bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mesenteric Lymph Derived from Ischemia/Reperfusion Gut Promotes Neutrophil Activation and Lung Infiltration
Authors: Ma Yonggang , Zabell Taylor , Creasy Alexandra , Yang Xiaoyuan , Chatterjee Victor , Yuan Sarah Y. 
Published: April 2020

Intestinal ischemia/reperfusion (I/R) can induce systemic inflammation and remote organ injury, where cytokine production and bacteria translocation are traditionally thought to be the major underlying mechanisms. The objective of our study was to determine whether the lymphatic circulation participates in this process. We focused on mesenteric lymph duct, as it drains gut interstitial fluid, proteins, absorbed lipids, and immune cells into the blood circulation through subclavian vein; the lungs are the first organ directly exposed to lymph contents. In a mouse model of intestinal I/R, we found that I/R‐induced lung inflammation, as evidenced by neutrophil infiltration and myeloperoxidase production, was inhibited by mesenteric lymph duct ligation. We then compared the direct effects of lymph collected from pre‐I/R vs. post‐I/R mesenteric lymph duct on isolated neutrophils from normal animals. Flow cytometric analysis revealed that neutrophils treated with post‐I/R lymph exhibited increased surface expression of CD11b. Additionally, upregulated mRNA expression of neutrophil specific chemokines (e.g. Cxcl1, Cxcl2, and Cxcl5) and pro‐inflammatory markers (e.g. Il1b, Il6, and Tnfa) was detected in post‐I/R lymph‐treated neutrophils. These effects were not seen in neutrophils treated with pre‐I/R lymph. Furthermore, because intestinal I/R injury is known to be associated with gut barrier leakage and bacteria translocation into the blood, we thought to determine whether bacteria translocation occurred to the lymphatic circulation during I/R. However, PCR experiments confirmed that all lymph samples tested, regardless of I/R status, were free of bacterial 16s rRNA, excluding the direct involvement of gut bacteria in I/R lymph‐induced neutrophil activation. In conclusion, our study suggests that mesenteric lymph derived from I/R gut contributes to systemic and lung inflammation by activating neutrophils.Support or Funding InformationThe work is supported by the American Heart Association 15SDG22930009 (to YM) and by the National Institutes of Health HL070752 (to SYY).
An siRNA screen for endothelial PAR1‐specific deubiquitinases regulating p38 inflammatory signaling
Authors: Patwardhan Anand , Cheng Norton , Trejo JoAnn 
Published: April 2020

Endothelial dysfunction is a hallmark of vascular disease and induced by various inflammatory mediators which signal through GPCRs pathways that are poorly understood. Thrombin induces endothelial inflammatory responses through the activation of protease‐activated receptor 1 (PAR1), a GPCR. Previously, we showed that thrombin‐activated PAR1 is modified by ubiquitin and drives recruitment of transforming growth factor‐β–activated kinase‐1–binding protein 2 (TAB2), an adaptor protein that binds TAB1 to trigger p38‐dependent endothelial barrier disruption, a hallmark of inflammation. However, the regulatory processes that control PAR1‐deubiquitination and associated p38 inflammatory signaling are not known. To fill this gap in knowledge, we sought to identify PAR1‐specific deubiquitinases (DUBs) that removes K63‐linked ubiquitin by conducting an unbiased comprehensive genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells using multiple PAR1 ubiquitin‐dependent functional readouts. We hypothesize that a PAR1‐specific DUB is essential for terminating thrombin‐induced p38 inflammatory signaling and might be altered in endothelial dysfunction. In an initial screen, endothelial cells were transfected with siRNA pools targeting specific DUBs and PAR1‐ubiquitin dependent responses were assessed. We specifically examined PAR1 cell surface expression, controlled by basal PAR1 ubiquitination, and thrombin‐stimulated p38 activation and downstream inflammatory signaling, which is dependent on agonist‐triggered PAR1 ubiquitination. In preliminary studies have identified several candidates PAR1‐specific DUBs. Currently, we are now investigating the function of top candidate DUBs in regulating the status of PAR1 ubiquitination, cell surface expression and p38 inflammatory signaling. The molecular mechanism responsible for PAR1 deubiquination and relation to controlling the spatio‐temporal dynamics of pro‐inflammatory signaling will be further investigated. Taken together, we anticipate that these studies using a comprehensive siRNA library screening approach will reveal the identity of several DUBS that have important functions in regulating PAR1‐induced ubiquitin driven p38 MAPK endothelial inflammatory signaling.Support or Funding InformationAuthors acknowledge NIH/NIGMS funding support.
Investigating the pathobiology of vocal fold dehydration
Authors: Cox Abigail , Sivasankar Preeti M. 
Published: April 2020

