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2.
Eur J Endocrinol ; 182(6): P1-P15, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32268295

RESUMEN

Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.


Asunto(s)
Trastornos del Desarrollo Sexual/diagnóstico , Trastornos del Desarrollo Sexual/terapia , Inmunoensayo/normas , Hormonas Peptídicas/sangre , Hormona Antimülleriana/sangre , Cromatografía Liquida/normas , Manejo de la Enfermedad , Europa (Continente) , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Inhibinas/sangre , Hormona Luteinizante/sangre , Masculino , Guías de Práctica Clínica como Asunto , Enfermedades Raras , Estándares de Referencia , Espectrometría de Masas en Tándem/normas
3.
BMJ ; 369: m1326, 2020 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-32245846
4.
EMBO J ; 39(10): e105114, 2020 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-32246845

RESUMEN

The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis.


Asunto(s)
Bronquios/citología , Expresión Génica , Pulmón/citología , Peptidil-Dipeptidasa A/genética , Serina Endopeptidasas/genética , Análisis de la Célula Individual , Adulto , Envejecimiento , Bronquios/metabolismo , Células Cultivadas , Enfermedad Crónica/epidemiología , Infecciones por Coronavirus/genética , Células Epiteliales/metabolismo , Femenino , Perfilación de la Expresión Génica , Alemania , Células Caliciformes/metabolismo , Humanos , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/genética , Estándares de Referencia , Análisis de Secuencia de ARN , Caracteres Sexuales , Fumar , Bancos de Tejidos
5.
Einstein (Sao Paulo) ; 18: eAO4948, 2020.
Artículo en Inglés, Portugués | MEDLINE | ID: mdl-32159604

RESUMEN

OBJECTIVE: To develop a computational algorithm applied to magnetic resonance imaging for automatic segmentation of brain tumors. METHODS: A total of 130 magnetic resonance images were used in the T1c, T2 and FSPRG T1C sequences and in the axial, sagittal and coronal planes of patients with brain cancer. The algorithms employed contrast correction, histogram normalization and binarization techniques to disconnect adjacent structures from the brain and enhance the region of interest. Automatic segmentation was performed through detection by coordinates and arithmetic mean of the area. Morphological operators were used to eliminate undesirable elements and reconstruct the shape and texture of the tumor. The results were compared with manual segmentations by two radiologists to determine the efficacy of the algorithms implemented. RESULTS: The correlated correspondence between the segmentation obtained and the gold standard was 89.23%. CONCLUSION: It is possible to locate and define the tumor region automatically with no the need for user interaction, based on two innovative methods to detect brain extreme sites and exclude non-tumor tissues on magnetic resonance images.


Asunto(s)
Algoritmos , Neoplasias Encefálicas/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Encéfalo/diagnóstico por imagen , Diagnóstico por Computador/métodos , Humanos , Estándares de Referencia , Reproducibilidad de los Resultados
6.
JAMA ; 323(8): 746-756, 2020 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-32207768

