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1.
Pharmacol Rev ; 72(4): 862-898, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32929000

RESUMO

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.


Assuntos
RNA/efeitos dos fármacos , RNA/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/uso terapêutico , Betacoronavirus , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Desenvolvimento de Medicamentos/organização & administração , Descoberta de Drogas , Humanos , MicroRNAs/farmacologia , MicroRNAs/uso terapêutico , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Pandemias , Pneumonia Viral/tratamento farmacológico , RNA/efeitos adversos , RNA Antissenso/farmacologia , RNA Antissenso/uso terapêutico , RNA Guia/farmacologia , RNA Guia/uso terapêutico , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/farmacologia , RNA Ribossômico/efeitos dos fármacos , RNA Ribossômico/farmacologia , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , RNA Viral/efeitos dos fármacos , Ribonucleases/metabolismo , Riboswitch/efeitos dos fármacos
2.
J Chromatogr A ; 1628: 461438, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822978

RESUMO

A fast, simple, environmentally friendly and sensitive on-line concentration method using microemulsion system as background solution (BGS) was developed for the trace detection of propazine, atrazine, simazine in food samples. The electrokinetic injection assisted micelle to cyclodextrin stacking (MCDS) was designed for the enrichment of target compounds. The factors affected enrichment performance, such as the kind of CDs, the amount of CDs, the concentration of methanol in BGS, the concentration of micelle in sample matrix, the concentration of phosphoric acid in BGS and the sample injection time were optimized. The optimized electrophoretic condition was obtained as following: 50 mM α-CD, 20 mM SDS in sample matrix., 80 mM PA and 20% MeOH (v/v) in BGS, sample solution by electrokinetic injection at -10 kV for 80 s. Under the optimized conditions described above, the linear range was 0.1-20 ug/mL with a good linear relationship with a correlation coefficient (r) ≥ 0.9985. The SEFs for the propazine, atrazine, simazine were found to be 123, 85 and 62 respectively. The proposed MCDS-MEEKC method provided an efficient method for trace analysis of triazine herbicides in honey and dendrobium officinale samples.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Capilar Eletrocinética Micelar , Ciclodextrinas/química , Herbicidas/isolamento & purificação , Micelas , Triazinas/isolamento & purificação , Atrazina , Ácidos Fosfóricos , Simazina
3.
J Chromatogr A ; 1628: 461439, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822979

RESUMO

Numerous structurally different amides and imides including succinimide derivatives exhibit diverse bioactive potential. The development of new compounds requires rationalization in the design in order to provide structural changes that guarantee favorable physico-chemical properties, pharmacological activity and safety. In the present research, a comprehensive study with comparison of the chromatographic lipophilicity and other physico-chemical properties of five groups of 1-arylsuccinimide derivatives was conducted. The chemometric analysis of their physico-chemical properties was carried out by using unsupervised (hierarchical cluster analysis and principal component analysis) and supervised pattern recognition methods (linear discriminant analysis), while the correlations between the in silico molecular features and chromatographic lipophilicity were examined applying linear and non-linear Quantitative Structure-Retention Relationship (QSRR) approaches. The main aim of the conducted research was to determine similarities and dissimilarities among the studied 1-arylsuccinimides, to point out the molecular features which have significant influence on their lipophilicity, as well as to establish high-quality QSRR models which can be used in prediction of chromatographic lipophilicity of structurally similar 1-arylsuccinimides. This study is a continuation of analysis and determination of the physico-chemical properties of 1-arylsuccinimides which could be important guidelines in further in vitro and eventually in vivo studies of their biological potential.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia em Camada Delgada , Relação Quantitativa Estrutura-Atividade , Solventes/química , Succinimidas/química , Análise por Conglomerados , Simulação por Computador , Análise de Componente Principal
4.
J Chromatogr A ; 1628: 461442, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822981

