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1.
Proc Natl Acad Sci U S A ; 117(31): 18216-18223, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32680962

RESUMO

Cysteine disulfides, which constitute an important component in biological redox buffer systems, are highly reactive toward the hydroxyl radical (•OH). The mechanistic details of this reaction, however, remain unclear, largely due to the difficulty in characterizing unstable reaction products. Herein, we have developed a combined approach involving mass spectrometry (MS) and theoretical calculations to investigate reactions of •OH with cysteine disulfides (Cys-S-S-R) in the gas phase. Four types of first-generation products were identified: protonated ions of the cysteine thiyl radical (+Cys-S•), cysteine (+Cys-SH), cysteine sulfinyl radical (+Cys-SO•), and cysteine sulfenic acid (+Cys-SOH). The relative reaction rates and product branching ratios responded sensitively to the electronic property of the R group, providing key evidence to deriving a two-step reaction mechanism. The first step involved •OH conducting a back-side attack on one of the sulfur atoms, forming sulfenic acid (-SOH) and thiyl radical (-S•) product pairs. A subsequent H transfer step within the product complex was favored for protonated systems, generating sulfinyl radical (-SO•) and thiol (-SH) products. Because sulfenic acid is a potent scavenger of peroxyl radicals, our results implied that cysteine disulfide can form two lines of defense against reactive oxygen species, one using the cysteine disulfide itself and the other using the sulfenic acid product of the conversion of cysteine disulfide. This aspect suggested that, in a nonpolar environment, cysteine disulfides might play a more active role in the antioxidant network than previously appreciated.


Assuntos
Antioxidantes/química , Cisteína/química , Radical Hidroxila/química , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Compostos de Sulfidrila/química
2.
Nat Commun ; 11(1): 3250, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591525

RESUMO

Biocompatible polymers are widely used in tissue engineering and biomedical device applications. However, few biomaterials are suitable for use as long-term implants and these examples usually possess limited property scope, can be difficult to process, and are non-responsive to external stimuli. Here, we report a class of easily processable polyamides with stereocontrolled mechanical properties and high-fidelity shape memory behaviour. We synthesise these materials using the efficient nucleophilic thiol-yne reaction between a dipropiolamide and dithiol to yield an α,ß - unsaturated carbonyl moiety along the polymer backbone. By rationally exploiting reaction conditions, the alkene stereochemistry is modulated between 35-82% cis content and the stereochemistry dictates the bulk material properties such as tensile strength, modulus, and glass transition. Further access to materials possessing a broader range of thermal and mechanical properties is accomplished by polymerising a variety of commercially available dithiols with the dipropiolamide monomer.


Assuntos
Elastômeros/química , Fenômenos Mecânicos , Nylons/química , Materiais Inteligentes/química , Animais , Materiais Biocompatíveis/farmacologia , Varredura Diferencial de Calorimetria , Linhagem Celular , Masculino , Teste de Materiais , Camundongos , Nylons/síntese química , Polimerização , Ratos Sprague-Dawley , Estresse Mecânico , Compostos de Sulfidrila/química , Temperatura
3.
Food Chem ; 331: 127207, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32569964

RESUMO

Reconstituted polyphenolic and aromatic fractions (PAFs) from 33 different Garnacha and Tempranillo grapes were incubated in strict anoxia (75 °C × 24 h). Obtained hydrolyzates were characterized by sensory analysis, gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). Five different aroma categories emerged. Garnacha may develop specific tropical/citrus fruit, kerosene and floral and Tempranillo toasty-woody and red-fruit characteristics. Those notes seem to mask alcoholic and fruit-in-syrup descriptors and the common vegetal background. Twenty-seven odorants were detected by GC-O. GC-MS data showed a clustering closely matching the one found by sensory analysis, suggesting the existence of five specific metabolomic profiles behind the five specific sensory profiles. Overall results suggest that 3-mercaptohexanol is responsible for tropical/citrus fruit, TDN for kerosene, volatile phenols for woody/toasty, ß-damascenone and massoia lactone, likely with Z-1,5-octadien-3-one for fruit-in-syrup and alcoholic notes. Nine lipid-derived unsaturated aldehydes and ketones may be responsible for the vegetal background.


