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Quantification of thioether-linked glutathione modifications in human lens proteins.
Wang Z, Schey KL
(2018) Exp Eye Res 175: 83-89
MeSH Terms: Adolescent, Alanine, Aminobutyrates, Cataract, Cellular Senescence, Chromatography, Liquid, Crystallins, Cysteine, Glutathione, Humans, Lens, Crystalline, Middle Aged, Protein Processing, Post-Translational, Serine, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Sulfides, Threonine, Tissue Donors, Young Adult
Show Abstract · Added April 4, 2019
Dehydroalanine (DHA) and dehydrobutyrine (DHB) intermediates, formed through β-elimination, induce protein irreversible glutathionylation and protein-protein crosslinking in human lens fiber cells. In total, irreversible glutathionylation was detected on 52 sites including cysteine, serine and threonine residues in 18 proteins in human lenses. In this study, the levels of GSH modification on three serine residues and four cysteine residues located in seven different lens proteins isolated from different regions and different aged lenses were quantified. The relative levels of modification (modified/nonmodified) were site-specific and age-related, ranging from less than 0.05% to about 500%. The levels of modification on all of the sites quantified in the lens cortex increased with age and GSH modification also increased from cortex to outer nucleus region suggesting an age-related increase of modification. The levels of modification on sites located in stable regions of the proteins such as Cys117 of βA3, Cys80 of βB1 and Cys27 of γS, continued increasing in inner nucleus, but modification on sites located in regions undergoing degradation with age decreased in the inner nucleus suggesting GSH modified proteins were more susceptible to further modification. Irreversible GSH modification in cataract lenses was typically higher than in age-matched normal lenses, but the difference did not reach statistical significance for a majority of sites, with the exception Cys117 of βA3 crystallin in WSF. Except for S59 of αA and αB crystallins, GSH modification did not induce protein insolubility suggesting a possible role for this modification in protection from protein-protein crosslinking.
Copyright © 2018 Elsevier Ltd. All rights reserved.
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DehydroalanylGly, a new post translational modification resulting from the breakdown of glutathione.
Friedrich MG, Wang Z, Schey KL, Truscott RJW
(2018) Biochim Biophys Acta Gen Subj 1862: 907-913
MeSH Terms: Alanine, Amino Acid Sequence, Crystallins, Dipeptides, Glutathione, Glutathione Disulfide, Humans, Lens, Crystalline, Lysine, Middle Aged, Molecular Structure, Peptides, Protein Conformation, Protein Processing, Post-Translational, Proteins, Tandem Mass Spectrometry, Young Adult
Show Abstract · Added April 3, 2018
BACKGROUND - The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues.
METHODS - Human lens digests were examined by mass spectrometry for the presence of (DHA)Gly (+144.0535 Da) adducts to Lys residues. Peptide model studies were undertaken to elucidate the mechanism of formation.
RESULTS - In the lens, this PTM was detected at 18 lysine sites in 7 proteins. Using model peptides, a pathway for its formation was found to involve initial formation of the glutathione degradation product, γ-Glu(DHA)Gly from oxidised glutathione (GSSG). Once the Lys adduct formed, the Glu residue was lost in a hydrolytic mechanism apparently catalysed by the ε-amino group of the Lys.
CONCLUSIONS - This discovery suggests that within cells, the functional groups of amino acids in proteins may be susceptible to modification by reactive metabolites derived from GSSG.
GENERAL SIGNIFICANCE - Our finding demonstrates a novel +144.0535 Da PTM arising from the breakdown of oxidised glutathione.
Copyright © 2018. Published by Elsevier B.V.
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Hotspots of age-related protein degradation: the importance of neighboring residues for the formation of non-disulfide crosslinks derived from cysteine.
