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1.
Forensic Sci Int ; 306: 110077, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31821940

RESUMO

Forensic samples are commonly influenced by various environmental factors, including ultraviolet (UV) irradiation; thus, forensic applications of DNA repair (e.g., PreCR™, Restorase®) have been investigated, focusing on short tandem repeat typing. However, current DNA-based examinations are used for both human and body fluid identification. This study thus aims to clarify the efficacy of a DNA repair approach for Streptococcus salivarius DNA-based identification of saliva from UV-damaged samples. Artificial UV-damaged genomic DNA of S. salivarius, drop saliva stains, and buccal swabs were used to evaluate the effects of DNA repair on S. salivarius DNA detection by using PreCR™ repair reagent. To evaluate forensic applications, we prepared mock forensic samples by exposing them to environmental conditions. Melting curve analysis following real-time PCR was applied for qualitatively detecting S. salivarius DNA with a specific melting peak of 80.5°C±0.4°C (n=10, mean ± 3SD). Single PCR was used for quantitative and qualitative analyses, whereas dual PCR was used for S. salivarius DNA qualitative detection. DNA repair experiments using artificial UV-damaged samples revealed a significant increase of only the quantitative value of genomic DNA samples by DNA repair. Moreover, significant quantitative DNA repair effects were not observed in all mock forensic samples, indicating the limitations of DNA repair for actual cell-derived DNA samples. Whereas, differences of qualitative results (with or without detection) were generated for mock forensic samples; thus, we consider the DNA repair strategy as an additional approach for S. salivarius DNA-based identification of saliva from environmentally damaged evidence.


Assuntos
Reparo do DNA , Saliva/microbiologia , Streptococcus salivarius/genética , Raios Ultravioleta/efeitos adversos , Dano ao DNA , DNA Bacteriano/genética , Humanos , Indicadores e Reagentes , Repetições de Microssatélites , Reação em Cadeia da Polimerase em Tempo Real
2.
Talanta ; 206: 120219, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514888

RESUMO

A new method to determine the total titratable acidity of orange, lemon and passion fruit, based on a spot test obtained from digital images and using anthocyanins as the biodegradable indicator, is presented for the first time. The colorimetric reactions were carried out by acid-base titration on a microscale, employing anthocyanin with a microplate for spot test purposes, with detection by digital imaging. To obtain highly precise data, a chamber based on a diffuser was developed to control the illumination supplied by the light emitting diodes, and coupled to a smartphone to acquire adequate digital images. High precision was obtained with a relative standard deviation of 0.758% for n = 95. The RGB values were extracted from the digital images and used as analytical signals, the values being correlated with the micro-volume of the titrant and used to construct the titration curves and obtain the first and second derivatives, respectively. For comparative purposes, the official AOAC (Association of Official Analytical Chemists) and MAPA (Ministry of Agriculture, Livestock and Food Supply of Brazil) methods were used and the results compared by applying the paired t-test at the 95% confidence level (n = 3). No difference was found between the values and the relative errors were less than 2.8%. The micro-titrimetric method was fast, uses anthocyanins as the natural indicator, is practical, and permits a reduction of 922 times or 99.9% of the volume required in a conventional titration. It is therefore ideal for routine analyses leading to a reduction in the waste generated, according to the principles of green chemistry.


Assuntos
Citrus sinensis/química , Frutas/química , Passiflora/química , Antocianinas/química , Colorimetria/instrumentação , Colorimetria/métodos , Química Verde/métodos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes/química , Phaseolus/química , Smartphone , Titulometria/instrumentação , Titulometria/métodos
3.
Vet Parasitol ; 276: 108975, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31775102