Assuntos
Vitis/química , Compostos Orgânicos Voláteis/análise , Frutas/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Hexanóis/química , Norisoprenoides/química , Odorantes/análise , Olfatometria , Análise de Componente Principal , Compostos de Sulfidrila/química
4.
Food Chem ; 330: 127212, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32526650

RESUMO

In this work, a facile solid phase extraction (SPE) method was developed for the analysis of trace Pb2+ and Cd2+ by using chitosan/thiol modified metal-organic frameworks (CS/MOF-SH) composite as adsorbent followed by graphite furnace atomic absorption spectrometer (GF-AAS) detection. The potential influencing factors, such as solution pH, adsorbent dosage, and extraction time, were fully estimated. Under the optimized extraction conditions, the detection limits of Pb2+ and Cd2+ were 0.033 µg L-1 and 0.008 µg L-1, respectively. Compared to other studies, CS/MOF-SH not only possessed superior adsorption performance, but also had the advantages of ease of handling and recyclability. Encouragingly, the developed method was of high accuracy and could monitor trace Pb2+ and Cd2+ in various certified reference materials (rice, wheat and tea) with complicated matrices, demonstrating its practical potential for regular monitoring of trace heavy metal ions in real food samples.


Assuntos
Cádmio/análise , Quitosana/química , Chumbo/análise , Estruturas Metalorgânicas/química , Compostos de Sulfidrila/química , Cádmio/química , Cátions Bivalentes , Análise de Alimentos , Chumbo/química , Oryza/química , Extração em Fase Sólida/métodos , Chá/química , Triticum/química
5.
Food Chem ; 326: 126982, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32413762

RESUMO

Glutathione (GSH) is a potential inhibitor for acrylamide (AA) in heated food. In the present study, the inhibition pathways of GSH on AA were investigated in the asparagine(Asn)/glucose(Glc)/GSH model system. In comparison to the Asn/Glc model system, three unique molecular ions (m/z 470, 379, and 193) were identified in the Asn/Glc/GSH model system. Those molecular ions were confirmed as the Amadori rearrangement products which formed in the reaction between Glc and GSH, as well as the addition reaction products between AA and the sulfhydryl group (-SH) of GSH and cysteine (Cys). The competition between Asn and GSH for Glc in the competitive reactions was assumed to be the major pathway. Additionally, the elimination reaction between AA and GSH or between AA and Cys also played a minor role in the inhibition of AA. The variances of precursors, intermediates, and final products provided quantitative evidence for the above pathways.


Assuntos
Acrilamida/antagonistas & inibidores , Glutationa/química , Acrilamida/metabolismo , Asparagina/química , Glucose/química , Glutationa/metabolismo , Compostos de Sulfidrila/química , Espectrometria de Massas em Tandem , Temperatura
6.
Nat Commun ; 11(1): 2558, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444769

RESUMO

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction. We report that this goal can be achieved in a traceless manner during ligation by appending a simple N-selenoethyl group to cysteine. While in synthetic organic chemistry the cleavage of carbon-nitrogen bonds is notoriously difficult, we describe that N-selenoethyl cysteine (SetCys) loses its selenoethyl arm in water under mild conditions upon reduction of its selenosulfide bond. Detailed mechanistic investigations show that the cleavage of the selenoethyl arm proceeds through an anionic mechanism with assistance of the cysteine thiol group. The implementation of the SetCys unit in a process enabling the modular and straightforward assembly of linear or backbone cyclized polypeptides is illustrated by the synthesis of biologically active cyclic hepatocyte growth factor variants.


Assuntos
Cisteína/química , Proteínas/síntese química , Compostos de Sulfidrila/química , Oxirredução , Peptídeos/química , Proteínas/química
7.
Food Chem ; 328: 127098, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32470775

RESUMO

Poly(1H-1,2,4-triazole-3-thiol) (poly(T3T)) conductive film was coated successfully on the gold electrode (Au). The electrochemical behavior of vanillin (VAN) was studied on the 1H-1,2,4-triazole-3-thiol-Au (T3T-Au) electrode. The determination of VAN was performed on the T3T-Au electrode using a differential pulse voltammetry (DPV) technique. In order to detect the concentration of VAN, suitable supporting electrolyte solution and pH value were determined. At pH 3 in HClO4 solution, the anodic peak current of VAN obtained with the T3T-Au electrode is 4.3 times greater than the bare Au electrode. The response oxidation peak current and concentration of VAN showed a good linear relationship in the range of 0.1-11.3 µM. The limit of detection was found as 0.04 µM. Besides, the reproducibility, repeatability, stability, and interference measurements were also assayed. This sensor was applied successfully for the detection of VAN in synthetic samples and various food samples.


