Microarray tools to unveil viral-microbe interactions in nature

The interactions between viruses and their microbial hosts play a central role in the control of microbial communities in nature. However, the study of such interactions within the uncultured majority is technically very challenging. Here, we review how microarray tools can be used to analyze the interactions between viruses and their microbial hosts in nature, away from laboratory pure culture-based models. We show examples of how DNA arrays have been used to study the expression of viral assemblages in natural samples, and to assign viruses to hosts within uncultured communities. Finally, we briefly discuss the possibilities of protein and glycan arrays to gain insight into the ways microbes interact with their viruses.Our current studies with viral microarrays are supported by projects CGL2012-39627-C03-01 (to Josefa Antón) and AYA2011-24803 (to Víctor Parro) of the Spanish Ministry of Science and Innovation, which are co-financed with FEDER support from the European Union

Environmental transcriptome analysis reveals physiological differences between biofilm and planktonic modes of life of the iron oxidizing bacteria Leptospirillum spp. in their natural microbial community

<p>Abstract</p> <p>Background</p> <p>Extreme acidic environments are characterized by their high metal content and lack of nutrients (oligotrophy). Macroscopic biofilms and filaments usually grow on the water-air interface or under the stream attached to solid substrates (streamers). In the Río Tinto (Spain), brown filaments develop under the water stream where the Gram-negative iron-oxidizing bacteria <it>Leptospirillum </it>spp. (<it>L. ferrooxidans </it>and <it>L. ferriphilum</it>) and <it>Acidithiobacillus ferrooxidans </it>are abundant. These microorganisms play a critical role in bioleaching processes for industrial (biominery) and environmental applications (acid mine drainage, bioremediation). The aim of this study was to investigate the physiological differences between the free living (planktonic) and the sessile (biofilm associated) lifestyles of <it>Leptospirillum </it>spp. as part of its natural extremely acidophilic community.</p> <p>Results</p> <p>Total RNA extracted from environmental samples was used to determine the composition of the metabolically active members of the microbial community and then to compare the biofilm and planktonic environmental transcriptomes by hybridizing to a genomic microarray of <it>L. ferrooxidans</it>. Genes up-regulated in the filamentous biofilm are involved in cellular functions related to biofilm formation and maintenance, such as: motility and quorum sensing (<it>mqsR, cheAY, fliA, motAB</it>), synthesis of cell wall structures (<it>lnt, murA, murB</it>), specific proteases (<it>clpX/clpP</it>), stress response chaperons (<it>clpB, clpC, grpE-dnaKJ, groESL</it>), etc. Additionally, genes involved in mixed acid fermentation (<it>poxB</it>, <it>ackA</it>) were up-regulated in the biofilm. This result, together with the presence of small organic acids like acetate and formate (1.36 mM and 0.06 mM respectively) in the acidic (pH 1.8) water stream, suggests that either <it>L. ferrooxidans </it>or other member of the microbial community are producing acetate in the acidophilic biofilm under microaerophilic conditions.</p> <p>Conclusions</p> <p>Our results indicate that the acidophilic filaments are dynamic structures in which different mechanisms for biofilm formation/dispersion are operating. Specific transcriptomic fingerprints can be inferred for both planktonic and sessile cells, having the former a more active TCA cycle, while the mixed acid fermentation process dominate in the latter. The excretion of acetate may play a relevant ecological role as a source of electron donor for heterotrophic Fe³⁺reducers like some Alphaproteobacteria, <it>Acidobacterium </it>spp. and <it>Sulfobacillus </it>spp., also present in the biofilm. Additionally, acetate may have a negative effect on bioleaching by inhibiting the growth of chemolithotrophic bacteria.</p