RESUMO

The suitability of acetic acid as a safer alternative to formalin in the modified Knott test was evaluated for the diagnosis of canine heartworm (Dirofilaria immitis). Microfilaria concentration was measured by both methods and found to agree within reasonable limits (-5.84 % bias; -88.1-76.4 % limits of agreement). The level of agreement was lower when samples were prepared with a 24 h delay, but this was due to the formalin method tending to yield lower counts (-20.1 % bias; -90.5-50.2 % limits of agreement). Clearing the sample of hemoglobin improves readability and is a key feature of the modified Knott test. Hemolysis was significantly lower in the acetic acid method than the formalin method as measured by red blood cell count (6.83 × 106 and 8.79 × 106 cells/ml, respectively; p = 0.015) and absorbance at 415 nm (33.20 and 34.75, respectively; p < 0.001). Visual assessment, however, revealed little practical difference in readability. Finally, lengths of microfilariae were measured to ensure the validity of species identification by the acetic acid method; mean length was significantly shorter after acetic acid treatment (273 µm) than formalin treatment (316 µm; p < 0.001). Length reduction was also observed in acetic acid-treated Acanthocheilonema reconditum (254 µm versus 262 µm; p = 0.035), though these samples were stored prior to testing and are not directly comparable. We conclude that, while the readability of samples is similar for both methods, species differentiation must still be accomplished by other means. For most clinical purposes in determining the presence or absence of blood circulating microfilariae, however, acetic acid appears to be a suitable alternative to formalin in the modified Knott test.


Assuntos
Ácido Acético , Dirofilariose/diagnóstico , Doenças do Cão/diagnóstico , Indicadores e Reagentes , Microfilárias/crescimento & desenvolvimento , Animais , Dirofilariose/parasitologia , Doenças do Cão/parasitologia , Cães , Formaldeído , Hemólise/efeitos dos fármacos
4.
Org Biomol Chem ; 17(47): 10097-10102, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31754683

RESUMO

New open-chain and water-soluble hypervalent iodine reagents were synthesized and used for the transfer of fluoroalkyl groups to sulfur atoms of cysteine and cysteine-containing peptides under biocompatible conditions. Some of the reagents displayed excellent reactivity despite their limited stability in aqueous media. In reactions with a short cysteine-containing peptide, in addition to the expected S-fluoroalkylated product, a range of side-products were obtained. The amount of side-products depended on the conditions used (type of reagent, concentration, and pH). With highly activated hypervalent iodine reagents, a new reactive mode was observed - reaction with disulfides to form fluoroalkyl thiols.


Assuntos
Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/síntese química , Indicadores e Reagentes/química , Indicadores e Reagentes/síntese química , Iodo/química , Compostos de Sulfidrila/química , Água/química , Alquilação , Estrutura Molecular , Solubilidade
5.
BMC Bioinformatics ; 20(1): 542, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675914

RESUMO

BACKGROUND: In biological experiments, comprehensive experimental metadata tracking - which comprises experiment, reagent, and protocol annotation with controlled vocabulary from established ontologies - remains a challenge, especially when the experiment involves multiple laboratory scientists who execute different steps of the protocol. Here we describe Annot, a novel web application designed to provide a flexible solution for this task. RESULTS: Annot enforces the use of controlled vocabulary for sample and reagent annotation while enabling robust investigation, study, and protocol tracking. The cornerstone of Annot's implementation is a json syntax-compatible file format, which can capture detailed metadata for all aspects of complex biological experiments. Data stored in this json file format can easily be ported into spreadsheet or data frame files that can be loaded into R ( https://www.r-project.org/ ) or Pandas, Python's data analysis library ( https://pandas.pydata.org/ ). Annot is implemented in Python3 and utilizes the Django web framework, Postgresql, Nginx, and Debian. It is deployed via Docker and supports all major browsers. CONCLUSIONS: Annot offers a robust solution to annotate samples, reagents, and experimental protocols for established assays where multiple laboratory scientists are involved. Further, it provides a framework to store and retrieve metadata for data analysis and integration, and therefore ensures that data generated in different experiments can be integrated and jointly analyzed. This type of solution to metadata tracking can enhance the utility of large-scale datasets, which we demonstrate here with a large-scale microenvironment microarray study.


Assuntos
Biologia Computacional/métodos , Curadoria de Dados/métodos , Indicadores e Reagentes/provisão & distribução , Metadados , Bancos de Espécimes Biológicos/estatística & dados numéricos , Software , Vocabulário Controlado
6.
Bull Cancer ; 106(11): 1008-1022, 2019 Nov.
Artigo em Francês | MEDLINE | ID: mdl-31606139

