bluuebunny/crossref_metadata_2025 huggingface dataset:
How do enzymes evolve?
Authors: Tawfik Dan S.
Published: June 2021

I will describe some insights regarding how new proteins functions evolve, via gradual, small changes in sequence. However, the mechanisms of such evolutionary micro‐transitions do not explain macro evolutionary transitions – how completely new proteins emerge, let alone how the very first enzyme(s) evolve. In the second part of my talk I will discuss our recent findings with respect to the de novo emergence of functional, globular proteins.

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 characterization and design of regulator RNAs using novel high‐throughput tools
Authors: Contreras Lydia M. , Haning Katie , Leistra Abigail N.
Published: June 2021

Regulatory RNAs enable bacteria to dynamically respond to stresses caused by changes in environmental conditions. Specifically, bacterial small RNAs, a class of RNA regulators, exert dynamic control on complex networks by regulating gene expression. Understanding their functions is a goal in both medicine and metabolic engineering given their relevance to pathogenesis and their potential to manage global regulatory networks that affect biological production of industrially‐relevant compounds. Given the importance of molecular structure to RNA functioning, fundamental sRNA characterization and applied engineering efforts depend heavily on the understanding and design of their specific shapes. Specifically, knowledge of the RNA structural landscape supports identification of interfaces relevant to regulation. In this talk, we will describe the development of a high throughput tool that allows for the simultaneous in vivo characterization of thousands of potential interacting interfaces in RNA molecules, as determined based on their molecular accessibility. We will describe how RNA structural insights obtained from this synthetic probing approach can be used in the functional characterization of newly discovered RNAs and in the rational design of bacterial sRNAs to achieve a tunable gradient of global control for metabolic engineering applications.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic functional assembly of the Torsin AAA+ ATPase and its modulation by LAP1: a novel mode of regulation for AAA+ ATPases
Authors: Chase A. R. , Laudermilch E. R. , Wang J. , Shigematsu H. , Yokoyama T. R. , Schlieker C.
Published: June 2021

TorsinA is an essential AAA+ ATPase requiring LAP1 or LULL1 as cofactors. The dynamics of the Torsin/cofactor system remain poorly understood, with previous models invoking Torsin/cofactor assemblies with fixed stoichiometries. Here we demonstrate that TorsinA assembles into homotypic oligomers in the presence of ATP. Torsin variants mutated at the “back” interface disrupt homo‐oligomerization but still show robust ATPase activity in the presence of its cofactors. These Torsin mutants are severely compromised in their ability to rescue nuclear envelope defects in Torsin‐deficient cells, suggesting that TorsinA homo‐oligomers play a key role in vivo. Engagement of the oligomer by LAP1 triggers ATP hydrolysis and rapid complex disassembly. Thus the Torsin complex is a highly dynamic assembly whose oligomeric state is tightly controlled by distinctively localized cellular cofactors. Our discovery that LAP1 serves as a modulator of the oligomeric state of an AAA+ protein establishes a novel means of regulating this important class of oligomeric ATPases. Notably, both the homo‐oligomerization and the ATPase activation are offset by a TorsinA mutation implicated in the movement disorder DYT1 dystonia, possibly contributing to the molecular etiology of this disease.

Support or Funding Information

Dystonia Medical FoundationNIH NIGMS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caspase‐6 Self‐Activation Enables Distinct Helix‐Strand Interconversion Upon Substrate Binding
Authors: MacPherson Derek J. , Dagbay Kevin B. , Hardy Jeanne A.
Published: June 2021

Caspases are cysteine proteases that are major players in key cellular processes, including apoptosis and inflammation. Amongst the human caspases, caspase‐6 has been implicated in playing a unique and critical role in the neurodegenerative pathways of Alzheimer's, Huntington's and Parkinson's disease. Unfortunately, structural similarities between caspase‐6 and other caspases have hampered precise targeting of caspase‐6 uniquely. All caspases can exist in a canonical conformation, in which the substrate binds atop a beta‐strand platform in the 130's region. This caspase‐6 region can also adopt a helical conformation that has not been seen in any other caspases. We have shown that caspase‐6 is inherently and dramatically more conformationally dynamic than closely related caspase‐7. In contrast to caspase‐7, which rests constitutively in the strand conformation before and after substrate binding, hydrogen/deuterium exchange data for the L2’ and 130's regions suggested that prior to substrate binding, caspase‐6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase‐6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu‐135, which showed a noticeably different calculated pKas in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. We have also mapped the local changes in the conformational flexibility of procaspase‐6 at the discrete states that reflect series of cleavage events that ultimately lead to the fully active, substrate‐bound state. Intramolecular self‐cleavage at Asp‐193 evoked higher solvent exposure in the regions of the substrate binding loops L1, L3, L4 and in the 130's, the intersubunit linker region, and the 26–32 region, as well as stabilized loop 2. Further removal of the linker allowed caspase‐6 to gain more flexibility in the 130's region and in the L2 region converting caspase‐6 to a competent substrate‐binding state. The prodomain region was found to be intrinsically disordered independent of the activation step of caspase‐6; however, its complete removal resulted in the protection of the adjacent 26–32 region, suggesting that this region may play a regulatory role, likely as an exosite for substrate recognition. The molecular details of caspase‐6 dynamics in solution provide a comprehensive scaffold for strategic design of therapeutic approaches for neurodegenerative disorders.

