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1.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946969

RESUMO

The cytogenetic and molecular assessment of deletions, amplifications and rearrangements are key aspects in the diagnosis and therapy of cancer. Not only the initial evaluation and classification of the disease, but also the follow-up of the tumor rely on these laboratory approaches. The therapeutic choice can be guided by the results of the laboratory testing. Genetic deletions and/or amplifications directly affect the susceptibility or the resistance to specific therapies. In an era of personalized medicine, the correct and reliable molecular characterization of the disease, also during the therapeutic path, acquires a pivotal role. Molecular assays like multiplex ligation-dependent probe amplification and droplet digital PCR represent exceptional tools for a sensitive and reliable detection of genetic alterations and deserve a role in molecular oncology. In this manuscript we provide a technical comparison of these two approaches with the golden standard represented by fluorescence in situ hybridization. We also describe some relevant targets currently evaluated with these techniques in solid and hematologic tumors.


Assuntos
Variações do Número de Cópias de DNA , DNA de Neoplasias/genética , Rearranjo Gênico , Proteínas de Neoplasias/genética , Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Aberrações Cromossômicas , Emulsões , Determinação de Ponto Final/métodos , Fluorometria , Humanos , Hibridização in Situ Fluorescente/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Proteínas de Fusão Oncogênica/genética , Sensibilidade e Especificidade
2.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947115

RESUMO

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.


Assuntos
Técnicas Biossensoriais , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência/métodos , Hidrocortisona/análise , Óleos Voláteis/farmacologia , Receptores de Glucocorticoides/agonistas , Atrofia , Acetato de Ciproterona/farmacologia , Dexametasona/farmacologia , Estradiol/farmacologia , Fluorometria , Células HeLa , Humanos , Inteínas , Limoneno/farmacologia , Proteínas Luminescentes/análise , Mentol/farmacologia , Mifepristona/farmacologia , Estrutura Molecular , Músculo Esquelético/patologia , Mioblastos/efeitos dos fármacos , Óleos Vegetais/farmacologia , Processamento de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 258: 119805, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-33957453

RESUMO

In recent years, global efforts have been directed towards the development of water safety routines, consequently demanding cost-effective sensors capable of detecting outbreaks at early stages. This work reports the development and study of an original in-field tryptophan fluorimetric sensor as a potential indicator of real-time microbial contamination in water. The sensor excitation and emission wavelengths were selected with respect to the coliform bacteria tryptophan peak; 280 nm for excitation and 330 nm for emission. The in-lab tests with standard samples show a detection limit of 4.89 nM (≈0.1 µg/l) for L-tryptophan. The sensor exhibited good linearity over three orders of magnitude and considerable detection reproducibility, which was confirmed during calibration tests. Small-scale in situ tests showed that the sensor was better correlated with coliform bacteria than other online sensors such as turbidity. This suggests that the fluorimetric tryptophan sensor can be integrated into early warning systems that quickly assess changes in water microbial quality.


Assuntos
Triptofano , Água , Fluorometria , Reprodutibilidade dos Testes
4.
Curr Protoc ; 1(5): e146, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34033698

RESUMO

Human papillomaviruses (HPVs), specifically high-risk HPVs, are responsible for up to 3% of all cancers in women and up to 2% of all cancers in men. They have been identified as the etiological agent of cervical cancer and have been increasingly found to be the driver behind head and neck cancers of the oropharynx. A system in which we can simultaneously observe transcriptional changes to both a host's tumor microenvironment and its associated oncogenic driver (e.g., HPV) would be highly valuable for understanding HPV's role in tumorigenesis. This article describes a detailed methodology for utilizing high-throughput RNA analysis to study viral transcription in formalin-fixed, paraffin-embedded clinical tumor samples. Although our lab utilizes these methods for the study of head and neck cancer, the principles contained within are widely applicable to all fields of HPV study. © 2021 Wiley Periodicals LLC. Basic Protocol: HPV16 transcript analysis using NanoString Support Protocol 1: Preparation of RNA from formalin-fixed, paraffin-embedded slides Support Protocol 2: Preparation of RNA from cell lysates Support Protocol 3: Fluorometric RNA concentration and RNA integrity analysis Support Protocol 4: Determination of input RNA based on DV300 calculation.


