bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of the Structure and Function of Phosphoserine Phosphatases
Authors: Brousseau Merissa C. , Ray Leah C. , Allen Karen N. 
Published: June 2021

With the ever increasing availability of new genome sequences every year, there is a growing need for functional annotation both on a structural and cellular level. In the case of large superfamilies of proteins with a great deal of sequence divergence, assignment of function based on sequence alone remains difficult. For instance, although Bacillus subtilis is the model gram positive bacterium, only 30% of the genome has assigned activity. YsaA (BSU28940), a B. subtilis protein discovered in a knockout screen for metabolic mutants, has been confirmed to have phosphoserine phosphatase activity by determining the steady‐state kinetic constants. Despite this, YsaA shares only 12% sequence identity to the known E. coli phosphoserine phosphatase SerB. We are currently working to determine the molecular structure of YsaA, along with a Bacillus halodurans protein BH2155 (60% similarity with YsaA) which will provide better understanding of the structure‐function relationship between these new proteins and SerB. It is very possible that although YsaA and SerB are within the same super family, they may have independently evolved the same function.BH2155 has been over‐expressed in E.coli, purified, and subjected to sparse‐matrix high throughput screens yielding initial crystallization conditions. Additionally, experiments have been performed using a phosphate assay to obtain the steady state kinetic constants for BH2155 and compare them to YsaA and known phosphoserine phosphatase SerB. By determining the structures of these proteins as well as profiling their activity we hope to gain insight into the structure‐function relationship and the possibility of pseudo‐convergent evolution in phosphoserine phosphatases.Support or Funding InformationWe thank the Undergraduate Research Opportunities Program at Boston University for funding supporting this project (to MCB).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure/Function Analysis of a Novel Myeloperoxidase Inhibitor from Staphylococcus delphini
Authors: Ploscariu Nicoleta T. , de Jong Nienke , van Strijp Jos , Geisbrecht Brian V. 
Published: June 2021

Staphylococcus aureus and closely related species are highly adapted to their hosts and have evolved many strategies to resist opsonization and phagocytosis. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium can actively evade specific intracellular killing mechanisms used by neutrophils. We recently discovered a highly conserved S.aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that specifically binds and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils [de Jong et al., PNAS, 2017]. S. aureus cells deleted for SPIN gene showed decreased survival compared to wild‐type bacteria after phagocytosis by neutrophils. A crystal structure of SPIN bound to MPO suggests that it acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. To further our understanding of the structure/function relationships of SPIN proteins, we cloned, overexpressed, purified, and characterized a series of SPIN orthologs of Staphylococcal origin.We found that SPIN originating from S. delphini although shares only ~53% identity to SPIN‐aureus, it still retained a strong affinity and inhibitory capacity for human MPO. A 2.4 Å crystal structure of SPIN‐delphini bound to human MPO provided a basis for comparing the similarities and differences between SPIN‐aureus and SPIN‐delphini. Together, these studies shed light on the molecular features which determine the specificity of SPIN proteins for MPO, and suggest potential avenues for using this information toward the design of synthetic MPO inhibitors.Support or Funding InformationNIH Grants AI111203 and GM121511This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantification of total HIV‐1 DNA in seminal and blood samples in men under cART
Authors: Torres‐Strubbe Rafael J. , Godoy‐Muñoz Lenin J. , Figueroa Lysmarie J. , Hill Martin J. 
Published: June 2021

Although antiretroviral therapy (ART) exists there are some anatomical sites that serve as drug‐sanctuaries for HIV‐1 treatment. Semen is the principal vector for HIV‐1 transmissions worldwide. Infectious HIV‐1 is found in seminal plasma and in seminal leukocytes. Previous studies have shown that copies of total HIV‐1 DNA are higher in blood than in semen; however, these studies only focus in men with less than two years living with the disease. Studies suggest that semen can be a reservoir promoting the transcription of the virus when it reaches non‐detectable levels. Therefore, we hypothesize that patients undergoing antiretroviral therapy and with non‐detectable viremia should present higher HIV‐1 DNA copies in semen than in blood. Patients with paired semen and blood samples were selected from a previous longitudinal study based on the inclusion criteria. Patient selection criteria included non‐detectable viremia and more than two years under ART. Peripheral blood mononuclear cells and seminal cells were isolated from blood and semen samples from each patient to extract genomic DNA. rtPCR was used to assess total HIV‐1 DNA on each compartment. Our results show higher number of HIV‐1 copies in blood that in seminal cells. Despite the theory of semen being a drug sanctuary and a reservoir for HIV‐1, there may be other factors involve in the ongoing viral replication in blood.Support or Funding InformationThis work was supported by NIGMS‐NIH grants: 5SC3GM088032‐08 (MH), UPR‐Ponce Research Initiative for Scientific Enhancement (R25GM096955) The authors also acknowledge the support from the Molecular and Genomics Core (MAGIC) at Ponce Health Sciences University (NIMHD Grant MD007579).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRAPPC6AΔ, TIAF1 and SH3GLB2 are initiators for amyloid beta plaque formation and tau aggregation in vivo
Authors: Chang Nan‐Shan , Chang Jean‐Yun , Yap Ye Vone , Sze Chun‐I 
Published: June 2021

