ESCCAR international congress on Rickettsia and other intracellular bacteria.

The European Society for the study of Chlamydia, Coxiella, Anaplasma and Rickettsia (ESCCAR) held his triennial international meeting in Lausanne. This meeting gathered 165 scientists from 28 countries and all 5 continents, allowing efficient networking and major scientific exchanges. Topics covered include molecular and cellular microbiology, genomics, as well as epidemiology, veterinary and human medicine. Several breakthroughs have been revealed at the meeting, such as (i) the presence of CRISPR (the "prokaryotic immune system") in chlamydiae, (ii) an Anaplasma effector involved in host chromatin remodelling, (iii) the polarity of the type III secretion system of chlamydiae during the entry process revealed by cryo-electron tomography. Moreover, the ESCCAR meeting was a unique opportunity to be exposed to cutting-edge science and to listen to comprehensive talks on current hot topics.

Heme oxygenase-1 induction by endogenous nitric oxide: influence of intracellular glutathione.

To investigate the influence of glutathione (GSH) on cellular effects of nitric oxide (NO) formation, human colon adenocarcinoma cells were transfected with a vector allowing controlled expression of inducible nitric oxide synthase (iNOS). Protein levels of oxidative stress-sensitive heme oxygenase-1 (HO-1) were analyzed in the presence or absence of GSH depletion using L-buthionine-[S,R]-sulfoximine and iNOS induction. While no effect was observed in the presence of iNOS activity alone, a synergistic effect on HO-1 expression was observed in the presence of iNOS expression and GSH depletion. This effect was prevented by addition of N-methyl-L-arginine. Therefore, targeting of endogenous NO may be modulated by intracellular GSH.

Sensitive photonic system to measure oxidative potential of airborne nanoparticles and ROS levels in exhaled air

A photonic system has been developed that enables sensitive quantitative determination of reactive oxygen species (ROS) - mainly hydrogen peroxide (H2O2) - in aerosol samples such as airborne nanoparticles and exhaled air from patients. The detection principle relies on the amplification of the absorbance under multiple scattering conditions due to optical path lengthening [1] and [2]. In this study, the presence of cellulose membrane that acts as random medium into the glass optical cell considerably improved the sensitivity of the detection based on colorimetric FOX assay (FeII/orange xylenol). Despite the loss of assay volume (cellulose occupies 75% of cell volume) the limit of detection is enhanced by one order of magnitude reaching the value of 9 nM (H2O2 equivalents). Spectral analysis is performed automatically with a periodicity of 5 to 15 s, giving rise to real-time ROS measurements. Moreover, the elution of air sample into the collection chamber via a micro-diffuser (impinger) enables quantitative determination of ROS contained in or generated from airborne samples. As proof-of-concept the photonic ROS detection system was used in the determination of both ROS generated from traffic pollution and ROS contained in the exhaled breath as lung inflammation biomarkers.

Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1β processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1β secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation.

Disassembly of a Medial Transenvelope Structure by Antibiotics during Intracellular Division.

Chlamydiales possess a minimal but functional peptidoglycan precursor biosynthetic and remodeling pathway involved in the assembly of the division septum by an atypical cytokinetic machine and cryptic or modified peptidoglycan-like structure (PGLS). How this reduced cytokinetic machine collectively coordinates the invagination of the envelope has not yet been explored in Chlamydiales. In other Gram-negative bacteria, peptidoglycan provides anchor points that connect the outer membrane to the peptidoglycan during constriction using the Pal-Tol complex. Purifying PGLS and associated proteins from the chlamydial pathogen Waddlia chondrophila, we unearthed the Pal protein as a peptidoglycan-binding protein that localizes to the chlamydial division septum along with other components of the Pal-Tol complex. Together, our PGLS characterization and peptidoglycan-binding assays support the notion that diaminopimelic acid is an important determinant recruiting Pal to the division plane to coordinate the invagination of all envelope layers with the conserved Pal-Tol complex, even during osmotically protected intracellular growth.

Sex- and melanism-specific variations in the oxidative status of adult tawny owls in response to manipulated reproductive effort.