Friedrich MG, Wang Z, Oakley AJ, Schey KL, Truscott RJW
(2017) Biochem J 474: 2475-2487
MeSH Terms: Age Factors, Alanine, Cysteine, Databases, Protein, Disulfides, Eye Proteins, Humans, Lens, Crystalline, Models, Molecular, Oligopeptides, Proteolysis, Tandem Mass Spectrometry, beta-Crystallin A Chain
Show Abstract · Added April 3, 2018
Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemized, truncated, and covalently cross-linked. One reaction responsible for age-related protein cross-linking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence for this was found in many lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent cross-links than others. Foremost among them was Cys5 in βA4 crystallin. The reason for this enhanced reactivity was investigated using peptides. Oxidation of Cys to cystine was a prerequisite for DHA formation, and DHA production was accelerated markedly by the presence of a Lys, one residue separated from Cys5. Modeling and direct investigation of the N-terminal sequence of βA4 crystallin, as well as a variety of homologous peptides, showed that the epsilon amino group of Lys can promote DHA production by nucleophilic attack on the alpha proton of cystine. Once a DHA residue was generated, it could form intermolecular cross-links with Lys and Cys. In the lens, the most abundant cross-link involved Cys5 of βA4 crystallin attached via a thioether bond to glutathione. These findings illustrate the potential of Cys and disulfide bonds to act as precursors for irreversible covalent cross-links and the role of nearby amino acids in creating 'hotpsots' for the spontaneous processes responsible for protein degradation in aged tissues.
© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
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Factors Associated With Persistent Increase in Level of Alanine Aminotransferase in Patients With Chronic Hepatitis B Receiving Oral Antiviral Therapy.
Jacobson IM, Washington MK, Buti M, Thompson A, Afdhal N, Flisiak R, Akarca US, Tchernev KG, Flaherty JF, Aguilar Schall R, Myers RP, Subramanian GM, McHutchison JG, Younossi Z, Marcellin P, Patel K
(2017) Clin Gastroenterol Hepatol 15: 1087-1094.e2
MeSH Terms: Administration, Oral, Adolescent, Adult, Aged, Aged, 80 and over, Alanine Transaminase, Antiviral Agents, Biopsy, Clinical Trials, Phase III as Topic, Fatty Liver, Female, Hepatitis B e Antigens, Hepatitis B, Chronic, Histocytochemistry, Humans, Liver, Male, Middle Aged, Tenofovir, Young Adult
Show Abstract · Added March 14, 2018
BACKGROUND & AIMS - Despite complete suppression of viral DNA with antiviral agents, in some patients with chronic hepatitis B (CHB), serum levels of alanine aminotransferase (ALT) do not normalize. We investigated factors associated with persistent increases in ALT level in patients with CHB given long-term tenofovir disoproxil fumarate.
METHODS - We analyzed data from 471 hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients with CHB participating in 2 phase 3 trials. We identified patients with an increased level of ALT (above the upper limit of normal range) after 5 years (240 weeks) of tenofovir disoproxil fumarate therapy. We analyzed findings from liver biopsy specimens collected from 467 patients (99%) at baseline and 339 patients (72%) at year 5 of treatment; biopsy specimens were evaluated by an independent pathologist. We performed stepwise, forward, multivariate regression analyses of specified baseline characteristics and on-treatment response parameters to identify factors associated with persistent increases in ALT level.
RESULTS - Of the 471 patients, 87 (18%) still had an increased ALT level at year 5 of treatment. Factors associated significantly with a persistent increase in ALT level were a steatosis score of 5% or greater (grade 1 or more) at baseline (odds ratio [OR], 2.236; 95% confidence interval [CI], 1.031-4.852; P = .042) and at year 5 (OR, 3.392; 95% CI, 1.560 ≥ 7.375; P = .002), HBeAg seropositivity at baseline (OR, 3.297; 95% CI, 1.653-6.576; P < .001), and age 40 years or older (OR, 2.099; 95% CI, 1.014-4.342; P = .046). Of the 42 HBeAg-positive patients with steatosis at baseline, 21 (50%) had an increased ALT level at year 5 of treatment. Patients with persistent increases in ALT level were more likely to have an increase in steatosis at year 5 than those with a normal ALT level.
CONCLUSIONS - HBeAg seropositivity and hepatic steatosis contribute to persistent increases in ALT level in patients with CHB receiving suppressive antiviral treatment. ClinicalTrials.gov registration numbers: NCT00117676 and NCT00116805.
Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.
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20 MeSH Terms
D-amino acid inhibits biofilm but not new bone formation in an ovine model.