RESUMO

With more than 3300 new cases and almost 2500 deaths each year, cervical cancer (CC) ranks second among female cancers in Moroccan women. The majority of cases occurs in women aged 50 and over. In absence of a national cancer registry, data published in Morocco are limited to the number of cases recorded in some oncology centers, so the incidence of this cancer is likely much higher than estimated. A Moroccan national program against CC based on the practice of visual inspection after application of acetic acid was set up in 2010, allowing both screening and possibly immediate treatment of (pre)cancerous lesions. However, this program has not been implemented in all regions of the country. The CC develops slowly and most often without any symptoms, and so it is diagnosed at an advanced stage of the disease. Virtually, all CC are associated with persistent infection of high risk human papillomavirus (HPV), particularly HPV16 and 18. For more than ten years, two prophylactic vaccines targeting these two HPV genotypes have been marketed. They have proved their excellent immunogenicity and efficacy and they are well tolerated. However, HPV vaccine is not yet recommended by health authorities in Morocco. In this literature review, we focused on the current situation of CC, the prevalence of HPV infection and the prevention strategies against CC in Morocco.


Assuntos
Infecções por Papillomavirus/prevenção & controle , Lesões Pré-Cancerosas/prevenção & controle , Neoplasias do Colo do Útero/prevenção & controle , Ácido Acético/administração & dosagem , Adulto , Algoritmos , Coinfecção/epidemiologia , Detecção Precoce de Câncer/métodos , Feminino , Infecções por HIV/epidemiologia , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Humanos , Incidência , Indicadores e Reagentes/administração & dosagem , Pessoa de Meia-Idade , Marrocos/epidemiologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Vacinas contra Papillomavirus/administração & dosagem , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/epidemiologia , Lesões Pré-Cancerosas/virologia , Prevenção Primária , Sistema de Registros/estatística & dados numéricos , Gestão de Riscos , Prevenção Secundária , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologia
8.
Nature ; 574(7777): 228-232, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31597972

RESUMO

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.


Assuntos
Indicadores e Reagentes/análise , Microfluídica/métodos , Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Testes Diagnósticos de Rotina , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Fluorometria , Glucosefosfato Desidrogenase/análise , Glucosefosfato Desidrogenase/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Microfluídica/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos
9.
Rev Lat Am Enfermagem ; 27: e3194, 2019 Oct 14.
Artigo em Português, Inglês, Espanhol | MEDLINE | ID: mdl-31618387

RESUMO

OBJECTIVE: using the urinary cotinine biomarker to verify the occurrence of green tobacco sickness in workers who cultivate Burley tobacco. METHOD: paired case-control study, based on smoking status and on the 1:4 ratio, with participation of 20 case workers and 91 controls. Data collection included household surveys and urine collection for cotinine examination. Student's T-Test, the Mann-Whitney test, Pearson's chi-square or Fisher's exact tests were used. RESULTS: of the 23 suspected cases, 20 showed elevated levels of cotinine, signs and symptoms of headache, skin irritation, nausea, sickness and general malaise, especially in the morning. Most had worked with tobacco that was wet from the morning dew and when the weather was warm. CONCLUSION: there are signs suggestive of green tobacco sickness in Burley tobacco workers. The action of health professionals is necessary for the development of health promotion and preventive actions addressing work-related illness.


Assuntos
Doenças dos Trabalhadores Agrícolas/diagnóstico , Cotinina/urina , Exposição Ocupacional/análise , Adulto , Doenças dos Trabalhadores Agrícolas/induzido quimicamente , Doenças dos Trabalhadores Agrícolas/epidemiologia , Doenças dos Trabalhadores Agrícolas/urina , Biomarcadores/urina , Brasil/epidemiologia , Estudos de Casos e Controles , Feminino , Cefaleia/induzido quimicamente , Humanos , Indicadores e Reagentes/análise , Masculino , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Nicotina/envenenamento , Exposição Ocupacional/efeitos adversos , Equipamento de Proteção Individual , Inquéritos e Questionários , Tabaco/envenenamento
10.
Forensic Sci Int ; 303: 109931, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31546160

RESUMO

Clinical and research-based tests in molecular biology require a substantial amount of DNA and RNA, unfortunately, a considerable number of cells is needed for this amount of sample. Blood is one of the best and easiest source of cells, but is not used due to its invasive drawing methods and the needed volume. Another considerable point is the low amount of samples detected in crime scenes. TRI reagent is one of the most available methods for DNA, RNA and protein extraction. However, based on unsuccessful results, this method has not been widely used on blood samples. In this study, for the first time, the use of TRI reagent on micro scale blood volume was reported, resulting in high yield of DNA and RNA with great quality.