RESUMO

An analytical method for the quantification of thirty-three perfluoroalkyl and polyfluoroalkyl substances (PFASs) in animal liver was developed applying the isotopic dilution methodology with twenty-one labelled isotopologues of native compounds. The proposed protocol involved the determination of short and long aliphatic chain PFASs (C4C18) extracting liver with acetonitrile followed by two clean-up steps. The instrumental analysis was performed with liquid chromatography coupled to high-resolution mass spectrometry. The acquisition method combined full MS/dd-MS2, t-SIM/dd-MS2 and SIM experiments with variable resolution in order to maximize in one chromatographic run accuracy, sensitivity and selectivity. An eight-level validation study was performed evaluating linearity, trueness, precision, quantification and detection limits. Trueness was from 94 to 126% with intra-laboratory reproducibility lower than 20%. Limits of quantification were in the range 2-100 pg g-1, except for 2,3,3,3-tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)-propanoic acid, HFPO-DA (500 pg g-1). The analysis of a certified reference material (IRMM-427) and participation in a proficiency test scheme (FAPAS - 0687) confirmed these satisfactory performances. Finally, the application of the developed procedure to detect PFASs in sixteen liver samples of farm animals revealed that chicken was the less contaminated species.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Fluorcarbonetos/análise , Fígado/química , Espectrometria de Massas em Tandem , Animais , Reprodutibilidade dos Testes
5.
J Chromatogr A ; 1628: 461443, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822982

RESUMO

Sodium dodecyl sulfate (SDS) in proteomics samples needs to be removed and estimated prior to mass spectrometry (MS)-based analysis and to avoid MS ion-source contamination. Here, we describe an organic solvent free method to remove SDS using a simple apparatus that mainly consists of an agarose gel inside a 1 mL plastic micropipette tip and a voltage power supply with electrodes. A small volume of sample (e.g., 50 µL) is loaded on top of the gel and then voltage (cathode at the sample side) is applied with an acidic solution at the other end of the micropipette tip. Within 25 min, SDS was removed (e.g., ≥99% SDS in 3.5 mM SDS) and the peptides were retained in the sample solution. The strategy was compared to the commercially available and expensive Pierce spin column for the removal of SDS and recovery of peptides from a digested bovine serum albumin sample.


Assuntos
Técnicas de Química Analítica/métodos , Técnicas Eletroquímicas , Proteômica/métodos , Dodecilsulfato de Sódio/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Peptídeos/química , Soroalbumina Bovina/química , Dodecilsulfato de Sódio/química
6.
J Chromatogr A ; 1628: 461447, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822986

RESUMO

Waterfowl populations have been decreasing in Europe for the last years and pollution appears to be one of the main factors. This study was conducted to develop a single sensitive and robust analytical method for the monitoring of 2 fungicides, 15 herbicides, 3 insecticides and 24 transformation products in wild bird eggs. One of the major challenges addressed was the characterization of chemicals with large logP range (from -1.9 to 4.8). A total of 11 different extraction parameters were tested in triplicate to optimize the extraction protocol, on generic parameters, buffer addition and use of clean-up steps. Quantification was based on matrix-match approach with hen eggs as reference matrix (34 analytes with r²>0.99). Particular attention was payed to matrix effects (-28% on average), quantification limits (0.5 to 25 ng.g-1 dry mass / 0.2 to 7.5 ng.g-1 fresh mass) and extraction yields (46 to 87% with 25 analytes up to 70%) to ensure the relevance of the method and its compatibility with ultra-trace analysis. It led to a simple solid/liquid low temperature partitioning extraction method followed by LC-MS/MS. Analysis of 29 field samples from 3 waterfowl species revealed that eggs were slightly contaminated with pesticides as only one egg presented a contamination (terbutryn, herbicide, 0.7 ng.g-1) and confirmed the relevance of the method.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida , Ovos/análise , Praguicidas/análise , Espectrometria de Massas em Tandem , Animais , Aves , Poluentes Ambientais/análise , Europa (Continente) , Praguicidas/química
7.
J Chromatogr A ; 1627: 461414, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823112