Assuntos
Benzaldeídos/análise , Benzaldeídos/química , Eletroquímica/instrumentação , Ouro/química , Polímeros/química , Compostos de Sulfidrila/química , Triazóis/química , Eletrodos , Oxirredução , Reprodutibilidade dos Testes
8.
J Chromatogr A ; 1622: 461133, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32354557

RESUMO

This work reports procedures for the immobilization of vinyl ligands on silica particles by UV-initiated thiol-ene radical addition reaction (photo-click immobilization). tert­Butylcarbamoyl quinine was the functional ligand (ene component) for the synthesis of chiral stationary phases. Two distinct surface chemistries were evaluated. In one approach, the ligand was directly attached to 3-mercaptopropyl-silica triggered by radicals generated by UV irradiation from a photoinitiator. In another approach, the ligand was immobilized onto vinyl silica via poly(3-mercaptopropyl-methylsiloxane) (PMPMS) as crosslinker by a photoinitiated double click reaction in which functionalization with chiral ligand and crosslinking to vinylsilica occurred simultaneously in one synthesis step. PMPMS-bonded CSPs were prepared from suspension (slurry method) or solventless after coating of the polythiol onto the vinylsilica surface (film method). Optimization by a design of experiment approach showed that the reaction time is the prime variable to optimize the surface coverage of chiral selector which also increased with PMPMS concentration. When the film formation of the latter approach was assisted by a minute volume of toluene during photo-click immobilization, selector coverage could be significantly increased to 0.73 µmol/m2 in a 2 h synthesis compared to 0.44 µmol/m2 by the solventless film method and 0.47 µmol/m2 by the slurry method under otherwise comparable conditions. The solvent assistance improved the chromatographic efficiency of the film method and resulted equal minimal reduced plate height of 2.6 as the slurry method for a chiral probe (Fmoc-Phe). The mass transfer resistance was around factor 2 smaller with the solvent-assisted film method as compared to the film approach without toluene, presumably due to a more homogenous distribution of the thin polymer film owing to lower dynamic viscosity in presence of toluene.


Assuntos
Cromatografia/métodos , Química Click/métodos , Luz , Dióxido de Silício/química , Compostos de Sulfidrila/química , Ligantes , Concentração Osmolar , Estereoisomerismo , Compostos de Sulfidrila/síntese química , Fatores de Tempo
9.
J Chromatogr A ; 1621: 461078, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32312546

RESUMO

A new derivatization reagent, N-(naphthalen-1-yl)-2-oxopropanehydrazonoyl chloride (UOSA54), was prepared and coupled with four drugs, bearing primary amino, secondary amino or mercapto functional groups. Glucosamine sulfate (GLU), cysteine (CYS), captopril (CAP), and vildagliptin (VIL), were used as representative reactive analytes. The prepared reagent was successfully coupled with the targeted analytes in the presence of triethylamine (TEA) as hydrochloride acceptor and acetonitrile as solvent. The resulting reaction products were separated by high-performance liquid chromatography and monitored simultaneously by diode array and triple quad mass spectrometry detectors. Enhanced DAD and electrospray ionization-MS (ESI-MS) responses were observed for the derivatized products. Complete derivatization of VIL was achieved after heating at 65 ± 3 °C for 4 min, while other analytes were derivatized instantaneously at room temperature. Both, the ESI-ionization suppression, due to the excess reagent, and matrix effect, due to co-eluted biogenic plasma constituents, were negligible. The derivatized GLU, CYS, CAP, and VIL showed a maximum absorption wavelength at 376, 417, 340, and 376 nm, with MS-limit of quantification value of 250.0, 2.0, 2.5, and 3.0 pg/µL, respectively. The relative ESI-MS response of UOSA54 derivatization products was within the range of 0.6-4.1 compared with dansylated products. The method was optimized and validated for optimal reaction product stability, sensitivity, linearity, range, precision, and accuracy. The percentage recovery was exceeding 97.2%, with an RSD value of less than 4.0%. The limit of quantification of targeted analytes was ranged from 80.0 to 0.7 pg/µL.