Assuntos
Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Papillomavirus Humano 16/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Transcrição Genética , Microambiente Tumoral/genética , Fluorometria , Formaldeído , Regulação Viral da Expressão Gênica , Humanos , Inclusão em Parafina , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Robótica , Fixação de Tecidos
5.
Molecules ; 26(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809185

RESUMO

INTRODUCTION: Alpha-galactosidase (α-Gal) is an enzyme responsible for the hydrolyzation of glycolipids and glycoprotein commonly found in dietary sources. More than 20% of the general population suffers from abdominal pain or discomfort caused by intestinal gas and by indigested or partially digested food residuals. Therefore, α-Gal is used in dietary supplements to reduce intestinal gases and help complex food digestion. Marketed enzyme-containing dietary supplements must be produced in accordance with the Food and Drug Administration (FDA) regulations for Current Good Manufacturing Practice (cGMPs). AIM: in this work we illustrated the process used to develop and validate a spectrophotometric enzymatic assay for α-Gal activity quantification in dietary supplements. METHODS: The validation workflow included an initial statistical-phase optimization of materials, reagents, and conditions, and subsequently a comparative study with another fluorimetric assay. A final validation of method performance in terms of specificity, linearity, accuracy, intermediate-precision repeatability, and system precision was then executed. RESULTS AND CONCLUSIONS: The proven method achieved good performance in the quantitative determination of α-Gal activity in commercial food supplements in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals (ICH) guidelines and is suitable as a rapid in-house quality control test.


Assuntos
Suplementos Nutricionais/análise , alfa-Galactosidase/análise , Suplementos Nutricionais/normas , Estabilidade Enzimática , Fluorometria/métodos , Análise de Alimentos/métodos , Análise de Alimentos/normas , Análise de Alimentos/estatística & dados numéricos , Humanos , Laboratórios , Controle de Qualidade , Espectrofotometria/métodos , Estados Unidos , United States Food and Drug Administration , alfa-Galactosidase/normas
6.
Molecules ; 26(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924894

RESUMO

A phosphate-substituted, zwitterionic berberine derivative was synthesized and its binding properties with duplex DNA and G4-DNA were studied using photometric, fluorimetric and polarimetric titrations and thermal DNA denaturation experiments. The ligand binds with high affinity toward both DNA forms (Kb = 2-7 × 105 M-1) and induces a slight stabilization of G4-DNA toward thermally induced unfolding, mostly pronounced for the telomeric quadruplex 22AG. The ligand likely binds by aggregation and intercalation with ct DNA and by terminal stacking with G4-DNA. Thus, this compound represents one of the rare examples of phosphate-substituted DNA binders. In an aqueous solution, the title compound has a very weak fluorescence intensity (Φfl < 0.01) that increases significantly upon binding to G4-DNA (Φfl = 0.01). In contrast, the association with duplex DNA was not accompanied by such a strong fluorescence light-up effect (Φfl < 0.01). These different fluorimetric responses upon binding to particular DNA forms are proposed to be caused by the different binding modes and may be used for the selective fluorimetric detection of G4-DNA.


Assuntos
Berberina/análogos & derivados , Quadruplex G , Fosfatos/química , Berberina/química , DNA/química , Corantes Fluorescentes/química , Fluorometria , Estrutura Molecular , Desnaturação de Ácido Nucleico , Análise Espectral , Relação Estrutura-Atividade , Temperatura
7.
Medicine (Baltimore) ; 100(15): e25301, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33847629