RESUMO

Various techniques have been evaluated for the extraction and cleanup of pesticides from environmental samples. In this work, a Selective Pressurized Liquid Extraction (SPLE) method for pesticides was developed using a Thermo Fisher Scientific Accelerated Solvent Extraction (ASE) system. This instrument was compared to the newly introduced (2017) extraction instrument, the Energized Dispersive Guided Extraction (EDGE) system, which combines Pressurized Liquid Extraction (PLE) and dispersive Solid Phase Extraction (dSPE). We first optimized the SPLE method using the ASE instrument for pesticide extraction from alfalfa leaves using layers of Florisil and graphitized carbon black (GCB) downstream of the leaf homogenate in the extraction cell (Layered ASE method). We then compared results obtained for alfalfa and citrus leaves with the Layered ASE method to those from a method in which the leaf homogenate and sorbents were mixed (Mixed ASE method) and to similar methods modified for use with EDGE (Layered EDGE and Mixed EDGE methods). The ASE and EDGE methods led to clear, colorless extracts with low residual lipid weight. No significant differences in residual lipid masses were observed between the methods. The UV-Vis spectra showed that Florisil removed a significant quantity of the light-absorbing chemicals, but that GCB was required to produce colorless extracts. Recoveries of spiked analytes into leaf homogenates were generally similar among methods, but in several cases, significantly higher recoveries were observed in ASE extracts. Nonetheless, no significant differences were observed among pesticide concentrations in field samples when calculated with the isotope dilution method in which labelled surrogates were added to samples before extraction. The extraction time with the ASE methods was ~45 minutes, which was ~4.5 times longer than with the EDGE methods. The EDGE methods used ~10 mL more solvent than the ASE methods. Based on these results, the EDGE is an acceptable extraction instrument and, for most compounds, the EDGE had a similar extraction efficiency to the ASE methods.


Assuntos
Técnicas de Química Analítica/métodos , Praguicidas/análise , Folhas de Planta/química , Solventes/química , Lipídeos/química , Medicago sativa/química , Resíduos de Praguicidas/análise , Extratos Vegetais/química , Espectrofotometria Ultravioleta
8.
J Chromatogr A ; 1622: 461096, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32359779

RESUMO

The main focus of the present research was the on-line coupling of two separation techniques, namely liquid chromatography (LC) and gas chromatography (GC). For such an analytical combination, a dedicated interface is required to remove solvent from the sample, leaving the latter in a sharp band at the head of the GC column. Considering such an objective, a lab-developed LC-GC interface is herein presented, based on the use of a six-port two-position valve and a programmed-temperature-vaporizing (PTV) injector. The PTV injector was derived from a commercial split/splitless injector body, heated using a resistance heating wire, and enabled a satisfactory recovery of low boiling compounds (≤ C13), working in the normal-phase mode. The lab-developed PTV injector allowed the use of a larger-volume liner (compared to the commercial one initially used), it being characterized by dimensions 95 mm length × 5.0 mm O.D. × 3.4 mm I.D. and a volume of 862 µL, thus facilitating the transfer of larger LC fractions. The developed system is fully automatized and controlled without the use of additional software. The interface was evaluated and used for the analysis of mineral oil saturated hydrocarbons in vegetable oils. Detection was carried out by using a flame ionization detector (FID), with quantification performed through external calibration, across the 5-1000 mg kg-1 range. The LC-GC-FID method linearity, limits of detection and quantification, accuracy and precision were measured. The resulting limits of detection and quantification values were 0.4 and 1.3 mg kg-1, respectively. The average accuracy at the 100 mg kg-1 level was 95.5% (ranging between 93.3 and 99.7%). Intra-day repeatability at levels of 5 and 100 mg kg-1 were 2.4% and 3.5%, respectively.