Biosensor comprising metal nanoparticles

[ES] La presente invención se refiere a un biosensor donde la detección del analito se realiza de forma visual por el cambio de color en las zonas del soporte en que el analito esté presente producido por las nanopartículas al ser irradiadas con una fuente de luz externa[EN] The present invention discloses a biosensor for visual detection of an analyte, based on the light to heat conversion properties of metal nanoparticles: the analyte is visually detected by the colour change in the support areas (where the analyte is present), produced as a result of the heat generated by the metal nanoparticles where they are irradiated with an external light source. Use of said biosensor in a method for the detection of analytes is also claimed.Peer reviewedUniversidad de Zaragoza, Fundación Agencia Aragonesa para la Investigación y el Desarrollo, Consejo Superior de Investigaciones Científicas (España)B1 Patente sin examen previ

Integration of an Optical Ring Resonator Biosensor into a Self-Contained Microfluidic Cartridge with Active, Single-Shot Micropumps

While there have been huge advances in the field of biosensors during the last decade, their integration into a microfluidic environment avoiding external tubing and pumping is still neglected. Herein, we show a new microfluidic design that integrates multiple reservoirs for reagent storage and single-use electrochemical pumps for time-controlled delivery of the liquids. The cartridge has been tested and validated with a silicon nitride-based photonic biosensor incorporating multiple optical ring resonators as sensing elements and an immunoassay as a potential target application. Based on experimental results obtained with a demonstration model, subcomponents were designed and existing protocols were adapted. The newly-designed microfluidic cartridges and photonic sensors were separately characterized on a technical basis and performed well. Afterwards, the sensor was functionalized for a protein detection. The microfluidic cartridge was loaded with the necessary assay reagents. The integrated pumps were programmed to drive the single process steps of an immunoassay. The prototype worked selectively, but only with a low sensitivity. Further work must be carried out to optimize biofunctionalization of the optical ring resonators and to have a more suitable flow velocity progression to enhance the system’s reproducibility.The authors would like to thank the European Union for their funding of the project PBSA “Photonic Biosensor for Space Application” within the FP7-program (FP7 program Grant Agreement No. 312942-PBSA. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI

Transposase interaction with the β sliding clamp: Effects on insertion sequence proliferation and transposition rate

Insertion sequences (ISs) are ubiquitous and abundant mobile genetic elements in prokaryotic genomes. ISs often encode only one protein, the transposase, which catalyzes their transposition. Recent studies have shown that transposases of many different IS families interact with the β sliding clamp, a DNA replication factor of the host. However, it was unclear to what extent this interaction limits or favors the ability of ISs to colonize a chromosome from a phylogenetically-distant organism, or if the strength of this interaction affects the transposition rate. Here we describe the proliferation of a member of the IS1634 family in Acidiphilium over ~600 generations of cultured growth. We demonstrate that the purified transposase binds to the β sliding clamp of Acidiphilium, Leptospirillum and E. coli. Further, we also demonstrate that the Acidiphilium IS1634 transposase binds to the archaeal sliding clamp (PCNA) from Methanosarcina, and that the transposase encoded by Methanosarcina IS1634 binds to Acidiphilium β. Finally, we demonstrate that increasing the strength of the interaction between β and transposase results in a higher transposition rate in vivo. Our results suggest that the interaction could determine the potential of ISs to be mobilized in bacterial populations and also their ability to proliferate within chromosomesThis work was funded by the Subdirección General de Proyectos de Investigación of the Spanish Ministry for Economy and Competitiveness Grants No. CGL2010-17384 and AYA2011-24803, and by the European Research Council (ERC) Advanced Grant No. 250350 (IPBSL). HDM has a FPI fellowship from the Spanish Governmen

Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

<p>Abstract</p> <p>Background</p> <p>The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite <it>Leishmania</it>. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom <it>L. infantum </it>genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome.</p> <p>Results</p> <p>Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings.</p> <p>Conclusions</p> <p>The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into <it>Leishmania </it>physiology.</p

Método y aparato para la detección de sustancias o analitos a partir del análisis de una o varias muestras

