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1.
Sci Rep ; 10(1): 16608, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024174

RESUMO

The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Coronavirus/virologia , Testes Diagnósticos de Rotina , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Pandemias , Pneumonia Viral/virologia , Kit de Reagentes para Diagnóstico/provisão & distribução
2.
Exp Parasitol ; 218: 107984, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32871143

RESUMO

The fascioliasis is a parasitic disease of importance in veterinary medicine and public health. For this parasitosis, the treatment by synthetic fasciolicides is used and due to their intense use although they have been shown less effective because of the establishment of resistant Fasciola hepatica population to these drugs, with a global concern. The use of derived products of plants with biological activity has been shown promising in the control of parasites. In this context, we evaluated the chemical composition and action of ovicidal in vitro fixed oil of Helianthus annuus L. (FOH) and essential oil of Cuminum cyminum L. (EOC), as well as their combination (FOH + EOC) of F. hepatica. In the assay in vitro of F. hepatica were submitted to different concentrations of oils, such as FOH (2.3 mg/mL + 0,017 mg/mL); EOC (2.07 mg/mL + 0,004 mg/mL) and the combination of (1.15 mg/mL + 1.03 mg/mL to 0,0085 mg/mL + 0,008 mg/mL) as well as a positive control of thiabendazole (0.025 mg/mL) and a negative control with distilled water and tween. The identification of the majority chemical compounds was performed by gas chromatography. The -cell viability of the oils was tested in MDBK cellular line by the MTT method. The majority compounds in the FOH were the linoleic (53.6%) and oleic (35.85%) unsaturated fatty acids, and the majority phytochemicals compounds in the EOC were the Cumaldehyde (26.8%) and the 2-Caren 10-al (22.17%). The EOC and the combination presented effectiveness of 99% (±1) and of 94% (±1) in the concentration of 0.03 mg/mL and 0.035 mg/mL+0.03 mg/mL, respectively, and the FOH was insufficiently active as ovicidal. The cell viability at this concentration of EOC was 93%. From the results above we could infer that the EOC is promising as a new alternative for the fascioliasis control.


Assuntos
Cuminum/química , Fasciola hepatica/efeitos dos fármacos , Helianthus/química , Óleos Voláteis/farmacologia , Óleos Vegetais/farmacologia , Análise de Variância , Animais , Anti-Helmínticos/farmacologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Gasosa , Cães , Combinação de Medicamentos , Indicadores e Reagentes , Fígado/parasitologia , Células Madin Darby de Rim Canino/efeitos dos fármacos , Óleos Voláteis/química , Óvulo/efeitos dos fármacos , Óleos Vegetais/química , Sais de Tetrazólio , Tiabendazol/farmacologia
5.
Nature ; 585(7826): 530-537, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32968259

RESUMO

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature1,2. Although synthetic protein functionalization strategies allow mimicry of PTMs3,4, as well as formation of unnatural protein variants with diverse potential functions, including drug carrying5, tracking, imaging6 and partner crosslinking7, the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C• radicals that form the native (ß-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C• radicals that form equivalent ß-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF2-labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized 'alkylator proteins' with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function.


Assuntos
Luz , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteínas/química , Proteínas/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Sítios de Ligação , Carbono/química , Carbono/metabolismo , Enzimas/química , Enzimas/metabolismo , Ésteres/síntese química , Ésteres/química , Células HeLa , Humanos , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/metabolismo , Indicadores e Reagentes/química , Oxirredução , Processos Fotoquímicos/efeitos da radiação , Domínios e Motivos de Interação entre Proteínas
6.
Yakugaku Zasshi ; 140(8): 1063-1069, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741864

RESUMO

Quantitative NMR (qNMR) has been developed as an absolute quantitation method to determine the purity or content of organic compounds including marker compounds in crude drugs. The "qNMR test" has been introduced into the crude-drug section of the Japanese Pharmacopoeia (JP) for determining the purity of reagents used for the assay in the JP. In Supplement II to the JP 17th edition published in June 2019, fifteen compounds adopted qNMR test were listed as the reagents for the assay. To establish the "qNMR test" in the crude drug section of the JP, there were several problems to be solved. Previously, we reported that the handling impurity signals from reference substances and targeted marker compounds, chemical shifts of reference substances, and peak unity of signals of targeted marker compounds are important factors to conduct qNMR measurements with intended accuracy. In this study, we investigated that the hygroscopicity of reagents could cause the changes in the compounds' purity depending on increasing their water content. Twenty-one standard products used for the crude-drug test in JP were examined by water sorption-desorption analysis, and ginsenosides and saikosaponins were found to be hygroscopic. To prepare a sample solution of saikosaponin b2 for qNMR analysis, samples need to be maintained for 18 h at 25°C and 76% relative humidity; further, samples need to be weighed at the same humidity for the qNMR analysis.