RESUMEN

Importance: Improved screening methods for women with dense breasts are needed because of their increased risk of breast cancer and of failed early diagnosis by screening mammography. Objective: To compare the screening performance of abbreviated breast magnetic resonance imaging (MRI) and digital breast tomosynthesis (DBT) in women with dense breasts. Design, Setting, and Participants: Cross-sectional study with longitudinal follow-up at 48 academic, community hospital, and private practice sites in the United States and Germany, conducted between December 2016 and November 2017 among average-risk women aged 40 to 75 years with heterogeneously dense or extremely dense breasts undergoing routine screening. Follow-up ascertainment of cancer diagnoses was complete through September 12, 2019. Exposures: All women underwent screening by both DBT and abbreviated breast MRI, performed in randomized order and read independently to avoid interpretation bias. Main Outcomes and Measures: The primary end point was the invasive cancer detection rate. Secondary outcomes included sensitivity, specificity, additional imaging recommendation rate, and positive predictive value (PPV) of biopsy, using invasive cancer and ductal carcinoma in situ (DCIS) to define a positive reference standard. All outcomes are reported at the participant level. Pathology of core or surgical biopsy was the reference standard for cancer detection rate and PPV; interval cancers reported until the next annual screen were included in the reference standard for sensitivity and specificity. Results: Among 1516 enrolled women, 1444 (median age, 54 [range, 40-75] years) completed both examinations and were included in the analysis. The reference standard was positive for invasive cancer with or without DCIS in 17 women and for DCIS alone in another 6. No interval cancers were observed during follow-up. Abbreviated breast MRI detected all 17 women with invasive cancer and 5 of 6 women with DCIS. Digital breast tomosynthesis detected 7 of 17 women with invasive cancer and 2 of 6 women with DCIS. The invasive cancer detection rate was 11.8 (95% CI, 7.4-18.8) per 1000 women for abbreviated breast MRI vs 4.8 (95% CI, 2.4-10.0) per 1000 women for DBT, a difference of 7 (95% CI, 2.2-11.6) per 1000 women (exact McNemar P = .002). For detection of invasive cancer and DCIS, sensitivity was 95.7% (95% CI, 79.0%-99.2%) with abbreviated breast MRI vs 39.1% (95% CI, 22.2%-59.2%) with DBT (P = .001) and specificity was 86.7% (95% CI, 84.8%-88.4%) vs 97.4% (95% CI, 96.5%-98.1%), respectively (P < .001). The additional imaging recommendation rate was 7.5% (95% CI, 6.2%-9.0%) with abbreviated breast MRI vs 10.1% (95% CI, 8.7%-11.8%) with DBT (P = .02) and the PPV was 19.6% (95% CI, 13.2%-28.2%) vs 31.0% (95% CI, 17.0%-49.7%), respectively (P = .15). Conclusions and Relevance: Among women with dense breasts undergoing screening, abbreviated breast MRI, compared with DBT, was associated with a significantly higher rate of invasive breast cancer detection. Further research is needed to better understand the relationship between screening methods and clinical outcome. Trial Registration: ClinicalTrials.gov Identifier: NCT02933489.


Asunto(s)
Densidad de la Mama , Neoplasias de la Mama/diagnóstico por imagen , Carcinoma Intraductal no Infiltrante/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Mamografía/métodos , Adulto , Anciano , Biopsia , Mama/diagnóstico por imagen , Mama/patología , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/patología , Estudios Transversales , Detección Precoz del Cáncer , Femenino , Estudios de Seguimiento , Humanos , Imagen por Resonancia Magnética/efectos adversos , Mamografía/efectos adversos , Persona de Mediana Edad , Invasividad Neoplásica , Valor Predictivo de las Pruebas , Estándares de Referencia , Sensibilidad y Especificidad
7.
Braz Oral Res ; 34: e021, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32187306

RESUMEN

This study aimed to assess the reliability and validity of Brazilian-Portuguese versions of the Health Literacy in Dentistry (HeLD) scale in a sample of elderly Brazilian participants. HeLD was initially translated into and cross-culturally adapted to the Brazilian Portuguese language. The reliability and validity of HeLD were then assessed in a sample of 535 non-institutionalized older persons who also completed a questionnaire containing sociodemographic and health information. Data were then randomly separated into two sub-datasets, and Confirmatory Factor Analysis was performed through structural equation modelling, with a maximum likelihood estimate to test the fit of the data to the factor structure of the long-and short-form HeLD (HeLD-29 and HeLD-14) versions of the instrument. The models were compared using the Akaike Information Criterion to assess goodness-of-fit and to determine which models were preferred. Internal consistency of HeLD was evaluated using Cronbach´s coefficient α. Both versions of HeLD were observed to demonstrate high internal reliability (Cronbach´s α ≥ 0.87 for all seven subscales), acceptable convergent (estimates of ≥ 0.50 for AVE and ≥ 0.70 for CR) and discriminant validity. However, the goodness-of-fit of the confirmatory factor analysis models demonstrated satisfactory results only for HeLD-14 subsamples (x2/df = 1.8-2.3; CFI = 0.97-0.98; GFI/NFI = 0.98-0.99; RMSEA = 0.05 and SRMR = 0.03). In conclusion, HeLD-14 was shown to be a reliable and valid instrument to measure oral health literacy in elderly Brazilian participants.