RESUMO

ABSTRACT: Carbohydrate antigen 24-2 (CA24-2) is usually used as a biomarker for the diagnosis of pancreatic cancer and colorectal cancer. Currentlly, a new quantitative assay kit for CA242 by flow fluorometry assay (FFA) was developed by Shanghai Tellgen Cooperation Co. Ltd. China. Therefore, we conducted the performance evaluation for it.According to the "Guiding principles on performance analysis of diagnostic reagents in vitro" and "American association of clinical laboratory standardization guidelines EP15-A2", the accuracy, precision, linear range, reportable range, biological reference interval verification, carry-over contamination rate, anti-interference capability and cross reaction of the assay kit used in TESMI F3999-Luminex200 automatic immunoassay system were evaluated. In addition, the assay kit was performed in parallel to CanAg kit (CanAg Diagnostics Products Beijing Co., Ltd.) to analyze the correlation between the 2 kits.The bias of accuracy of the new assay kit was less than 12.5% and the coefficient of variations (CVs) of precision were all less than 10.0%. The linear range of CA242 concentration of the testing kit was between 3.46 U/ml and 434.76 U/ml and the reportable range was 6.00 to 535.13 U/ml. The CA242 reference interval 0.00 to 20.00 U/ml was suitable for use in laboratory. The carry-over contamination rate was -0.14%. Correlation analysis showed a satisfactory relevance and consistency (r = 0.982, P < .001) between the new assay kit and CanAg kit, with a regression equation Y = 1.0012X to 0.878 (R2 = 0.9647, P < .001). No statistically significant difference between serum samples without interferences and samples containing lipemia, bilirubin and hemoglobin. And no cross reaction existed between the assay kit and the other tumor markers, such as carbohydrate antigen 125 (CA125), alpha-fetoprotein (AFP), and cytokeratin-19 soluble fragment (CYFRA21-1).The new CA242 quantitative assay kit possesses good detection performance when it is used in TESMI F3999-Luminex200 automatic immunoassay system, which can be used for the examination of CA242 in clinical practice.


Assuntos
Antígenos Glicosídicos Associados a Tumores/análise , Fluorometria/métodos , Fluorometria/normas , Humanos , Sensibilidade e Especificidade
8.
J Med Chem ; 64(7): 4059-4070, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33730493

RESUMO

Fibroblast activation protein (FAP) has become a favored target for imaging and therapy of malignancy. We have synthesized and characterized two new (4-quinolinoyl)-glycyl-2-cyanopyrrolidine-based small molecules for imaging of FAP, QCP01 and [111In]QCP02, using optical and single-photon computed tomography/CT, respectively. Binding of imaging agents to FAP was assessed in six human cancer cell lines of different cancer types: glioblastoma (U87), melanoma (SKMEL24), prostate (PC3), NSCLC (NCIH2228), colorectal carcinoma (HCT116), and lung squamous cell carcinoma (NCIH226). Mouse xenograft models were developed with FAP-positive U87 and FAP-negative PC3 cells to test pharmacokinetics and binding specificity in vivo. QCP01 and [111In]QCP02 demonstrated nanomolar inhibition of FAP at Ki values of 1.26 and 16.20 nM, respectively. Both were selective for FAP over DPP-IV, a related serine protease. Both enabled imaging of FAP-expressing tumors specifically in vivo. [111In]QCP02 showed high uptake at 18.2 percent injected dose per gram in the U87 tumor at 30 min post-administration.


Assuntos
Fibroblastos/metabolismo , Corantes Fluorescentes/química , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Fluorometria , Xenoenxertos/metabolismo , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Pirrolidinas/síntese química , Pirrolidinas/química , Quinolinas/síntese química , Quinolinas/química
9.
J Med Chem ; 64(7): 4020-4033, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33745280

RESUMO

Abnormally high levels of class I histone deacetylases (HDACs) are associated with triple-negative breast cancer (TNBC) proliferation, malignant transformation, and poor prognosis of patients. Herein, we report a near-infrared imaging probe for TNBC detection via visualizing class I HDACs. Conjugating Cy5.5 to a cyclic depsipeptide inhibitor, we obtained the probe (20-Cy5.5) that retained desirable class I HDAC affinity and selectivity. Then, this probe could visualize epigenetic changes by class I HDACs in TNBC MDA-MB-231 cells and in xenograft tumor models in real time. Treatment with suberoylanilide hydroxamic acid (SAHA) significantly reduced the uptake of the probe in tumors, suggesting its potential use in evaluation of therapeutic responses of HDACi-mediated therapy. Moreover, 20-Cy5.5 could detect class I HDAC expression in TNBC lung metastasis. This novel NIR probe that achieves tumor class I HDAC imaging not only leads to a better understanding of epigenetic regulation in tumors but also has great potential for improving the TNBC diagnosis and treatment.