Harmata AJ, Ma Y, Sanchez CJ, Zienkiewicz KJ, Elefteriou F, Wenke JC, Guelcher SA
(2015) Clin Orthop Relat Res 473: 3951-61
MeSH Terms: Amino Acids, Animals, Anti-Bacterial Agents, Biofilms, Biomarkers, Bone Transplantation, Cell Differentiation, Cell Proliferation, Cells, Cultured, Dose-Response Relationship, Drug, Female, Femur, Mesenchymal Stem Cells, Methicillin-Resistant Staphylococcus aureus, Methionine, Mice, Models, Animal, Osseointegration, Osteoblasts, Osteoclasts, Osteogenesis, Phenylalanine, Proline, Sheep, Domestic, Time Factors, X-Ray Microtomography
Show Abstract · Added May 16, 2017
BACKGROUND - Infectious complications of musculoskeletal trauma are an important factor contributing to patient morbidity. Biofilm-dispersive bone grafts augmented with D-amino acids (D-AAs) prevent biofilm formation in vitro and in vivo, but the effects of D-AAs on osteocompatibility and new bone formation have not been investigated.
QUESTIONS/PURPOSES - We asked: (1) Do D-AAs hinder osteoblast and osteoclast differentiation in vitro? (2) Does local delivery of D-AAs from low-viscosity bone grafts inhibit new bone formation in a large-animal model?
METHODS - Methicillin-sensitive Staphylococcus aureus and methicillin-resistant S aureus clinical isolates, mouse bone marrow stromal cells, and osteoclast precursor cells were treated with an equal mass (1:1:1) mixture of D-Pro:D-Met:D-Phe. The effects of the D-AA dose on biofilm inhibition (n = 4), biofilm dispersion (n = 4), and bone marrow stromal cell proliferation (n = 3) were quantitatively measured by crystal violet staining. Osteoblast differentiation was quantitatively assessed by alkaline phosphatase staining, von Kossa staining, and quantitative reverse transcription for the osteogenic factors a1Col1 and Ocn (n = 3). Osteoclast differentiation was quantitatively measured by tartrate-resistant acid phosphatase staining (n = 3). Bone grafts augmented with 0 or 200 mmol/L D-AAs were injected in ovine femoral condyle defects in four sheep. New bone formation was evaluated by μCT and histology 4 months later. An a priori power analysis indicated that a sample size of four would detect a 7.5% difference of bone volume/total volume between groups assuming a mean and SD of 30% and 5%, respectively, with a power of 80% and an alpha level of 0.05 using a two-tailed t-test between the means of two independent samples.
RESULTS - Bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation were inhibited at D-AAs concentrations of 27 mmol/L or greater in a dose-responsive manner in vitro (p < 0.05). In methicillin-sensitive and methicillin-resistant S aureus clinical isolates, D-AAs inhibited biofilm formation at concentrations of 13.5 mmol/L or greater in vitro (p < 0.05). Local delivery of D-AAs from low-viscosity grafts did not inhibit new bone formation in a large-animal model pilot study (0 mmol/L D-AAs: bone volume/total volume = 26.9% ± 4.1%; 200 mmol/L D-AAs: bone volume/total volume = 28.3% ± 15.4%; mean difference with 95% CI = -1.4; p = 0.13).
CONCLUSIONS - D-AAs inhibit biofilm formation, bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation in vitro in a dose-responsive manner. Local delivery of D-AAs from bone grafts did not inhibit new bone formation in vivo at clinically relevant doses.
CLINICAL RELEVANCE - Local delivery of D-AAs is an effective antibiofilm strategy that does not appear to inhibit bone repair. Longitudinal studies investigating bacterial burden, bone formation, and bone remodeling in contaminated defects as a function of D-AA dose are required to further support the use of D-AAs in the clinical management of infected open fractures.
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26 MeSH Terms
An Iron-Regulated Autolysin Remodels the Cell Wall To Facilitate Heme Acquisition in Staphylococcus lugdunensis.
Farrand AJ, Haley KP, Lareau NM, Heilbronner S, McLean JA, Foster T, Skaar EP
(2015) Infect Immun 83: 3578-89
MeSH Terms: Bacterial Proteins, Cell Wall, Fluorescent Antibody Technique, Immunoblotting, Iron, Mass Spectrometry, N-Acetylmuramoyl-L-alanine Amidase, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Staphylococcal Infections, Staphylococcus lugdunensis
Show Abstract · Added February 8, 2016
Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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11 MeSH Terms
HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry.