Asunto(s)
Odontología , Alfabetización en Salud/normas , Encuestas y Cuestionarios/normas , Anciano , Anciano de 80 o más Años , Brasil , Comparación Transcultural , Estudios Transversales , Análisis Factorial , Femenino , Humanos , Masculino , Psicometría , Estándares de Referencia , Reproducibilidad de los Resultados , Factores Socioeconómicos , Traducciones
8.
Artículo en Español | PAHO-IRIS | ID: phr-51862

RESUMEN

[RESUMEN]. La Comisión Lancet de Hipertensión determinó que una medida clave para responder a la carga mundial que representa la hipertensión arterial era mejorar la calidad de las mediciones de la presión arterial, mediante la utilización de dispositivos cuya exactitud haya sido validada. En la actualidad existen 3000 dispositivos comercializados, pero muchos no tienen datos publicados sobre pruebas de exactitud conformes a las normas científicas establecidas. La falta de regulación o su ineficiencia, que permiten la autorización de dispositivos para uso comercial sin una validación oficial, posibilitan este problema. Además, han surgido tecnologías nuevas de medición de la presión arterial (por ejemplo, los sensores sin brazalete) sobre las cuales no existe unanimidad en la comunidad científica con respecto a las normas de exactitud de la medición. En conjunto, estos aspectos contribuyen a la disponibilidad generalizada de tensiómetros de consultorio o domiciliarios que ofrecen una exactitud limitada o incierta, que llevan a diagnósticos, manejo y farmacoterapia inapropiados de la hipertensión a escala mundial. Los problemas más importantes relacionados con la exactitud de los dispositivos de medición de la presión arterial se pueden resolver mediante el requisito regulatorio de una validación independiente obligatoria de los dispositivos, en consonancia con la norma ISO universalmente aceptada. Esta es una recomendación básica y constituye una necesidad internacional acuciante. Otras recomendaciones clave son la elaboración de normas de validación específicas para las tecnologías nuevas de medición de la presión arterial y la publicación en línea de listas de los dispositivos nuevos exactos que están a la disposición de los usuarios y los profesionales de salud. Las recomendaciones están en consonancia con las políticas de la Organización Mundial de la Salud sobre los dispositivos médicos y la atención universal de la salud. El cumplimiento de las recomendaciones aumentará la disponibilidad mundial de dispositivos de medición de la presión arterial que sean exactos y tendrá como efecto un mejor diagnóstico y tratamiento, reduciendo así la carga mundial de la hipertensión.


[ABSTRACT]. The Lancet Commission on Hypertension identified that a key action to address the worldwide burden of high blood pressure (BP) was to improve the quality of BP measurements by using BP devices that have been validated for accuracy. Currently, there are over 3 000 commercially available BP devices, but many do not have published data on accuracy testing according to established scientific standards. This problem is enabled through weak or absent regulations that allow clearance of devices for commercial use without formal validation. In addition, new BP technologies have emerged (e.g. cuffless sensors) for which there is no scientific consensus regarding BP measurement accuracy standards. Altogether, these issues contribute to the widespread availability of clinic and home BP devices with limited or uncertain accuracy, leading to inappropriate hypertension diagnosis, management and drug treatment on a global scale. The most significant problems relating to the accuracy of BP devices can be resolved by the regulatory requirement for mandatory independent validation of BP devices according to the universally-accepted International Organization for Standardization Standard. This is a primary recommendation for which there is an urgent international need. Other key recommendations are development of validation standards specifically for new BP technologies and online lists of accurate devices that are accessible to consumers and health professionals. Recommendations are aligned with WHO policies on medical devices and universal healthcare. Adherence to recommendations would increase the global availability of accurate BP devices and result in better diagnosis and treatment of hypertension, thus decreasing the worldwide burden from high BP.