Oxidative stress, determined by the balance between the production of damaging reactive oxygen species (ROS) and antioxidant defences, is hypothesized to play an important role in shaping the cost of reproduction and life history trade-offs. To test this hypothesis, we manipulated reproductive effort in 94 breeding pairs of tawny owls (Strix aluco) to investigate the sex- and melanism-specific effects on markers of oxidative stress in red blood cells (RBCs). This colour polymorphic bird species shows sex-specific division of labour and melanism-specific history strategies. Brood sizes at hatching were experimentally enlarged or reduced to increase or decrease reproductive effort, respectively. We obtained an integrative measure of the oxidative balance by measuring ROS production by RBCs, intracellular antioxidant glutathione levels and membrane resistance to ROS. We found that light melanic males (the sex undertaking offspring food provisioning) produced more ROS than darker conspecifics, but only when rearing an enlarged brood. In both sexes, light melanic individuals had also a larger pool of intracellular antioxidant glutathione than darker owls under relaxed reproductive conditions (i.e. reduced brood), but not when investing substantial effort in current reproduction (enlarged brood). Finally, resistance to oxidative stress was differently affected by the brood size manipulation experiment in males and females independently of their plumage coloration. Altogether, our results support the hypothesis that reproductive effort can alter the oxidative balance in a sex- and colour-specific way. This further emphasizes the close link between melanin-based coloration and life history strategies.

Approches diagnostiques des bactéries intracellulaires et des germes fastidieux [Diagnostic approach of intracellular bacteria and fastidious microorganisms].

Obligate or facultative intracellular bacteria are fastidious organisms that do not or poorly grow on conventional culture media. Some of them may be the cause of frequent and potentially severe infections, such as tuberculosis (Myco- bacterium tuberculosis), community-acquired respiratory infections (Legionella spp., Mycoplasma pneumoniae, Chlamydia pneumoniae) or blood culture-negative endocarditis (Coxiella burnetii, Bartonella spp., Tropheryma whipplei). The objective of this paper is to provide a comprehensive summary of the available and recommended diagnostic tests for the detection of these fastidious organisms in clinical practice.

Caracterización física, química e hidromorfológica de los ríos altoandinos tropicales de Ecuador y Perú

Para comprender la variabilidad física, química e hidromorfológica de los ríos en la zona altoandina tropical (sobre los 2000 msnm) se muestrearon 123 ríos de ocho cuencas hidrográficas de Ecuador y Perú, seleccionados según criterios de distribución en latitud y altitud, así como el grado de alteración antrópica. Los muestreos se realizaron entre octubre 2007 y octubre 2008, coincidiendo con la época seca en ambos paises. En cada localidad se midieron tanto parámetros físicos y químicos (i.e., temperatura, oxígeno disuelto, conductividad, fosfatos, nitritos, nitratos, amonio, etc.) como hidromorfológicos (i.e., altitud, índice de calidad riparia (QBR), calidad y naturalidad de la cubierta vegetal de la ribera, naturalidad del canal fluvial, índice del hábitat fluvial (IHF), frecuencia de rápidos, composición del sustrato). A nivel regional (entre cuencas) la mineralización, las características hidromorfológicas y la heterogeneidad del hábitat mostraron ser los factores de mayor importancia para explicar la variabilidad encontrada. La temperatura, oxígeno disuelto y heterogeneidad del hábitat fueron los parámetros relevantes en el gradiente altitudinal, mientras que la mineralización lo fue en el gradiente latitudinal. La significación o importancia de un factor u otro parecen estar determinados en gran medida por el nivel espacial estudiado (localidad, cuenca, región). Sin embargo, se determinó que a nivel regional la altitud y las variables que cambiaron con ella, como temperatura y oxígeno disuelto, son siempre significativas independientemente de la ubicación latitudinal de la cuenca.

Guia para el reconocimiento de las larvas de Chironomidae (DIPTERA) de los ríos mediterráneos.

Este volumen especial de la col·lección F.E.M.- Guies está dedicado a la familia Chironomidae (INSECTA: DIPTERA). Esta guía tiene por objeto reconocer los principales morfotipos larvarios de quironómidos de los ríos mediterráneos utilizando caracteres macroscópicos que pueden ser observados a la lupa binocular (hasta 100x) ya que así es posible identificar las larvas a nivel de género o grupos de géneros. De esta manera, sin realizar preparaciones microscópicas se puede llegar a tener una cierta información de los taxones presentes. Se han incluido tanto los taxa propios de pequeños ríos, incluyendo los temporales, como los de las partes medias y bajas de los ríos, especialmente del río Llobregat.

Guia para el reconocimiento de las larvas de Chironomidae (DIPTERA) de los ríos mediterráneos.

Este volumen especial de la col·lección F.E.M.- Guies está dedicado a la familia Chironomidae (INSECTA: DIPTERA). Esta guía tiene por objeto reconocer los principales morfotipos larvarios de quironómidos de los ríos mediterráneos utilizando caracteres macroscópicos que pueden ser observados a la lupa binocular (hasta 100x) ya que así es posible identificar las larvas a nivel de género o grupos de géneros. De esta manera, sin realizar preparaciones microscópicas se puede llegar a tener una cierta información de los taxones presentes. Se han incluido tanto los taxa propios de pequeños ríos, incluyendo los temporales, como los de las partes medias y bajas de los ríos, especialmente del río Llobregat.

