic effects by irreversibly inhibiting acetylcholinesterase (AChE). Currently available antidotes for OP toxicity can reactivate AChE, but reactivation rates are slow which can lead to irreversible nervous system damage or death. We hypothesize that better antidotes with accelerated reactivation rates can be developed by understanding the structural dynamics of OP‐AChE conjugates. Previous steady‐state small‐angle X‐ray scattering (SAXS) experiments showed that POX and certain other OPs induce quaternary structural changes in human AChE (hAChE) that were reversible by oxime antidotes. To characterize the dynamics of these OP‐induced structural changes we have used time‐resolved SAXS (TR‐SAXS) and inelastic neutron scattering (INS). Stopped‐flow TR‐SAXS experiments were performed at BL‐4 at the Stanford Synchrotron Radiation Lightsource by mixing a molar excess of POX (500 μM) with hAChE (50 μM). Scattering data were collected every 20 msec during the first 1000 msec after mixing. Changes in the solution structure of hAChE during this period occurred with kobs= 0.13(±0.01) s−1. This rate of structural change is comparable to the rate of hAChE inactivation observed using biochemical assays. INS data were collected at the Cold Neutron Chopper Spectrometer at the Spallation Neutron Source at Oakridge National Laboratory using 100 mg samples of hAChE and POX‐hAChE that were lyophilized and rehydrated in D2O. INS data collected at 120 K (to prevent translational motions) were used to compare the vibrational density states of native and POX‐conjugated hAChE. The POX‐conjugated hAChE exhibited an increased number of vibrational states, particularly those associated with regional motions such as loop dynamics, whereas global collective motions remained unchanged. Molecular dynamics simulations were used to simulate the vibrational spectra and identify the region(s) in hAChE that contributed to the differences in the INS experimental data. In aggregate, these studies provide experimental evidence for OP conjugate‐induced changes in hAChE dynamics that has to be considered in the design of antidotes for OP poisoning that have improved efficacy and patient outcomes.

Support or Funding Information

This work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS, and by grants to SSRL and ORNL from the U.S. Department of Energy, and the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Genomics and Gene Annotation of a Region of Drosophila eugracilis Chromosome 4
Authors: Gayle Ashley , Van Stry Melanie
Published: June 2021

The common fruit fly, Drosophilia melanogaster, has around 1,300 genes in its genome. Chromosome 4 is 3.5% of its genome and contains approximately 80 genes within large heterochromatic domains. Interestingly, these genes are expressed at similar levels to genes found in euchromatin. The goal of the Genomics Education Partnership (GEP) project is to understand the gene expression within this heterochromatic environment. For the GEP project, we are identifying conserved DNA regulatory sequences around transcription start sites using the fourth chromosome from other species of Drosophila and D. melanogaster. I annotated genes located within a 35,000 base‐pair section of Drosophilia eugracilis chromosome 4 using BLASTX, Gene predictors, and RNA‐seq evidence within the UCSC Genome Browser. I annotated coding exons and splice sites for 2 genes with 3 isoforms each, Cubitis interruptus (Ci) and Ribosomal protein S3A (RpS3A) and the transcription start sites for each gene. GEP students are annotating many megabases of DNA and their pooled results are furthering an understanding of heterochromatic gene structure and regulation.

Support or Funding Information

Supported by NSF IUSE #1431407 and NIH R25GM119157 to Sarah C.R. Elgin (GEP) and NSF HRD#1623340 to MVS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Annotation of Genes on Contig 31 of the Drosophila eugracilis Chromosome 4
Authors: Hobson Joanna , Van Stry Melanie
Published: June 2021

