Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21.645
Filtrar
1.
Nat Commun ; 13(1): 108, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013165

RESUMO

Biomolecular function is based on a complex hierarchy of molecular motions. While biophysical methods can reveal details of specific motions, a concept for the comprehensive description of molecular dynamics over a wide range of correlation times has been unattainable. Here, we report an approach to construct the dynamic landscape of biomolecules, which describes the aggregate influence of multiple motions acting on various timescales and on multiple positions in the molecule. To this end, we use 13C NMR relaxation and molecular dynamics simulation data for the characterization of fully hydrated palmitoyl-oleoyl-phosphatidylcholine bilayers. We combine dynamics detector methodology with a new frame analysis of motion that yields site-specific amplitudes of motion, separated both by type and timescale of motion. In this study, we show that this separation allows the detailed description of the dynamic landscape, which yields vast differences in motional amplitudes and correlation times depending on molecular position.


Assuntos
Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Tampões (Química) , Isótopos de Carbono , Espectroscopia de Ressonância Magnética , Membranas Artificiais , Movimento (Física) , Soluções
2.
Microbiol Spectr ; 9(2): e0068321, 2021 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-34668722

RESUMO

Antigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation. IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer's pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.


Assuntos
Antígenos Virais/análise , Teste Sorológico para COVID-19/métodos , Água Potável/virologia , Alimentos/virologia , SARS-CoV-2/isolamento & purificação , Tampões (Química) , COVID-19/diagnóstico , Comunicação , Reações Falso-Positivas , Humanos , SARS-CoV-2/imunologia
3.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361707

RESUMO

The mechanism by which proteins are solvated in hydrated ionic liquids remains an open question. Herein, the photoexcitation dynamics of photoactive yellow protein dissolved in hydrated choline dihydrogen phosphate (Hy[ch][dhp]) were studied by transient absorption and transient grating spectroscopy. The photocyclic reaction of the protein in Hy[ch][dhp] was similar to that observed in the buffer solution, as confirmed by transient absorption spectroscopy. However, the structural change of the protein during the photocycle in Hy[ch][dhp] was found to be different from that observed in the buffer solution. The known change in the diffusion coefficient of the protein was apparently suppressed in high concentrations of [ch][dhp], plausibly due to stabilization of the secondary structure.


Assuntos
Proteínas de Bactérias/química , Líquidos Iônicos/química , Fosforilcolina/química , Fotorreceptores Microbianos/química , Água/química , Tampões (Química) , Difusão , Luz , Solubilidade , Análise Espectral/métodos
4.
J Immunol Methods ; 498: 113125, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34450115

RESUMO

Food allergy prevalence is increasing worldwide, therefore there is a high demand for reliable tests to correctly diagnose this disease. Knowledge of proteins allergenicity and how they react both in the body and in diagnostic tests is necessary to adequately assess the potential immunogenicity of both natural foods and those produced through biotechnological processes. Thus, our aim was to analyze the factors that influence the protein extraction of foods in terms of, immunogenicity and immunoassays sensitivity. Peanut proteins were extracted using four distinct extraction buffers with different pH values (physiological saline, tris buffer, borate buffer with and without ß-mercaptoethanol), the protein concentration was determined by the Lowry method and polyacrylamide electrophoresis (SDS-PAGE) was used to compare the protein profile of each extract. The immunogenicity of each extract was verified by sensitizing two mouse strains (Balb/c and C57Bl/6) with a solution containing 100 µg of the extracted proteins and was determined by ELISA. Results show that extraction with the distinct buffers resulted in protein solutions with different yields and profiles. The immunogenicity of the different extracts also demonstrated distinct patterns that varied depending on the extraction methods, mouse strain and in vitro test. Immunoreactivity varied in accordance with the protein extract used to coat the microtitration plates. In conclusion, the protein profile in the extracts is critically influenced by the salt composition and pH of the extraction buffers, this in turn influences both in vivo immunogenicity and in vitro immunoreactivity.