[RESUMO]. A Comissão Lancet sobre Hipertensão Arterial identificou que uma iniciativa central para enfrentar a carga mundial da hipertensão arterial seria a melhoria na qualidade da mensuração da pressão arterial pelo uso aparelhos de pressão arterial validados quanto à acurácia. Atualmente, existem mais de 3 000 aparelhos de pressão arterial disponíveis comercialmente; entretanto, muitos não têm dados publicados sobre testes de acurácia realizados de acordo com padrões científicos estabelecidos. Este problema resulta de regulamentação fraca ou inexistente, o que permite a aprovação para uso comercial de dispositivos sem validação formal. Além disso, surgiram novas tecnologias de mensuração da pressão arterial (por exemplo, sensores sem algemas) sem consenso científico quanto aos padrões de acurácia. No conjunto, essas questões contribuem para a oferta generalizada de dispositivos de pressão arterial clínica e domiciliar com acurácia limitada ou incerta, levando a diagnóstico, gerenciamento e tratamento inadequados da hipertensão em escala global. Os problemas mais significativos relacionados com a acurácia dos dispositivos de pressão arterial podem ser resolvidos por regulamentação que imponha a obrigatoriedade de validação independente dos aparelhos de pressão arterial, de acordo com a norma universalmente aceita pela Organização Internacional de Normalização. Esta é uma recomendação fundamental para a qual existe uma necessidade internacional urgente. Outras recomendações essenciais incluem o desenvolvimento de padrões de validação especificamente para novas tecnologias de mensuração da pressão arterial e listas on-line de aparelhos com acurácia adequada que sejam acessíveis aos consumidores e profissionais de saúde. As recomendações estão alinhadas com as políticas da Organização Mundial da Saúde (OMS) sobre dispositivos médicos e atenção universal à saúde. A adesão às recomendações aumentaria a oferta global de dispositivos de pressão arterial com acurácia adequada e resultaria em melhor diagnóstico e tratamento da hipertensão arterial, diminuindo assim a carga mundial dessa doença.


Asunto(s)
Salud Global , Tecnología Biomédica , Estándares de Referencia , Equipo para Diagnóstico , Salud Global , Tecnología Biomédica , Estándares de Referencia , Equipo para Diagnóstico , Salud Global , Tecnología Biomédica , Estándares de Referencia , Equipo para Diagnóstico
9.
Zhonghua Yu Fang Yi Xue Za Zhi ; 54(3): 314-319, 2020 Mar 06.
Artículo en Chino | MEDLINE | ID: mdl-32187938

RESUMEN

Objective: To systematically evaluate the quality of gastric cancer screening guidelines/recommendations, and provide a reference for the update of gastric cancer screening guidelines/recommendations in China. Methods: "guidelines/consensus/specifications/standards" , "stomach/gastric tumors" , "screening/diagnosis" , "guideline/recommendation" , "gastric cancer/gastric tumor," "early detection of cancer/screening" were searched as keywords in PubMed, Embase, Web of knowledge, China Knowledge Network, Wanfang, China Biomedical Literature Database, and Cochrane Library, as well as the US Preventive Services Working Group, the American Cancer Society, the International Agency for Research on Cancer, the Australia Cancer Council and the International Guide Collaboration Network at the end of July 2018. The inclusion criteria were independent guidelines/recommendation documents for gastric cancer screening. The exclusion criteria were guideline abstracts, interpretation and evaluation literature, duplicate publications, updated original guidelines, and clinical treatment or practice guidelines for gastric cancer. The language was limited to Chinese and English. The European Guide to Research and Evaluation Tools (AGREE Ⅱ) and Practice Guideline Reporting Standard (RIGHT) for Gastric Cancer Screening Guidelines/Recommendations were used to compare and evaluate the quality and reporting standard of gastric cancer screening guidelines/recommendations. Results: A total of five guides/recommendations were included. The results of the AGREE Ⅱ quality evaluation showed that the overall quality of five guides/recommendations was different, including one recommended for "A", one for "B", and three for "C". Each guide/recommendation scored higher in the scope and purpose, clarity, and scores were more significant in the areas of rigor and independence. In the participants, the application field scores were generally low. The RIGHT evaluation results showed that the quality of five guides/recommendations should be improved. The six items with poor report quality were background, evidence, recommendations, review and quality assurance, funding and conflict of interest statement and management, and other aspects. Conclusion: The quality of the included gastric cancer screening guidelines/recommendations is generally low, and the standardization should be strengthened.