Referencia OEPM: P200301292.-- Fecha de solicitud: 30/05/2003.-- Titulares: Instituto Nacional de Técnica Aeroespacial “Esteban Terradas”, Sener ingeniería y Sistemas S.A. y Consejo Superior de Investigaciones Científicas (CSIC).El método comprende mezclar la muestra con un líquido tampón apropiado, homogeneizar dicha muestra, añadir reactivos a la misma, filtrarla, inyectar la muestra a una cámara de incubación, dejar reaccionar la muestra con un biosensor, lavar el exceso de muestra no reaccionada y detectar la muestra retenida en el biosensor. El aparato incluye un módulo homogeneizador de muestras con un dispositivo piezoeléctrico de ultrasonidos formado por un convertidor y una bocina; un módulo de procesamiento de muestras, que incluye un recipiente de homogeneización y un bastidor móvil; un módulo de gestión de reactivos y soluciones, que incluye una jeringa motorizada, un módulo de reacción, compuesto por un soporte que forma una cámara de reacción, y un módulo de lectura de datos que incluye un diodo láser y una cámara CCD.Peer reviewe

Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once-living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules, making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L-aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1–10 ppb level and some even at the part-per-trillion level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (ca. 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside EarthThis work was supported by granted projects AYA2011-24803, ESP2014-51989-P, and ESP2015-69540-R, from the Ministry of Economy and Competitiveness (MINECO) of Spain, and by Grant No. ST/N000803/1 (United Kingdom). A. G-C was a fellow of ‘‘Plan de Formación from INTA.Peer reviewe

Nursing workforce characteristics and control of diabetes mellitus in primary care: A multilevel analysis. Spain

Fundamentos: La actividad de enfermería está condicionada por las características de la plantilla. El objetivo fue determinar cómo afectan las características de la plantilla de enfermería de atención primaria (AP) al control de la diabetes mellitus (DM) en personas adultas. Método: Estudio analítico transversal. Instrumentos para la recogida de datos: sistemas de información de AP y cuestionario PES-Nursing Work Index. Participantes: 44.214 pacientes diabéticos en dos zonas de salud de la Comunidad de Madrid: Zona Noroeste (ZNO) con mejor situación socioeconómica y Zona Suroeste (ZSO) con peor situación socioeconómica y los 507 profesionales de enfermería de referencia. Se realizaron análisis multivariantes multinivel de regresión logística. La variable dependiente fue la DM estaba mal controlada (cuando los valores de Hb1Ac eran ≥ a 7%) Resultados: La prevalencia DM mal controlada fue de 40,1% (IC95%:38,2-42,1). Existía un riesgo de un 25% más de peor control si el paciente cambiaba de centro de salud y de un 27% si cambiaba de pareja médico de cabaecera y enfermera. En los modelos de regresión logística multivariante multinivel: para la ZSO a mayor ratio de pacientes mayores de 65 años aumentaba el riesgo de mal control (OR=1,00008 [IC95%:1,00006-1,001]); a mayor proporción de pacientes sin seguimiento por centro de salud peor control (OR=5,1 [IC95%:1,6-15,6]). En los dos modelos por zona de salud, la condición de ser inmigrante económico aumentó el riesgo de mal control, ZSO (OR=1,3 [IC95%:1,03-1,7]); y ZNO (OR=1,29 [IC95%:1,03-1,6]). Conclusiones: Son factores de riesgo de tener mal controlada la diabetes mellitus la mayor proporción de pacientes mayores de 65 años por enfermera, ser inmigrante y la proporción de pacientes sin seguimientoBackground: Nurse activity is determined by the characteristics of nursing staff. The objective was to determine the impact of Primary Health Care (PHC) nursing workforce characteristics on the control of Diabetes Mellitus (DM) in adults. Method: Cross-sectional analytical study. Administrative and clinical registries and questionnaire PES-Nursing Work Index from PHC nurses. Participants 44.214 diabetic patients in two health zones within the Community of Madrid, North-West Zone (NWZ) with higher socioeconomic situation and South-West Zone (SWZ) with lower socioeconomic situation, and their 507 reference nurses. Analyses were performed to multivariate multilevel logistic regression models. Primary outcome measure: Poor DM control (figures ≥ 7% HbA1c) Results: The prevalence of poor DM control was 40.1% [CI95%: 38.2-42.1]. There was a risk of 25% more of poor control if the patient changed centre and of 27% if changed of doctor-nurse pair. In the multilevel multivariate regression models: in SWZ increasing the ratio of patients over 65 years per nurse increased the poor control (OR=1.00008 [CI95%:1.00006-1.001]); and higher proportion of patients whose Hb1Ac was not measured at the centre contributed to poor DM control (OR=5.1 [CI95%:1.6-15.6]). In two models for health zone, the economic immigration condition increased poor control, in SWZ (OR=1.3 [CI95%:1.03-1.7]); and in NWZ (OR=1.29 [CI95%:1.03-1.6]). Conclusions: Higher 65 years old patients ratio per nurse, economic immigration condition and a higher proportion of patients whose Hb1Ac was not measured contribute to worse DM controlLos resultados presentados en este manuscrito forman parte de un proyecto financiado parcialmente por el Premio Nacional Marqués de Valdecilla (Santander) 2010 (12ª edición