Assuntos
Alérgenos/imunologia , Alérgenos/isolamento & purificação , Arachis/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/sangue , Hipersensibilidade a Amendoim/diagnóstico , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Alérgenos/administração & dosagem , Animais , Biomarcadores/sangue , Tampões (Química) , Fracionamento Químico , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Concentração de Íons de Hidrogênio , Injeções Subcutâneas , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Hipersensibilidade a Amendoim/imunologia , Proteínas de Plantas/administração & dosagem , Valor Preditivo dos Testes , Reprodutibilidade dos Testes
5.
Biomolecules ; 11(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34439803

RESUMO

Developing multifunctional systems for the biomimetic remineralization of human enamel is a challenging task, since hydroxyapatite (HAP) rod structures of tooth enamel are difficult to replicate artificially. The paper presents the first report on the simultaneous use of chitosan (CS) and agarose (A) in a biopolymer-based hydrogel for the biomimetic remineralization of an acid-etched native enamel surface during 4-10-day immersion in artificial saliva with or without (control group) fluoride. Scanning electron microscopy coupled with energy-dispersive X-ray spectrometry, Fourier transform infrared and Raman spectroscopies, X-ray diffraction, and microhardness tests were applied to investigate the properties of the acid-etched and remineralized dental enamel layers under A and CS-A hydrogels. The results show that all biomimetic epitaxial reconstructed layers consist mostly of a similar hierarchical HAP structure to the native enamel from nano- to microscale. An analogous Ca/P ratio (1.64) to natural tooth enamel and microhardness recovery of 77.4% of the enamel-like layer are obtained by a 7-day remineralization process in artificial saliva under CS-A hydrogels. The CS component reduced carbonation and moderated the formation of HAP nanorods in addition to providing an extracellular matrix to support growing enamel-like structures. Such activity lacked in samples exposed to A-hydrogel only. These data suggest the potential of the CS-A hydrogel in guiding the formation of hard tissues as dental enamel.


Assuntos
Materiais Biomiméticos/farmacologia , Quitosana/farmacologia , Esmalte Dentário/efeitos dos fármacos , Durapatita/química , Sefarose/farmacologia , Remineralização Dentária/métodos , Condicionamento Ácido do Dente/métodos , Materiais Biomiméticos/química , Tampões (Química) , Quitosana/química , Esmalte Dentário/fisiologia , Esmalte Dentário/ultraestrutura , Durapatita/metabolismo , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Teste de Materiais/métodos , Dente Molar/cirurgia , Saliva/química , Sefarose/química , Extração Dentária
6.
Crit Care ; 25(1): 314, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34461963

RESUMO

This article is one of ten reviews selected from the Annual Update in Intensive Care and Emergency Medicine 2021. Other selected articles can be found online at https://www.biomedcentral.com/collections/annualupdate2021 . Further information about the Annual Update in Intensive Care and Emergency Medicine is available from https://link.springer.com/bookseries/8901 .


Assuntos
Acidose/terapia , Terapia de Substituição Renal/normas , Bicarbonato de Sódio/uso terapêutico , Acidose/epidemiologia , Tampões (Química) , Humanos , Unidades de Terapia Intensiva/organização & administração , Unidades de Terapia Intensiva/estatística & dados numéricos , Terapia de Substituição Renal/instrumentação , Terapia de Substituição Renal/métodos
7.
J Immunol Methods ; 496: 113099, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34224737

RESUMO

Bispecific antibodies (BsAbs) are engineered to simultaneously bind two different antigens, and offer promising clinical outcomes for various diseases. The dual binding properties of BsAbs may enable superior efficacies and/or potencies compared to standard monoclonal antibodies (mAbs) or combination mAb therapies. Characterizing BsAb binding properties is critical during biotherapeutic development, where data is leveraged to predict efficacy and potency, assess critical quality attributes and improve antibody design. Traditional single-target, single-readout approaches (e.g., ELISA) have limited usefulness for interpreting complex bispecific binding, and double the benchwork. To address these deficiencies, we developed and implemented a new dual-target/readout binding assay that accurately dissects the affinities of both BsAb binding domains directly and simultaneously. This new assay uses AlphaPlex® technology, which eliminates traditional ELISA wash steps and can be miniaturized for automated workflows. The optimized BsAb AlphaPlex assay demonstrates 99-107% accuracy within a 50-150% linear range, and detected >50% binding degradation from photo- and thermal stress conditions. To the best of our knowledge, this is the first instance of a dual-target/readout BsAb AlphaPlex assay with GMP-suitable linear range, accuracy, specificity, and stability-indicating properties. As a highly customizable and efficient assay, BsAb AlphaPlex may be applicable to numerous bispecific formats and/or co-formulations against a variety of antigens beyond the clinical therapeutic space.