Assuntos
Aminas/análise , Cromatografia Líquida/métodos , Iminas/análise , Espectrometria de Massas/métodos , Compostos de Sulfidrila/análise , Cromatografia Líquida de Alta Pressão , Compostos de Dansil/química , Fluorenos/síntese química , Fluorenos/química , Reprodutibilidade dos Testes , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/química
10.
Food Chem ; 322: 126754, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32283367

RESUMO

During storage of coffee, the key aroma 2-furfurylthiol becomes less active, the mechanisms of this loss and ways to mitigate it were investigated. Aroma profiles were analyzed using GC-MS and sensory properties were evaluated by Quantitative Descriptive Analysis. Quinones, as the oxidation products of hydroxydroquinone, was found to actively bind 2-furfurylthiol, which accounted for the loss of 2-furfurylthiol. To mitigate this loss, ingredients were screened for their ability to prevent 2-furfurylthiol from loss. Cysteine had the highest 2-furfurylthiol releasing efficiency and ascorbic acid was also selected due to its 2-furfurylthiol releasing ability in Fenton reaction system. Concentrations were optimized and the addition of 0.045 g/L cysteine and 0.05 g/L ascorbic acid directly protected aroma during storage, these included 2-furfurylthiol, dimethyltrisulfide, methyl furfuryl disulfide, 4-ethylguaiacol and 4-vinylguaiacol. Ultimately, sensory testing showed a direct enhancement in nutty, sulfurous and roasted aroma attributes, an increase in flavour intensity and preference over shelf life.


Assuntos
Café/química , Furanos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Compostos de Sulfidrila/metabolismo , Compostos Orgânicos Voláteis/análise , Ácido Ascórbico/química , Café/metabolismo , Culinária/métodos , Cisteína/química , Armazenamento de Alimentos , Furanos/química , Análise dos Mínimos Quadrados , Quinonas/química , Quinonas/metabolismo , Compostos de Sulfidrila/química , Paladar , Compostos Orgânicos Voláteis/química
11.
Acta Virol ; 64(1): 44-58, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32180418

RESUMO

Protein disulfide isomerase (PDI) is an enzyme that catalyzes disulfide bond reduction or formation and rearrangements of disulfide bridges, and also functions as a chaperone. During entry of some of the viruses PDI participates in thiol-disulfide exchange. Previous reports show that rotavirus entry is interfered by impermeant thiol/disulfide exchange inhibitors and antibodies against PDI. Our objective was to assess the interaction between PDI and triple-layered particles (TLPs) from rotavirus strains ECwt and RRV and from a human rotavirus isolate (HI) during the early steps of virus entry in a system of isolated small intestinal villi. Purified soluble PDI was incubated with either isolated intestinal villi or cell membrane-enriched fractions in the presence or absence of thiol/disulfide inhibitors such as bacitracin, DTNB or N- ethylmaleimide followed by the assessment of the PDI interactions with TLPs and rotavirus structural proteins in terms of their redox state changes. Soluble and membrane-bound PDI was found to interact with TLPs from all the rotaviruses assayed and also with the isolated structural proteins represented by the recombinant rVP5* (a tryptic cleavage product of VP4), rVP6 and the native VP7. PDI interaction with TLPs and rotavirus structural proteins was decreased by the presence of thiol/disulfide exchange inhibitors. Interactions of cell membrane-enriched fractions with TLPs produced rearrangements in the disulfide bridges of rotavirus structural proteins. We conclude that PDI interacts with rotavirus virions through redox reactions that could facilitate the rotavirus entry into the host cell. Keywords: cell surface PDI; thiol-disulfide exchange; rotavirus TLPs; virus entry; bacitracin; DTNB.


Assuntos
Dissulfetos/química , Mucosa Intestinal/virologia , Rotavirus/fisiologia , Compostos de Sulfidrila/química , Proteínas Estruturais Virais/química , Membrana Celular/virologia , Humanos , Técnicas In Vitro , Isomerases de Dissulfetos de Proteínas , Internalização do Vírus
12.
Science ; 367(6482): 1147-1151, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139546

RESUMO

Mycobacterium tuberculosis has an unusual outer membrane that lacks canonical porin proteins for the transport of small solutes to the periplasm. We discovered that 3,3-bis-di(methylsulfonyl)propionamide (3bMP1) inhibits the growth of M. tuberculosis, and resistance to this compound is conferred by mutation within a member of the proline-proline-glutamate (PPE) family, PPE51. Deletion of PPE51 rendered M. tuberculosis cells unable to replicate on propionamide, glucose, or glycerol. Growth was restored upon loss of the mycobacterial cell wall component phthiocerol dimycocerosate. Mutants in other proline-glutamate (PE)/PPE clusters, responsive to magnesium and phosphate, also showed a phthiocerol dimycocerosate-dependent growth compromise upon limitation of the corresponding substrate. Phthiocerol dimycocerosate determined the low permeability of the mycobacterial outer membrane, and the PE/PPE proteins apparently act as solute-specific channels.