Assuntos
Contaminação de Medicamentos/prevenção & controle , Higroscópicos/química , Higroscópicos/normas , Indicadores e Reagentes/normas , Espectroscopia de Ressonância Magnética/métodos , Farmacopeias como Assunto/normas , Ginsenosídeos/química , Ginsenosídeos/normas , Umidade , Japão , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Ácido Oleanólico/normas , Psicoterapia Breve , Saponinas/química , Saponinas/normas , Temperatura , Água/análise
7.
J Chromatogr A ; 1626: 461324, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797817

RESUMO

Sorption of PFASs onto surfaces of laboratory materials has been frequently reported. Due to the often complex and poorly understood nature of such sorption, workarounds have often included use of whole samples only, accompanied by sample vessel rinsing to desorb active surfaces. The resulting methods tend to require considerable sample preparation times and preclude typical activities such as aliquoting and dilution of water samples prior to extraction. This manuscript reports an approach for PFAS analysis which uses subsampling of water matrices from vessels including centrifuge tubes and autosampler vials, through the optimized use of solvent to reduce PFAS retention on subsampling vessels. Online solid phase extraction (SPE) using a weak anion exchange resin is then used to concentrate sample aliquots to improve sensitivity and allow for removal of matrix interferences. With the technique of ultra performance liquid chromatography (UPLC) coupled to isotope dilution tandem mass spectrometry, statistically based quantitation limits ranged from sub ng/L to single digit ng/L for carboxylate, sulfonate, and sulfonamide PFASs analytes from C4 to C12. Linear calibration ranges were from 0.25 to 4000 ng/L. Matrix effects relevant for drinking water treatment studies, such as cations, organic carbon, and competing PFAS compounds, were evaluated and found to not impact method performance within QC criteria consistent with study data quality objectives.


Assuntos
Fluorcarbonetos/análise , Indicadores e Reagentes/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão , Fluorcarbonetos/isolamento & purificação , Água Doce/análise , Indicadores e Reagentes/isolamento & purificação , Marcação por Isótopo , Sais/química , Extração em Fase Sólida , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/isolamento & purificação
8.
Nat Commun ; 11(1): 4170, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820174

RESUMO

Sulfur-sulfur motifs widely occur in vital function and drug design, which yearns for polysulfide construction in an efficient manner. However, it is a great challenge to install desired functional groups on both sides of sulfur-sulfur bonds at liberty. Herein, we designed a mesocyclic bilateral disulfurating reagent for sequential assembly and modular installation of polysulfides. Based on S-O bond dissociation energy imparity (mesocyclic compared to linear imparity is at least 5.34 kcal mol-1 higher), diverse types of functional molecules can be bridged via sulfur-sulfur bonds distinctly. With these stable reagents, excellent reactivities with nucleophiles including C, N and S are comprehensively demonstrated, sequentially installing on both sides of sulfur-sulfur motif with various substituents to afford six species of unsymmetrical polysulfides including di-, tri- and even tetra-sulfides. Life-related molecules, natural products and pharmaceuticals can be successively cross-linked with sulfur-sulfur bond. Remarkably, the cyclization of tri- and tetra-peptides affords 15- and 18-membered cyclic disulfide peptides with this reagent, respectively.


Assuntos
Dissulfetos/química , Indicadores e Reagentes/química , Peptídeos/química , Sulfetos/química , Enxofre/química , Produtos Biológicos/química , Técnicas de Química Sintética/métodos , Ciclização , Indicadores e Reagentes/síntese química , Modelos Químicos , Estrutura Molecular , Oxirredução , Preparações Farmacêuticas/química
10.
J Clin Microbiol ; 58(11)2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-32839250

RESUMO

The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.


Assuntos
Betacoronavirus/efeitos dos fármacos , Indicadores e Reagentes/farmacologia , Inativação de Vírus/efeitos dos fármacos , Animais , Betacoronavirus/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Filtração/instrumentação , Humanos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Células Vero
11.
Lab Invest ; 100(11): 1485-1489, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32647285

RESUMO

Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes.


Assuntos
Betacoronavirus/isolamento & purificação , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Feminino , Humanos , Indicadores e Reagentes , Rim/virologia , Pulmão/virologia , Inclusão em Parafina , Placenta/virologia , Gravidez
12.
Nat Commun ; 11(1): 2756, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488003

RESUMO

Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z'.