Optimal hydration is thought to be necessary for maintaining healthy vocal folds. Whether in vivo systemic dehydration induces vocal fold dehydration is not fully understood. Furthermore, whether systemic rehydration of vocal folds following systemic dehydration has not been investigated. Due to the ubiquitous nature of water in the body, the complex physiological mechanisms in place to maintain body hydration, and the often insensitive markers of hydration status, we set out to design an animal model to study how systemic dehydration affects the vocal folds. The objectives of this study include (1) identifying the optimal animal model for investigations of systemic dehydration and systemic rehydration, (2) developing a reliable and physiologically relevant methodology of systemic dehydration, (3) developing markers of systemic dehydration and (4) demonstrating whether systemic dehydration induces vocal fold dehydration through a combination of methodologies. Sprague Dawley (SD) rats (males and females) and New Zealand White rabbits (males) were used for systemic dehydration and rehydration studies. Rats were systemically dehydrated to an average of 10% reduction in body weight by withholding water. Rabbits were given 5 mg/kg furosemide IP to induce a moderate level of systemic dehydration based on 5% body weight loss. Body weight loss due to dehydration was corroborated by parameters of hemoconcentration. Proton‐density weight MRI at variable dehydration states, genomic analysis of vocal folds, and histopathology were all investigated to determine effects of systemic dehydration. Proton‐density weighted MR imaging can be used to demonstrate that systemic dehydration reliably induces vocal fold dehydration as detected by signal intensity changes. These changes however, are only detected at high body weight loss levels (> 6% body weight loss). Acute episodes of systemic dehydration do not produce reliable, adverse pathological changes to the vocal folds as assessed by histopathology and gene expression studies. Access to water does not induce rehydration if defined by body weight. There are challenges in inducing physiologically‐relevant systemic dehydration and developing a robust and reliable animal model to study the pathobiology of vocal fold dehydration. A combination of techniques are necessary to confirm that dehydration, in an otherwise healthy animal, is occurring; and that dehydration of the body is dehydrating the vocal folds. The sequelae of chronic versus acute dehydration, and systemic dehydration versus surface dehydration also need to be parsed out.Support or Funding InformationNIH R01DC015545
Human Primary Osteoarthritic Chondrocytes Revert to a Healthier Extracellular Matrix Composition Following Long‐term, Serum‐free, Three‐dimensional Alginate Culture
Authors: Morris Gregory Philip , Popper Hannah , Laird Patrick , Mattioli Patrisia , Healy Andrea , Otto Ashley , Milton LaBraya , Pollard Mark , Miller Lawrence , Behling Kathryn C. , D’Angelo Marina 
Published: April 2020

