Iron-tracking strategies: Chaperones capture iron in the cytosolic labile iron pool.

Cells express hundreds of iron-dependent enzymes that rely on the iron cofactors heme, iron-sulfur clusters, and mono-or di-nuclear iron centers for activity. Cells require systems for both the assembly and the distribution of iron cofactors to their cognate enzymes. Proteins involved in the binding and trafficking of iron ions in the cytosol, called cytosolic iron chaperones, have been identified and characterized in mammalian cells. The first identified iron chaperone, poly C-binding protein 1 (PCBP1), has also been studied in mice using genetic models of conditional deletion in tissues specialized for iron handling. Studies of iron trafficking in mouse tissues have necessitated the development of new approaches, which have revealed new roles for PCBP1 in the management of cytosolic iron. These approaches can be applied to investigate use of other nutrient metals in mammals.

Nucleic acid-based biosensors: analytical devices for prevention, diagnosis and treatment of diseases / Biosensores basados en ácidos nucleicos: dispositivos analíticos para la prevención, el diagnóstico y tratamiento de enfermedades

BACKGROUND: Biosensing techniques have been the subject of exponentially increasing interest due to their performance advantages such as high selectivity and sensitivity, easy operation, low cost, short analysis time, simple sample preparation, and real-time detection. Biosensors have been developed by integrating the unique specificity of biological reactions and the high sensitivity of physical sensors. Therefore, there has been a broad scope of applications for biosensing techniques, and nowadays, they are ubiquitous in different areas of environmental, healthcare, and food safety. Biosensors have been used for environmental studies, detecting and quantifying pollutants in water, air, and soil. Biosensors also showed great potential for developing analytical tools with countless applications in diagnosing, preventing, and treating diseases, mainly by detecting biomarkers. Biosensors as a medical device can identify nucleic acids, proteins, peptides, metabolites, etc.; these analytes may be biomarkers associated with the disease status. Bacterial food contamination is considered a worldwide public health issue; biosensor-based analytical techniques can identify the presence or absence of pathogenic agents in food. OBJECTIVES: The present review aims to establish state-of-the-art, comprising the recent advances in the use of nucleic acid-based biosensors and their novel application for the detection of nucleic acids. Emphasis will be given to the performance characteristics, advantages, and challenges. Additionally, food safety applications of nucleic acid-based biosensors will be discussed. METHODS: Recent research articles related to nucleic acid-based biosensors, biosensors for detecting nucleic acids, biosensors and food safety, and biosensors in environmental monitoring were reviewed. Also, biosensing platforms associated with the clinical diagnosis and food industry were included. RESULTS: It is possible to appreciate that multiple applications of nucleic acid-based biosensors have been reported in the diagnosis, prevention, and treatment of diseases, as well as to identify foodborne pathogenic bacteria. The use of PNA and aptamers opens the possibility of developing new biometric tools with better analytical properties. CONCLUSIONS: Biosensors could be considered the most important tool for preventing, treating, and monitoring diseases that significantly impact human health. The aptamers have advantages as biorecognition elements due to the structural conformation, hybridization capacity, robustness, stability, and lower costs. It is necessary to implement biosensors in situ to identify analytes with high selectivity and lower detection limits

ANTECEDENTES: Las técnicas de biodetección han sido objeto de un interés cada vez mayor debido a ventajas, tales como alta selectividad y sensibilidad, facilidad de manejo, bajo costo, tiempo de análisis corto, preparación sencilla de muestras y detección en tiempo real. Los biosensores se han desarrollado integrando la especificidad única de las reacciones biológicas y la alta sensibilidad de los sensores físicos. Por lo tanto, las técnicas de biodetección han tenido un amplio campo de aplicación y hoy en día son omnipresentes en diferentes áreas del medio ambiente, la salud y la seguridad alimentaria. Se han utilizado biosensores para estudios ambientales, detectando y cuantificando contaminantes en el agua, el aire y el suelo. Los biosensores también mostraron un gran potencial para desarrollar herramientas analíticas con innumerables aplicaciones en el diagnóstico, prevención y tratamiento de enfermedades, principalmente mediante la detección de biomarcadores. Los biosensores como dispositivo médico pueden utilizarse para identificar ácidos nucleicos, proteínas, péptidos, metabolitos, etc. Estos analitos pueden ser biomarcadores asociados al estado de la enfermedad. La contaminación bacteriana de los alimentos se considera un problema de salud pública mundial; se pueden utilizar técnicas analíticas basadas en biosensores para determinar la presencia o ausencia de agentes patógenos en los alimentos. OBJETIVOS: La presente revisión tiene por objeto establecer los últimos adelantos en la utilización de biosensores basados en ácidos nucleicos y su novedosa aplicación para la detección de ácidos nucleicos. Se hará hincapié en las características del desempeño, las ventajas y los desafíos. Además, se examinarán las aplicaciones de los biosensores basados en ácidos nucleicos para la inocuidad de los alimentos. MÉTODOS: Se examinaron artículos de investigación recientes relacionados con los biosensores a base de ácidos nucleicos, los biosensores para la detección de ácidos nucleicos, los biosensores y la inocuidad de los alimentos, y los biosensores para la vigilancia del medio ambiente. También se incluyeron plataformas de biosensores asociadas al diagnóstico clínico y a la industria alimentaria. RESULTADOS: Es posible apreciar que se han reportado múltiples aplicaciones de biosensores basados en ácido nucleico para el diagnóstico, prevención y tratamiento de enfermedades, así como para identificar bacterias patógenas transmitidas por los alimentos. El uso de PNA y aptámeros abre la posibilidad de desarrollar nuevas herramientas biométricas con mejores propiedades analíticas. CONCLUSIONES: Los biosensores pueden ser considerados como los instrumentos más importantes para la prevención, el tratamiento y la vigilancia de las enfermedades que tienen un impacto significativo en la salud humana. Los aptámeros tienen ventajas como elemento de biorreconocimiento debido a la conformación estructural, capacidad de hibridación, robustez, estabilidad y menores costos. Es necesario implementar biosensores in situ para identificar analitos con alta selectividad y menores límites de detección