Assuntos
Carbocianinas/farmacologia , Depsipeptídeos/farmacologia , Epigênese Genética/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Carbocianinas/síntese química , Linhagem Celular Tumoral , Depsipeptídeos/síntese química , Feminino , Fluorometria , Regulação Neoplásica da Expressão Gênica/fisiologia , Xenoenxertos/metabolismo , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/análise , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos Nus , Neoplasias de Mama Triplo Negativas/patologia , Vorinostat/farmacologia
10.
Mar Pollut Bull ; 165: 112059, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33677415

RESUMO

Chlorophyll a fluorescence is increasingly being used as a rapid, non-invasive, sensitive and convenient indicator of photosynthetic performance in marine autotrophs. This review presents the methodology, applications and limitations of chlorophyll fluorescence in marine studies. The various chlorophyll fluorescence tools such as Pulse-Amplitude-Modulated (PAM) and Fast Repetition Rate (FRR) fluorometry used in marine scientific studies are discussed. Various commonly employed chlorophyll fluorescence parameters are elaborated. The application of chlorophyll fluorescence in measuring natural variations, stress, stress tolerance and acclimation/adaptation to changing environment in primary producers such as microalgae, macroalgae, seagrasses and mangroves, and marine symbiotic invertebrates, namely symbiotic sponges, hard corals and sea anemones, kleptoplastic sea slugs and giant clams is critically assessed. Stressors include environmental, biological, physical and chemical ones. The strengths, limitations and future perspectives of the use of chlorophyll fluorescence technique as an assessment tool in symbiotic marine organisms and seaplants are discussed.


Assuntos
Organismos Aquáticos , Clorofila , Animais , Clorofila A , Fluorescência , Fluorometria , Fotossíntese
11.
Biosens Bioelectron ; 181: 113136, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33714857

RESUMO

For understanding the status of intestinal flora non-invasively, methanol (MeOH) has been attracting the attention. In this study, we have developed and compared two different liquid-phase methanol biosensors. One, referred to as the AOD electrosensor, utilized alcohol oxidase (AOD) and an oxygen electrode. It electrochemically measured the decrease in oxygen through AOD-catalyzed oxidation of MeOH. The other, referred to as the AOD-FALDH fluorosensor, exploited a cascade reaction of AOD and formaldehyde dehydrogenase (FALDH) in conjunction with a fiber-optic sensor. It measured increase in the fluorescence from reduced form of ß-nicotinamide adenine dinucleotide (NADH) that was a final product of the two-enzyme cascade reaction. Due to the cascade reaction, the AOD-FALDH fluorosensor showed 3 times better sensitivity along with 335 times wider dynamic range (494 nM-100 mM) than those of the AOD electrosensor (1.5-300 µM). The selectivity to MeOH was also improved by the cascade reaction with AOD-FALDH as no sensor output was observed from other aliphatic alcohols than MeOH in contrast to the AOD electrosensor. Although the use of FALDH resulted in the increase in the sensor output from aldehydes, such as acetaldehyde and formaldehyde, considering their concentrations in body fluids, the influence on the sensor output is limited. These results indicate that incorporating the cascade reaction into fluorometry enables enhanced biosensing of MeOH that will be useful for assessment of intestinal flora with little burden.