Osteoarthritis (OA) is a common condition involving the loss of articular cartilage which is primarily made up of type II collagen, proteoglycans, and water. In OA, there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage and leads to the abnormal production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free, three‐dimensional alginate culture and components of the extracellular matrix (ECM) can be measured. However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. In this study, we obtained HOACs from the femoral condyles and the tibial plateau of patients undergoing total knee arthroplasty. HOACs of greater or least pathology, determined by gross observation, were isolated and plated in 12‐well cultures at a density of 1.8 × 106 cells/0.5 mL alginate. Collagens I and II degradation and proteoglycan synthesis were measured in conditioned media and alginate‐associated matrix of the cultures at days 2, 5, 8 and 11. During long term HOAC culture, collagen degradation was reduced (p<0.001 for coll I) while proteoglycans were retained in the ECM. This trend suggests that long term, three‐dimensional, serum‐free culture of HOACs may revert to a healthier phenotype. The largest changes in extracellular matrix production were demonstrated between days 2 and 5 in HOAC culture giving insight of a treatment window to test possible therapeutics.Support or Funding InformationSupport was provided by New Jersey Health Foundation Research Award, Cooper Foundation Research Grant and the Division of Research, PCOM.
A Continuous Fluorescent Assay for β‐Amylase Activity
Authors: Ozcan Kenan , Monroe Jonathan , Berndsen Christopher 
Published: April 2020

The production and degradation of starch is key for the survival of plants during the day/night cycle. The stored energy is harnessed through the work of many different enzymes including the β‐Amylase (BAM) family of proteins. BAMs break down long chains of starch into the disaccharide maltose (4‐O‐α‐D‐Glucopyranosyl‐D‐glucose). The model organism Arabidopsis thaliana has 9 β‐Amylase proteins with apparently distinct functions, though not all have the ability to hydrolyze starch. Each BAM seems to have its own unique characteristics, although the specific functional differences are not clear. The objective of our research is to create an assay that can show the kinetics of BAM proteins, that is continuous and sensitive while able to work with synthetic and natural substrates of the BAM proteins. We aim to apply this assay to determine the unique biochemical features of the BAMs. To detect BAM activity, we used a maltose binding sensor composed of Maltose Binding Protein fused to Green Fluorescence Protein (MBP‐GFP). When maltose is bound to MBP it causes conformational changes to GFP resulting in fluorescence. We have tested the assay with maltodextrin chains from 4 to 20 residues in length and with soluble starch finding that we can detect maltose production from all of these substrates with the MBP‐GFP biosensor in the single micromolar range. Moreover, we can detect amylase activity in a continuous measurement format using a plate reader allowing for higher throughput sampling with reduced assay time.Support or Funding InformationNSF‐RUI MCB‐1616467 to J.M.
Novel Bioluminescent Tools to Study GPCR Pharmacology in Living Cells
Authors: Edenson Steven 
Published: April 2020

G‐protein coupled receptors (GPCRs) continue to be prominent targets for new therapeutics. To support these efforts, we have developed a suite of bioluminescent assays to study GPCR pharmacology in real time in living cells. Quantification of ligand binding and receptor internalization relies on N‐terminal fusion to High BiT (HiBiT), an 11 a.a. peptide. HiBiT binds with high affinity to Large BiT (LgBiT) to form NanoBiT Luciferase. For both applications, a non‐lytic detection reagent is added containing cell‐impermeable LgBiT and furimazine substrate. Ligand binding dynamics at the cell surface can be measured in real time through bioluminescent resonance energy transfer (BRET) between NanoBiT Luciferase and a fluorescent tracer. Receptor internalization and recycling are monitored in real time by measuring changes in the cell surface density of HiBiT‐tagged GPCRs. Notably, the bright signal of NanoBiT Luciferase allows both assays to be run at endogenous levels of expression using CRISPR/Cas9 to introduce the HiBiT tag. Similarly, fusion proteins to LgBiT and Small BiT (SmBiT), an 11 a.a. peptide with low affinity binding to LgBiT, can be used to measure β‐arrestin‐1/2 recruitment to activated GPCRs in real time. Furthermore, changes in intracellular cAMP concentration can be sensitively measured using endpoint (cAMP‐Glo) or real time formats (GloSensor cAMP) using thermal stable or circularly permuted forms of luciferase, respectively. These homogenous, cell‐based assays, which are readily adaptable to laboratory automation, should be immensely useful in the study of GPCR pharmacology.Support or Funding InformationPromega Corporation
Semaphorin 3F: A Novel Biologic Treatment for Oral Squamous Cell Carcinoma
Authors: Almazyad Asma , Gao Yao , Shahrabi-Farahani Shokoufeh , Sui Lufei , Gartung Allison , Watnick Randy , Panigrahy Dipak , Adam Rosalyn M. , Bielenberg Diane R. 
Published: April 2020