Assuntos
Anticorpos Biespecíficos/imunologia , Especificidade de Anticorpos , Antígenos/imunologia , Antígeno CTLA-4/imunologia , Imunoensaio , Receptor de Morte Celular Programada 1/imunologia , Anticorpos Biespecíficos/metabolismo , Complexo Antígeno-Anticorpo , Antígenos/metabolismo , Sítios de Ligação de Anticorpos , Tampões (Química) , Antígeno CTLA-4/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Valor Preditivo dos Testes , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes
8.
J Oleo Sci ; 70(7): 911-918, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34193668

RESUMO

Monoammonium glycyrrhizinate is produced by the neutralization of glycyrrhizic acid from plant licorice with ammonia. In this study, the physicochemical properties of aqueous monoammonium glycyrrhizinate were investigated from the viewpoint of surface chemistry. The structure of the amphiphilic molecule is bola type, comprising two glucuronic acid moieties having two carboxylic acids groups and an aglycone part having a carboxylic acid at the opposite end of the molecule from the glucuronic acids. We found that the physicochemical properties of aqueous monoammonium glycyrrhizinate are dependent on the ionization of the carboxylic acid groups. The solubility of monoammonium glycyrrhizinate gradually increased above pH 4 in the buffer solution. The critical micelle concentration (CMC) and surface tension at the CMC (γCMC) of monoammonium glycyrrhizinate were determined by the surface tension method to be 1.5 mmol L-1 and 50 mN m-1 in pH 5 buffer and 3.7 mmol L-1 and 51 mN m-1 in pH 6 buffer, respectively. The surface tension gradually decreased with increasing concentration of monoammonium glycyrrhizinate in the pH 7 buffer, but the CMC was not defined by the curve. Light scattering measurements also did not reveal a clear CMC in the pH 7 buffer. The ionization of the carboxylic acid groups in the bola-type amphiphilic molecule with increasing pH is disadvantageous for micelle formation. Cryo-transmission electron microscopy showed that monoammonium glycyrrhizinate forms rod-like micelles in pH 5 buffer, and small angle X-ray scattering experiments confirmed that the average micellar structure was rod-like in pH 5 buffer. Thus, it was found that monoammonium glycyrrhizinate can form micelles only in weakly acidic aqueous solutions.


Assuntos
Ácido Glicirrízico/química , Micelas , Tampões (Química) , Concentração de Íons de Hidrogênio , Solubilidade , Tensão Superficial
9.
J Synchrotron Radiat ; 28(Pt 4): 1237-1244, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34212889

RESUMO

During the COVID-19 pandemic, synchrotron beamlines were forced to limit user access. Performing routine measurements became a challenge. At the Life Science X-ray Scattering (LiX) beamline, new instrumentation and mail-in protocols have been developed to remove the access barrier to solution scattering measurements. Our efforts took advantage of existing instrumentation and coincided with the larger effort at NSLS-II to support remote measurements. Given the limited staff-user interaction for mail-in measurements, additional software tools have been developed to ensure data quality, to automate the adjustments in data processing, as users would otherwise rely on the experience of the beamline staff, and produce a summary of the initial assessments of the data. This report describes the details of these developments.


Assuntos
Espalhamento a Baixo Ângulo , Soluções/efeitos da radiação , Síncrotrons/instrumentação , Difração de Raios X/instrumentação , Tampões (Química) , COVID-19 , Coleta de Dados , Conjuntos de Dados como Assunto , Processamento Eletrônico de Dados , Pandemias , Robótica , SARS-CoV-2 , Software , Manejo de Espécimes , Água
10.
Elife ; 102021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34236318