Assuntos
Técnicas Biossensoriais , Metanol , Acetaldeído , Bactérias , Fluorometria
12.
Biochem Biophys Res Commun ; 545: 125-131, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33548625

RESUMO

Bromodomain and PHD finger containing transcription factor (BPTF) is a multidomain protein that regulates the transcription of chromatin and is related to many cancers. Herein, we report the screening-based discovery of Cpd1, a compound with micromolar affinity to the BPTF bromodomain. Through structure-guided optimization, we synthesized a variety of new inhibitors. Among these compounds, Cpd8 and Cpd10 were highly potent and selective inhibitors, with KD values of 428 nM and 655 nM in ITC assays, respectively. The high activity was explained by the cocrystal structure of Cpd8 in complex with the BPTF bromodomain protein. Cpd8 and Cpd10 were able to stabilize the BPTF bromodomain protein in cells in a cellular thermal shift assay (CETSA). Cpd8 downregulated c-MYC expression in A549 cells. All experiments prove that these two compounds are potential BPTF inhibitors.


Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Células A549 , Antígenos Nucleares/química , Antígenos Nucleares/genética , Calorimetria , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Fluorometria , Regulação da Expressão Gênica/efeitos dos fármacos , Genes myc , Células HEK293 , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Domínios Proteicos , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética
13.
Analyst ; 146(7): 2144-2151, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33538722

RESUMO

Biogenic amines are involved in physiological roles in living organisms, but their excessive production or intake can induce undesired toxicological effects. As biogenic amines can be found in the process of food spoilage, they are considered an indicator of food quality and freshness, and their detection is of crucial importance in food safety. In this contribution, we report the fast and direct colorimetric and fluorometric sensing of biogenic amines by means of a dinuclear Zn(ii) Schiff-base complex. The selective and sensitive detection involves the formation of stable adducts between the dinuclear complex, acting as the Lewis acidic molecular tweezer, and biogenic di- or polyamines. The selectivity towards biogenic amines, even in the presence of common aliphatic, primary, secondary, or tertiary monoamines, heterocyclic amines, and amino acids, is demonstrated by competitive experiments. The quantitation of histamine in a fish matrix is easily achieved using a standard extraction procedure followed by simple colorimetric or fluorometric measurements.


Assuntos
Aminas Biogênicas , Colorimetria , Animais , Aminas Biogênicas/análise , Fluorometria , Qualidade dos Alimentos , Histamina
14.
Food Chem ; 349: 129160, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33550018

RESUMO

Indigo carmine (IC) dye is hazardous and allergenic for humans even though it has been excessively used in a wide range of industries. Therefore, the quantitative determination of IC is still challenging. Herein, for the first time, we have developed fluorometric and colorimetric dual-mode nanoprobe derived from the ion-pair association complex between the negatively charged IC and positively charged N@C-dots in pH = 3.0. Consequently, the binding between N@C-dots and IC resulted in cyan blue and quenching of N@C-dots fluorescence. The dependence of the fluorescence response on IC concentrations was linear over the range of 0.73-10.0 µM (R2 = 0.9989) with LOD of 0.24 µM. On the other hand, the linearity of the colorimetric method ranged from 9.97 to 80.0 µM (R2 = 0.9986) with LOD of 3.3 µM. The sensor was applied for estimation of IC in fruit juice and soft drink without the need for exhaustive extraction steps.


Assuntos
Bebidas/análise , Carbono/química , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Índigo Carmim/análise , Limite de Detecção , Nitrogênio/química , Colorimetria , Fluorometria , Humanos
15.
Spectrochim Acta A Mol Biomol Spectrosc ; 252: 119419, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33524816

RESUMO

In this study, a series of green, interference-free fluorimetric detection methods of the excitation-emission matrix coupled with the second-order calibration methods were proposed for the determination of ibrutinib and pralatrexate in various complicated biological fluids. The second-order advantage of the proposed method can overcome the problem of poor selectivity caused by the wide spectra of the fluorescence method. Even in the presence of uncalibrated interferences and severe peak overlap, the signal of pure substance and accurate quantitative results were still obtained. The average recoveries of the three methods were 94.5-104.9% for Alternating Trilinear Decomposition (ATLD) algorithm, 95.5-105.8% for Alternating Normalization Weighted Error (ANWE) algorithm and 94.4-105.7% for Parallel Factor Analysis (PARAFAC) algorithm, respectively. For ATLD, ANWE and PARAFAC, the relative standard deviations (RSD) were lower than 9.2%, 6.8% and 9.2%, and the RMSEPs were less than 8.1, 8.4 and 8.6 ng mL-1, respectively. In addition, the elliptic joint confidence region (EJCR) was adopted to further prove the accuracy of the three methods. The results showed that the three methods can accurately be quantified without significant difference. Good figures of merit parameters were also obtained. Among them, the limit of detection (LOD) and limit of quantification (LOQ) of ibrutinib and pralatrexate were in the range of 0.11-0.76 ng mL-1 and 0.21-1.12 ng mL-1, respectively, which were lower than the corresponding blood concentrations. These results indicate that the proposed method provides a promising, alternative and universal analysis strategy for clinical drug monitoring.