Assuntos
Etanol/análogos & derivados , Etanol/química , Compostos de Organossilício/química , Trifluoretanol/química , Álcoois/química , Alquilação , Técnicas de Química Sintética , Indicadores e Reagentes/química , Estrutura Molecular
13.
Mol Cell Proteomics ; 19(9): 1503-1522, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32591346

RESUMO

As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC-MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/genética , Interações Hospedeiro-Patógeno/genética , Pneumonia Viral/genética , Proteômica/métodos , Proteínas Virais/genética , Células A549 , Sequência de Aminoácidos , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Betacoronavirus/patogenicidade , Células CACO-2 , Estudos de Casos e Controles , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Indicadores e Reagentes , Anotação de Sequência Molecular , Fases de Leitura Aberta , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Proteômica/instrumentação , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Transdução de Sinais , Células Vero , Proteínas Virais/classificação , Proteínas Virais/metabolismo , Internalização do Vírus
14.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 664-672, May-June, 2020. tab
Artigo em Português | LILACS, VETINDEX | ID: biblio-1128613

RESUMO

Objetivou-se avaliar a condição metabólica e estrutural das células espermáticas bovinas após congelação, com adição prévia de IGF-I e insulina no meio diluidor seminal. Os ejaculados de seis touros Nelore foram submetidos a quatro tratamentos: controle; insulina (100µUI/mL); IGF-I (150ng/mL) e insulina + IGF-I (50µUI/mL e 75ng/mL, respectivamente). Após a congelação, realizaram-se os testes de termorresistência rápida, coloração pelo corante azul de tripan e Giemsa, além da análise computadorizada da motilidade espermática, da integridade das membranas plasmática e acrossomal, e da peça intermediária por meio de sondas fluorescentes. O teste de termorresistência rápida apresentou efeito dentro do tempo de cada tratamento, mas não entre os tratamentos. Na análise computadorizada da motilidade espermática, foram observados movimento, motilidade e velocidade espermáticos; não houve efeitos dos tratamentos sobre qualquer uma dessas variáveis. Respostas iguais foram obtidas com as sondas fluorescentes e o corante azul de tripan/Giemsa. A adição de insulina e IGF-I, de forma isolada ou combinada, ao meio diluidor para congelação de sêmen não produziu efeitos sobre as condições metabólica e estrutural das células espermáticas.(AU)


This study aimed to evaluate the metabolic and structural condition of the spermatic bovine cells after the freezing, with addition, previously, of IGF-I and Insulin in the seminal thinner medium. The semen of 6 Nellore bulls were submitted to four treatments: Control, Insulin (100µUI/mL); IGF-I (150ng/mL) and Insulin + IGF-I (50µUI/mL and 75ng/mL, respectively). After freezing, rapid resistance tests, Tripan and Giemsa Blue staining, and computerized analysis of sperm motility and integrity of the plasma and acrosomal membranes and the intermediate part were performed by fluorescent probes. The term rapid resistance test had effect within the time of each treatment, but not between treatments. In the computer analysis of sperm motility, sperm movement, motility and velocity no effects of treatments were observed on any of these variables. The same results were obtained with the fluorescent probes and the Blue dye Trypan / Giemsa. The addition of Insulin and IGF-I, alone or in combination, to the semen freezing dilution medium had no effect on the metabolic and structural condition of sperm cells.(AU)


Assuntos
Sêmen/metabolismo , Fator de Crescimento Insulin-Like I/administração & dosagem , Criopreservação/veterinária , Insulina/administração & dosagem , Bovinos , Indicadores e Reagentes
15.
Yakugaku Zasshi ; 140(6): 809-818, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32475931

RESUMO

Standard analytical methods for the detection of dieldrin and 4,6-dichloro-7-(2,4,5-trichlorophenoxy)-2-trifluoromethylbenzimidazole (DTTB) in textiles, which are regulated by Japanese law ("Act on the Control of Household Products Containing Harmful Substances"), have been in place for more than 30 years. In this study, we developed an improved analytical method, based on GC-MS, that uses safe reagents and can simultaneously detect dieldrin and DTTB analytes. In the standard (existing) analytical method, dimethyl sulfate, which is a potential carcinogen, is used to derivatize DTTB. In the developed method, phenyltrimethylammonium hydroxide, as an alternative reagent, was used to derivatize DTTB in good results. Dieldrin and the derivatized DTTBs gave highly linear calibration curves when analyzed by GC-MS. Moreover, we found that both analytes are adequately extracted from textiles by refluxing in hydrochloric acid and methanol. Furthermore, we established a purification method using the Bond Elut PRS column that effectively removed interfering substances in woolen products. Finally, we developed an improved analysis method by combining the above-mentioned techniques; the developed method exhibited a recovery rate of 94-104% and a relative standard deviation of less than 7% for both analytes. In addition, the limits of quantitation (dieldrin: 1.3 µg/g, DTTB: 0.72 µg/g) were sufficiently lower than the Japanese regulatory value of 30 µg/g.