Support or Funding Information

This work was supported by the National Institutes of Health (NIH) GM 080532.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biological function of TIMP‐2 is regulated by the composition of the tissue microenvironment
Authors: peeney david , Stetler‐Stevenson William
Published: June 2021

The activity of mammalian matrix metalloproteinases (MMPs) is regulated by a family of four endogenous inhibitors known as the tissue inhibitor of metalloproteinases (TIMPs). Since their original discovery and characterization several additional physiological functions have been ascribed to TIMP family members, including MMP‐independent biological functions, such as regulation of cell growth and migration. TIMP‐2 is abundantly expressed in normal tissues with low levels of MMP expression and demonstrates broad spectrum inhibition of MMPs, binding to and inhibiting all 23 members of this family and ADAM12 with varying affinities. In addition, TIMP‐2 has been shown to interact with several membrane proteins including MMP14, insulin‐like growth factor‐1 receptor (IGF‐I‐R), low density lipoprotein receptor‐related protein 1 (LRP1) and alpha3 beta1 integrin (a3b1). Recent studies in our lab have shown that TIMP‐2 is a promising candidate for biological cancer therapy. However, the role of TIMP‐2 in tumorigenesis has been subject to debate with varying accounts purporting both anti‐ and pro‐tumor effects. In addition, TIMP‐2 and its putative receptors have been shown to exhibit multiple interactions with components of the tissue microenvironment. Using various cell‐lines we demonstrate that TIMP‐2 cellular binding, signaling and uptake in tumor cells can be differentially regulated by components of the tissue microenvironment resulting in opposing effects on cell behavior, such as intracellular kinase activity in response to growth factors. Our goal is to understand the regulatory factors/conditions that influence the different cellular responses to TIMP‐2 in order to effectively target the aberrant extracellular environment that is associated with many human diseases.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Visualizing translation by ensemble cryo‐EM
Authors: Korostelev Andrei
Published: June 2021

Translation of mRNA is a key step of gene expression regulation and a therapeutic target. Translation is governed by ribosome dynamics. To visualize the translational machinery in action, we determine high‐resolution ensembles of structures by cryo‐EM. I will discuss how this approach provides insights into the essential aspects of gene expression, such as translation initiation, translation fidelity and ribosome‐induced stress signaling.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Approaches for Localization of Proteins and Interaction Partners in Cells with High Spatial Resolution
Authors: Carr Steven A. , Udeshi Namrata , Myers Samuel
Published: June 2021

Labeling of subproteomes within the living cell using genetically‐encoded peroxidase enzyme (APEX; developed in the lab of Alice Ting, Stanford) together with quantitative liquid chromatography‐mass spectrometry is new technology that targets, labels enriches and identifies proteins in subcellular region of interest. In our initial application to the mitochondrial matrix we demonstrated the high specificity of live‐cell peroxidase‐mediated proteomic mapping and the utility for finding novel, previously unknown mitomatrix proteins (Rhee Science 2013). More recently we have extended this powerful new approach to define the molecular composition of unbounded compartments in living cells (Hung Mol Cell 2014; Hung Nature Protocols 2016). Here we illustrate the application of the technology, and recent advances, to define the proteomes of additional sub‐compartments such as excitatory and inhibitory synapses of living neurons. A limitation of the approach has been that few biotinylated sites are identified, reducing the ability to directly obtain information on protein orientation and topology as this requires identifying the sites of biotinylation and not just the proteins that have been modified. This is a known issue with the use of Streptavidin for protein enrichment. We have now developed a new workflow employing anti‐biotin antibodies that increases the numbers of identified sites by >30‐fold and that has manifold potential for studies of biotinylated peptides in a number of other posttranslational modification contexts (Udeshi Nature Methods 2017). Finally, we will describe new work combining recent advances in proximity labeling, genome targeting, and quantitative mass spectrometry to develop genomic locus proteomics (GloPro), a method able to identify proteins that occupy specific genomic loci.