RESUMO

Biomolecular condensates are formed by liquid-liquid phase separation (LLPS) of multivalent molecules. LLPS from a single ("homotypic") constituent is governed by buffering: above a threshold, free monomer concentration is clamped, with all added molecules entering the condensed phase. However, both experiment and theory demonstrate that buffering fails for the concentration dependence of multicomponent ("heterotypic") LLPS. Using network-free stochastic modeling, we demonstrate that LLPS can be described by the solubility product constant (Ksp): the product of free monomer concentrations, accounting for the ideal stoichiometries governed by the valencies, displays a threshold above which additional monomers are funneled into large clusters; this reduces to simple buffering for homotypic systems. The Ksp regulates the composition of the dilute phase for a wide range of valencies and stoichiometries. The role of Ksp is further supported by coarse-grained spatial particle simulations. Thus, the solubility product offers a general formulation for the concentration dependence of LLPS.


Assuntos
Fenômenos Bioquímicos , Transição de Fase , Biofísica , Tampões (Química) , Solubilidade
11.
Int J Pharm ; 605: 120857, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34229072

RESUMO

There are many hurdles in the development of generic formulations. In vitro biopredictive dissolution conditions together with alternative in vitro - in vivo relationship (IVIVR) approaches can be a powerful tool to support the development of such formulations. In this study, we hypothesized that the release profile of enteric coated (EC) formulations of pantoprazole in physiologically relevant bicarbonate buffer (BCB) would detect possible performance differences between test and reference formulations resulting in more accurate IVIVR results and predictability when compared to a pharmacopeial dissolution test. We correlated the in vitro performance of test and reference formulations (both in BCB and pharmacopeial phosphate buffer) with the in vivo data from a failed bioequivalence study. Test and reference formulations of EC pantoprazole tablets passed the USP dissolution criteria. However, they failed statistical similarity in vitro both in compendial and BCB. Bicarbonate buffer was additionally more discriminative while being more physiologically relevant. Having BCB as an additional test to evaluate EC products in vitro might improve the comparison of formulations. This can de-risk the development of generic EC formulations.


Assuntos
Química Farmacêutica , Tampões (Química) , Concentração de Íons de Hidrogênio , Pantoprazol , Solubilidade , Comprimidos , Comprimidos com Revestimento Entérico
12.
J Chromatogr A ; 1652: 462077, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34214832

RESUMO

Stepwise change between low and high salt concentration buffers of the same pH results in pH transition, the length of which was demonstrated to be proportional to the quantity of ion-exchange groups present on the matrix. In this work, we analyzed the effect of the ligand type, density, and buffer concentration on the pH transition shape for typical ion-exchange groups (QA, DEAE, SO3, and COOH) and ligands acting as metal-chelators, such as IDA, TAEA, and EDA. It was demonstrated that pH transition can occur either as a chromatographic or flat-top peak. pH transition peaks were evaluated by their length, height, and peak center parameters. While no parameter can describe the ligand density accurately with a single linear correlation for both peak types, all parameters can be used for the description of one peak type. Peak length and height exhibited the same accuracy, while their sensitivity depended on the pH transition shape: length being more sensitive for the flat-top peaks, while height for the chromatographic peaks. pH height can be obtained faster, at lower elution volume, and seems to be more suitable for the determination of low amounts of ligand, when typically chromatographic peak type pH transitions occur.


Assuntos
Técnicas de Química Analítica , Ligantes , Polímeros , Tampões (Química) , Quelantes/química , Técnicas de Química Analítica/métodos , Cromatografia Líquida , Emulsões/química , Concentração de Íons de Hidrogênio , Polímeros/química
13.
J Chromatogr A ; 1650: 462247, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34087520

RESUMO

The adsorptive loss of acidic analytes in liquid chromatography was investigated using metal frits. Repetitive injections of acidic small molecules or an oligonucleotide were made on individual 2.1 or 4.6 mm i.d. column frits. Losses were observed for adenosine 5'-(α,ß-methylene) diphosphate, 2-pyridinol 1-oxide and the 25-mer phosphorothioate oligonucleotide Trecovirsen (GEM91) on stainless steel and titanium frits. Analyte adsorption was greatest at acidic pH due to the positive charge on the metal oxide surface. Analyte recovery increased when a series of injections was performed; this effect is known as sample conditioning. Nearly complete recovery was achieved when the metal adsorptive sites were saturated with the analyte. A similar effect was achieved by conditioning the frits with phosphoric, citric or etidronic acids, or their buffered solutions. These procedures can be utilized to mitigate analyte loss. However, the effect is temporary, as the conditioning agent is gradually removed by the running mobile phase. Metal frits modified with hybrid organic/inorganic surface technology were shown to mitigate analyte-to-metal surface interactions and improve recovery of acidic analytes. Quantitative recovery of a 15-35 mer oligodeoxythymidine mixture was achieved using column hardware modified with hybrid surface technology, without a need for column conditioning prior to analysis.


