bluuebunny/crossref_metadata_2025_split huggingface dataset:
Design of Novel Lectins by Computer‐Guided Directed Evolution
Authors: Sharma Prerna , Kazan I. Can , Ozkan Sefika Banu , Ghirlanda Giovanna 
Published: June 2021

Changes in the level and type of glycosylation are implicated in mediating countless physiological and disease processes, yet the glycome is poorly defined. To overcome this barrier, we integrate state of the art computational methods with guided directed evolution for the design of selective glycan binding reagents based on a natural lectin, cyanovirin.Cyanovirin‐N (CV‐N) is a small (11 kDa) lectin that displays potent antiviral activity by binding to the oligomannosides of gp120. The solution structure shows two quasi‐symmetric domains, defined as A (residues 1‐38/90‐101) and B (residues 39–89), which are connected on each side by a short helical linker. Each domain contains a carbohydrate binding pocket, which binds selectively Mana(1®2)Mana termini in branched high‐mannose oligosaccharides. We assessed the binding affinity of WT CV‐N through ITC titrations with dimannose revealing a high‐affinity binding site (Kd=15.3 mM) and a low‐affinity site (Kd=0.400 mM). To increase the binding affinity for glycosylated gp120, we exploited avidity effects by designing a dimeric CV‐N in which a CV‐N sequence is spliced into another. The construct contains four glycan binding domains in two sets (A, A′, B, B′), and binds dimannose with Kd=24.5 mM and 0.95 mM respectively. Four indistinguishable binding events, with Kd=1.1 mM, are observed with Man9.We recently developed computational tools to predicted protein‐glycan interactions accurately by using multiple sequence alignment (MSA) and flexibility analysis to identify co‐evolved positions, and coupling this analysis to our flexible docking tool (BP‐Dock). These tools were validated by assessing the energetic contribution of polar residues within the binding pocket, docking dimannose to single‐point CVN mutant models. We found that the E41A/G and T57A mutations led to a significant decrease in binding energy scores due to rearrangements of the hydrogen‐bond network that reverberated throughout the binding cavity. N42A decreased binding by affecting the integrity of the local protein structure. In contrast, N53S resulted in a high binding energy score. Experimental characterization of the five mutants by NMR and ITC confirmed the binding affinity pattern predicted computationally.Computational analysis also yielded a set of mutations at several positions beyond the immediate binding pocket that impact glycan recognition. These predictions were validated by directed evolution in yeast display; screening resulted in mutants with increased affinity and selectivity for dimannose. We are currently using the same library to identify mutants with high affinity and selectivity towards glycan biomarkers.Support or Funding InformationThis work is supported by NIH‐IR21AI129895 grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of Bioorthogonal N‐Acetylcysteamine (SNAc) Analogues and Peptidoglycan O‐Acetyltransferase B (PatB) to Label Peptidoglycan
Authors: Lazor Klare M. , Wang Yiben , DeMeester Kristen E. , Liang Hai , Heiss Tyler K. , Grimes Catherine L. 
Published: June 2021

In Gram (−) bacteria, peptidoglycan O‐acetyltransferase B (PatB) is responsible for the O‐acetylation of peptidoglycan to prevent degradation by lysozyme and other lytic transglycosylases generated by its host. In this work, PatB was exploited as a tool to incorporate bioorthogonal functionality on the carbohydrate backbone of the peptidoglycan by using a variety of donors, either p‐nitrophenol (pNP) or N‐acetylcysteamine (SNAc). Using tri‐N‐acetylglucosamine (GlcNAc)3 as a peptidoglycan mimic, a variety of functionalities were installed, including an alkyne and azide handle, on this mimic. Further studies in vitro showed that these modifications have a protective effect on isolated peptidoglycan from B. subtilis, E. coli, P. putida, and V. parahaemolyticus against lysozyme degradation. Intrigued by these results, an alkyne handle was subsequently installed on whole bacterial cells of B. subtilis using the SNAc alkyne derivative and the carbohydrate backbone was fluorescently labeled via click chemistry. Altering the peptidoglycan post‐synthetically with these derivatives will aid in the isolation and identification of immune stimulatory fragments from bacteria, as well as in the development of antibiotics and immune modulators.Support or Funding InformationThe Delaware COBRE and INBRE programs supported this project with a grant from the NIGMS (5 P30GM110758‐02, P20GM104316‐01A1 and P20GM103446) from the NIH.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 Role of Sphingomyelin Synthase on the Healthspan of Organisms Via Modulating Neurotransmission
Authors: Chuh Steven Sung Hwan , Wentz Curtis , Chan Jason , Dries Daniel 
Published: June 2021