Support or Funding Information

This work is funded by NCI CPTAC PCC 1U24CA210986‐01 to S.A.C. and a Broad Institute SPARC grant to S.M. Portions of this work were also supported by an HHMI Collaborative Innovation Awardsto S.A.C. (Norbert Perrimon PI).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing membrane‐mediated forces between proteins in cells using super‐resolution fluorescence localization imaging.
Authors: Veatch Sarah L. , Shelby Sarah A.
Published: June 2021

Transmembrane and peripheral membrane proteins can interact directly via mechanisms common to soluble proteins, but also can feel forces mediated through their embedding membrane through mechanisms involving membrane curvature, electrostatics, as well as their preference for phase‐like membrane domains. In this presentation, I will discuss our recent efforts to probe effective interactions between plasma membrane proteins using super‐resolution fluorescence localization microscopy paired with a quantitative cross‐correlation analysis in both live and chemically fixed cells. In particular, we find that, in a B cell line, ordered phase‐like domains can sort plasma membrane kinases and phosphatases, establishing localized hotspots for tyrosine phosphorylation. We also use peptide probes that contain membrane proximal charged residues to explore the roles of membrane electrostatics in modulating the lateral distribution of proteins on the cell surface. Overall, we argue that cells use these interaction modalities that are unique to membranes in order to influence the biochemical networks that occur on the cell surface in ways that contribute a broad array of cellular sensing and signaling cascades.

Support or Funding Information

Research was funded by the National Institutes of Health (NIH) (R01 GM110052) and the the National Science Foundation (NSF) (MCB 1552439).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relevance of Keratin Alterations and Desmoglein 3 Internalization in the Autoimmune Skin Disease Pemphigus Vulgaris
Authors: Schlögl Elisabeth , Radeva Mariya Y. , Vielmuth Franziska , Schinner Camilla , Waschke Jens , Spindler Volker
Published: June 2021

Pemphigus vulgaris (PV) is a potentially lethal autoimmune disease characterized by blister formation of the skin and mucous membranes and is caused by autoantibodies against desmoglein (Dsg) 1 and Dsg3. Dsg1 and Dsg3 are linked to keratin filaments in desmosomes, adhering junctions abundant in tissues exposed to high levels of mechanical stress. The binding of the autoantibodies leads to internalization of Dsg3 and a collapse of the keratin cytoskeleton – yet, the relevance and interdependence of these changes for loss of cell‐cell adhesion and blistering is poorly understood. In live‐cell imaging studies, loss of the keratin network at the cell periphery was detectable starting after 60min of incubation with immunoglobulin G fractions of PV patients (PV‐IgG). These rapid changes correlated with loss of cell‐cell adhesion detected by dispase‐based dissociation assays and were followed by a condensation of keratin filaments into thick bundles after several hours. Dsg3 internalization started at 90min of PV‐IgG treatment, thus following the early keratin changes. By inhibiting casein kinase 1 (CK1), we provoked keratin alterations resembling the effects of PV‐IgG. Although CK1‐induced loss of peripheral keratin network correlated with loss of cell cohesion and Dsg3 clustering in the membrane, it was not sufficient to trigger the internalization of Dsg3. However, additional incubation with PV‐IgG was effective to promote Dsg3 loss at the membrane, indicating that Dsg3 internalization is independent from keratin alterations. Vice versa, inhibiting Dsg3 internalization did not prevent PV‐IgG‐induced keratin retraction and only partially rescued cell cohesion. Together, keratin changes appear very early after autoantibody binding and temporally overlap with loss of cell cohesion, suggesting a crucial role for initial loss of cell cohesion in PV.

Support or Funding Information

The study was supported by grants of the Deutsche Forschungsgemeinschaft SP1300/1‐3 and SP1300/3‐1 to VS as well as the Föfole program of the LMU.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prolonged Activation of STAT3 Mediates the IL6‐Induced Loss of Stress Fibers and Increase in Endothelial Permeability
Authors: Alsaffar Hiba , Martino Nina , Leyden Kara , Adam Alejandro
Published: June 2021

Interleukin 6 (IL6) promotes endothelial barrier breakdown in vitro, as well as, in vivo. However, the signaling mechanisms that mediate this response are not fully understood. IL6 trans‐signaling promotes a prolonged activation of JAK/STAT3 in human umbilical vein endothelial cells (HUVECs) that lasts for at least 24 hours, leading to a sustained increase in endothelial permeability. IL6‐induced STAT3 phosphorylation and loss of barrier function is prevented when cells are pre‐treated with the JAK inhibitor, ruxolitinib, suggesting that JAK activity is required for these responses. Furthermore, siRNA‐mediated STAT3 knockdown attenuates the loss of IL6‐induced loss of barrier function, demonstrating a critical role for STAT3 in this response. Immunofluorescence analysis showed that this change in barrier function correlates with a marked loss of junctional ZO‐1 (but not changes in ZO‐1 protein expression). Phalloidin staining demonstrates that IL‐6 also promotes a JAK, and STAT3, dependent loss of actin stress fibers and a marked reduction of the cortical actin signal. A prolonged activation of STAT3 is required for this response, because treatment of HUVECs with ruxolitinib up to 4 hours after activation of IL6 trans‐signaling, completely reverses the IL6‐induced loss of barrier function. STAT3 activation kinetics are regulated by a negative feedback loop that is mediated by the suppressor of cytokine signaling 3 (SOCS3). Consistently, siRNA‐mediated SOCS3 knockdown increases IL6‐induced STAT3 phosphorylation in HUVECs and exacerbates the IL6‐induced increase in permeability. Collectively, our data shows that prolonged activation of STAT3 is required for the IL6‐induced barrier breakdown, and loss of junctional ZO‐1, cortical actin and actin stress fibers in endothelial cells.