Drosophila melanogaster has a small fourth chromosome, the Muller F element (3.5% of the genome), that has very large heterochromatin domains which are rich in HP1. Our goal for this project is to understand the mechanisms of gene expression in this heterochromatic environment. In the Genomics Education Partnership Project, we are using comparative genomics to identify conserved DNA regulatory sequences, by comparing the D. melanogaster Muller F element to those of other fly species that diverged from D. melanogaster ~10 million years ago, aiming to find regions of homology surrounding the transcription start sites. I have annotated the genes on a 40,000 base‐pair piece from the Drosophila eugracilis chromosome four. Sequence comparisons using BLASTX, gene predictors like Gene Scans, and RNA sequence data were used to annotate coding genes in this region. I identified two genes: myo (3 isoforms) and ey (4 isoforms) and annotated protein coding exons and splice sites for each isoform. The myo amino acid sequence is identical for each isoform in D. eugracilis, with 70.3% amino acid identity compared to the D. melanogaster myo protein. The D. eugracilis ey isoforms are also similar to the D. melanogaster ey protein sequences, with a range from 73% to 78% identity to their corresponding D. melanogaster isoform. Putative Transcription Start Sites for each gene were investigated, including a search for known transcription factor binding sites. By pooling results, GEP students are annotating several megabases of DNA, and contributing to an understanding of heterochromatic genes.

Support or Funding Information

Supported by NSF IUSE #1431407 and NIH R25GM119157 to Sarah C.R. Elgin (GEP) and NSF HRD#1623340 to MVS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PY489‐β‐Catenin Phosphorylation Involved in the Intestinal Regeneration of the Sea Cucumber H. glaberrima
Authors: Torres‐Gutierrez Vanessa , Bello Samir A. , García‐Arrarás Jose E.
Published: June 2021

Organ regeneration is a rare phenomenon in higher vertebrates. However, members of closely related groups such as echinoderms have striking regeneration capacities. In studies of intestinal regeneration using the sea cucumber Holothuria glaberrima, recent results have shown the involvement of the Wnt signaling pathway. One important component in this intracellular cascade is β‐catenin, that, depending on its phosphorylation state, activates transcription of Wnt target genes. In one case, phosphorylation of the tyrosine residue 489 (Y489) increases transcriptional activity of the β‐catenin/Tcf complex. To determine if Y489‐β‐catenin phosphorylation is involved in intestinal regeneration, normal and regenerating intestines of H. glaberrima specimens were dissected at different post‐evisceration stages. Tissues were processed for immunohistochemistry using anti‐PY489‐β‐catenin antibody and assessed by counting the temporal and spatial appearance of labeled cells in the distal, medial and proximal ends of the mesentery and gut rudiment. In normal non‐eviscerated animals, the antibody labeled about the 14% of cells in the coelomic lining and 31.2% of cells in the luminal epithelium. In regenerating specimens, PY489‐β‐catenin was expressed at all stages of regeneration from 3‐dpe up to 14‐dpe, with a peak increment of 40.6% of cells labeled in the mesothelium of the 7‐dpe intestinal rudiment. To determine the role of activating or inhibiting the WNT/β‐catenin pathway on the expression of the PY489 labeling, we treated tissue explants with the Wnt pathway activator LiCl and inhibitor iCRT14. When compared to controls there was no significant difference in the number of PY489‐β‐catenin immunoreactive cells observed in the LiCl‐treated explants, while there was a significant increase in immunoreactive cells in the iCRT14‐treated explants. Our results suggest that the Wnt/β‐catenin signaling is activated during intestinal regeneration in H. glaberrima and that phosphorylation in the Y489 residue of β‐catenin is important for the Wnt role in intestinal regeneration. In particular, the results suggest a possible role for Wnt pathway in the dedifferentiation status of the mesothelium once the initial intestinal blastema is formed. Our study might provide the basis for the role of the Wnt signaling pathway during organogenesis in holothurians and thus provide a better understanding of general regenerative processes.