Individuals are living longer due to medical interventions; however, simply increasing lifespan without proportionally increasing healthspan will result in longer periods of decrepitude. Healthspan is defined as the length of time before the onset of morbidity. One metric of healthspan that decreases as organisms age is neuronal function. To determine signaling molecules that may mediate healthy neuronal function, we examined neuromuscular function in the model organism C. elegans. In particular, we systematically analyzed a class of lipids called sphingolipids. Sphingolipids mediate for a variety of cellular signaling functions such as cell proliferation and apoptosis, but also involved in neurotransmission by interacting with neurotransmitter receptors and promoting neurotransmitter release. Sphingolipids are interconverted by metabolic enzymes that balance the levels of lipids within cells. We examined changes in neuromuscular function with age of C. elegans knockouts in several sphingolipid converting enzymes, including sphingomyelin (SM) synthases (sms‐1, sms‐3), ceramidase (asah‐2) and ceramide synthase double mutant (lagr‐1, hyl‐1). Sphingomyelin synthase converts ceramide to sphingomyelin. Additionally, ceramide synthase converts sphingosine to ceramide. We experimented with these mutants because both sphingomyelin and ceramide play important roles in the production of neurotransmitter receptors. To examine neuromuscular changes with age, we examined worm trashing behavior in 5, 10, and 15 day old worms. We found that mutants lacking sms‐1/SM synthase had decreased thrashing in comparison to wildtype worms at all ages. As stated, SM affects neurotransmission and development, but it is still unclear at which age window SM levels will have the most prominent effect. Therefore, we aim to further observe the role of SM during development and adulthood. We will expose wildtype and mutant worms with a pharmacological agent, D609 which inhibits sphingomyelin synthases. We will treat D609 specifically during development or adult time frames. Lastly, we wanted to further investigate this phenomena by investigating changes in aspects of synaptic vesicle cycling such as exocytosis and endocytosis. To do this, we will examine changes in the synapses of the neuromuscular junction (NMJ) by analyzing changes in GFP‐tagged synaptic markers with age and when exposed to the D609 drug treatments. These results will help identify how sphingomyelin processing enzymes might affect neurotransmission that may have a cascading effect on health span in aging animals.Support or Funding InformationASBMBThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Investigation into the Antibacterial Properties of Citrus and Plant Essential Oils
Authors: Axup Antonetta , Sostarecz Audra Goach 
Published: June 2021

Langmuir Monolayers of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and E.coli lipid extract were used to investigate the antibacterial properties of essential oils, in particular sweet orange oil. DPPC served as the control membrane while DPPG served as the gram positive bacteria model and E.coli lipid extract was the gram negative bacteria model. The Langmuir Monolayer technique is useful for forming model cell membranes and allows for the analysis of the interactions between phospholipids and their molecular organization in the presence of another molecule. The main chemical in sweet orange oil, limonene, was found to disrupt phospholipid‐phospholipid interactions in all models except for DPPG simply because PG headgroups hydrogen bond with each other and there is no space for the hydrophobic limonene molecule. The largest disruption was observed for the E.coli extract lipids due to the unsaturated tails in these lipids resulting in less tight packing. Monolayers were seen in all three membrane systems to be destabilized as evidenced by the decrease in collapse pressure of the monolayer film but the E.coli lipid extract monolayer was the most affected in terms of fluidity as seen by the compression modulus. The antibacterial effects of orange oil on these systems is currently being compared to that of peppermint oil.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stainless Imaging To Identify The Biochemical changes during Bleomycin‐Induced Pulmonary Fibrosis By Fourier Transform Infrared Technique
Authors: Suryadevara Vidyani , Nazeer Shaiju Suhara , Sreedhar Hari , Natarajan Viswanathan , Walsh Micheal 
Published: June 2021