Substantial evidence shows that tumor suppressor WW domain‐containing oxidoreductase, designated WWOX, FOR or WOX1, is involved in neurodegenerative diseases, neural disorders, cancer progression, and metabolic diseases. WWOX protects neurons from degeneration by binding tau and tau‐hyperphosphorylating enzymes GSK3β, ERK, and JNK1, and promotes neuronal differentiation. WWOX is frequently downregulated in the hippocampi of patients with Alzheimer's disease (AD). Indeed, WWOX may start to downregulate in the cortex and hippocampus in mid‐aged normal individuals. Both in vivo and in vitro evidence show that upon WWOX downregulation, a cascade of protein aggregation occurs, involving spontaneous relocation of TRAPPC6AΔ (trafficking protein particle complex 6A delta, TPC6AΔ) and TGFβ1‐induced anti‐apoptotic factor 1 (TIAF1) to the mitochondria. TPC6AΔ aggregates first and then TIAF1, followed by activating caspases and leading to amyloid precursor protein (APP) degradation and formation of amyloid beta (Aβ) plaques and tau tangles. Here we identified SH3GLB2 (SH3‐Domain GRB2‐Like Endophilin B2 ), whose aggregation is downstream that of TIAF1. The aggregates can be found in the AD hippocampi. A 31‐amino‐acid protein Zfra is highly effective in blocking and preventing the aggregation of TPC6AΔ, TIAF1, SH3GLB2, Aβ, and Tau. To characterize how the BAR domain‐containing SH3GLB2 aggregates, here we show that alteration of Tyr77 to Phe77 abolishes TGF‐β‐mediated polymerization, suggesting that Tyr77 phosphorylation is needed for SH3GLB2 aggregation. Activation of p53 by PRIMA‐1 abolishes SH3GLB2 aggregation, whereas inhibition of p53 activation by Pifithrin‐μ induces the aggregation. Induction of autophagy by thapsigargin accelerates SH3GLB2 aggregation. Transiently overexpressed SH3GLB2 causes apoptosis. Neurotoxin MPP+ induces neuronal death in vivo due in part to SH3GLB2 aggregation. Tumor suppressors p53 and WWOX are partners in cancer suppression. Loss of WWOX causes p53 instability. Conceivably, dysregulation of both p53 and WWOX results in initiation of the TPC6AΔ/TIAF1/SH3GLB2 aggregation cascade and leading to the formation of Aβ plaques and tau tangles of the neurodegeneration pathway.Support or Funding InformationMOST, Taiwan, and DoD, USA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
P21‐Activated Kinase 1 is Necessary for Autophagy and Mitophagy in Cardiomyocytes
Authors: Clements Kevin , Tin Kathy , Kobayashi Tamayo , Huang Yuan , Kobayashi Satoru , Liang Qiangrong 
Published: June 2021