Oral squamous cell carcinoma (OSCC) accounts for the majority of all oral malignancies. The overall survival rate for patients diagnosed with advanced‐stage OSCC (with metastasis) is only 39%, and the prognosis for these patients has not improved dramatically despite advances in chemotherapeutic, radiotherapeutic, and surgical protocols. As such, there remains an urgent need to develop new therapies for OSCC to improve outcome. Recently, our laboratory identified the Neuropilin‐2 (NRP2) transmembrane receptor as a novel target in OSCC and its associated vasculature in human patient biopsies, human OSCC xenografts, and mouse carcinogenesis models of OSCC. NRP2 is an ideal drug target as it is not expressed in the oral epithelium nor the normal vasculature after birth, hence systemic toxicities are not expected. However, NRP2 is dramatically upregulated on the cell surface in OSCC and its associated tumor endothelium. In embryonic endothelium, NRP2 acts as a co‐receptor with VEGFR2 for the angiogenic factor VEGF‐A and as a co‐receptor with VEGFR3 for the lymphangiogenic factors VEGF‐C/D to regulate the developmental processes of angiogenesis and lymphangiogenesis, respectively. Alternately, NRP2 can bind Semaphorin‐3F (SEMA3F), a secreted chemorepulsive and inhibitory protein, which mediates cellular guidance and vascular patterning. SEMA3F is an inhibitor of the VEGFA/C/D signaling axes as it competes with binding to NRP2 as well as directly signals through the NRP2/PlexinA1 complex to collapse the cellular cytoskeleton and inhibit motility. Our hypothesis is that SEMA3F may potently inhibit OSCC progression by targeting NRP2 in tumor cells and neovessels in the tumor microenvironment. Our in vitro results demonstrate that SEMA3F exogenous protein inhibits the migration, invasion, and proliferation of Nrp2‐expressing mouse OSCC cells isolated from 4NQO‐induced tumors. In vivo, systemic SEMA3F protein delivered continuously via slow‐release mini‐osmotic pumps induces regression, as confirmed by bioluminescence imaging and histologic examination, in established syngeneic luciferase‐labeled OSCC implanted in the tongue of C57BL/6 mice. In human OSCC xenografts implanted orthotopically in immunodeficient mice, systemic SEMA3F treatment inhibits tumorigenicity and reduces tumor‐associated lymphangiogenesis compared to control (vehicle) treatment. Taken together, these preclinical data suggest that SEMA3F may be a powerful anti‐angiogenic and anti‐tumor therapy specifically targeting the NRP2 axis in carcinoma cells and stromal cells in the tumor microenvironment.Support or Funding InformationNone
Getting to the (Turning) Point: A Systematic Interactive Approach for Teaching Identification of Cross‐Sectional Cadaveric Structures
Authors: Nist Kayla M. , Afifi Linda M. , Zumwalt Ann C. 
Published: April 2020