Assuntos
Amidas/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Permeabilidade da Membrana Celular , Farmacorresistência Bacteriana/genética , Deleção de Genes , Lipídeos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia
13.
Nat Commun ; 11(1): 1207, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139700

RESUMO

The emergence of antibiotic resistant bacteria is a major threat to the practice of modern medicine. Photobactericidal agents have obtained significant attention as promising candidates to kill bacteria, and they have been extensively studied. However, to obtain photobactericidal activity, an intense white light source or UV-activation is usually required. Here we report a photobactericidal polymer containing crystal violet (CV) and thiolated gold nanocluster ([Au25(Cys)18]) activated at a low flux levels of white light. It was shown that the polymer encapsulated with CV do not have photobactericidal activity under white light illumination of an average 312 lux. However, encapsulation of [Au25(Cys)18] and CV into the polymer activates potent photobactericidal activity. The study of the photobactericidal mechanism shows that additional encapsulation of [Au25(Cys)18] into the CV treated polymer promotes redox reactions through generation of alternative electron transfer pathways, while it reduces photochemical reaction type-ІІ pathways resulting in promotion of hydrogen peroxide (H2O2) production.


Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/efeitos da radiação , Ouro/farmacologia , Luz , Nanopartículas/química , Compostos de Sulfidrila/química , Violeta Genciana/farmacologia , Testes de Sensibilidade Microbiana , Espectroscopia Fotoeletrônica , Espécies Reativas de Oxigênio/metabolismo
14.
Food Chem ; 317: 126454, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32113140

RESUMO

The reaction efficiency of o-benzoquinones with amines (L-lysine, Nα-acetyl-L-lysine, glycine, L-methionine and L-arginine), thiols (L-cysteine and Nα-acetyl-L-cysteine) and protein (bovine serum albumin) were determined at pH 5.0, 7.0 and 8.0 and scan rate of 10, 50 and 100 mV/s by cyclic voltammetry. Nucleophiles containing multiple nucleophilic groups and nucleophilic group possessing low pKa value would enhance the reactivity of nucleophiles towards o-benzoquinones. The reactivity of different o-benzoquinones with L-lysine/L-cysteine followed the order: protocatechuic acid quinone ≈ catechol quinone > 4-methylbenzoquinone ≈ caffeic acid quinone > rosmarinic acid quinone > chlorogenic acid quinone. The reactivity of quinones would be decreased by the steric hindrance of substituents on quinone ring, and it would also be weakened by enhancing electron cloud density of quinone ring. Adducts generated by the interaction of 4-methylbenzoquinone with amines and thiols were tentatively identified as amine-quinone adduct and thiol-phenol adduct respectively by UPLC-QTOF-MS/MS and cyclic voltammetry.


Assuntos
Aminoácidos/química , Benzoquinonas/química , Técnicas Eletroquímicas/métodos , Aminas/química , Catecóis/química , Cromatografia Líquida , Cisteína/química , Hidroxibenzoatos/química , Fenóis , Quinonas/química , Compostos de Sulfidrila/química , Espectrometria de Massas em Tandem
15.
Nanoscale Horiz ; 5(3): 507-513, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32118225

RESUMO

Silver nanoparticles (AgNPs) are efficient biocides increasingly used in consumer products and medical devices. Their activity is due to their capacity to release bioavailable Ag(i) ions making them long-lasting biocides but AgNPs themselves are usually easily released from the product. Besides, AgNPs are highly sensitive to various chemical environments that triggers their transformation, decreasing their activity. Altogether, widespread use of AgNPs leads to bacterial resistance and safety concerns for humans and the environment. There is thus a crucial need for improvement. Herein, a proof of concept for a novel biocide based on AgNP assemblies bridged together by a tri-thiol bioinspired ligand is presented. The final nanomaterial is stable and less sensitive to chemical environments with AgNPs completely covered by organic molecules tightly bound via their thiol functions. Therefore, these AgNP assemblies can be considered as safer-by-design and innovative biocides, since they deliver a sufficient amount of Ag(i) for biocidal activity with no release of AgNPs, which are insensitive to transformations in the nanomaterial.