Assuntos
Benzimidazóis/análise , Dieldrin/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Têxteis/análise , Indicadores e Reagentes , Compostos de Amônio Quaternário
16.
J Vis Exp ; (160)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32568242

RESUMO

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.


Assuntos
Análise de Dados , Proteômica , Manejo de Espécimes , Espectrometria de Massas em Tandem , Animais , Clorofórmio/química , Indicadores e Reagentes , Metanol/química , Camundongos , Peptídeos/metabolismo , Proteínas/isolamento & purificação , Proteoma/análise
17.
Chemosphere ; 251: 126626, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32443247

RESUMO

Three spectrophotometric methods have been developed and compared for the quantification of low concentrations (0.03-63 µM) of aqueous permanganate in neutral pH conditions. Although permanganate is a widely used oxidant in drinking water and wastewater treatment, no widely accepted method of quantification has been reported to date. While one method presented does not require the need for any reagent chemicals (direct spectrophotometric analysis), it yielded a relatively low molar absorption coefficient of 3340 M-1 cm-1 at 525 nm and a level of detection (LOD) and quantification (LOQ) of 0.45 and 1.51 µM, respectively. Some instability of permanganate species during direct quantification was found to occur over 60 min, with a total decrease of 0.002 (arbitrary units) of absorbance, equivalent to a decrease in concentration of 0.6 µM. Beyond 60 min, no further degradation was observed. Indirect spectrophotometric analyses using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and sodium iodide (NaI) provided a significantly more sensitive method for permanganate quantification, yielding molar absorption coefficients of 140,030 and 61,130 M-1 cm-1, respectively. The LOD and LOQ were determined to be 0.01 and 0.03 µM for the ABTS method and 0.02 and 0.08 µM for the NaI method, respectively. Although conservative and accurate limits of quantification for both the ABTS and NaI methods are presented, which should be sufficient of most practical applications, lower limits may be possible with further refinement of the methods.


Assuntos
Compostos de Manganês/análise , Óxidos/análise , Espectrofotometria/métodos , Águas Residuárias/química , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Limite de Detecção , Padrões de Referência , Espectrofotometria/instrumentação
18.
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
19.
Klin Lab Diagn ; 65(6): 362-367, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32459895

RESUMO

A new original domestic set of reagents has been developed for the determination of class G immunoglobulins to individual human herpes virus antigens of type 7 by the method of immune blotting in the "Western-blot" format. Preliminary clinical trials were conducted using 134 serums of healthy children aged 1-16 years who underwent diagnostic testing. Stud y of diagnostic efficiency ofthe new kit showed high sensitivity, comparable to the sensitivity of the reaction indirect immunofluorescence and high specificity, which is manifested in the absence of false positive results when testing samples containing immunoglobulin G to herpes virus 6 type.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Western Blotting , Herpesvirus Humano 7 , Imunoglobulina G/isolamento & purificação , Indicadores e Reagentes , Infecções por Roseolovirus/diagnóstico , Adolescente , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Humanos , Lactente , Sensibilidade e Especificidade
20.
J Vet Diagn Invest ; 32(4): 577-580, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32450762

RESUMO

Rift Valley fever virus (RVFV) causes Rift Valley fever (RVF), resulting in morbidity and mortality in humans and ruminants. Evidence of transboundary outbreaks means that RVFV remains a threat to human health and livestock industries in countries that are free from the disease. To enhance surveillance capability, methods for detection of RVFV are required. The generation of reagents suitable for the detection of RVFV antigen in formalin-fixed, paraffin-embedded tissues from infected animals have been developed and are described herein. Recombinant nucleoprotein (rNP) was expressed in Escherichia coli and purified using immobilized metal ion affinity chromatography. Purified rNP was used as an immunogen to produce anti-NP polyclonal antisera in rabbits for use in detection of RVFV NP in experimentally infected animals by immunohistochemistry. Antisera raised in rabbits against rNP were able to recognize viral NP antigen in fixed infected Vero cell pellets and sheep liver. Therefore, the methods and reagents described herein are useful in assays for detection of RVFV infections in animals, for research and surveillance purposes.


Assuntos
Febre do Vale de Rift/diagnóstico , Vírus da Febre do Vale do Rift/isolamento & purificação , Doenças dos Ovinos/diagnóstico , Animais , Indicadores e Reagentes/química , Ovinos
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