IntroductionThe ability to interpret cross‐sectional anatomy in radiological scans is a necessary diagnostic skill for clinicians. The skills to spatially translate three‐dimensional (3‐D) anatomy into a two‐dimensional (2‐D) plane of section is challenging for beginner anatomy students. However, the capability to build these skills is critical for their medical and research training.AimThe following lesson was employed with the objective to aid first year medical and graduate students at Boston University School of Medicine in applying their understanding of 3‐D anatomy to interpret 2‐D transverse sections of the thorax, abdomen, and pelvis of human cadavers. Turning Technologies “Turning Point” application was used to measure students’ change in understanding of the material before and after.MethodsThe lesson began with an assessment of cross‐sectional knowledge using the Turning Point application on students’ phones. The students then participated in a lecture designed to systematically guide them through a general screening of osteological landmarks, muscles, blood vessels, organs, and other visceral structures at each vertebral level on cross‐sectional images of the thorax, abdomen, and pelvis. Students were guided on how to recognize the general topography and obviously identifiable organs in each section. They were then taught more specifically how to identify increasingly challenging anatomical features, such as organ‐specific structures, based on their previous 3‐D and 2‐D knowledge of the larger structures in the section. This lecture portion of the lesson gave students a system for how to apply and interpret 3‐D anatomy and transpose it into a 2‐D cross‐section. After the lecture portion of the lesson, students were asked to answer a series of practical‐style questions using the Turning Point app. Question types included multiple choice, short answer, and “hot
spot” style.ResultsIn total, 42 students participated in the lesson. The overall class pre‐test average was 60.31 ± 26.79% while the post‐test average was 72.40 ± 13.28%. A comparison of individual pre‐test vs. post‐test scores using Wilcoxon Signed‐Ranks test revealed a significant improvement in student’s performance (p=0.0098, two‐tailed). Average scores of questions in each anatomical region were assessed (thorax: 67.21 ± 32.46%, abdomen: 71.71 ± 18.82%, pelvis: 70.76 ± 34.43%).ConclusionBased on student performance and feedback, this teaching method is an effective tool for improving students’ confidence and knowledge in interpretation of cross‐sectional anatomy. By using a systematic teaching approach with robust identification markers, Turning Point was an effective addition to help build students understanding and confidence in the material.Support or Funding InformationNo funding was received
Structural Basis of 7‐Deazaguanine Modification of DNA in Bacteria and Phage
Authors: Hellie Eric , Alkuzweny Sana , Bayooz Susan , Phan Phuc , Savage Joshua , Luthra Amit , Iwata-Reuyl Dirk , Swairjo Manal 
Published: April 2020

Two important modifications of tRNA, the 7‐deazaguanine nucleosides queuosine (Q) and archaeosine (G+), are biosynthesized from GTP in bacteria and archaea, respectively, in a well characterized multi‐enzyme pathway leading to the shared advanced intermediate, 7‐cyano‐7‐deazaguanine (preQ0). In bacteria, preQ0 is converted to an aminomethyl derivative that is then inserted in tRNA by the bacterial tRNA‐guanine transglycosylase (TGT) enzyme. In archaea, preQ0 is inserted directly in tRNA by the archaeal TGT before conversion to G+. Recently, in 230 bacterial and phage genomes, a genomic island that contains a paralog of TGT was identified, which led to the discovery of Q, G+ and preQ0 deoxy derivatives in the DNA of some of these organisms. This gene cluster was renamed DpdA‐K, for “7‐deazapurine in DNA.” The G+‐modified DNA of E. coli bacteriophage 9g has been shown to resist restriction by >140 Type II restriction endonucleases (REases), consistent with a role of the modification as a defense mechanism. Recent experiments have shown that DpdA is a DNA‐guanine transglycosylase that catalyzes the insertion of preQ0 at a specific palindromic sequence in DNA. Here we report overexpression, purification, and crystallographic analysis of S. Montevideo DpdA (SmDpdA, ~47 kDa). The crystal structure, determined at 2.25‐Å resolution by multi‐wavelength anomalous diffraction methods, reveals a TIM‐barrel enzyme similar to the bacterial TGT in overall fold, active site and structural Zn2+ site. However, a large DpdA‐specific insertion in the TIM barrel provides an extended positively charged 26‐Å wide surface groove, consistent with a DNA binding surface. Free docking of a DNA duplex using the HADDOCK server places DNA to this surface in a fashion reminiscent of DNA binding to the ubiquitous Zn2+‐dependent DNA repair enzyme apurinic/apyrimidinic endonuclease IV. The structure and docking model suggest that DpdA bends its substrate DNA by 60° and flips the modification‐site guanine base out of the helix for transglycosylation. The results also inform the design of a substrate DNA duplex for future crystallization of the nucleoprotein complex.Support or Funding InformationNIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).