Assuntos
Técnicas de Química Analítica , Cromatografia Gasosa , Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Ionização de Chama , Gases/análise , Hidrocarbonetos/análise , Limite de Detecção , Óleo Mineral/análise , Óleos Vegetais/química , Reprodutibilidade dos Testes , Solventes/análise , Temperatura
9.
J Chromatogr A ; 1622: 461100, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32359780

RESUMO

The aim of the present investigation was application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the study of antisense oligonucleotides. The influence of several mobile phases, differing with the salt type, their concentration and pH value on the retention and the separation of antisense oligonucleotides has been examined for this purpose. Four different stationary phases were also applied including unmodified silica, silica modified with the use of sulfobetaine groups, polyhydroxy and aminopropyl groups. Such wide range of tested conditions has been useful in better understanding of the retention mechanism of tested compounds. The results obtained during this investigation indicated that greater retention, greater peaks symmetry, as well as more effective separation of oligonucleotides, were obtained for the zwitterionic stationary phase. Moreover, the optimization of tandem mass spectrometry parameters with the use of Central Composite Design was performed and different mobile phases were tested to choose that one, which provided the greatest antisense oligonucleotides peak areas in Multiple Reaction Monitoring mode and consequently, the greatest possible sensitivity. Hydrophilic interaction liquid chromatography was compared with the ion pair chromatography, commonly used in the analysis of oligonucleotides. Both techniques were compared in terms of selectivity of separation as well as the sensitivity of their determination. Obtained results proved that ion pair chromatography provided better results in terms of separation efficiency and peak areas in Multiple Reaction Monitoring for tested conditions. However, these results do not preclude application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the oligonucleotides analysis especially when a mobile phase without ion pair reagents is required.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Oligonucleotídeos Antissenso , Espectrometria de Massas em Tandem , Betaína/análogos & derivados , Betaína/química , Indicadores e Reagentes , Oligonucleotídeos Antissenso/isolamento & purificação , Oligonucleotídeos Antissenso/metabolismo , Dióxido de Silício/química
10.
J Chromatogr A ; 1622: 461094, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32362359

RESUMO

This work describes the development of a solid-phase extraction method capable of detecting common fracturing fluid additives in flowback and produced water with mass spectrometry. Dissolved organic carbon (DOC) was used as a bulk measurement to investigate the retentive capacity of seven sorbents and to determine a loading volume. Conductivity was used to determine rinse volume. Based on this, four sorbents (HLB, PPL, Carbon, and C-18) were selected for further investigation of their ability to recover common fluid additives. Enrichment factors were calculated for poly(ethylene) glycols (PEGs), PEG-amines, and their metabolites PEG-carboxylates and PEG-carboxylate-amines, poly(propylene) glycols (PPGs), and linear alkyl ethoxylates (LAEs). The sorbent HLB gave the greatest enrichment for all of these compounds, with an average of 8.0× for PEGs, 11.9× for PEG-amines, 4.9× for PEG-carboxylates, and 21.6× for LAEs, though enrichment was highly dependent on sample composition. The effect was more pronounced for higher molecular weight compounds and enabled detection of some compounds in saltier samples. Then, HLB was used to recover these additives from 1:200 and 1:1000 dilutions in groundwater, illustrating the ability of solid-phase extraction to detect these compounds at low levels (<100 ppb) and highlighting the utility of desalting. This method was used to identify ethoxylated amines in flowback and produced waters from across the country.


Assuntos
Técnicas de Química Analítica , Fraturamento Hidráulico , Extração em Fase Sólida , Águas Residuárias , Poluentes Químicos da Água , Carbono/análise , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Água Subterrânea/química , Espectrometria de Massas , Águas Residuárias/química , Poluentes Químicos da Água/química , Poluentes Químicos da Água/isolamento & purificação
11.
J Chromatogr A ; 1622: 461091, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32376022

