bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationResearch 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 InformationThe 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 InformationThis 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 InformationSupport by The University of Texas Rio Grande Valley School of MedicineThis 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 InformationThis 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 InformationSupported by the National Institutes of HealthThis 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 InformationThis 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.