Asunto(s)
Detección Precoz del Cáncer/normas , Guías de Práctica Clínica como Asunto/normas , Neoplasias Gástricas/diagnóstico , China , Consenso , Detección Precoz del Cáncer/métodos , Humanos , Estándares de Referencia
10.
PLoS One ; 15(2): e0228493, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32027673

RESUMEN

Domestic yak (Bos grunniens) is the most crucial livestock in the Qinghai-Tibetan Plateau, providing meat and other necessities for local people. The skeletal muscle of adult livestock is composed of muscle fibers, and fiber composition in muscle has influence on meat qualities, such as tenderness, pH, and color. Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful tool to evaluate the gene expression of muscle fiber, but the normalization of the data depends on the stability of expressed reference genes. Unfortunately, there is no consensus for an ideal reference gene for data normalization in muscle tissues of yak. In this study, we aimed to assess the stability of 14 commonly used candidate reference genes by using five algorithms (GeNorm, NormFinder, BestKeeper, Delat Ct and Refinder). Our results suggested UXT and PRL13A were the most stable reference genes, while the most commonly used reference gene, GAPDH, was most variably expressed across different muscle tissues. We also found that the extensor digitorum lateralis (EDL), trapezius pars thoracica (TPT), and psoas major (PM) muscle had the higher content of type I muscle fibers and the lowest content of type IIB muscle fibers, while gluteobiceps (GB) muscle had the highest content of type IIB muscle fibers. Our study provides the suitable reference genes for accurate analysis of yak muscle fiber composition.


Asunto(s)
Bovinos/genética , Genes Esenciales , Músculo Esquelético/metabolismo , Cadenas Pesadas de Miosina/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Animales , Animales Domésticos/genética , Expresión Génica , Masculino , Carne , Fibras Musculares Esqueléticas/metabolismo , Isoformas de Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de Referencia
11.
Anal Bioanal Chem ; 412(8): 1847-1861, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32020317

RESUMEN

Biomonitoring for human exposure to lead, arsenic, mercury, and other toxic metal(loid)s often relies on analyzing traditional biospecimens such as blood and urine. While biomonitoring based on blood and urine is well-established, non-traditional biospecimens such as hair and nails can offer the potential to explore past exposures as well as the advantages of non-invasive collection and ease of storage. The present study describes the production of four reference materials (NYS RMs 18-01 through 18-04) based on caprine horn, a keratinized tissue similar to human hair and nails, intended to serve as a resource for calibration, quality control, and method validation purposes. The elemental content and homogeneity of these candidate reference materials were characterized for 17 elements using inductively coupled plasma mass spectrometry (ICP-MS). Commutability between two or more of the NYS caprine horn RMs and human nails was established for 8 elements (Ba, Ca, Cr, Cu, Mn, Pb, Sr, and Zn) based on analysis by ICP-MS/MS and ICP-optical emission spectrometry. The development and optimization of an ICP-MS/MS instrumental method for the determination of 17 elements in keratinized tissues is described. The method was validated against three certified reference materials based on human hair showing good accuracy and method repeatability better than 25% for all analytes. This study also describes sample preparation issues and addresses common challenges including surface contamination, microwave digestion, matrix effects, and spectral interferences in inorganic mass spectrometry. New York State Department of Health Keratin Matrix Reference Materials. Graphical abstract.