RESUMO

Immobilized protein makes a profound impact on the development of assays for drug discovery, diagnosis and in vivo biological interaction analysis. Traditional methods are enormously challenged by the G-protein coupled receptor ascribed to the loss of receptor functions. We introduced a ß2-adrenergic receptor (ß2-AR) aptamer into the immobilization of the receptor. This was achieved by mixing the receptor conjugated silica gel with cell lysates containing the receptor. We found that the aptamer-directed method makes immobilized ß2-AR good stability in seven days and high specificity of ligand recognition at the subtype receptor level. Feasibility of the immobilized ß2-AR in drug-receptor interaction analysis was evaluated by injection amount-dependent method, nonlinear chromatography, and peak decay analysis. Salbutamol, methoxyphenamine, ephedrine hydrochloride, clorprenaline, tulobuterol, bambuterol, propranolol and ICI 118551 bound to the receptor through one type of binding sites. The association constants presented good agreement within the three methods but exhibited clear differences from the data by radio-ligand binding assay. Regarding these results, we concluded that the aptamer-directed method will probably become an alternative for reversible and site-specific immobilization of GPCRs directly from complex matrices; the immobilized receptor is qualitative for drug-receptor interaction analysis.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/isolamento & purificação , Agonistas Adrenérgicos beta/metabolismo , Sítios de Ligação , Interações Medicamentosas , Ligantes , Receptores Adrenérgicos beta 2/metabolismo
12.
J Chromatogr A ; 1622: 460895, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32408991

RESUMO

Baseline separation and analysis of multicomponent mixtures of closely related pharmaceuticals using single column selectivity can often be challenging, requiring the combination of orthogonal stationary and mobile phase methods to monitor all the species and optimize reaction outcomes. In recent years, two-dimensional liquid chromatography (2D-LC) has become a valuable tool for improving peak capacity and selectivity. Though powerful, standard 2D-LC instrumentation and software can often lead to tedious method development and has a requirement for very specific expertise that is poorly suited for a fast-paced industrial environment. In this regard, the introduction of an automated online 2D-LC setup that could screen multiple columns in both dimensions without manual intervention will undeniably serve to streamline column/mobile phase selection and secure the viability of 2D-LC as a mainstay instrument for industrial applications. Herein, we introduce and investigate a multicolumn online 2D-LC approach that simplifies column screening and method development dramatically. This setup incorporates 6-position column selection valve technology whose functionality enables us to combine multiple columns in the first and second dimensions. This strategy in conjunction with diode array detection (DAD) in both dimensions and mass spectrometry (MS) acquisition in the second dimension serves to explore different columns and mobile phases as a framework for screening targeted compounds in multicomponent mixtures without having to perform chromatographic purification. Multiple online heart cutting achiral RPLC - achiral RPLC and achiral RPLC - chiral RPLC coupled to DAD and ESI-MS methods combining several stationary phase selectivity in an automated fashion are successfully applied to the separation and analysis of complex mixtures of drug substances, where in many instances, traditional 1D-ultra-high performance liquid chromatography (UHPLC) fails or delivers sub-optimal results. This automated online multicolumn 2D-LC workflow enables rapid and efficient identification of column/eluent combinations, as well as sample analysis across multiple columns in both dimensions overnight with a single click.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão , Técnicas de Química Analítica/instrumentação , Sistemas On-Line , Preparações Farmacêuticas/química
13.
PLoS One ; 15(5): e0233350, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437441