Assuntos
Cromatografia Líquida , Metais , Adsorção , Tampões (Química) , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Ácido Cítrico/química , Ácido Etidrônico/química , Indicadores e Reagentes , Metais/química , Ácidos Fosfóricos/química , Aço Inoxidável/química , Propriedades de Superfície , Titânio/química
14.
J Chromatogr A ; 1651: 462294, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34098249

RESUMO

Few articles are reported for the simultaneous separation and sensitive detection of the kynurenine pathway (KP) metabolites. This work describes a capillary electrochromatography-mass spectrometry (CEC-MS) method using acrylamido-2-methyl-1-propanesulfonic acid (AMPS) functionalized stationary phase. The AMPS column was prepared by first performing silanization of bare silica with gamma-maps, followed by polymerization with AMPS. The CEC-MS/MS methods were established for six upstream and three downstream KP metabolites. The simultaneous separation of all nine KP metabolites is achieved without derivatization for the first time in the open literature. Numerous parameters such as pH and the concentration of background electrolyte, the concentration of the polymerizable AMPS monomer, column length, field strength, and internal pressure were all tested to optimize the separation of multiple KP metabolites. A baseline separation of six upstream metabolites, namely tryptophan (TRP), kynurenine (KYN), 3-hydroxykynurenine (HKYN), kynurenic acid (KA), anthranilic acid (AA), and xanthurenic acid (XA), was possible at pH 9.25 within 26 min. Separation of six downstream and related metabolites, namely: tryptamine (TRPM), hydroxy­tryptophan (HTRP), hydroxyindole-3 acetic acid (HIAA), 3-hydroxyanthranilic acid (3-HAA), picolinic acid (PA), and quinolinic acid (QA), was achieved at pH 9.75 in 30 min. However, the challenging simultaneous separation of all nine KP metabolites was only accomplished by increasing the column length and simultaneous application of internal pressure and voltage in 114 min. Quantitation of KP metabolites in commercial human plasma was carried out, and endogenous concentration of five KP metabolites was validated. The experimental limit of quantitation ranges from 100 to 10,000 nM (S/N = 8-832, respectively), whereas the experimental limit of detection ranges from 31 to 1000 nM (S/N = 2-16, respectively). Levels of five major KP metabolites, namely TRP, KYN, KA, AA, and QA, and their ratios in patient plasma samples previously screened for inflammatory biomarkers [C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α)] was measured. Pairs of the level of metabolites with significant positive correlation were statistically evaluated.


Assuntos
Eletrocromatografia Capilar/métodos , Cinurenina/metabolismo , Redes e Vias Metabólicas , Metaboloma , Ácidos Alcanossulfônicos/química , Tampões (Química) , Calibragem , Eletricidade , Humanos , Concentração de Íons de Hidrogênio , Cinurenina/sangue , Limite de Detecção , Modelos Lineares , Pressão , Ácido Quinolínico/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos
15.
J Chromatogr A ; 1651: 462257, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34090057

RESUMO

This study assesses the potential of using ionic liquids (ILs) as mobile phase additives to control the retention mechanism of four cytostatic drugs: doxorubicin hydrochloride (DOX), epirubicin hydrochloride (EPI), daunorubicin hydrochloride (DAU) and idarubicin hydrochloride (IDA). Chromatographic separations were performed on a C18 analytical column (Discovery C18 150 × 4.6 mm, 5 µm) using six IL anions and four methyl-substituted IL cations with different alkyl chain lengths (alone or with the additional methyl group on the aromatic ring), or with an allyl group added as a cationic substituent. Thus, a total of 17 different ILs were assessed. The aqueous formic acid solution and phosphate buffer were used to compare how mobile phase composition affected the behavior of the analyzed cytostatic agents in the presence of ILs. In addition, the impacts of IL concentration, phosphate buffer concentration, and phosphate buffer pH on the final results were also considered. The ability to change analyte retention without negatively impacting peak shape or analytical efficiency was also controlled via the tailing factor and number of theoretical plates. Based on the results, the tested ILs were classified as either effective or ineffective mobile phase additives for separation of anthracyclines and identification by LC-FL technique.