Support or Funding Information

This project was supported by an American Heart Association Science Development Grant 13SDG17100110 and funds from the Department of Molecular and Cellular Physiology, Albany Medical Center.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Embracing Active Learning: A Novel Approach to Teaching Embryology at the Larner College of Medicine
Authors: McCarthy Sarah A. , Holterman Leigh Ann
Published: June 2021

The Larner College of Medicine at the University of Vermont is in the process of transitioning to an entirely active learning‐based curriculum by 2019. With this goal in mind, we flipped all of the embryology lectures given in our Foundations of Clinical Science course to large group workshops. We replaced 12 hours of lecture material with 8 self‐directed learning modules and 5 large group workshops. After each learning module, students were given a multiple‐choice quiz. During the workshops, students were asked to develop a linear timeline of embryological development and work through clinical questions related to each topic. We hypothesized that these active learning sessions would assist students in better understanding the main concepts of embryology.

Student groups (n=17) each developed a timeline based on embryological development in a OneNote notebook shared by the entire class. Each group's timeline was visible to other groups and faculty. A master timeline was then assembled using parts from each group's timeline and then groups worked together to answer clinically‐relevant questions. Examination scores and end‐of‐course evaluations between 2016, when only traditional lectures were given, and 2017, with the new active learning curriculum, were compared. There was no difference in overall examination scores between 2016 and 2017. While the sessions were well‐reviewed by the students, they would have preferred to have more time during the session to compile the timeline. The students reported that the clinical questions were helpful in integrating embryology. To our knowledge, this is an innovative approach to teaching embryology as well as utilizing OneNote within the classroom. In the future, students will complete a version of their own timeline before each session to better utilize class time. In conclusion, this type of workshop is an excellent approach to teaching a difficult topic and the development of a timeline allows for a better overall view of human development.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Teaching Clinical Anatomy without Lectures in an Integrated Medical School Curriculum
Authors: Spencer Marc R. , DeSantis Mark E. , Stadler Justin R. , Woodcock Jade , Saltarelli William
Published: June 2021

With the wholesale push to move medical curricula toward integration and organ system‐based courses, many basic medical science disciplines have had to compensate for loss of didactic time. Anatomy has perhaps seen the most reduction overall due to the extensive hours used in traditional curricula dedicated to lecture and dissection labs. The lab portion of the anatomy curriculum has borne most of the reduction. Various solutions have been implemented to deal with this reduction (e.g., prosection). Here, we describe an approach whereby the majority of anatomy content is taught and assessed in lab rather than in lectures. We use a modified jigsaw approach within the lab to maximize the amount of content delivered in the curricular time allotted for anatomy. Each lab is split into small groups with different sets of learning objectives that fall under the larger session objectives. Instructors facilitate each group's topic during one hour of the lab session (learning hour). Each group must assemble a presentation prior to the next lab session. The presentation is no more than 10 minutes long while delivering the bulk of the learning objectives. During the first hour of the next lab session (teaching hour), each group member is responsible for teaching a subset of their peers for 10 minutes. After that, group members and peer learners rotate stations, so each member of a group spends 10 minutes teaching their peers, and then rotates to different stations learning from their peers over the remaining course of the teaching hour. The learning objectives are crafted to facilitate teaching via clinical applications. For example, a group responsible for the supraclavicular brachial plexus may teach some of the anatomical structures by describing the deficits noted in Erb's palsy. Following the teaching hour, we provide a brief review session with clicker‐style questions. At the end of the week, the course has formative quizzes that include one anatomy‐based clinical vignette question per group. We assess students via a traditional lab practical with 30 pinned and 15 follow‐up questions—follow‐ups are typically more clinically oriented than the pinned questions. The method outlined here has shown impressive results relative to the other techniques used in the curriculum (e.g., case‐, team‐based learning). Indeed, among practice tests (i.e., National Board of Medical Examiners' Comprehensive Basic Science Examination) and on the United States Medical Licensing Examination Step 1 examination, our students have performed better in anatomy than in any other basic science discipline. Despite the inevitable reduction in time for medical anatomy within integrated curricula, these results suggest that this method is a viable alternative focused on the lab‐based science of clinical anatomy.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“For of those to whom much is given…”
Authors: Laitman Jeffrey T.
Published: June 2021

On January 9, 1961, some weeks before being inaugurated the 35th President of the United States, John F. Kennedy exhorted the Massachusetts State Legislature, “For of those to whom much is given, much is required.” His words, internationally distributed, gave new platform and audience to the Biblical message from Luke (KJV, 12:48) from whence it came. Kennedy would often return to versions of this ancient missive in speeches during his all‐too‐brief time as President.