Asunto(s)
Queratinas/química , Oligoelementos/análisis , Animales , Calibración , Cabras , Cabello/química , Humanos , Límite de Detección , Uñas/química , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos , Oligoelementos/normas
12.
PLoS One ; 15(2): e0226668, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32017769

RESUMEN

To accurately evaluate expression levels of target genes, stable internal reference genes is required for normalization of quantitative real-time PCR (qRT-PCR) data. However, there have been no systematical investigation on the stability of reference genes used in the bedstraw weed, Galium aparine L. (BGA). In this study, the expression profiles of seven traditionally used reference genes, namely 18S, 28S, ACT, GAPDH, EF1α, RPL7 and TBP in BGA were assessed under both biotic (developmental time and tissue), and abiotic (temperature, regions and herbicide) conditions. Four analytical algorithms (geNorm, Normfinder, BestKeeper and the ΔCt method) were used to analyze the suitability of these genes as internal reference genes. RefFinder, a comprehensive analytical software, was used to rank the overall stability of the candidate genes. The optimal normalization internal control genes were ranked as: 28S and RPL7 were best for all the different experimental conditions (developmental stages, tissues, temperature, regions and herbicide treatment); 28S and RPL7 for developmental stages; TBP and GAPDH for different tissues; 28S and GAPDH were relatively stable for different temperature; 28S and TBP were suitable for herbicide treatment. A specific set of reference genes were recommended for each experimental condition in BGA.


Asunto(s)
Galium/genética , Perfilación de la Expresión Génica/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Programas Informáticos
13.
PLoS One ; 15(2): e0229423, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32084228

RESUMEN

RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit-blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.


Asunto(s)
Leucocitos Mononucleares/metabolismo , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Manejo de Especímenes/métodos , Actinas/genética , Humanos , ARN/análisis , ARN/genética , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Reproducibilidad de los Resultados
14.
Euro Surveill ; 25(6)2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32046815

RESUMEN

Timely detection of novel coronavirus (2019-nCoV) infection cases is crucial to interrupt the spread of this virus. We assessed the required expertise and capacity for molecular detection of 2019-nCoV in specialised laboratories in 30 European Union/European Economic Area (EU/EEA) countries. Thirty-eight laboratories in 24 EU/EEA countries had diagnostic tests available by 29 January 2020. A coverage of all EU/EEA countries was expected by mid-February. Availability of primers/probes, positive controls and personnel were main implementation barriers.


Asunto(s)
Betacoronavirus , Técnicas de Laboratorio Clínico/normas , Infecciones por Coronavirus/diagnóstico , Coronavirus/genética , Coronavirus/aislamiento & purificación , Laboratorios/normas , Neumonía Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Técnicas de Laboratorio Clínico/métodos , Coronavirus/clasificación , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/virología , Brotes de Enfermedades , Unión Europea , Humanos , ARN Viral/genética , Estándares de Referencia , Sensibilidad y Especificidad , Vigilancia de Guardia , Análisis de Secuencia
15.
Anal Bioanal Chem ; 412(7): 1685-1692, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32008083

RESUMEN

The 5α-reductase converts testosterone to dihydrotestosterone (DHT), and excess DHT could cause androgen-related diseases such as androgenetic alopecia and benign prostatic hyperplasia (BPH). To discover new 5α-reductase inhibitors, effective drug screening method with high throughput is thus essential. In this study, fully automated chip-based nanoelectrospray ionization-mass spectrometry (nano-ESI-MS) was innovatively utilized as a screening tool for 5α-reductase inhibitory assay in direct infusion mode, which simplified sample pretreatment and greatly improved experimental efficiency. The preliminary data indicated that curcumin, a natural anti-inflammatory compound, exhibited notably 5α-reductase inhibition activity. Moreover, the obtained results of the chip-based nano-ESI-MS were well consistent with those of HPLC-MS, which suggested that the chip-based nano-ESI-MS could be treated as a rapid and high-throughput drugs screening strategy in pharmaceutical development. Graphical abstract.