RESUMO

BACKGROUND: Serum-ascites albumin gradient (SAAG) remains the most sensitive and specific marker for the differentiation of ascites due to portal hypertension from ascites due to other causes. SAAG has some limitations and may fail in selected conditions. Voltammetric analysis (VA) has been used for the detection of electroactive species of biological significance and has proven effective for detection infections in biological fluids. AIMS: In this study, we compared the accuracy of voltammetric analysis (VA) with that of SAAG to differentiate ascites due to portal hypertension from that having a different origin. METHODS: 80 ascites samples were obtained from patients undergoing paracentesis at the Campus Bio-Medico Hospital of Rome. VA was performed using the BIONOTE device. The ability of VA to discriminate ascitic fluid etiology and biochemical parameters was evaluated using Partial Least Square Discriminant Analysis (PLS-DA), with ten-fold cross-validations. RESULTS: Mean age was 68.6 years (SD 12.5), 58% were male. Ascites was secondary to only portal hypertension in 72.5% of cases (58 subjects) and it was secondary to a baseline neoplastic disease in 27.5% of cases (22 subjects). Compared to SAAG≥1.1, e-tongue predicted ascites from portal hypertension with a better accuracy (92.5% Vs 87.5%); sensitivity (98.3% Vs 94.8%); specificity (77.3% Vs 68.2%); predictive values (PPV 91.9% Vs 88.7% and NPV 94.4% Vs 83.3%). VA correctly classified ascites etiology in 57/58 (98.2%) of cases with portal hypertension and in 17/22 (77.2%) of cases with malignancy. Instead, VA showed poor predictive capacities towards total white blood count and polymorphonuclear cell count. CONCLUSIONS: According to this proof of concept study, VA qualifies as a promising low-cost and easy method to discriminate between ascites secondary to portal hypertension and ascites due to malignancy.


Assuntos
Ascite/diagnóstico , Ascite/etiologia , Técnicas Eletroquímicas/métodos , Hipertensão Portal/complicações , Hipertensão Portal/diagnóstico , Neoplasias/complicações , Neoplasias/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Albuminas/análise , Líquido Ascítico/química , Biomarcadores/análise , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/estatística & dados numéricos , Diagnóstico Diferencial , Técnicas Eletroquímicas/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reconhecimento Automatizado de Padrão , Estudo de Prova de Conceito , Albumina Sérica Humana/análise
14.
Phys Chem Chem Phys ; 22(16): 8461-8466, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32281996

RESUMO

A method for efficient prediction of the relative stability of a protein due to a single amino acid point mutation is presented. In this approach, we calculate the free energy change due to an arbitrary point mutation of a protein from a single MD trajectory of the wild type protein. The method is tested on 27 diverse protein systems with a total of 853 mutations and the calculated relative free energies show a generally good correlation with the experimental values (a correlation coefficient of 0.63). Comparison with the free energy perturbation (FEP) method and the recently developed machine learning methods on two different benchmark data sets shows that the current method is computationally efficient and also numerically reliable for predicting the changes in thermostability upon an arbitrary point mutation of a protein. A discussion is provided on how to further improve the accuracy of the method for the prediction of thermostability of proteins.


Assuntos
Técnicas de Química Analítica/métodos , Química Computacional , Mutação Puntual , Estabilidade Proteica , Proteínas/genética , Temperatura
15.
J Chromatogr A ; 1622: 461095, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32334852

RESUMO

A two-membrane electrodialytic carbonate eluent generator (EDG) for ion chromatography (IC) is described. It is in a sandwich-like configuration, in which the central eluent channel is spatially isolated from two outer regenerant channels by stacked cation exchange membranes (CEMs) and anion exchange membranes (AEMs). A platinum screen electrode is placed in each of two outer regenerant channels. The electrode at the CEMs side is set as an anode with respect to the electrode at the AEMs side being cathode. Potassium carbonate and/or bicarbonate solution is pumped into the regenerant channel as feed solution. The electromigration of carbonate and/or bicarbonate and potassium from feed solution respectively through AEMs and CEMs will form a gas-free eluent. With this configuration, ion transport behavior through AEMs was explored. The device demonstrated good reproducibility, as indicated by the relative standard deviation of retention time of less than 0.08%.