This historical admonition speaks not only to those benefited by material wealth or comfort; he was pointedly addressing those placed at the nexus of sacred opportunities and those they would influence. Indeed, a theme throughout Kennedy's life was his passionate transmission of the existential need for giving and giving back. The most well known of his quotes, forcefully spoken through the cold January air at his inauguration,“…ask not what your country can do for you, but what you can do for your country,” epitomizes his world‐view of the centrality of service to your home and the greater good.

We in the academic world, particularly those of us given the gift of sharing the core science of anatomy sensu lato, have much: we have learned from caring teachers who stimulated our careers; have been afforded great freedom to explore science without many limitations; have unfettered openness through our journals and forums; and have been allowed to share our thoughts with bright, eager, and pure minds at the outset of their journey. The pressures of our daily world ‐ grants, curricular changes, a seemingly never‐ending ladder of administrators – can at times jade our spirit and dull our senses to the dual obligations we hold: to serve our science and mentor those who have knocked at our door. To paraphrase President Kennedy, perhaps we should focus a bit more on what we can do for the future of our discipline and those who will carry the torch then sit in expectation or disappointment of what we should have received. We have, indeed, been given much; good service to our next generation is a blessing in return, not a burden.

(American Association of Anatomists A.J. Ladman Award Symposium Lecture).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vision Cycle Proteins: Their Function, Structure and Links to Retinal Disease
Authors: Tsin Andrew
Published: June 2021

There are many structural and functional disorders of the eye. The human eye demands a large percentage of the metabolic needs of the body compared to other tissues. Inherent in this demand is the function of the retina and its conversion of light to electrical impulses which are partially processed in the retina and then sent on to the brain where they are interpreted as a visual image. The conversion of light to electrical impulses has been heavily studied, but many mysteries remain in the explanation of the structure of molecules involved and their functional role in retinal disease.

In this mini‐review, the first goal is to provide a current understanding of the biochemical and molecular events of vision cycle molecules in normal function and disease. Selected enzymes and binding proteins will be highlighted for their roles in the visual cycle. Genetic mutations and malfunctions of these proteins provide etiological evidence for many ocular diseases from anomalies of retinoid (vitamin A) metabolism and mutation of visual cycle proteins. Genetic retinal diseases such as retinitis pigmentosa, and Lebers congenital amaurosis are linked to changes in the structure of the molecules involved in the vision cycle. Further explanation will be given on how vision cycle proteins and their derivatives play a role in age‐related macular degeneration (AMD). AMD is the leading cause of blindness in individuals 50 years and older. Recently vision cycle proteins have been found to play a role in diabetic eye disease.

While cures for these maladies remains elusive progress has been made on many of these fronts. Basic science has offered the ground work for potential strategies in curing many of these blinding afflictions, but much work remains. Some of these strategies have advanced to the stage of clinical trials, but many are not yet at that stage, and still more are only ideas yet to be elucidated in the laboratory. Some examples of these will be discussed. Recent and future understanding of the vision cycle will afford novel intervention strategies to preserve human vision and to treat blindness.

Support or Funding Information

Support by The University of Texas Rio Grande Valley School of Medicine

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Super‐resolution imaging of transcription in live mammalian cells
Authors: Cissé Ibrahim I.
Published: June 2021

Gene activation is thought to involve a multistep process whereby transcription factors bind to distal enhancer sites, recruit the Mediator complex which contacts the pre‐initiating RNA Polymerase II (Pol II) complex assembled at the start site of the gene. The interaction of Mediator and Pol II has yet to be observed in the nucleus of living cells and the dynamics of this interaction are not yet elucidated. Here we use quantitative live cell super‐resolution and light sheet imaging to study the organization and dynamics of endogenous Mediator and Pol II directly in living mouse embryonic stem cells. In addition to forming transient clusters with average lifetimes of 11.1 (± 0.9) s, and 12.1 (± 1.4) s respectively, Mediator and Pol II also form previously uncharacterized large and stable clusters in stem cells (~15 stable clusters per cell). The large and stable Mediator and Pol II clusters gradually disappear within hours after induction of stem cell differentiation. Stable Mediator and Pol II clusters co‐localize with each other, as well as with pluripotency factors. Inhibition of Brd4 bromodomains necessary for enhancer association eliminates both Mediator and Pol II stable clusters, and inhibition of transcription elongation selectively eliminates stable Pol II but not stable Mediator clusters. Tracking of Mediator and Pol II stable clusters suggests they are chromatin associated and they coalesce upon contact, a property associated with phase separated droplets. We conclude that Mediator and Pol II associate in diffraction‐sized condensates with a define lifetime dependent on active transcription in living stem cells.