Prokaryotic Community Structure and Metabolisms in Shallow Subsurface of Atacama Desert Playas and Alluvial Fans After Heavy Rains: Repairing and Preparing for Next Dry Period

The Atacama Desert, the oldest and driest desert on Earth, displays significant rains only once per decade. To investigate how microbial communities take advantage of these sporadic wet events, we carried out a geomicrobiological study a few days after a heavy rain event in 2015. Different physicochemical and microbial community analyses were conducted on samples collected from playas and an alluvial fan from surface, 10, 20, 50, and 80 cm depth. Gravimetric moisture content peaks were measured in 10 and 20 cm depth samples (from 1.65 to 4.1% w/w maximum values) while, in general, main anions such as chloride, nitrate, and sulfate concentrations increased with depth, with maximum values of 13–1,125; 168–10,109; and 9,904–30,952 ppm, respectively. Small organic anions such as formate and acetate had maximum concentrations from 2.61 to 3.44 ppm and 6.73 to 28.75 ppm, respectively. Microbial diversity inferred from DNA analysis showed Actinobacteria and Alphaproteobacteria as the most abundant and widespread bacterial taxa among the samples, followed by Chloroflexi and Firmicutes at specific sites. Archaea were mainly dominated by Nitrososphaerales, Methanobacteria, with the detection of other groups such as Halobacteria. Metaproteomics showed a high and even distribution of proteins involved in primary metabolic processes such as energy production and biosynthetic pathways, and a limited but remarkable presence of proteins related to resistance to environmental stressors such as radiation, oxidation, or desiccation. The results indicated that extra humidity in the system allows the microbial community to repair, and prepare for the upcoming hyperarid period. Additionally, it supplies biomarkers to the medium whose preservation potential could be high under strong desiccation conditions and relevant for planetary exploration.This work was funded by the Spanish Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional Grants no. ESP2014-58494-R, ESP2015-69540-R (MINECO/FEDER), ESP2017-89053-C2-2-P, and “María de Maeztu” program project no. MDM-2017-0737; the NASA ASTEP “Life in the Atacama (LITA)” project no. NNX11AJ87G, and NASA Astrobiology Institute Colaborative Agreement 7 (CAN-7) project No. NNX15BB01A. MF-M had a posdoctoral grant from the Comunidad de Madrid/European Union YEI program, and IG-C had a FPI grant from the Spanish Ministry of Economy and Competitivity (MINECO).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (MDM-2017-0737).Peer reviewe