Asunto(s)
Inhibidores de 5-alfa-Reductasa/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Nanotecnología , Espectrometría de Masa por Ionización de Electrospray/métodos , Andrógenos/análisis , Automatización , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Células HEK293 , Humanos , Masculino , Estándares de Referencia
16.
Anal Bioanal Chem ; 412(7): 1653-1661, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32008082

RESUMEN

As quantitative analysis of biotherapeutics in cerebrospinal fluid (CSF) with LC-MS becomes increasingly widespread, there is a need for method developments towards higher sensitivity. By using artificial CSF (aCSF) in the development phase, the consumption of costly and sparsely available CSF can be limited. The aCSF compositions tested here were made from various dilutions of bovine serum albumin (BSA) or rat plasma to mimic the total protein concentration found in CSF. Focusing on monoclonal antibodies, the aCSF was spiked with human immunoglobulin (hIgG) and prepared with the bottom-up analysis technique using LC-MS. Assuming that the composition of the aCSF would affect the digest, the response from aCSF matrices was compared with CSF from rat, monkey, and dog in terms of estimated sample concentration and matrix effects. The samples were spiked with hIgG in the range of 10 to 1000 ng/mL and volumes of 10 µL were transferred to sample preparation. The results indicate that BSA dilutions from 300 to 2000 µg/mL and rat plasma dilutions of 0.5-2% provide the most accurate concentration estimates when compared with rat CSF. 1000 µg/mL BSA did not produce significantly different concentration estimates for 500 ng/mL samples when compared with CSF from rat, monkey, and dog, and can therefore be used as aCSF for several different species.


Asunto(s)
Anticuerpos/líquido cefalorraquídeo , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Calibración , Perros , Haplorrinos , Humanos , Ratas , Estándares de Referencia
17.
Anal Bioanal Chem ; 412(7): 1469-1481, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32034456

RESUMEN

Due to their omnipresence in consumer products, there is a growing concern about the potential effects of nanoparticles on human health. Toxicological assessment and NP end-product studies require proper quantification of these materials in biological fluids. However, their quantifications in these media require stable predispersed NP solutions in aqueous media to enable the fortification in the matrices of interest or the preparation of calibration standards. In this study, a sample preparation scheme was developed by studying various dispersion media (polyvinylpyrrolidone and polyethylene glycol) and sonication strategies (bath and ultrasonic probe) to ensure homogeneous dispersion of titanium dioxide nanoparticles. Optimization of the various parameters was performed using SRM NIST 1898 NP reference material, composed of rutile and anatase phases. Number-based size distribution for titanium dioxide NPs was determined by dynamic light scattering and single-particle inductively coupled plasma mass spectrometry to evaluate the procedure efficiency. Changes in mean size and most frequent size distribution were also studied to determine if the agglomeration of nanoparticles occurs at the various dispersion conditions tested. Among the different dispersion parameters tested herein, the use of polyvinylpyrrolidone combined with a sonication process generated by a probe leads to a significant improvement in terms of suspension efficiency and stability over 72 h. The dispersion efficiency of the proposed methodology was assessed by single-particle inductively coupled plasma mass spectrometry with spiked biological fluids such as urine and blood. Graphical abstract.


Asunto(s)
Líquidos Corporales/química , Nanopartículas del Metal/química , Titanio/química , Humanos , Nanopartículas del Metal/normas , Estándares de Referencia , Titanio/normas , Agua
18.
Clin Chim Acta ; 505: 31-33, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32084381