Support or Funding Information

Funded by NIH R15NS081686, NSF IOS‐1252679 and the University of Puerto Rico.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Role of Myc Transcript as Active Player in the Canonical Wnt Pathway During Intestinal Regeneration
Authors: Alicea‐Delgado Miosotis , Garcia Arraras Jose E.
Published: June 2021

Molecular approaches have been widely used to explore the role of genes associated with regenerative processes in different animal models. Our own studies in the sea cucumber Holothuria glaberrima have identified several genes that appear to play a role in the regeneration of the digestive system. We have focused on two of these genes, Wnt and Myc, whose expression has been localized to the intestinal rudiment during regeneration. Myc and Wnt are interrelated, as the former is known to serve as an active player in the canonical Wnt signaling pathway. We now have quantified the levels of myc transcript at different stages of regeneration. In addition, we designed Dicer‐substrate short interfering RNAs (DsiRNAs)‐targeting Myc transcripts. DsiRNAs‐targeting Myc are electroporated in regenerating intestinal tissue explants of H. glaberrima to evaluate their effects on cell proliferation and cell dedifferentiation. Quantification of myc transcripts shows a decrease following electroporation of specific DsiRNAs when compared to explants electroporated with control DsiRNAs. Moreover, a decrease in cell proliferation is observed following the reduction in myc transcript, suggesting that Myc has a functional role on cell proliferation during the intestinal regeneration process. Instead, cell dedifferentiation does not change with the interference of the expression of Myc, suggesting that Myc does not play an important functional role on cell dedifferentiation during the intestinal regeneration process. In summary, this project not only presents data on Wnt‐Myc interactions, but presents three important technical advances: (1) the use of intestinal tissue explants, (2) the implementation of the electroporation as a transfection method and (3) the use of DsiRNAs to manipulate the gene expression in H. glaberrima explants.

Support or Funding Information

Funded by NIH R15GM124595, NSF IOS‐1252679 and the University of Puerto Rico

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution of TLR Signaling: A Sea Anemone Model has a Single Toll‐like Receptor with Roles in Pathogen Detection, NF‐κB Signal Transduction, and Development
Authors: Messerschmidt Jonathan L. , Brennan Joseph J. , Gilmore Thomas D.
Published: June 2021

The innate immune system is the most primitive branch of the immune system and is responsible for recognizing pathogens and eliciting responses such as inflammation and antimicrobial peptide production in many organisms. Toll‐like receptors (TLRs) are transmembrane receptors that have been characterized in organisms from insects to humans. TLRs detect molecules expressed by microbial pathogens, and then activate the NF‐κB transcription factor signaling pathway to turn on the expression of innate immune effector genes. Recent genome and transcriptome sequencing efforts have led to the identification of TLR‐like genes in organisms more basal to insects, but the biological roles of these basal TLRs are not well understood. In this project, we are characterizing a TLR‐like protein in the sea anemone Nematostella vectensis (Nv), a model organism for the phylum Cnidaria. The single Nv‐TLR harbors leucine‐rich extracellular, transmembrane, and intracellular TIR domains, consistent with TLR structures in higher organisms, including humans. In cell culture‐based reporter assays, we show that Nv‐TLR can activate the human NF‐κB signaling pathway and this pathway activation can be stimulated by the biologically relevant bacterium Vibrio coralliilyticus and purified flagellin from Salmonella typhimurium. In biochemical studies, the intracellular TIR domain of Nv‐TLR can bind to human NF‐κB signal transduction adapter proteins MAL and MyD88, and the extracellular domain of Nv‐TLR can directly bind to flagellin. In anemones, Nv‐TLR and Nv‐NF‐κB (and most intermediate TLR to NF‐κB signaling proteins) are expressed in cnidocytes, both in the body column and in a Nematostella‐specific circulating organ called the nematosome. Nv‐TLR is also required for embryonic development in N. vectensis. Ongoing studies are characterizing the biochemical aspects of the flagellin‐TLR interaction. Taken together, these studies suggest that a TLR‐to‐NF‐κB pathway is functionally conserved from cnidarians to humans. Moreover, these studies provide insight into the evolutionary basis of vertebrate innate immunity, and shed light on how simple organisms respond at the molecular level to environmental and biological stressors.