Assuntos
Desinfetantes/química , Nanopartículas Metálicas/química , Prata/química , Compostos de Sulfidrila/química , Desenho de Fármacos , Estabilidade de Medicamentos , Prata/farmacocinética
16.
Org Biomol Chem ; 18(14): 2696-2701, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32206767

RESUMO

S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this context, this work focuses on the introduction of an S-linkage into arabinoxylan oligosaccharides (AXs) in order to obtain a small collection of synthetic tools for the study of AXs degrading enzymes. The key step for the introduction of the S-glycosidic linkage involved anomeric thiol S-alkylation of an orthogonally protected l-arabinopyranoside triflate. The resulting S-linked disaccharide was subsequently employed in a series of glycosylation reactions to obtain a selectively protected tetrasaccharide. This could be further elaborated through chemoselective deprotection and glycosylation reactions to introduce branching l-arabinofuranosides.


Assuntos
Glicosídeos/química , Oligossacarídeos/química , Xilanos/química , Arabinose/análogos & derivados , Arabinose/química , Reagentes para Ligações Cruzadas/química , Dissacarídeos/síntese química , Glicosilação , Compostos de Sulfidrila/química
17.
Nat Methods ; 17(3): 335-342, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066960

RESUMO

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids.


Assuntos
Biomimética , Neoplasias Colorretais/patologia , Regulação da Expressão Gênica , Intestino Delgado/citologia , Organoides/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Técnicas de Cocultura/métodos , Neoplasias Colorretais/metabolismo , Citofotometria/métodos , Enterócitos/citologia , Células Enteroendócrinas/citologia , Feminino , Fibroblastos/citologia , Células Caliciformes/citologia , Humanos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Celulas de Paneth/citologia , Análise de Célula Única/métodos , Compostos de Sulfidrila/química , Proteína Supressora de Tumor p53/metabolismo
18.
J Mol Biol ; 432(7): 2067-2079, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32061935

RESUMO

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential-driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulfide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2-SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis.


Assuntos
Proteínas de Transporte/metabolismo , Cobre/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Compostos de Sulfidrila/química , Proteínas de Transporte/genética , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Metalochaperonas , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Mutação , Transporte Proteico
19.
J Oleo Sci ; 69(3): 271-276, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32051359

RESUMO

In this study, we investigated the direct detection of DNA, without pretreatment, using a quartz crystal microbalance (QCM) sensor. This sensor is modified by a self-assembled monolayer of a thiol derivative that has an amino group as the terminal functional group. Contact angle values and the attenuated total reflectance Fourier transform infrared (ATR/FT-IR) spectra of the QCM sensors after immersion into an ethanol solution of thiol derivatives clearly showed that self-assembled monolayers of the derivatives were formed on the QCM sensors. Although QCM measurements using unmodified and carboxylic group-modified sensors could not detect DNA-Na salt, the sensor modified with amino groups could detect the DNA. This system can be used for the analysis of the interaction between DNA and DNAbinding proteins.


Assuntos
DNA/análise , Técnicas de Microbalança de Cristal de Quartzo/métodos , Compostos de Sulfidrila/química , Etanol , Psicoterapia Breve , Espectroscopia de Infravermelho com Transformada de Fourier
20.
J Agric Food Chem ; 68(9): 2738-2746, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32020795

RESUMO

A homologous series (C6-C10) of 2-mercapto-4-alkanols was obtained by the addition of thioacetic acid to the respective alkenones and subsequent reduction with LiAlH4. Gas chromatographic separation of the stereoisomers was achieved using chiral stationary phases. Their absolute configurations were assigned by the correlation of 1H NMR data and enzyme-catalyzed kinetic resolutions. Odor thresholds and odor qualities were determined by capillary gas chromatography/olfactometry. Compared to the odor qualities reported for the isomeric 4-mercapto-2-alkanols, the homologous series of 2-mercapto-4-alkanols lacked fruity, tropical notes. There was no consistent correlation between the configurations and the odor qualities. However, the observed odor thresholds indicated the importance of the configuration of the asymmetric center bearing the hydroxyl group and the alkyl substituent. The length of this alkyl chain is a main driver for the odor properties, ranging from pungent, vegetable to earthy, mushroom notes.


Assuntos
Aromatizantes/química , Odorantes/análise , Compostos de Sulfidrila/química , Cromatografia Gasosa , Estrutura Molecular , Olfatometria , Estereoisomerismo
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