P21‐Activated Kinase 1 (PAK1) is a serine‐threonine kinase that regulates multiple signal transduction pathways in cardiomyocytes. Despite the large body of evidence indicating a key role for PAK1 in cardiac protective signalling, the mechanisms underlying the cardioprotective effects of PAK1 remain elusive. Autophagy is a cellular process in which unwanted organelles and proteins are degraded and recycled to maintain cellular homeostasis. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria from the cell via the lysosome system to control mitochondrial quality. Autophagy and mitophagy are essential for cell survival in response to starvation, but may become detrimental under other conditions. How autophagy and mitophagy are regulated for adaptation in different contexts is under intense investigation. In this study, we explored whether PAK1 regulates autophagy and mitophagy in cardiomyocytes. PAK1 was knocked down by using siRNA in H9c2 cardiac myoblasts and then the expression levels of the autophagosome marker LC3‐II were analysed by Western blot. Interestingly, downregulation of PAK1 reduced LC3‐II levels in the total cell lysates, which was not significantly increased by the lysosomal protease inhibitors pepstatin A (pepA) and E64d, suggesting that autophagy flux is reduced by downregulation of PAK1. Similarly, PAK1 knockdown reduced LC3‐II expression levels in mitochondria either in the presence or absence of pepA and E64d, indicating limited mitochondrial degradation or mitophagy in the absence of PAK1. These results were reproduced in mouse hearts lacking PAK1. Together, our results suggest that PAK1 is essential to maintain the activities of autophagy and mitophagy in cardiomyocytes in vitro and in vivo. Current studies in our lab are investigating the functional significance of autophagy and mitophagy in PAK1‐induced myocardial protection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiac remodeling in obesity is associated with high gene expression of TLR‐4 and TNF‐α
Authors: Alves Pedro Henrique Rizzi , Hasimoto Fabiana , Kitawara Koody , Francisqueti Fabiane , Ferron Artur , Bazan Silméia Garcia Zanati , Campos Djon , Ferreira Ana Lúcia , Correa Camila Renata 
Published: June 2021

AimEvaluate the correlation between the gene expression of inflammatory markers and cardiac remodeling in obese animals.Methods16 male Wistar rats were assigned to receive water plus standard diet (group C, n = 8) and Ob diet with water plus 25% sucrose (Ob group, n = 8). Weekly body weight (BW) of the animals was assessed and, after 30 weeks of experiment, adiposity index (AI). Cardiac structures measured: diastolic diameter (LVDD); diameter of the aorta (AO), left atrium (LA); The MVE index (LVMI) was calculated by normalizing to body weight estimated LV mass. The LV systolic function was assessed by cardiac output; The LV diastolic function was evaluated by the following indices: peak velocity of early diastolic filling (E wave); peak velocity of late diastolic filling (A wave); ratio between the E and A waves (E/A); deceleration time of E wave (DTE); The study was supplemented by evaluation by tissue Doppler early diastolic (E′) and late (A ′) of the mitral annulus (arithmetic average travel speeds of lateral and septal walls), and the ratio the waves (E/E′ and E′/A′). Left ventricular fragment of the animals was homogenized in TRIzol® for extraction of ribonucleic acid (RNA). After wards, the RNA was submitted to reverse transcription, conversion of RNA to complementary deoxyribonucleic acid (cDNA). cDNA was used in the polymerase chain reaction (PCR) using ready‐made assays containing TaqMan MGB (FAM) probe and primer specific for tumoral necrosis factor‐ alpha (TNF‐α) and toll‐like receptor 4 (TLR‐4).ResultsThe Ob group showed significantly higher weight (C = 493.008 ± 50.775, Ob = 562.95 ± 53.69; p < 0.05) and higher adiposity index (C = 4.79 ± 0.73, Ob = 8.68 ± 1.76; p < 0.05). Gene expression of TRL‐4 (C = 1.00 ± 0.337, Ob = 1.584 ± 0.559; p < 0.05) and TNF‐α (C = 1.0 ± 0.485, Ob = 2.897 ± 2.596; p < 0.05) presented significant increase in ob group. Results of cardiac evaluation are described in Table 1 followed by the graphs of correlation.ConclusionHigh gene expression of TLR‐4 and TNF‐α in heart is correlated with cardiac remodeling in obese animals.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of the Prostaglandin E2 EP3 Receptor Does Not Affect Beta Adrenergic Signaling in the Heart
Authors: Bryson Timothy D. , Pandrangi Teja S. , Khan Safa Z. , Xu Jiang , Peterson Edward , Harding Pamela 
Published: June 2021