Support or Funding Information

Undergraduate Research Opportunities Program (Boston University), National Science Foundation, Arnold and Mabel Beckman Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MK‐STYX‐Induced PC‐12 Neurites are Dopaminergeric
Authors: Swack Ben D. , Banks Dallas , Dahal Arya
Published: June 2021

MK‐STYX [MAPK (mitogen‐activated protein kinase) phosphoserine/threonine/tyrosine‐binding protein] is a pseudophosphatase. There is compelling evidence that this unique catalytically inactive member of the protein tyrosine phosphatase family is a regulator in neuronal processes such as neurite outgrowth. We previously reported that MK‐STYX dramatically increases the number of primary neurites in rat pheochromocytoma (PC‐12) cells through the RhoA signaling pathway. Transmission electron microscopy data demonstrate that MK‐STYX‐ induced neurites form synapse‐like structures. PC‐12 cells have been shown to communicate through synaptic release of dopamine and norepinephrine. Dopamine release is modulated by phosphatases IA‐2 (islet antigen 2) and calcineurin. Because MK‐STYX is an atypical phosphatase, we wanted to determine if neurites induced in the presence of MK‐STYX retain their dopaminergic qualities. In this report, we show that this is the case. Using an ELISA assay for dopamine, we found that PC‐12 cells expressing MX‐STYX had similar dopamine concentrations as control cells, suggesting the synapses are dopaminergic in both conditions. These data provide evidence that MK‐STYX has a role in neuronal signaling.

Support or Funding Information

This work was supported by the National Science Foundation Grant MCB1113167 to S.D.H.; Coco Award to S.D.H.; Howard Hughes Medical Research Institute grant through the Undergraduate Science Education Program to the College of William and Mary; a DuPont Fund Student‐Faculty Research Team Stipend awarded to A.D. and S.D.H. through a grant by the Jessie Ball Grant Fund to the William and Mary Scholars for Undergraduate Research Experience program; Ferguson Awards to D.A.B., A.D., and C.S., and the Dintersmith Honors Fellowship to A.D.

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 TRPV4‐TAZ signaling axis in matrix stiffness‐ and TGFβ1‐induced epithelial‐mesenchymal transition
Authors: Rahaman Shaik O. , Sharma Shweta , Goswami Rishov
Published: June 2021

Epithelial to mesenchymal transition (EMT) is a critical event in development, tissue regeneration, fibrogenesis, and oncogenesis. Emerging data support a role for both a mechanical signal, and a biochemical signal, e.g., transforming growth factor β1 (TGFβ1) in EMT. However, the mechanisms by which these signals regulate EMT are not well understood. Here, we report evidence that transient receptor potential vanilloid 4 (TRPV4), a calcium‐permeable channel sensitive to both matrix stiffness and TGFβ1 stimuli, is a likely mediator of EMT. We found that antagonism of TRPV4 blocked both matrix stiffness‐ and TGFβ1‐induced EMT in normal human primary epidermal keratinocytes (NHEKs), as determined by changes in morphology and alterations in expression of EMT markers including E‐cadherin, N‐cadherin, and α‐smooth muscle actin (α‐SMA). In a murine skin fibrosis model, TRPV4 deletion resulted in decreased expression of the mesenchymal marker, α‐SMA, and increased expression of epithelial marker, E‐cadherin. Mechanistically, we found that: i) TRPV4 activity was essential for the nuclear translocation of TAZ (transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1; ii) Antagonism of TRPV4 activity inhibited both matrix stiffness‐induced and TGFβ1‐induced expression of TAZ proteins; and iii) TRPV4 antagonism suppressed both matrix stiffness‐induced and TGFβ1‐induced activation of Smad2/3, but not of AKT. Altogether, these data identify a novel role for TRPV4 in regulating EMT in response to both matrix stiffness and TGFβ1.

Support or Funding Information

AHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to SOR.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.