Support or Funding Information

This project is supported by the NIH Director's New Innovator award #DP2CA195769 to I.I.C. and The Pew Charitable Trusts through the PEW Biomedical Scholars grant to I.I.C.

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 dynamic epitranscriptome: Control of mRNA fate and function by nucleotide modifications
Authors: Jaffrey Samie
Published: June 2021

The fate and function of each mRNA is determined by both its nucleotide sequence, as well as by its “epitranscriptomic code.” This code refers to the presence and distribution of methylation marks in the transcript. The discovery of the epitranscriptomic code was made possible by our initial development of MeRIP‐Seq, a method for mapping N6‐methyladenosine (m6A) throughout the transcriptome. MeRIP‐Seq revealed that m6A is found in at least 25% of mRNAs in the transcriptome, differentially expressed in development and disease, and enriched in specific regions of the transcript body. The second most prevalent modified nucleotide in mRNA is N6,2′‐O‐dimethyladenosine (m6Am), which is exclusively located adjacent to the 7‐methylguanosine‐cap at the first encoded nucleotide in up to 40% of mRNAs. This modification is formed when adenosine residues at the first encoded nucleotide position in mRNAs are constitutively modified with a single methyl modification to form 2′‐O‐methyladenosine, Am. Am may then be subjected to an additional methylation to form m6Am. Although initial mapping technologies could not discriminate between m6A and m6Am, we developed miCLIP, a single‐nucleotide resolution mapping technique that definitively discriminates between these highly abundant modifications. We find m6A and m6A have different effects on mRNA. m6A has diverse effects on transcripts, and when found in the 5′ untranslated region, can divert transcripts into a new form of translation that bypasses normal cap‐binding proteins. This form of m6A‐regulated translation appears to promote translation of specific mRNAs, especially in disease‐states associated with inhibition of cap‐binding proteins. In addition, we find that m6A influences the subcellular localization of transcripts in normal and disease cells. In contrast, we find that m6Am primarily enhances mRNA stability and translation. m6Am‐initiated transcripts are markedly more stable due to resistance to the mRNA‐decapping enzyme DCP2. m6Am promotes translation via recruitment of specific m6Am‐binding proteins leading to a unique form of translation initiation, that like m6A, does not require conventional cap‐binding proteins. Notably, m6Am can be demethylated to Am by FTO (fat mass and obesity‐associated protein). FTO was previously thought to demethylate m6A; however, FTO shows a markedly higher activity towards m6Am compared to m6A in vitro and no detectable demethylation of m6A in vivo. FTO demethylates m6Am to Am, and thereby switches off the stability of these transcripts. Overall, our findings reveal that epitranscriptomic information is stored in both internal nucleotides and mRNAs caps, with the “cap epitranscriptome” defining a novel reversible form of methylation that influences a large subset of cellular transcripts.

Support or Funding Information

Supported by 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.
Proteasomal regulation of hormone signaling in Mycobacterium tuberculosis
Authors: Darwin K. Heran
Published: June 2021

Tuberculosis (TB) kills about 1.5 million people globally every year, making it the leading causes of death by an infectious agent. The lack of an effective TB vaccine or the development of immunity after TB infection suggests antimicrobial drugs may be our best chance for controlling this disease. The Mycobacterium tuberculosis (Mtb) proteasome is essential to cause lethal infections in animals and, therefore, it is currently being targeted for tuberculosis chemotherapy.

My lab is working to understand how a bacterial proteasome allows Mtb to persist in animals by characterizing substrates of the Mtb proteasome. Dozens if not hundreds of proteins that are degraded by the bacterial proteasome must first be post‐translationally modified with the small protein Pup (prokaryotic ubiquitin‐like protein). We reported the identification of a pupylated protein called Lonely Guy (Log) that catalyzes the synthesis of cytokinins, which have previously only been characterized as plant hormones. Log levels are maintained at very low levels in Mtb by the Pup‐proteasome system, suggesting the presence of cytokinins in Mtb requires tight regulation. In my talk, I will discuss exciting new data that addresses the function of Log and cytokinins in Mtb physiology.