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal pulmonary disease due to scarring of the lung tissue. While biopsies are the gold standard for diagnosis of IPF, there is definitive need for increased efforts towards identifying new molecular markers associated with the disease for efficient diagnosis. Fourier Transform Infrared imaging (FT‐IR) combines imaging at a spatial resolution that approaches light microscopy coupled with biochemical sensitivity of spectroscopy. In this study, bleomycin induced pulmonary fibrosis in rodents, a pre‐clinical model of IPF was used to detect the biochemical changes that occur during different stages of fibrosis. Based on the time point after bleomycin challenge, the animals were grouped as 1. Control; 2. Day 7 (Inflammation); 3. Day 14 (Onset of fibrosis); 4. Day 21 (fibrosis); 5. Day 28 (recovery phase). FT‐IR images were acquired from the lung tissues in each group in reflectance mode using an Agilent Cary 600 Series FT‐IR spectrometer. The spectral profile across the entire lung was obtained and pre‐processed for further analysis using multivariate data analysis. Non‐hierarchical k‐means cluster analysis of the spectrum resulted in delineation of the entire lung tissue based on the biochemical changes into different clusters that reflects the histological changes in the tissue. Further, specific changes in the biochemical composition of the fibrotic and non‐fibrotic regions in the lung during various stages of fibrosis was quantified by the changes in the spectral ratios of the biomolecules of interest. PCA‐LDA analysis could score the probability of occurrence of fibrosis based on the spectral changes. FT‐IR imaging will be a potentially powerful clinical tool, giving new insights into the biochemistry of tissue in a label‐free and non‐perturbing manner.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resolving the structure of metallochaperone AztD
Authors: Fullam Stephanie , Neupane Durga , Yukl Erik 
Published: June 2021

Zinc is an essential nutrient, and proteins mediating zinc homeostasis are important virulence factors among pathogenic bacteria, making them attractive targets for the development of novel antibiotics. Of particular interest are the ATP binding cassette (ABC) transporters, which utilize extracellular solute binding proteins (SBPs) to bind zinc with high affinity and specificity for delivery into the cell via integral membrane permeases. We are studying the zinc import machinery of Paracoccus denitrificans as a model for highly homologous systems in pathogenic bacteria including the carbapenem resistant enterobacteraceae (CRE) species Klebsiella pneumoniae and Citrobacter koseri. Specifically, these species encode an unusual zinc ABC transporter operon aztABCD that encodes a periplasmic metallochaperone (AztD) that transfers zinc specifically to the SBP (AztC). The structure for PdAztC has been solved and features a long, protruding loop containing three His residues. Deletion of the loop or mutation of any of the His residues to Ala completely abrogated zinc transfer from AztD but had no impact on metal binding from solution. From this data, we hypothesized that the loop must be required to access the zinc binding site in AztD, chelate the metal and return it to the AztC binding site. However, without structural information on the AztD chaperone the details of this mechanism remained unclear. We have recently solved high‐resolution crystal structures of AztD from both P. denitrificans and C. koseri using single wavelength anomalous dispersion (SAD) on a lead derivative crystal. This was necessary as there were no homologous of AztD in the protein databank. The protein is a 7‐bladed beta propeller, with zinc bound to three conserved His residues in the central pore. A second zinc binding site was also identified, consistent with solution data indicating that AztD can bind up to three zinc ions. A detailed mechanism for zinc transfer from the primary AztD binding site to AztC is proposed based on the existing solution data, new structures, accessibility of the AztD zinc binding site and electrostatic complementarity between AztD and AztC surfaces. Given the importance of dynamic zinc transfer for bacterial zinc homeostasis and the role of this process in pathogenesis, high‐resolution structural data on zinc binding and transport proteins may be very useful in the development of novel antibiotics for resistant bacteria such as C. koseri.Support or Funding InformationNational Institutes of Health under award number 1SC2GM111170‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Atherogenic diet induced cardiovascular disease and bone loss in ApoE knockout mice
Authors: Cheng Wei‐Yuan , Lan Ying‐Wei , Chen Hsiao‐Ling , Chen Chuan‐Mu 
Published: June 2021