Assuntos
DNA/isolamento & purificação , Medicina Legal/métodos , Indicadores e Reagentes , RNA/isolamento & purificação , Adolescente , Adulto , DNA/sangue , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , RNA/sangue , Adulto Jovem
11.
Chemistry ; 25(68): 15662-15679, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31549752

RESUMO

Sixteen cationic prodrugs of the antitumor alkylphospholipid (APL) erufosine were rationally synthesized to provide original gene delivery reagents with improved cytotoxicity profile. The DNA complexation properties of these cationic lipids were determined and associated transfection rates were measured. Furthermore, the self-assembly properties of the pro-erufosine compounds were investigated and their critical aggregation concentration was determined. Their hydrolytic stability under pH conditions mimicking the extracellular environment and the late endosome milieu was measured. Hemolytic activity and cytotoxicity of the compounds were investigated. The results obtained in various cell lines demonstrate that the prodrugs of erufosine display antineoplastic activity similar to that of the parent antitumor drug but are not associated with hemolytic toxicity, which is a dose-limiting side effect of APLs and a major obstacle to their use in anticancer therapeutic regimen. Furthermore, by using lipoplexes prepared from a prodrug of erufosine and a plasmid DNA encoding a pro-apoptotic protein (TRAIL), evidence was provided for selective cytotoxicity towards tumor cells while nontumor cells were resistant. This study demonstrates that the combination approach involving well tolerated erufosine cationic prodrugs and cancer gene therapy holds significant promise in tumor therapy.


Assuntos
Antineoplásicos/farmacologia , Cátions/química , Organofosfatos/farmacologia , Plasmídeos/química , Pró-Fármacos/farmacologia , Compostos de Amônio Quaternário/farmacologia , Humanos , Indicadores e Reagentes , Organofosfatos/química , Pró-Fármacos/química , Compostos de Amônio Quaternário/química , Transfecção
12.
Forensic Sci Int Genet ; 43: 102139, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31487605

RESUMO

Correct identification of probative samples is the first crucial step in the analysis of sexual assault kits (SAKs). We report a nucleic acid-based approach, as an alternative to the widely utilized p30 assay, to screening male DNA from SAKs collected from female victims by combining a rapid lysis protocol with an isothermal amplification method. The enzymatic lysis protocol efficiently digests biological material to release nuclear DNA in 10 min in a single closed tube, including resilient cell types such as sperm cells. The amplification and detection of human male specific DNA is achieved through loop-mediated isothermal amplification (LAMP) accompanied with hydroxynaphthol blue, a colorimetric indicator, producing a visually-distinctive color change in the presence of male DNA. The Y-screen approach demonstrated high specificity to human male DNA, can reliably detect target DNA as low as 50 pg, and correctly identified all probative samples from 14 single-blind mock sexual assault samples. In contrast with the widely used p30 assay which requires at least 2 h incubation time and manual application to a lateral flow pad, this Y-screen assay can be completed in half the time, and can be performed in a 96-well format without the need for a fluorescence detector, making facile high-throughput sample screening possible.


Assuntos
Colorimetria , Técnicas de Amplificação de Ácido Nucleico/métodos , Espermatozoides/química , Amelogenina/genética , Cromossomos Humanos Y , DNA/análise , Marcadores Genéticos , Humanos , Indicadores e Reagentes , Masculino , Naftalenossulfonatos , Reação em Cadeia da Polimerase , Delitos Sexuais
13.
Molecules ; 24(17)2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31480658

RESUMO

Due to their ability to inhibit viral DNA or RNA replication, nucleoside analogues have been used for decades as potent antiviral therapeutics. However, one of the major limitations of nucleoside analogues is the development of antiviral resistance. In that regard, flexible nucleoside analogues known as "fleximers" have garnered attention over the years due to their ability to survey different amino acids in enzyme binding sites, thus overcoming the potential development of antiviral resistance. Acyclic fleximers have previously demonstrated antiviral activity against numerous viruses including Middle East Respiratory Syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), and, most recently, flaviviruses such as Dengue (DENV) and Yellow Fever Virus (YFV). Due to these interesting results, a Structure Activity Relationship (SAR) study was pursued in order to analyze the effect of the pyrimidine functional group and acyl protecting group on antiviral activity, cytotoxicity, and conformation. The results of those studies are presented herein.