Support or Funding Information

This work was supported by NIH grant R01HL092774 awarded to K.H.D. K.H.D. holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. Trainees were supported by NIH grant T32 AT007180.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A crystallographic view of the reaction cycle in iron(II) and 2‐(oxo)‐glutarate‐dependent oxygenases
Authors: Boal Amie K.
Published: June 2021

Iron(II)‐ and 2‐(oxo)‐glutarate‐ (Fe/2OG) dependent oxygenases are a large and functionally diverse family of oxidative tailoring enzymes, the majority of which are hydroxylases. Many other outcomes are possible, including halogenation, stereoinversion, cyclization, and desaturation reactions, but the structural adaptations that enable alternative transformations are not fully understood. All Fe/2OG enzymes, regardless of outcome, proceed through an Fe(IV)‐oxo intermediate that has been isolated in solution but has not been characterized crystallographically. We have determined high‐resolution x‐ray crystal structures of four distinct states in the reaction pathway of an Fe/2OG hydroxylase, including a previously unobserved intermediate. The study reveals key second‐sphere structural changes during the reaction cycle that explain known reactivity patterns in Fe/2OG enzymes and provide new opportunities for functional prediction and protein engineering in the super family.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tyrosine kinase in ulcerative colitis associated compromised microglial functions.
Authors: Kumar Narendra , Kumar Premranjan , Mishra Jayshree
Published: June 2021

Microglia plays an essential role in the maintenance of brain homeostasis and compromise in microglia functions are well documented to trigger neurodegenerative disorders including Alzheimer's disease. However, the role of gut immunity and intestinal inflammation in microglia function and neurodegeneration remains unclear. Previously, we have established that the loss of Jak3 induces predisposition of compromised intestinal barrier function mediated colitis. In the present study, we demonstrate that loss of Jak3 induces chronic inflammation associated with increased circulating pro‐inflammatory cytokines that led to increased neuroinflammation and microglia dysfunction. Our data showed that DSS induced mucosal inflammation was more severe in Jak3 global knockout (KO) and intestinal epithelial‐specific knockout (Int‐KO) mouse compared to corresponding wild type mouse. Further, loss of Jak3 increased TLR signaling and associated pro‐inflammatory cytokines expression and together, exacerbated neuroinflammation in the brain. The increase in neuro‐inflammation triggered dysfunctional microglia (Iba1) and reduced TREM2 expression in brain further increased the β‐Amyloid and pTau accumulation in DSS treated KO and Int‐KO mice compared to corresponding control wild type group. Taken together these results demonstrate the essential role of intestinal‐Jak3 in mucosal homeostasis and microglial dysfunction associated Alzheimer's disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the effect of the WNT/b‐catenin pathway on the ischemic blood‐brain barrier using induced pluripotent stem cells
Authors: Page Shyanne , Al‐Ahmad Abraham
Published: June 2021

The blood‐brain barrier (BBB) is a selectively permeable barrier made up of tight junctions formed by endothelial cells that separates the circulating blood from the brain extracellular fluid. The primary function of this barrier is to keep toxins, bacteria, viruses, and drugs from entering the brain. In the event of an ischemic stroke, the BBB becomes leaky due to the activation of hypoxia inducible factor 1‐alpha (HIF1‐a) and neurons become apoptotic. It is known that activation of the WNT pathway results in the production of B‐catenin, which has been shown to enhance HIF1‐a mediated transcription, which promotes the survival of cells and their adaptation to hypoxia. Currently, there are no drugs that have been formulated to restore the BBB function as well as neuron survival following ischemic stress. The purpose of our study was to determine the effect of small molecules on the BBB/WNT pathway during ischemia/re‐oxygenation. Brain microvascular endothelial cells (BMECs) derived from iPSCs and the immortalized human brain endothelial cell line, HCMEC/D3, were cultured and placed in ischemic stress for 6hrs, followed by a 24 hour period of re‐oxygenation. At the time of re‐oxygenation, cells were treated with small molecules CHIR99021, TWS (WNT activators), IWP‐4, IWR, and XAV (WNT inhibitors) in order to determine the effect of the WNT pathway on cell survival. Transendothelial Electrical Resistance (TEER) and permeability were performed to assess the effect that the WNT pathway has barrier integrity following re‐oxygenation. Further, MTT and ICC were performed to determine cell viability and tight junction expression, respectively. Preliminary results of this study have shown that inhibition of the WNT pathway during re‐oxygenation results in up‐regulation of tight junction proteins and an increase in cell viability as well as an increase in barrier function.