Assuntos
Algoritmos , Aminopterina/análogos & derivados , Fluorometria , Adenina/análogos & derivados , Calibragem , Limite de Detecção , Piperidinas , Espectrometria de Fluorescência
16.
Anal Methods ; 13(9): 1084-1105, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33595559

RESUMO

Formaldehyde, a highly reactive carbonyl species, has been widely used in day-to-day life owing to its numerous applications in essential commodities, etc.; the extrusion of formaldehyde from these sources basically leads to increased formaldehyde levels in the environment. Additionally, formaldehyde is endogenously produced in the human body via several biological processes. Considering the adverse effects of formaldehyde, it is highly important to develop an efficient and reliable method for monitoring formaldehyde in environmental and biological samples. Several chemodosimeters (reaction-based sensing probes) have been designed and synthesized to selectively detect the presence of formaldehyde utilizing the photophysical properties of molecules. In this review, we have comprehensively discussed the recent advances in the design principles and sensing mechanisms of developed probes and their biological/environmental applications in selective formaldehyde detection and imaging endogenous formaldehyde in cells. We have summarized the literature based on three different categories: (i) the Schiff base reaction, (ii) the 2-aza-Cope sigmatropic rearrangement reaction and (iii) miscellaneous approaches. In all cases, reactions are accompanied by changes in color and/or emission that can be detected by the naked eye.


Assuntos
Colorimetria , Formaldeído , Fluorometria , Humanos
17.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498248

RESUMO

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.


Assuntos
Automação Laboratorial/métodos , Testes de Coagulação Sanguínea/métodos , Carbono-Nitrogênio Liases/metabolismo , Deficiência do Fator XIII/diagnóstico , Fator XIII/análise , Corantes Fluorescentes/normas , Automação Laboratorial/normas , Bilirrubina/metabolismo , Testes de Coagulação Sanguínea/normas , Compostos Cromogênicos/normas , Fator XIII/metabolismo , Deficiência do Fator XIII/sangue , Fluorometria/métodos , Fluorometria/normas , Hemólise , Humanos , Testes Imunológicos/métodos , Testes Imunológicos/normas , Reprodutibilidade dos Testes , Transglutaminases/metabolismo
18.
Aquat Toxicol ; 231: 105732, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33385847

RESUMO

River biofilms are a suitable indicator of toxic stress in aquatic ecosystems commonly exposed to various anthropogenic pollutants from industrial, domestic, and agricultural sources. Among these pollutants, heavy metals are of particular concern as they are known to interfere with various physiological processes of river biofilm, directly or indirectly related to photosynthetic performance. Nevertheless, only limited toxicological data are available on the mechanisms and toxicodynamics of heavy metals in biofilms. Pulse Amplitude Modulated (PAM) fluorometry is a rapid, non-disruptive, well-established technique to monitor toxic responses on photosynthetic performance, fluorescence-kinetics, and changes in yield in other non-photochemical processes. In this study, a new micro-PAM-sensor was tested to assess potential acute and chronic effects of heavy metals in river biofilm. Toxicity values across the three parameters considered in this study (photosynthetic yield YII, non-photochemical quenching NPQ, and basal fluorescence F0) were comparable, as determined EC50 were within one order of magnitude (EC50 ∼1-10 mg L-1). However, the stimulation of NPQ was more clearly associated with early acute effects, especially in illuminated samples, while depression of YII and F0 were more prevalent in chronic tests. These results have implications for the development of functional indicators for the biomonitoring of aquatic health, in particular for the use of river biofilm as a bioindicator of water quality. In conclusion, the approach proposed seems promising to characterize and monitor the exposure and impact of heavy metals on river periphyton communities. Furthermore, this study provides a fast, highly sensitive, inexpensive, and accurate laboratory method to test effects of pollutants on complex periphyton communities that can also give insights regarding the probable toxicological mechanisms of heavy metals on photosynthetic performance in the river biofilm.