Assuntos
Antivirais/química , Antivirais/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Linhagem Celular Tumoral , Ebolavirus/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Lipídeos/química , Conformação Molecular , Espectroscopia de Prótons por Ressonância Magnética , Relação Estrutura-Atividade
14.
J Chromatogr A ; 1604: 460502, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31492465

RESUMO

Parallel factor analysis 2 (PARAFAC2) is still being advocated for the processing of second-order chromatographic-spectral data, both for qualitative and quantitative applications. However, neither classical PARAFAC2 nor the newly developed flexible non-negative NN-PARAFAC2 version can adequately model these data in a general situation. In quantitative analysis, considerable bias may result in the estimation of analyte concentrations, due to the fact that both PARAFAC2 models apply an artificial constraint to the retrieved profiles, requiring constant cross-product, i.e., constant overlapping, between all pairs of component elution profiles in all samples. This only occurs under limited conditions. In this report, simulations help to understand, visualize and interpret these PARAFAC2 features. Experimental data are also studied concerning the determination of a fluoroquinolone antibiotic in bovine liver samples by liquid chromatography with multi-wavelength fluorescence detection. Both for simulated and experimental data, the PARAFAC2 versions provide poor analytical results, while correct data processing and reasonable analytical indicators can be achieved using multivariate curve resolution - alternating least-squares (MCR-ALS). For the simulated data sets, root mean square errors/relative errors of prediction were 0.01 concentration units/2% for MCR-ALS, compared to 0.02-0.06 units/4-12% for both PARAFAC2 and NN-PARAFAC2. For the experimental data sets, they were 0.025 µg mL-1/11% for MCR-ALS, 0.09 µg mL-1/40% for PARAFAC2 and 0.16 µg mL-1/71% for NN-PARAFAC2, with average recoveries (standard deviation) of 91(14)%, 185(135)% and 69(35)% respectively.


Assuntos
Cromatografia Líquida/métodos , Análise Fatorial , Modelos Teóricos , Animais , Calibragem , Bovinos , Simulação por Computador , Fluorescência , Indicadores e Reagentes , Análise dos Mínimos Quadrados , Análise Multivariada , Reprodutibilidade dos Testes , Soluções
15.
Microscopy (Oxf) ; 68(5): 417-421, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31415090

RESUMO

Correlative light and electron microscopy (CLEM) enables ultrastructural-level analysis of fluorescence-labeled proteins by combining images obtained from both fluorescence and electron microscopies. A technical challenge with the CLEM method is the effective detection of fluorescence from samples embedded in resins, which generally cause fluorescence decay. To overcome this issue, we developed a method for fluorescence recovery of green fluorescent protein (GFP) in resin-embedded semi-thin sections using commercially available antifade reagents. By applying this method, we successfully obtained CLEM images using field-emission scanning electron microscopy with moderately enhanced GFP signals, demonstrating the efficacy of this simple fluorescence recovery method.


Assuntos
Indicadores e Reagentes , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Microscopia , Coloração e Rotulagem , Arabidopsis/genética , Fluorescência , Proteínas de Fluorescência Verde/genética , Técnicas Histológicas , Processamento de Imagem Assistida por Computador , Plântula/genética , Manejo de Espécimes
16.
J Drugs Dermatol ; 18(8): 798-802, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31424710

RESUMO

Cutaneous fungal infections account for millions of office visits per year, yet their varied presentations often lead to misdiagnosis. If dermatology clinics are Clinical Laboratory Improvement Amendment (CLIA) certified, direct microscopy with potassium hydroxide or other stains can be used to inexpensively and rapidly diagnose fungal infections. In this survey, we examined dermatologists' perceptions of fungal preparations and CLIA certification to identify barriers that prevent the use of these bedside diagnostics. The response rate was 13% (n=308, based on the number of emails opened). When a cutaneous fungal infection is suspected, 20.94% rarely/never and 19.86% sometimes perform fungal preparations, often because they think clinical diagnosis is adequate or that preparations take too long. 21.32% reported not having CLIA certification, most frequently because the process requires too much work, or they do not know how to apply. Of providers with CLIA certification, over 25% thought it was difficult to obtain. Our results demonstrate that numerous barriers prevent the common use of fungal preparations, including the perception that clinical diagnosis is sufficient and the lack of required CLIA certification. These barriers emphasize the need for additional education about cutaneous fungal infections and use of bedside diagnostics. Additionally, policy-based interventions are necessary to ease the process of CLIA certification.