Zhou J, Price AJ, Halambage UD, James LC, Aiken C
(2015) J Virol 89: 9068-79
MeSH Terms: Amino Acid Substitution, Anti-HIV Agents, Binding Sites, CD4-Positive T-Lymphocytes, Capsid, Capsid Proteins, Cell Line, Drug Resistance, Viral, HIV Infections, HIV-1, Host-Pathogen Interactions, Humans, Indoles, Macrophages, Molecular Chaperones, Nuclear Pore Complex Proteins, Phenylalanine, Protein Binding, Protein Conformation, RNA Interference, RNA, Small Interfering, Virus Internalization, Virus Replication, beta Karyopherins, mRNA Cleavage and Polyadenylation Factors
Show Abstract · Added February 4, 2016
UNLABELLED - During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4(+) T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution.
IMPORTANCE - The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages--cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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25 MeSH Terms
Compensatory substitutions in the HIV-1 capsid reduce the fitness cost associated with resistance to a capsid-targeting small-molecule inhibitor.
Shi J, Zhou J, Halambage UD, Shah VB, Burse MJ, Wu H, Blair WS, Butler SL, Aiken C
(2015) J Virol 89: 208-19
MeSH Terms: Amino Acid Substitution, Anti-HIV Agents, Cells, Cultured, Drug Resistance, Viral, HIV Core Protein p24, HIV-1, Humans, Indoles, Macrophages, Mutant Proteins, Mutation, Missense, Phenylalanine, Selection, Genetic, Suppression, Genetic, Virus Replication
Show Abstract · Added February 19, 2015
UNLABELLED - The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.
IMPORTANCE - The HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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15 MeSH Terms
A conserved phenylalanine as a relay between the α5 helix and the GDP binding region of heterotrimeric Gi protein α subunit.
Kaya AI, Lokits AD, Gilbert JA, Iverson TM, Meiler J, Hamm HE
(2014) J Biol Chem 289: 24475-87
MeSH Terms: Crystallography, X-Ray, GTP-Binding Protein alpha Subunits, Guanosine Diphosphate, Models, Molecular, Phenylalanine, Protein Conformation
Show Abstract · Added January 20, 2015
G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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6 MeSH Terms
Discovery of a potent stapled helix peptide that binds to the 70N domain of replication protein A.
Frank AO, Vangamudi B, Feldkamp MD, Souza-Fagundes EM, Luzwick JW, Cortez D, Olejniczak ET, Waterson AG, Rossanese OW, Chazin WJ, Fesik SW
(2014) J Med Chem 57: 2455-61
MeSH Terms: Alanine, Amino Acid Sequence, Cell Line, Crystallization, Crystallography, X-Ray, DNA, Single-Stranded, Drug Discovery, Electrophoretic Mobility Shift Assay, Fluorescence Polarization, Magnetic Resonance Spectroscopy, Microscopy, Fluorescence, Models, Molecular, Molecular Sequence Data, Penetrance, Peptides, Protein Conformation, Replication Protein A, Structure-Activity Relationship, Tumor Suppressor Protein p53
Show Abstract · Added March 11, 2014
Stapled helix peptides can serve as useful tools for inhibiting protein-protein interactions but can be difficult to optimize for affinity. Here we describe the discovery and optimization of a stapled helix peptide that binds to the N-terminal domain of the 70 kDa subunit of replication protein A (RPA70N). In addition to applying traditional optimization strategies, we employed a novel approach for efficiently designing peptides containing unnatural amino acids. We discovered hot spots in the target protein using a fragment-based screen, identified the amino acid that binds to the hot spot, and selected an unnatural amino acid to incorporate, based on the structure-activity relationships of small molecules that bind to this site. The resulting stapled helix peptide potently and selectively binds to RPA70N, does not disrupt ssDNA binding, and penetrates cells. This peptide may serve as a probe to explore the therapeutic potential of RPA70N inhibition in cancer.
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19 MeSH Terms