Assuntos
Técnicas Biossensoriais , Exposição Ambiental , Fluorometria/instrumentação , Metais Pesados/toxicidade , Rios/química , Biofilmes/efeitos dos fármacos , Clorófitas/citologia , Clorófitas/efeitos dos fármacos , Diatomáceas/citologia , Diatomáceas/efeitos dos fármacos , Monitoramento Ambiental , Fluorescência , Perifíton/efeitos dos fármacos , Processos Fotoquímicos , Fotossíntese/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Poluição da Água/análise , Qualidade da Água
19.
Sensors (Basel) ; 21(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498154

RESUMO

The authors describe a novel, facile, and sensitive fluorometric strategy based on a Cu2+-thiamine (Cu2+-TH) system for the detection of alkaline phosphatase (ALP) activity and inhibition. The principle of the method is as follows. Under a basic conditions, TH, which does not exhibit a fluorescence signal, is oxidized into fluorescent thiochrome (TC) by Cu2+. Ascorbic acid 2-phosphate (AAP), which is the enzyme substrate, is hydrolyzed to produce ascorbic acid (AA) by ALP. The newly formed AA then reduces Cu2+ to Cu+, which prevents the oxidation of TH by Cu2+; as a result, the fluorescent signal becomes weaker. On the contrary, in the absence of ALP, AAP cannot reduce Cu2+; additions of Cu2+ and TH result in a dramatic increase of the fluorescent signal. The sensing strategy displays brilliant sensitivity with a detection limit of 0.08 U/L, and the detection is linear in the concentration range of 0.1 to 100 U/L. This approach was successfully applied to ALP activity in human serum samples, indicating that it is reliable and may be applied to the clinical diagnosis of ALP-related diseases.


Assuntos
Fosfatase Alcalina/análise , Bioensaio , Fluorometria , Humanos , Limite de Detecção , Tiamina
20.
Chemistry ; 27(16): 5142-5159, 2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33411942

RESUMO

We report four new luminescent tetracationic bis-triarylborane DNA and RNA sensors that show high binding affinities, in several cases even in the nanomolar range. Three of the compounds contain substituted, highly emissive and structurally flexible bis(2,6-dimethylphenyl-4-ethynyl)arene linkers (3: arene=5,5'-2,2'-bithiophene; 4: arene=1,4-benzene; 5: arene=9,10-anthracene) between the two boryl moieties and serve as efficient dual Raman and fluorescence chromophores. The shorter analogue 6 employs 9,10-anthracene as the linker and demonstrates the importance of an adequate linker length with a certain level of flexibility by exhibiting generally lower binding affinities than 3-5. Pronounced aggregation-deaggregation processes are observed in fluorimetric titration experiments with DNA for compounds 3 and 5. Molecular modelling of complexes of 5 with AT-DNA, suggest the minor groove as the dominant binding site for monomeric 5, but demonstrate that dimers of 5 can also be accommodated. Strong SERS responses for 3-5 versus a very weak response for 6, particularly the strong signals from anthracene itself observed for 5 but not for 6, demonstrate the importance of triple bonds for strong Raman activity in molecules of this compound class. The energy of the characteristic stretching vibration of the C≡C bonds is significantly dependent on the aromatic moiety between the triple bonds. The insertion of aromatic moieties between two C≡C bonds thus offers an alternative design for dual Raman and fluorescence chromophores, applicable in multiplex biological Raman imaging.


Assuntos
DNA , RNA , Sítios de Ligação , Fluorometria , Modelos Moleculares
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