Assuntos
Dermatomicoses/diagnóstico , Fungos/isolamento & purificação , Indicadores e Reagentes/química , Adulto , Idoso , Dermatologistas/estatística & dados numéricos , Dermatologia/métodos , Dermatologia/estatística & dados numéricos , Dermatomicoses/microbiologia , Dermatomicoses/patologia , Diagnóstico Diferencial , Feminino , Humanos , Hidróxidos/química , Masculino , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/estatística & dados numéricos , Microscopia , Pessoa de Meia-Idade , Visita a Consultório Médico/estatística & dados numéricos , Compostos de Potássio/química , Padrões de Prática Médica/estatística & dados numéricos , Pele/microbiologia , Pele/patologia , Inquéritos e Questionários/estatística & dados numéricos
17.
Anal Bioanal Chem ; 411(26): 6847-6856, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31440782

RESUMO

During drug development, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is used for visually elucidating the distribution of substances such as biomarkers, candidate compounds, and metabolites in the tissues. However, it is difficult to make relative comparisons between tissue sections and there are still many challenges. Here, we report a new method of "triple spray" for the comparison of analyte distribution in multiple tissue slices. This method targets amino acids and amines, and it incorporates the application of the internal standard in the on-tissue derivatization step. With further development, it has the potential to alleviate problems caused by the matrix effect. Initially, we measured three serial sections of rat brain to verify the efficacy of this method. In the hypothalamus, where gamma-aminobutyric acid (GABA) is known to be present in high concentration, the GABA levels of the three serial section showed little variation (CV = 1.62%). Subsequently, we compared the GABA level in the brain between stroke-prone spontaneous hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats with three individuals each. It showed significant differences between these models at the pre-selected region of interest (p < 0.05). Our results show that the triple spray allows for relative comparison among multiple tissue slices with high reproducibility. Graphical abstract.


Assuntos
Aminoácidos/análise , Química Encefálica , Neurotransmissores/análise , Animais , Indicadores e Reagentes , Marcação por Isótopo/métodos , Masculino , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ácido gama-Aminobutírico/análise
18.
J Chromatogr A ; 1605: 460371, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31375330

RESUMO

The genus Primula (Primulaceae) comprises species with high medicinal as well as ornamental values. Plants belonging to this genus are well recognized for their richness in bioactive constituents. The huge variety of secondary metabolites make their complete analysis impossible. In order to cope with this challenge, effect-directed analysis (EDA) via HPTLC coupled to structure elucidation techniques was applied on Primula species for the first time. As straightforward non-target bioanalytical technique, HPTLC-UV/Vis/FLD-EDA-ESI-HRMS hyphenates three different orthogonal dimensions, i.e. chromatography with spectrometric detection, biological/enzymatic assays and HRMS. The bioactive metabolites were determined in the middle polar extracts of two Primula species, P. veris (flower) and P. boveana (leaf). The bioactivity profiling comprised the antibacterial activity against Aliivibrio fischeri and Bacillus subtilis bacterial strains and acetyl-/butyrylcholinesterase (AChE/BChE) inhibition assays. The compounds were characterized and identified via their recorded spectral data (HRMS and 1H NMR). The results showed that linoleic and linolenic acids were the principle bioactive compounds present in the studied P. veris flower extract. In the P. boveana leaf extract, flavone, 2'-methoxy-, 2'-hydroxy- and 5,6,2',6'-tetramethoxyflavone (zapotin) were determined as active metabolites. The identification of zapotin, which was previously undescribed in the investigated plant, was considered as the strength of the straightforward non-target bioanalytical technique. Flavone turned out to be the highest potent metabolite, and at the same time, a multipotent compound referring to its various bioactivities discovered. An equivalency calculation of the HPTLC-AChE inhibition by flavone was performed with reference to the well-known inhibitor rivastigmine. As a result, the amount of flavone contained in 10.0 µg dry powder of P. boveana (corresponding to 0.1 µL extract) inhibited as strong as 4.5 µg rivastigmine. In other words, the flavone contained in P. boveana leaf extract powder turned out to be half as strong as the well-known AChE inhibitor rivastigmine.