Biomonitoreo de la calidad del agua en los ríos ibéricos: lecciones aprendidas.

Los autores resumen en un decálogo la experiencia de más de 30 años de estudio de los ríos españoles con dos focos principales: el uso de macroinvertebrados para la obtención de la calidad biológica y el diseño de sistemas de evaluación del estado ecológico de los ríos. Después de una introducción general y de un resumen de cómo se gestaron los estudios de biomonitoreo en España, los autores analizan los trabajos que se han publicado en la revista Limnetica sobre este tema. Se usan estos datos para ilustrar los puntos relevantes en el diseño y aplicación del biomonitoreo, que es posteriormente resumido en un decálogo. Este está pensado para contener todos los puntos necesarios para que el diseño de un sistema de biomonitoreo cumpla las especificaciones de la Directiva Marco del Agua (DMA). Los diez puntos analizados son: 1) establecimiento de la zona de estudio; 2) recursos humanos y materiales disponibles; 3) tipología de ríos; 4) establecimiento de las condiciones de referencia; 5) protocolos de muestreo; 6) métricas a utilizar; 7) establecimiento de los cortes de calidad y relación de las métricas con las presiones; 8) mapas de estado ecológico usando el valor relativo respecto a los valores de referencia (el EQR: Ecological Quality Ratio); 9) control de calidad; 10) uso de los datos para establecer los objetivos de calidad y el programa de medidas. En la revista Limnetica se han publicado desde su inicio en el año 1981 un total de 641 trabajos, de los cuales un 42% trataban de ríos y un 18% se centraban exclusivamente en el biomonitoreo. La mayor parte de los trabajos de biomonitoreo publicados utilizaron a los macroinvertebrados como elemento de calidad (88%), pero relativamente pocos (menos del 20%) usaron la tipología y las condiciones de referencia para establecer correctamente las clases de calidad. Se analiza por separado el período anterior al año 2002, y con un número similar de trabajos se advierte un mayor porcentaje de trabajos de biomonitoreo después de 2002. Sin embargo, y a pesar de la existencia de la DMA desde el año 2000, muchos trabajos publicados en Limnetica a partir de 2002 (cuando se hizo un número especial sobre biomonitoreo en ríos mediterráneos) no cumplen los requisitos que demanda esta Directiva. En este momento, después de la experiencia que se tiene por la implementación de la DMA, cualquier sistema de biomonitoreo desarrollado en Europa debe ser comparable a los que existen actualmente, especialmente a la métrica común de intercalibración (ICM).Este decálogo presenta los pasos necesarios para que cuando se diseñe un sistema de muestreo este cumpla los requisitos que demanda la DMA.

Clave para la determinación de exuvias pupales de los quironómidos (Diptera: Chironomidae) de ríos altoandinos tropicales

The Chironomidae is a cosmopolitan family of Nematoceran flies with more than 20 000 species described. However the diversity of genera and species of the family in the Andean region beyond the 2 000m.a.s.l are scarcely known. We conducted faunal surveys and biomonitoring research in different streams of Colombia, Ecuador and Peru from May 2005 to October 2011. Based on specimens collections, and a taxonomic key was developed to identify pupae and pupal exuviae of 46 genera of midges (Diptera, Chironomidae) collected from streams at high altitude areas on the Andean tropical mountains. We included illustrations and brief taxonomic descriptions for all genera, of which several ones have not yet been formally described; in this latter case we used the nomenclature of Roback & Coffman (1983). For two genera, Cricotopus and Genus 1, keys to the most common morphospecies were provided. Results showed that in this area the chironomid assemblages are dominated by the members of the subfamily Orthocladiinae (22 genera) followed by the Chironominae (13). Six genera of Tanypodinae were identified, while only three and two genera were present from subfamilies Podonominae and Diamesinae. This key may be very useful for both studies about drift in streams, and for biomonitoring purposes.

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, we developed nanovesicles containing bioactive cationic lysine-based amphiphiles, and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. We found different cytotoxic responses among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalized by HeLa cells, and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behavior after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute to reducing the uncertainty surrounding their potential health hazards.

La biota de los ríos : los microorganismos autótrofos

Capítol 12 del llibre 'Conceptos y técnicas en ecología fluvial' que es refereix als microorganismes autòtrofes dels sistemes fluvials