High blood pressure is a major risk factor for cardiovascular disease. Prostaglandin E2 (PGE2) is a metabolite of arachidonic acid and is known to mediate inflammation and blood pressure regulation. Previous data from our lab have shown that PGE2 reduces contractility when signaling via its EP3 receptor subtype and that EP3 expression increases in the Angiotensin II (Ang II) model of hypertension. Furthermore, we demonstrated in vivo that the hypertensive effect of Ang II could be abrogated using an EP3 inhibitor (EP3i), suggesting an interaction between the two pathways. Since stimulation of the sympathetic nervous system increases Ang II and is an important regulator of cardiovascular function, we therefore hypothesized that inhibition of EP3 would also antagonize beta adrenergic signaling. To test our hypothesis, we studied 4 groups of male 10–12 wk. old C57Bl/6 mice receiving: (1) vehicle, (2) the beta adrenergic agonist Isoproterenol, ISO 30 mg/kg/d S.C., (3) the EP3i L798,106 40 μg/kg/d, (4) ISO + L798,106 for 10 days. ISO or its vehicle were given via osmotic mini pump and L798,106 or its vehicle were administered via daily injections S.C. Systolic blood pressure (BP) was measured every 2 days via tail cuff and cardiac function was assessed by echo at the end of the study period. At baseline, there were no differences in BP between the 4 groups. As expected, ISO infusion significantly increased both BP (105 ± 3.4 mmHg vs. 130 ± 4.1 mmHg, p<0.01) and heart rate (600 ± 21 bpm vs. 749 ± 16 bpm, p<0.005) at end of study, however, there was no difference in BP between the ISO group and the ISO + EP3i group (130 ± 4.1 mmHg vs 125 ± 6.1 mmHg, respectively) nor heart rate (749 ± 16 bpm vs 729 ± 26 bpm). Consistent with the blood pressure data, myocyte cross sectional area increased with ISO infusion from 195.5 ± 9.1 μm2 to 234.0 ± 10.4 μm2, p < 0.05 and this was unchanged at 219.9 ± 13.1 μm2 in the ISO+EP3i group. Similar results were noted in the heart weight to body weight ratios. Echocardiography in conscious animals demonstrated no significant differences in either ejection fraction (EF), shortening fraction, or left ventricle dimensions between the ISO group and the ISO + EP3i group. In conclusion, our data suggest that contrary to our hypothesis, there is no interaction between the EP3 receptor signaling pathway and the beta adrenergic pathway after ten days of ISO infusion. Thus, EP3 receptor signaling appears to be uniquely linked to the angiotensin II pathway.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Clinically Relevant Non‐Small Cell Lung Cancer Model in Athymic Nude Mice using 3D Spheroids of A549‐iRFP Cells
Authors: Huang Yingbo , Guo Xin 
Published: June 2021

BackgroundLung cancer is the top killer cancer in the US. Monolayer cultures of lung cancer cells and their animal xenografts are major models for testing therapeutic modalities against lung cancer. Injection of suspended cancer cells into the lung is one of the most common approach to build an orthotopic lung cancer model but carry several major disadvantages, including the omission of a primary solid tumor and the artificial metastasis generated from the premature leakage of the injected cell suspension [1]. All such drawbacks could severely mislead the development of new therapies against lung cancer. Compared to cell monolayers or cell suspensions in culture, three‐dimension (3D) cancer cell spheroids better mimic the tumor microenvironment [2].PurposeThe purpose of this study is to build a more clinically relevant, orthotopic lung cancer model by inoculating 3D spheroids of lung cancer cells into the mouse lung.MethodThe 3D spheroids were cultured by seeding human lung cancer cell line A549‐iRFP in corning 96 multicellular microplate. The cells stably expressed infrared fluorescent protein, whose signal was detected on Odyssey CLx imaging machine (λex = 685 nm, λem = 700 nm) to image the tumor growth in vivo. Tumor inoculation was performed by transpleural surgery on the left side of the athymic nude mice [3].ResultsThe fluorescence of the spheroids was proportional to their volume and viability (R2=0.9845 and 0.9305). Early in Day 7, the mice inoculated with the 3D spheroids (right 2 and 3) showed confined tumor growth at the inoculation side of the lung whereas those inoculated with cancer cell suspension (left 1) displayed perfused signal on both sides of the lung, indicating premature leakage of the inoculated cancer cells.ConclusionThe mice inoculated with the cancer cell suspension displayed artificial metastasis due to premature leakage of the injected cells inside the lung, which decreased its clinical relevance of mimicking lung cancer progression. In contrast, mice inoculated with the 3D cancer cell spheroids better mimic the progression of non‐small cell lung cancer in clinic.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 rare, autism‐associated in‐frame deletion in the dopamine transporter exhibits profound functional deficits
Authors: Shekar Aparna , Campbell Nicholas , Aguilar Jenny , Peng Dungeng , Erreger Kevin , Matthies Heinrich , Sutcliffe James , Mchaourab Hassane , Galli Aurelio 
Published: June 2021