Assuntos
Cromatografia Líquida/métodos , Citostáticos/análise , Líquidos Iônicos/química , Dióxido de Silício/química , Ânions , Antraciclinas/análise , Tampões (Química) , Cátions , Cromatografia de Fase Reversa/métodos , Fosfatos/química , Fatores de Tempo
16.
PLoS One ; 16(6): e0253297, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34133472

RESUMO

Infectious salmon anaemia virus (ISAV) is the cause of an important waterborne disease of farmed Atlantic salmon. Detection of virus in water samples may constitute an alternative method to sacrificing fish for surveillance of fish populations for the presence of ISA-virus. We aimed to evaluate different membrane filters and buffers for concentration and recovery of ISAV in seawater, prior to molecular detection. One litre each of artificial and natural seawater was spiked with ISAV, followed by concentration with different filters and subsequent elution with different buffers. The negatively charged MF hydrophilic membrane filter, combined with NucliSENS® lysis buffer, presented the highest ISAV recovery percentages with 12.5 ± 1.3% by RT-qPCR and 31.7 ± 10.7% by RT-ddPCR. For the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter, combined with NucliSENS® lysis buffer, the ISAV recovery percentages were 3.4 ± 0.1% by RT-qPCR and 10.8 ± 14.2% by RT-ddPCR. The limits of quantification (LOQ) were estimated to be 2.2 x 103 ISAV copies/L of natural seawater for both RT-qPCR and RT-ddPCR. The ISAV concentration method was more efficient in natural seawater.


Assuntos
Filtração/métodos , Doenças dos Peixes/virologia , Isavirus , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Água do Mar/virologia , Animais , Tampões (Química) , Filtração/instrumentação , Doenças dos Peixes/prevenção & controle , Membranas Artificiais , Infecções por Orthomyxoviridae/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmo salar/virologia
17.
Ultrason Sonochem ; 76: 105609, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34157567

RESUMO

A novel method of thermoultrasound-assisted plasma functionalized buffer (PFB) for decontaminating grass carp was evaluated using the Box-Behnken design (BBD) with processing variables including PFB generating voltage (PV), ultrasound treatment time (UT) and temperature (TP). The predicted models were found to be significant (p < 0.05) and displayed sufficient fitness with experimental data as indicated by non-significant (p > 0.05) lack of fit and high coefficient of determination (R2≥0.97) values. The optimum decontamination conditions for the responses of S. putrefaciens and S. Typhimurium were PV of 66 V, UT of 14.90 min and TP of 60 ℃, achieving reductions of 4.40 and 3.97 log CFU/g, respectively, with a desirability of 0.998. Among the variables, temperature presented higher significance for inactivating bacteria and the production of volatile basic nitrogen and lipid peroxidation under the optimized conditions were within the limits of freshness for grass carp. Additionally, the effects of PFB and the optimized thermoultrasound-assisted PFB decontamination were mild on the microstructure of grass carp with slight ruptures and loose myofibril structures, indicating the potential of thermoultrasound-assisted PFB for seafood products decontamination with reduced processing time.