Support or Funding Information

Laura W. Bush Institute for Women's 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.
Platelet Activating Factor Receptor (PAFR) Regulates Colitis‐induced Pulmonary Inflammation
Authors: Liu Gang , Mateer Sean , Mathe Andrea , Goggins Bridie , Hsu Alan , Minahan Kyra , Bruce Jessica , Fricker Michael , Wark Peter , Hansbro Philip , Keely Simon
Published: June 2021

Inflammatory bowel disease (IBD) is a progressive disease that includes Crohn's diseases and ulcerative colitis. Over one third of IBD patients have secondary organ pathologies and pulmonary manifestations are common. The platelet activating factor receptor (PAFR) plays a critical role in regulating inflammation and it is expressed on epithelial cells and neutrophils in both colon and lung. Having identified increased expression of PAFR in both the gastrointestinal (GI) tract and lungs of mice with dextran sulfate sodium (DSS) colitis, we hypothesised that PAFR was a mediator of the pulmonary inflammation associated with colitis. We aimed to elucidate the role of PAFR in gut‐lung inflammatory cross‐talk using of the DSS‐induced experimental model of colitis (7 days). DSS mice were treated with PAFR antagonist CV6209 both intranasally and intravenously and lung, colon and blood were assessed for inflammatory cells and mediators by qPCR, immunoblot, immunohistochemistry and flow cytometry. We demonstrated that DSS‐induced colitis resulted in inflammation in mouse bronchoalveolar lavage fluid (BALF) and lungs, as well as increased PAFR protein levels in lungs and colons. Pulmonary neutrophils in DSS animals showed increased PAFR staining. Bacterial 16S mRNA expression were also increased in mouse lungs after 7 days DSS challenge. Both intravenous and intranasal inhibition of PAFR by CV6209 reduced colitis‐induced numbers of neutrophils, but not macrophages, in mouse lungs. Inhibition of PAFR also reduced levels of TNF and IL‐1b proteins and bacterial 16S expression in mouse lungs. Importantly, intravenous administration of CV6209 reduced colitis pathology, TNF and IL‐1b protein levels in mouse colons after 7 days DSS challenge. In the DSS model, increased PAFR protein expression was associated with inflammasome activation, characterized by increased NLRP3 and mature caspase‐1 proteins, however inhibition of PAFR by CV6209 reduced NLRP3 and caspase‐1 levels after DSS challenge in mouse lungs. In vitro, NLRP3, activated caspase‐1 and secreted IL‐1b protein levels were increased in human alveolar epithelial cell culture (A549) after 24h LPS stimulation, and this was inhibited by treatment with CV6209. These data suggest that PAFR regulates colitis‐induced lung inflammation by IL‐1b protein activation via the NLRP3 inflammasome signalling pathway. PAFR may act as an inflammasome‐activating pattern recognition receptor during mucosal inflammation thus is a potential therapeutic target for lung inflammation associated with colitis and bacteraemia.

Support or Funding Information

This study was supported by a grant from National Health Medical Research Council (NHMRC) Australia

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glucose limited enhance the cancer stem cell population through PKM2/AMPK‐dependent signaling
Authors: Yang Yi‐Chieh , Chien Ming‐Hsien , Hsiao Michael
Published: June 2021

Background

Solid tumor cells encounter stress from oxygen and glucose limitation as tumors continue to grow. This kind of metabolic stress in the solid tumor interiors affects cancer stem cell (CSC) formation. However, the mechanisms of how cancer stem cells are affected by glucose, a key nutrient in the microenvironment, exist many controversial reports and it still remain largely elusive.

Design

The enhancement of cancer stem cell population were detecting by the ability of sphere formation, percentage of CD133+ positive cells, and expression of cancer stem cell markers under glucose limitation condition. We investigated the detailed mechanism of nuclear translocation of PKM2 and AMP2 under glucose limited condition by Western blot and immunoprecipitation. We also assessed the effects of nuclear PKM2 in vitro and in vivo.

Results and Conclusion

In response to glucose limited, we found that nuclear translocation of the glycolic key enzyme, pyruvate kinase M2 (PKM2), helps cancer cells survive under metabolic stress. We demonstrate here that limited of glucose concentration stimulates AMPK activation, resulting in co‐translocation of AMPK and PKM2 through Ran‐mediated nuclear transport. Translocated PKM2 subsequently binds with OCT4 to promote expression of cancer stemness‐related genes. In this way, nuclear PKM2 is able to enrich a cancer stem cell population under metabolic stress. Nuclear PKM2 is also shown to promote cancer metastasis in an orthotopic xenograft model. In conclusion, our work not only reveals the critical role of nuclear PKM2 in CSC formation, but also identifies a detailed mechanism of PKM2 nuclear translocation, which cooperates with the cytosolic nutrient sensor AMPK, under a glucose limited environment. These details suggest the possibility of finding new targets for therapeutic intervention.

Support or Funding Information

Ministry of Science and Technology (ROC): MOST 105‐2320‐B‐038‐058‐MY3, MOST 106‐0210‐01‐15‐02

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