The neurotransmitter dopamine (DA) mediates fundamental behaviors such as reward, motivation, attention, and cognition. Important to dopamine neurotransmission is the dopamine transporter (DAT), a member of the solute carrier family 6 encoded by the SLC6A3 gene. DAT is a presynaptic membrane protein responsible for the reuptake and recycling of DA following calcium‐mediated vesicular release. DAT dysfunction has been associated with neuropsychiatric disorders including ADHD, schizophrenia, bipolar disorder, and autism spectrum disorders (ASD). DAT is also the target of commonly abused psychostimulants and controlled substances, namely cocaine and amphetamine (AMPH).Recently, our laboratory identified a novel, SLC6A3 variant identified in an ASD proband from whole exome sequencing. The genetic variant is an in‐frame deletion of three nucleotides resulting in a deletion of amino acid Asn336 (ΔN336). Located in the third intracellular loop, N336 is conserved from human to Drosophila, and in silico algorithms predict that ΔN336 confers a functionally damaging effect to DAT. Expression of human DAT (hDAT) ΔN336 in Chinese Hamster Ovary (CHO) cells revealed a near absence of DAT‐dependent DA uptake relative to wildtype DAT, yet surface expression was unchanged. This SLC6A3 variant is permissible to AMPH‐induced efflux, but exhibits diminished DAT‐mediated inward currents promoted by rapid application of AMPH. To understand the basis of the ΔN336 variant's effects on transporter conformational dynamics, we conducted electron paramagnetic resonance (EPR) spectroscopy analyses by introducing the correspondent deletion in the leucine transporter, LeuT. LeuT ΔV269 promotes a closed conformation of the extracellular gate and facilitates the intracellular gate to reside in a “half‐open and inward‐facing” conformation.The results of this study provide new structural and functional insights into the role of DAT structural domains controlling DA transport. In addition, they define how disruption of specific DAT structural interactions and gate conformations translate to deficits in molecular function. Most importantly, this work adds to the growing body of literature implicating altered regulation of DA homeostasis/transport as a potential biological mechanism underlying liability to ASD.Support or Funding InformationAmerican Heart Association Predoctoral Award 17PRE33410145 (A.S), NIH T32 Training Programs T32NS007491 (N.G.C.) and 4TR32GM007628‐29 (J.I.A.) and NIH grant DA038058 (A.G.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reinforcing, Antinociceptive, and Pruritic Effects of a G Protein‐Biased Mu Opioid Receptor Agonist, PZM21, in Primates
Authors: Ding Huiping , Kiguchi Norikazu , Perrey David A. , Nguyen Thuy , Czoty Paul W. , Zhang Yanan , Ko Mei‐Chuan 
Published: June 2021

PZM21 is a newly discovered G protein‐biased mu opioid peptide (MOP) receptor agonist which exerts antinociceptive effects with fewer side effects in rodents. The aim of this study was to determine the functional profile of systemic and spinal delivery of PZM21 in non‐human primates. Effects of PZM21 were compared with those of MOP agonists, such as oxycodone and morphine, in a series of behavioral assays established in rhesus monkeys (Macaca mutatta). Following systemic administration, PZM21 (1–6 mg/kg) and oxycodone (0.1–0.6 mg/kg) dose‐dependently produced antinociceptive effects against an acute noxious stimulus. Similar to a prescription opioid oxycodone, PZM21 produced reinforcing effects in primates under fixed ratio (FR30) and progressive‐ratio (PR) schedules of intravenous drug self‐administration. Following intrathecal administration, PZM21 (0.03–0.3 mg) and morphine (0.003–0.03 mg) dose‐dependently produced antinociceptive effects. Although intrathecal PZM21 has a slower onset for eliciting itch scratching activities, intrathecal PZM21 (0.3 mg) and morphine (0.03 mg) both elicited robust scratching responses lasting for more than 5 hours. In addition, intrathecal PZM21 (0.03–0.3 mg) dose‐dependently attenuated capsaicin‐induced thermal allodynia. Anti‐allodynic effects of intrathecal PZM21 or morphine could be blocked by a MOP receptor antagonist, naltrexone. Taken together, these pharmacological studies in primates indicate that PZM21 displays similar functional efficacy as an analgesic like clinically used MOP agonists, oxycodone and morphine. Although structure‐based discovery of PZM21 opens a new, exciting chapter for G protein‐biased MOP agonists, the abuse potential and pruritic effect of PZM21 are not significantly different from those of clinically used opioid analgesics in primates.Support or Funding InformationThe US‐PHS grants DA044775, DA032568, and DA040693.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.