RESUMEN

BACKGROUND: Vitamin A and E are routinely monitored to assess nutritional status. The most commonly used approach for their measurement involves laborious liquid-liquid extraction followed by high-performance liquid chromatography (HPLC) analysis on dedicated instrumentation. We describe a simple, rapid protocol for measurement of vitamin A and E and their integration into an existing online sample preparation liquid chromatography tandem mass spectrometry (SPLC-MS/MS) workflow. METHODS: We performed a method comparison between the SPLC-MS/MS and HPLC methods for vitamin A and E by measuring patient specimens across the concentration range 11-81 µg/dL for vitamin A and 1-18 mg/L for vitamin E. The analysis times on each platform were also compared. RESULTS: SPLC-MS/MS and HPLC methods were comparable with regards to analytical performance; mean bias across the measured range was 2.54% (95% CL: -11.56-16.64%) for vitamin A and -2.04% (95% CL: -18.20-14.12%) for vitamin E. Total analysis times were 7 min and 15 min for SPLC-MS/MS and HPLC respectively. CONCLUSIONS: The development of a simplified sample preparation protocol and the use of multiplexing SPLC-MS/MS have reduced sample analysis times for vitamin A and E. This method has also optimized clinical workflow through consolidation of previously independent benches.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Vitamina A/sangre , Vitamina E/sangre , 25-Hidroxivitamina D 2/análisis , Anticonvulsivantes/análisis , Busulfano/análisis , Humanos , Inmunosupresores/análisis , Laboratorios/organización & administración , Estándares de Referencia , Reproducibilidad de los Resultados , Flujo de Trabajo
20.
PLoS One ; 15(2): e0228069, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32053603

RESUMEN

BACKGROUND: Standardised packaging for factory made (FM) and roll your own (RYO) tobacco was fully implemented in the UK in May 2017. Around the same time, several changes to the tax system were applied (a Minimum Excise Tax (MET) for FM products and tax increases weighted towards RYO products). The tobacco industry claims that standardised packaging will lower prices (a disincentive for quitting) by commoditising the product, yet had itself taken advantage of the previous tax regime to achieve large profits from premium brands while also keeping some products' prices relatively low. Here we evaluate the impact of standardised packaging, the MET and the RYO focussed tax changes on price and industry profitability. METHODS AND FINDINGS: Nielsen electronic point of sale (EPOS) data (May 2015 to April 2018) were used to calculate real (inflation adjusted) monthly price per stick overall, by cigarette type (FM and RYO) and by seven market segments. Trend estimation, using additive mixed models, assessed weighted average price (weighted by volume of sales) and tobacco industry net revenue changes. The beginning and end of the data series were compared in terms of: (a) average monthly price growth, (b) average monthly net revenue growth, and (c) undershifting and overshifting patterns after tax changes. FM and RYO real prices changed little over the 3-year period-overall prices rose by about 1p per stick. There was no evidence of commoditisation with prices of all FM segments (but not RYO) rising faster after the implementation of standardised packaging than immediately beforehand. The prices of the cheapest FM brands rose with the implementation of the MET. RYO price increases did not close the gap to FM pricing levels despite RYO focussed tax increases. Tax changes following the implementation of standardised packaging and the MET were more widely and quickly passed on to smokers in the form of higher prices than the tax change pre-implementation. The main limitations are first that because we do not know the exact mechanism by which Nielsen scales up sample data to provide UK estimates, we could only use data for a set three year period during which the same adjustments are made. Second, the tax and standardised packaging events were sometimes too close in time to separate their consequences statistically. Third, tobacco prices may also be affected by external factors such as changes in smokers' disposable income or availability of electronic nicotine delivery systems. CONCLUSIONS: There was no long-term lowering of tobacco prices after the implementation of standardised packaging as predicted by the industry. The introduction of the MET was successful in increasing the price of the cheapest FM cigarettes and narrowing the price gap between FM brands. The RYO tax increases were, however, insufficient to narrow the price gap between RYO and FM. Overall, undershifting became less extensive indicating that tobacco industry manipulation of the tax system which had previously kept cheap products available had declined. This suggests that standardised packaging and a MET will likely contribute to further declines in UK tobacco use.


Asunto(s)
Embalaje de Productos/economía , Impuestos/estadística & datos numéricos , Tabaco , Análisis Multivariante , Estándares de Referencia , Industria del Tabaco/economía , Reino Unido
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