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis.

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma membrane showed features of high affinity/slow turnover ATPases, with enzymatic parameters KM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma mem-brane showed features of high affinity/slow turnover ATPases, with enzymatic parametersKM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

CtpA, a putative Mycobacterium tuberculosis P-type ATPase, is stimulated by copper (I) in the mycobacterial plasma membrane.

The transport of heavy-metal ions across the plasma membrane is essential for mycobacterial intracellular survival; in this context, P-type ATPases are pivotal for maintenance of ionic gradients and the plasma membrane homeostasis of mycobacteria. To date, the copper ion transport that is mediated by P-type ATPases in mycobacteria is poorly understood. In this work, the ion-specific activation of CtpA, a putative plasma membrane Mycobacterium tuberculosis P-type ATPase, with different heavy-metal cations was assessed. Mycobacterium smegmatis mc(2)155 cells heterologously expressing the M. tuberculosis ctpA gene displayed an increased tolerance to toxic levels of the Cu(2+) ion (4 mM) compared to control cells, suggesting that CtpA is possibly involved in the copper detoxification of mycobacterial cells. In contrast, the tolerance of M. smegmatis recombinant cells against other heavy-metal divalent cations, such as Co(2+), Mn(2+), Ni(2+) and Zn(2+), was not detected. In addition, the ATPase activity of plasma membrane vesicles that were obtained from M. smegmatis cells expressing CtpA was stimulated by Cu(+) (4.9 nmol of Pi released/mg of protein.min) but not by Cu(2+) ions; therefore, Cu(2+) reduction to Cu(+) inside mycobacterial cells is suggested. Finally, the plasma membrane vesicles of M. smegmatis that were enriched with CtpA exhibited an optimal activity at 37 °C and pH 7.9; the apparent kinetic parameters of the enzyme were a K(1/2) of 4.68 × 10(-2) µM for Cu(+), a Vmax of 10.3 U/mg of protein, and an h value of 1.91.

In silico identification and characterization of the ion transport specificity for P-type ATPases in the Mycobacterium tuberculosis complex.

BACKGROUND: P-type ATPases hydrolyze ATP and release energy that is used in the transport of ions against electrochemical gradients across plasma membranes, making these proteins essential for cell viability. Currently, the distribution and function of these ion transporters in mycobacteria are poorly understood. RESULTS: In this study, probabilistic profiles were constructed based on hidden Markov models to identify and classify P-type ATPases in the Mycobacterium tuberculosis complex (MTBC) according to the type of ion transported across the plasma membrane. Topology, hydrophobicity profiles and conserved motifs were analyzed to correlate amino acid sequences of P-type ATPases and ion transport specificity. Twelve candidate P-type ATPases annotated in the M. tuberculosis H37Rv proteome were identified in all members of the MTBC, and probabilistic profiles classified them into one of the following three groups: heavy metal cation transporters, alkaline and alkaline earth metal cation transporters, and the beta subunit of a prokaryotic potassium pump. Interestingly, counterparts of the non-catalytic beta subunits of Hydrogen/Potassium and Sodium/Potassium P-type ATPases were not found. CONCLUSIONS: The high content of heavy metal transporters found in the MTBC suggests that they could play an important role in the ability of M. tuberculosis to survive inside macrophages, where tubercle bacilli face high levels of toxic metals. Finally, the results obtained in this work provide a starting point for experimental studies that may elucidate the ion specificity of the MTBC P-type ATPases and their role in mycobacterial infections.