Cardiovascular diseases and bone loss are chronic degenerative diseases which have been considered to be independent pathway. Hyperlipidemia is the dominant risk of cardiovascular disease that caused atherosclerosis, seriously, it will cause vascular calcification. In recent years, growing evidences indicated that the hyperlipidemia also be a risk factor of bone loss which leads to an increased risk of bone fracture. In clinical studies, aortic calcification often exists in patients with osteoporosis, independently of age. Apolipoprotein E (ApoE) is a class of proteins mediated cholesterol metabolism in the body, which plays an important role in cardiovascular disease. The aim of this research is using the ApoE knockout mice to investigate the correlation of cardiovascular disease and bone loss under atherogenic diet treatment. In this study, B6:129P2‐Apoetm1Unc/J knockout mice were used as experimental animal that exhibiting lipid metabolism dysfunction. Mice were divided into two groups (n=6) and placed on either a chow diet or a atherogenic diet for 13 weeks. C57BL/6 mice at the same age were used as a wild‐type normal control. The analysis data were collected including blood chemistry, pathological section, and micro‐computed tomography (μ‐CT). In the blood biochemical examinations, result showed that total cholesterol (TC), oxidized low‐density lipoprotein (Ox‐LDL), calcium content, and carboxyl‐terminal cross‐linking telopeptides of type I collagen (CTX‐1) were significantly increased in atherogenic diet‐induced ApoE KO mice group; however, procollagen type 1 amino‐terminal propeptide (P1NP) was significantly decreased compared to chow diet‐fed ApoE KO mice group. Histopathological results revealed that the severe atherosclerosis and developing vascular calcification in the atherogenic diet‐induced ApoE KO mice group. In the analyses of femurs trabecular bone parameters, bone volume/tissue volume (BV/TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular bone mineral density (Tb.BMD) were obviously lost in the atherogenic diet‐induced ApoE KO mice group. In conclusion, we have successfully used the atherogenic diet to induce cardiovascular disease and bone loss in ApoE knockout mice. This study provides a platform to further investigate common pathophysiologic mechanism between cardiovascular disease and bone loss. It also provides a drug screening animal model test the prevention or therapy at both conditions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deficiency of Lysosomal Ceramide Hydrolysis Contributes to Enhanced Exosome Release and Calcification in Coronary Artery Myocytes
Authors: Yuan Xinxu , Bhat Owais M. , Lohner Hannah , Zhang Qinghua , Zhang Yang , Boini Krishna , Li Pin‐Lan 
Published: June 2021

Ceramide has been shown to contribute to vascular calcification under different pathological conditions. However, the precise mechanism mediating this process remains unknown. The present study investigated the role of lysosomal ceramide metabolism via acid ceramidase (AC) in the regulation of exosome excretion in coronary artery myocytes (CAMs) and the possible implication in the development of calcification. In cultured CAMs from smooth muscle (SM)‐specific Asah1 (mouse code of AC gene) knock‐out mice (Asah1fl/fl/SMcre), we observed that deletion of Asah1 gene increased lysosomal ceramide level compared to their control littermates, as detected by immunostaining of ceramide co‐localized with lysosome marker lamp‐1. Treatment of CAMs from wild type mice produced similar effects to reduce lysosomal ceramide given its effect to inhibit AC activity. Alizarin Red staining showed that more remarkable calcium deposition or calcification by 1.9 folds occurred in cultured CAMs from Asah1fl/fl/SMcre mice compared to cells from their control littermates, if these cells were treated with 3 mM NaH2PO3 and atherogenic stimulator, 7‐ket. Correspondingly, CAMs from Asah1fl/fl/SMcre mice were found to have significant phenotype transition as shown by Western blot analysis, increased expression of synthetic and osteogenic markers (more vimentin and osteopontin) and decreased contractile phenotypic marker (less SM22α), and this synthetic or osteogenic phenotype was enhanced by 7‐ket treatment. Using super‐resolution imaging with Structured Illumination Microscopy (SR‐SIM), we found that 7‐ket markedly reduced colocalization of Rab‐7a (multivesicular body (MVB) marker) with lamp‐1 in 7‐ket‐treated WT CAMs by 3 folds or CAMs from Asah1fl/fl/SMcre mice by 2.5 folds, suggesting inhibition of MVB‐lysosome encountering or fusion during inhibition of AC expression and activity. Using nanoparticle tracking analysis, 7‐ket‐induced excretion of exosomes (80–140 nm in size) was found to be significantly increased in CAMs of Asah1fl/fl/SMcre mice by 2.1 folds compared to CAMs from their control littermates. These results suggest that AC‐mediated ceramide hydrolysis in lysosomes critically controls MVB fate and exosome release and that insufficient AC expression and activity may lead to enhancement of exosome release, contributing to calcium deposition or calcification in CAMs or arterial media.Support or Funding Information(supported by NIH grants HL057244, HL075316 and HL122937).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disease‐associated mutation in PTH reveals molecular mechanisms in endosomal GPCR signaling
Authors: Jean‐Alphonse Frederic , Sutkeviciute Ieva , Xiao Kunhong , Vilardaga Jean‐Pierre 
Published: June 2021

Parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only PTH sustains GS–mediated cAMP production after PTHR internalization into early endosomes. The physiological relevance of this unexpected behavior for PTHR is unclear. Here we present evidence that endosomal PTHR signaling through cAMP also has disease relevance. We demonstrated that R25C mutation recently identified in PTH as a new cause of hypocalcemia in humans affects receptor binding at endosomal pH resulting in rapid ligand dissociation and recycling of PTHR preventing sustained endosomal cAMP signaling. Using hydrogen–deuterium exchange coupled with mass spectrometry (HDXMS) to map structural changes of PTHR upon PTH binding, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Arg25 binding and endosomal cAMP signaling. Determination of the dynamic of protein‐protein interaction using FRET approaches revealed that PTH R25C has a lower potency to form and stabilize the functional complex between β‐Arrestin, Gβγ and PTHR required for endosomal cAMP generation. Overall, these new findings indicate that PTH‐induced endosomal cAMP signaling is likely a primary signaling component regulating calcium homeostasis.Support or Funding InformationNIH R01‐DK102495 and R01‐DK087688 National Research Foundation of Korea NRF‐2013R1A1A1A05005629This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Live‐cell super‐resolution imaging of PKA activity reveals microdomain organization and regulation
Authors: MO Gary , Zhang Jin 
Published: June 2021

Compartmentalized biochemical activities are crucial to many signaling processes in the cell. However, it has been difficult to visualize dynamic protein activities in living cells at the super‐resolution required to see their sub‐diffraction‐limit spatial regulation. Recently, we introduced a novel class of fluorescent biosensors that can detect biochemical activities in living cells at a resolution up to three‐fold better than the diffraction limit. Harnessing a specific binding‐induced change in protein fluorescence dynamics called Fluorescent fLuctuation INcrease by Contact (FLINC), these biosensors translate kinase activities or protein‐protein interactions into changes in fluorescence fluctuations. These fluctuations can then be quantified through Stochastic Optical Fluctuation Imaging (SOFI), permitting us to follow the protein activity dynamics, over tens of minutes, at super‐resolution. Utilizing one such biosensor for Protein Kinase A (PKA), we resolved minute activity microdomains on the plasma membrane of living cells, and discovered the role of clustered anchoring proteins in organizing these active PKA microdomains. We further demonstrate a new version of FLINC – REmoved Dronpa FLINC (red‐FLINC) – and use it to contextualize the PKA activity microdomains with the location of an additional protein. With red‐FLINC, live cell super‐resolution activity imaging requires only a single channel of the visible spectrum, freeing the others for imaging other proteins, or perturbing their activities with optogenetics. Using this technology. we determined the spatial relationship between membrane PKA activity microdomains and important participants in the PKA pathway such as AKAP79 and L‐type calcium channel. Our studies suggest that biochemical activities of the cell are spatially organized into an activity architecture.Support or Funding InformationNIH DP1 CA174423, R35 CA197622 and R01 DK073368This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.