A bioluminescent microbial biosensor for in vitro pretreatment assessment of cytarabine efficacy in leukemia

BACKGROUND: The nucleoside analog cytarabine (Ara-C [cytosine arabinoside]) is the key agent for treating acute myeloid leukemia (AML); however, up to 30% of patients fail to respond to treatment. Screening of patient blood samples to determine drug response before commencement of treatment is needed. This project aimed to construct and evaluate a self-bioluminescent reporter strain of Escherichia coli for use as an Ara-C biosensor and to design an in vitro assay to predict Ara-C response in clinical samples. METHODS: Weused transposition mutagenesis to create a cytidine deaminase (cdd)-deficient mutant of E. coli MG1655 that responded to Ara-C. The strain was transformed with the luxCDABE operon and used as a whole-cell biosensor for development an 8-h assay to determine Ara-C uptake and phosphorylation by leukemic cells. RESULTS: Intracellular concentrations of 0.025 μmol/L phosphorylated Ara-C were detected by significantly increased light output (P < 0.05) from the bacterial biosensor. Results using AML cell lines with known response to Ara-C showed close correlation between the 8-h assay and a 3-day cytotoxicity test for Ara-C cell killing. In retrospective tests with 24 clinical samples of bone marrow or peripheral blood, the biosensor-based assay predicted leukemic cell response to Ara-C within 8 h. CONCLUSIONS: The biosensor-based assay may offer a predictor for evaluating the sensitivity of leukemic cells to Ara-C before patients undergo chemotherapy and allow customized treatment of drug-sensitive patients with reduced Ara-C dose levels. The 8-h assay monitors intracellular Ara-CTP (cytosine arabinoside triphosphate) levels and, if fully validated, may be suitable for use in clinical settings. © 2010 American Association for Clinical Chemistry

TIMSS 2011 : marcos de la evaluación

Incluye anexosCon el fin de ser especialmente relevante en lo que se refiere a la toma de decisiones y aplicación de las directrices escolares, TIMSS evalúa a los alumnos en dos hitos educativos importantes: al final de Cuarto curso de Educación Primaria y al final de Segundo curso de Educación Secundaria Obligatoria. Puesto que TIMSS estudia la eficacia del currículo y de la instrucción en relación con el rendimiento del estudiante, es importante que TIMSS evalúe el rendimiento en Matemáticas y Ciencias en el mismo punto de la escolarización en los distintos países. Esto quiere decir que, para que las comparaciones sean equitativas, los alumnos deben haber tenido la oportunidad de aprender Matemáticas y Ciencias durante un número equivalente de años de escolarización oficial. Los datos de TIMSS complementan el Estudio Internacional de Progreso en Comprensión Lectora (PIRLS) que se realiza en Cuarto curso de Educación Primaria. Los países que participan en TIMSS y PIRLS pueden tener información regular sobre cómo leen sus alumnos y sobre sus conocimientos, además de, sobre lo qué pueden hacer en Matemáticas y Ciencias. El año 2011 ha sido una oportunidad única para la evaluación internacional en Cuarto curso, ya que, el ciclo de cuatro años de TIMSS está alineado con el ciclo de cinco de PIRLS. Éste último se realiza por tercera vez en 2011 después de las evaluaciones de los años 2001 y 2006.MadridES

Investigation and verification of a bioluminescent biosensor for the quantitation of ara-CTP generation: A biomarker for cytosine arabinoside sensitivity in acute myeloid leukaemia

A novel whole cell bacterial biosensor, which emits light in response to the active metabolite of cytosine arabinoside (ara-C, cytarabine), ara-CTP, has been investigated and verified. The biosensor has been formulated as an ex vivo assay, designed for peripheral blood or bone marrow cells, which can produce a clinical result within a working day. The nucleoside analogue ara-C is a key agent for treatment of acute myeloid leukaemia (AML); treatment decisions are made rapidly with AML, patients often receiving same-day commencement of chemotherapy. Currently no rapid predictive test is available to select appropriate therapy for patients prior to treatment. Experiments were designed to determine optimal assay conditions using leukaemic cell lines. We observed a significant increase (~15 fold) in bioluminescence signal compared to control after 8-h incubation of the biosensor with ara-C. This corresponded to a &gt;2-log increase in light output per bacterial cell. Interestingly, bioluminescence conferred a survival advantage to the bacteria following ara-C treatment. The assay is sensitive (lower limit of quantitation of 0.05. μM), selective, accurate (≤15% RE) and precise (≤15% coefficient of variation) over a linear concentration range of ara-CTP (0.05-0.5. μM), and detection is independent of reaction volume. Recovery of added standard was tested using ex vivo patient leukaemic cells (n=5). Stability studies on lyophilized bacterial biosensor were performed to ensure maintenance of performance over 12 months. The biosensor assay could be invaluable to the clinician, assisting with treatment selection, and potentially mitigating the risks of resistance and toxicity observed with this drug. © 2013 Elsevier B.V

Investigation and verification of a bioluminescent biosensor for the quantitation of ara-CTP generation: A biomarker for cytosine arabinoside sensitivity in acute myeloid leukaemia

A novel whole cell bacterial biosensor, which emits light in response to the active metabolite of cytosine arabinoside (ara-C, cytarabine), ara-CTP, has been investigated and verified. The biosensor has been formulated as an ex vivo assay, designed for peripheral blood or bone marrow cells, which can produce a clinical result within a working day. The nucleoside analogue ara-C is a key agent for treatment of acute myeloid leukaemia (AML); treatment decisions are made rapidly with AML, patients often receiving same-day commencement of chemotherapy. Currently no rapid predictive test is available to select appropriate therapy for patients prior to treatment. Experiments were designed to determine optimal assay conditions using leukaemic cell lines. We observed a significant increase (~15 fold) in bioluminescence signal compared to control after 8-h incubation of the biosensor with ara-C. This corresponded to a >2-log increase in light output per bacterial cell. Interestingly, bioluminescence conferred a survival advantage to the bacteria following ara-C treatment. The assay is sensitive (lower limit of quantitation of 0.05. μM), selective, accurate (≤15% RE) and precise (≤15% coefficient of variation) over a linear concentration range of ara-CTP (0.05-0.5. μM), and detection is independent of reaction volume. Recovery of added standard was tested using ex vivo patient leukaemic cells (n=5). Stability studies on lyophilized bacterial biosensor were performed to ensure maintenance of performance over 12 months. The biosensor assay could be invaluable to the clinician, assisting with treatment selection, and potentially mitigating the risks of resistance and toxicity observed with this drug. © 2013 Elsevier B.V