Assuntos
Carbonatos , Técnicas de Química Analítica , Cromatografia por Troca Iônica , Bicarbonatos/química , Carbonatos/química , Cátions/química , Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Cromatografia por Troca Iônica/instrumentação , Eletrodos , Potássio/química , Reprodutibilidade dos Testes
16.
J Chromatogr A ; 1622: 461093, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32340726

RESUMO

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia de Fase Reversa , Bases de Dados Factuais , Peptídeos/isolamento & purificação , Animais , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Insulina/química , Peptídeos/química , Análise de Componente Principal , Dióxido de Silício/química
17.
Poult Sci ; 99(3): 1267-1274, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32111304

RESUMO

The chicken major histocompatibility B complex (MHC-B) region is of great interest owing to its very strong association with resistance to many diseases. Variation in the MHC-B was initially identified by hemagglutination of red blood cells with specific alloantisera. New technologies, developed to identify variation in biological materials, have been applied to the chicken MHC. Protein variation encoded by the MHC genes was examined by immunoprecipitation and 2-dimensional gel electrophoresis. Increased availability of DNA probes, PCR, and sequencing resulted in the application of DNA-based methods for MHC detection. The chicken reference genome, completed in 2004, allowed further refinements in DNA methods that enabled more rapid examination of MHC variation and extended such analyses to include very diverse chicken populations. This review progresses from the inception of MHC-B identification to the present, describing multiple methods, plus their advantages and disadvantages.


Assuntos
Técnicas de Química Analítica/veterinária , Galinhas/genética , Complexo Principal de Histocompatibilidade/genética , Animais , Técnicas de Química Analítica/métodos
18.
Nat Protoc ; 15(3): 1132-1157, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32005983

RESUMO

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.


Assuntos
Técnicas de Química Analítica/métodos , Espectrometria de Massas/métodos , Proteínas/química , Tampões (Química) , Cromatografia em Gel
19.
J Vis Exp ; (156)2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32090995

RESUMO

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%-20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.


Assuntos
Esclerose Amiotrófica Lateral/sangue , Esclerose Amiotrófica Lateral/diagnóstico , Plaquetas/metabolismo , Técnicas de Química Analítica/métodos , Proteínas de Ligação a DNA/sangue , Anticorpos/sangue , Anticorpos/imunologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Western Blotting/métodos , Proteínas de Ligação a DNA/metabolismo , Eletroforese Capilar/métodos , Humanos , Imunoensaio/métodos , Reprodutibilidade dos Testes
20.
J Chromatogr A ; 1619: 460918, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32008819

RESUMO

The anionic phospholipid class of cardiolipins (CL) is increasingly attracting scientific attention in the recent years. CL can be found as a functional component of mitochondrial membranes in almost all living organisms. Changes in the CL composition are favored by oxidative stress. Based on this finding, the investigation of CL and their oxidation products in relation to various disease patterns, including neurodegenerative ones, is moving into the focus of current research. The analysis of this diverse lipid class is still challenging and requires sensitive and selective methods. In this work, we demonstrate an online two-dimensional liquid chromatography (2D-LC) approach by means of a heart-cut setup. In the first dimension, a fast hydrophilic interaction liquid chromatography (HILIC) method was developed for the separation of CL and their oxidation products from other phospholipid classes, but more important from nonpolar lipid classes, such as triacylglycerol and cholesterol. Those classes can negatively affect the electrospray ionization and also the chromatography. For the heart-cut approach, the CL fraction was selectively transferred to a loop using a six-port valve followed by the transfer to a reversed phase (RP) column in second dimension. On the RP column, the transferred CL fraction including the oxidation products were separated according to the hydrophobicity of acyl chain moieties. Matrix effects were significantly reduced compared to the one-dimensional LC-MS method. In addition, the total separation time had not to be prolonged by shifting the equilibration step of the RP column parallel to the separation in first dimension. The heart-cut LC-LC approach was applied to artificially oxidized lipid extracts of bovine heart and yeast by means of Fenton reaction. In summary, 42 species have been identified by high resolution mass spectrometry and database matching. 31 species thereof have been further characterized by MS/MS experiments.


Assuntos
Cardiolipinas/análise , Técnicas de Química Analítica/métodos , Cromatografia Líquida , Espectrometria de Massas em Tandem , Animais , Bovinos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Fosfolipídeos/análise
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