Assuntos
Cromatografia em Camada Delgada/métodos , Flores/metabolismo , Metaboloma , Extratos Vegetais/metabolismo , Primula/metabolismo , Antibacterianos/análise , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Benzaldeídos/química , Inibidores da Colinesterase/análise , Inibidores da Colinesterase/farmacologia , Indicadores e Reagentes , Espectroscopia de Prótons por Ressonância Magnética , Ácidos Sulfúricos/química
19.
Forensic Sci Int Genet ; 42: 198-202, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31376647

RESUMO

Evidence of sexual aggression may be obtained from superabsorbent polymer (SAP) sanitary pads, which are used by forensic laboratories for semen evaluation. Semen can be extracted from their upper layers, which are free of SAPs. However, our previous results showed a need to optimize the protocol for semen analysis by considering its extraction from the lower core, often composed of sodium polyacrylate SAPs. SAPs generate a hydrogel, which traps the cellular components, hindering the possibility of obtaining cells and hence their genetic material. Simple filtration has been tried previously, but further maximization by application of a treatment has never been attempted. In this paper, we compare both chemical and physical shredding treatments for maximizing gel-trapped sperm and male cell DNA recaptures from hygienic superabsorbent substrates in sanitary pads, panty-liners or diapers. Our findings suggest that the lower core should be treated to induce a dewaterisation of the SAP hydrogels in order to maximize the extraction of bodily fluids.


Assuntos
Impressões Digitais de DNA/métodos , Produtos de Higiene Menstrual , Sêmen , Delitos Sexuais , Manejo de Espécimes/métodos , DNA/isolamento & purificação , Células Epiteliais , Feminino , Humanos , Indicadores e Reagentes , Masculino , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Polímeros , Manejo de Espécimes/instrumentação , Espermatozoides , Vagina/citologia
20.
Clin Appl Thromb Hemost ; 25: 1076029619867137, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31364394

RESUMO

To describe the effect of dabigatran on thrombin time (TT) reagents at different concentrations of thrombin. Pooled normal plasma enriched with dabigatran was dissolved in dimethylsulfoxide (DMSO) at concentrations of 0, 20, 50, 100, 200, 300, and 500 ng/mL. Samples with each concentration were evaluated using a semiautomatic coagulation analyzer to assess the effect of dabigatran on internal normalized ratio (INR), thromboplastin time (APTT), and TT, which were purchased from Instrument Laboratory (IL), Sysmex (SYS), and Stago (STA), respectively. Regarding INR, no reagent showed good sensitivity to increasing concentration of dabigatran, despite all reagents showing good linear response curves (P = .012). Regarding APTT, all reagents had low sensitivity to increasing dabigatran concentration, but SYS-APTT showed a better linear response curve (P = .001). Regarding TT, all reagents had a good linear response to the concentration of dabigatran; however, SYS-TT was very sensitive at low concentrations of dabigatran (0-100 ng/mL), while IL (TT-5 mL) and STA-TT were sensitive at medium concentrations of dabigatran (0-300 ng/mL), and IL (TT-2 mL) was less sensitive for a wide concentration of dabigatran (0-500 ng/mL; P = .007). Internal normalized ratio and APTT showed low sensitivity and SYS-TT showed high sensitivity to concentrations of dabigatran that were unsuitable to monitor. Both IL (TT-5 mL) and STA-TT were useful at medium concentrations of dabigatran by semiautomatic coagulation analyzer, which calculated results using the end point method of coagulation. Instrument Laboratory (TT-2 mL), which contains a higher concentration of thrombin, had better sensitivity to the concentration of dabigatran than APTT and was suitable for routine monitoring by an automatic analyzer.


Assuntos
Dabigatrana/farmacocinética , Monitoramento de Medicamentos/métodos , Tempo de Trombina , Antitrombinas/sangue , Antitrombinas/farmacocinética , Testes de Coagulação Sanguínea , Dabigatrana/sangue , Dabigatrana/normas , Monitoramento de Medicamentos/instrumentação , Monitoramento de Medicamentos/normas , Humanos , Indicadores e Reagentes/farmacologia , Tempo de Tromboplastina Parcial , Sensibilidade e Especificidade , Trombina/farmacologia
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