Assuntos
Carpas/microbiologia , Gases em Plasma/química , Gases em Plasma/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Shewanella putrefaciens/efeitos dos fármacos , Temperatura , Ondas Ultrassônicas , Animais , Tampões (Química) , Microbiologia de Alimentos , Viabilidade Microbiana/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Shewanella putrefaciens/fisiologia
18.
Sci Rep ; 11(1): 13193, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162990

RESUMO

We report the development of a lab-on-a-chip system, that facilitates coupled dielectrophoretic detection (DEP-D) and impedimetric counting (IM-C), for investigating drug resistance in K562 and CCRF-CEM leukemia cells without (immuno) labeling. Two IM-C units were placed upstream and downstream of the DEP-D unit for enumeration, respectively, before and after the cells were treated in DEP-D unit, where the difference in cell count gave the total number of trapped cells based on their DEP characteristics. Conductivity of the running buffer was matched the conductivity of cytoplasm of wild type K562 and CCRF-CEM cells. Results showed that DEP responses of drug resistant and wild type K562 cells were statistically discriminative (at p = 0.05 level) at 200 mS/m buffer conductivity and at 8.6 MHz working frequency of DEP-D unit. For CCRF-CEM cells, conductivity and frequency values were 160 mS/m and 6.2 MHz, respectively. Our approach enabled discrimination of resistant cells in a group by setting up a threshold provided by the conductivity of running buffer. Subsequent selection of drug resistant cells can be applied to investigate variations in gene expressions and occurrence of mutations related to drug resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Tampões (Química) , Linhagem Celular Tumoral , Condutividade Elétrica , Impedância Elétrica , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Desenho de Equipamento , Humanos , Concentração Inibidora 50 , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Técnicas Analíticas Microfluídicas/instrumentação , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia
19.
J Chromatogr A ; 1651: 462298, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34111678

RESUMO

In this work, novel stationary phase coatings by zeolite SiO2NPs coupled with ß-cyclodextrin (ß-CD) or ß-CD/L-phenylalanine were developed for chiral open-tubular capillary electrochromatography (OT-CEC). The OT columns were prepared taking advantage of the strong adhesion of polydopamine in one-step method. Scanning electron micrography and electroosmotic flow were used to characterize the prepared single/dual-selector OT columns. Chiral separation of four chiral analytes (catechin/epicatechin, ephedrine/pseudoephedrine, ritodrine and salbutamol) was carried out in order to evaluate the performance of the prepared columns in OT-CEC with amperometric detection system. In terms of migration time, peak area, resolution, and selectivity factor of catechin/epicatechin and salbutamol, the run-to-run, day-to-day, and column-to-column repeatability were within 8.9%. Under the optimum conditions, the developed methods were applied for the analyses of Chinese herbal medicine Catechu herbs and salbutamol aerosol samples.


Assuntos
Eletrocromatografia Capilar/métodos , Nanopartículas/química , Dióxido de Silício/química , Zeolitas/química , Tampões (Química) , Concentração de Íons de Hidrogênio , Estereoisomerismo , beta-Ciclodextrinas/química
20.
Appl Biochem Biotechnol ; 193(9): 2843-2857, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34019251

RESUMO

Tris is an extensively used buffer that presents a primary amine group on its structure. In the present work trypsin, chymotrypsin and penicillin G acylase (PGA) were immobilized/stabilized on glyoxyl agarose in presence of different concentrations of Tris (from 0 to 20 mM). The effects of the presence of Tris during immobilization were studied analyzing the thermal stability of the obtained immobilized biocatalysts. The results indicate a reduction of the enzyme stability when immobilized in the presence of Tris. This effect can be observed in inactivations carried out at pH 5, 7, and 9 with all the enzymes assayed. The reduction of enzyme stability increased with the Tris concentration. Another interesting result is that the stability reduction was more noticeable for immobilized PGA than in the other immobilized enzymes, the biocatalysts prepared in presence of 20 mM Tris lost totally the activity at pH 7 just after 1 h of inactivation, while the reference at this time still kept around 61 % of the residual activity. These differences are most likely due to the homogeneous distribution of the Lys groups in PGA compared to trypsin and chymotrypsin (where almost 50% of Lys group are in a small percentage of the protein surface). The results suggest that Tris could be affecting the multipoint covalent immobilization in two different ways, on one hand, reducing the number of available glyoxyl groups of the support during immobilization, and on the other hand, generating some steric hindrances that difficult the formation of covalent bonds.


Assuntos
Enzimas Imobilizadas/química , Glioxilatos/química , Penicilina Amidase/química , Sefarose/química , Trometamina/química , Tripsina/química , Tampões (Química) , Estabilidade Enzimática , Concentração de Íons de Hidrogênio
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...