Fruit residues as substrates for single-cell oil production by Rhodococcus species: physiology and genomics of carbohydrate catabolism.

Strains belonging to R. opacus, R. jostii, R. fascians, R. erythropolis and R. equi exhibited differential ability to grow and produce lipids from fruit residues (grape marc and apple pomace), as well as single carbohydrates, such as glucose, gluconate, fructose and sucrose. The oleaginous species, R. opacus (strains PD630 and MR22) and R. jostii RHA1, produced higher yields of biomass (5.1-5.6 g L-1) and lipids (38-44% of CDW) from apple juice wastes, in comparison to R. erythropolis DSM43060, R. fascians F7 and R. equi ATCC6939 (4.1-4.3 g L-1 and less than 10% CDW of lipids). The production of cellular biomass and lipids were also higher in R. opacus and R. jostii (6.8-7.2 g L-1 and 33.9-36.5% of CDW of lipids) compared to R. erythropolis, R. fascians, and R. equi (3.0-3.6 g L-1 and less than 10% CDW of lipids), during cultivation of cells on wine grape waste. A genome-wide bioinformatic analysis of rhodococci indicated that oleaginous species possess a complete set of genes/proteins necessary for the efficient utilization of carbohydrates, whereas genomes from non-oleaginous rhodococcal strains lack relevant genes coding for transporters and/or enzymes for the uptake, catabolism and assimilation of carbohydrates, such as gntP, glcP, edd, eda, among others. Results of this study highlight the potential use of the oleaginous rhodococcal species to convert sugar-rich agro-industrial wastes, such as apple pomace and grape marc, into single-cell oils.

Insights into the Metabolism of Oleaginous Rhodococcus spp.

Some species belonging to the Rhodococcus genus, such as Rhodococcus opacus, R. jostii, and R. wratislaviensis, are known to be oleaginous microorganisms, since they are able to accumulate triacylglycerols (TAG) at more than 20% of their weight (dry weight). Oleaginous rhodococci are promising microbial cell factories for the production of lipids to be used as fuels and chemicals. Cells could be engineered to create strains capable of producing high quantities of oils from industrial wastes and a variety of high-value lipids. The comprehensive understanding of carbon metabolism and its regulation will contribute to the design of a reliable process for bacterial oil production. Bacterial oleagenicity requires an integral configuration of metabolism and regulatory processes rather than the sole existence of an efficient lipid biosynthesis pathway. In recent years, several studies have been focused on basic aspects of TAG biosynthesis and accumulation using R. opacus PD630 and R. jostii RHA1 strains as models of oleaginous bacteria. The combination of results obtained in these studies allows us to propose a metabolic landscape for oleaginous rhodococci. In this context, this article provides a comprehensive and integrative view of different metabolic and regulatory attributes and innovations that explain the extraordinary ability of these bacteria to synthesize and accumulate TAG. We hope that the accessibility to such information in an integrated way will help researchers to rationally select new targets for further studies in the field.

Rhodococcus bacteria as a promising source of oils from olive mill wastes.

The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2-2.7 g/l) and lipids (77-83% of CDW, 1.8-2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1-1.6 g/l of cell biomass and 7.1-14.0% of CDW, 0.1-0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.

Formation of indigoidine derived-pigments contributes to the adaptation of Vogesella sp. strain EB to cold aquatic iron-oxidizing environments.

We investigated previously under explored cold aquatic environments of Andean Patagonia, Argentina. Oily sheens similar to an oil spill are frequently observed at the surface of water in creeks and small ponds in these places. Chemical analysis of a water sample revealed the occurrence of high concentrations of iron and the presence of a free insoluble indigoidine-derived pigment. A blue pigment-producing bacterium (strain EB) was isolated from the water sample and identified as Vogesella sp. by molecular analysis. The isolate was able to produce indigoidine and another derived-pigment (here called cryoindigoidine) with strong antifreeze properties. The production of the pigments depended on the cell growth at cold temperatures (below 15 °C), as well as on the attachment of cells to solid surfaces, and iron limitation in the media. The pigments produced by strain EB showed an inhibitory effect on the growth of diverse microorganisms such as Candida albicans, Escherichia coli and Staphylococcus aureus. In addition, pigmented cells were more tolerant to freezing than non-pigmented cells, suggesting a role of cryoindigoidine/indigoidine as a cold-protectant molecule. The possible roles of the pigments in strain EB physiology and its interactions with the iron-rich environment from which the isolate was obtained are discussed. Results of this study suggested an active role of strain EB in the investigated iron-oxidizing ecosystem.

Physiological and genetic differences amongst Rhodococcus species for using glycerol as a source for growth and triacylglycerol production.

We analysed the ability of five different rhodococcal species to grow and produce triacylglycerols (TAGs) from glycerol, the main byproduct of biodiesel production. Rhodococcus fascians and Rhodococcus erythropolis grew fast on glycerol, whereas Rhodococcus opacus and Rhodococcus jostii exhibited a prolonged lag phase of several days before growing. Rhodococcus equi only exhibited poor growth on glycerol. R. erythropolis DSMZ 43060 and R. fascians F7 produced 3.9-4.3 g cell biomass l(-1) and 28.4-44.6% cellular dry weight (CDW) of TAGs after 6 days of incubation; whereas R. opacus PD630 and R. jostii RHA1 produced 2.5-3.8 g cell biomass l(-1) and 28.3-38.4% CDW of TAGs after 17 days of growth on glycerol. Genomic analyses revealed two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2) amongst rhodococci. Those species that possessed cluster 1 (glpFK1D1) (R. fascians and R. erythropolis) exhibited fast growth and lipid accumulation, whereas those that possessed cluster 2 (glpK2D2) (R. opacus, R. jostii and R. equi) exhibited delayed growth and lipid accumulation during cultivation on glycerol. Three glycerol-negative strains were complemented for their ability to grow and produce TAGs by heterologous expression of glpK2 from R. opacus PD630. In addition, we significantly reduced the extension of the lag phase and improved glycerol assimilation and oil production of R. opacus PD630 when expressing glpK1D1 from R. fascians. The results demonstrated that rhodococci are a flexible and amenable biological system for further biotechnological applications based on the reutilization of glycerol.

Label-free and redox proteomic analyses of the triacylglycerol-accumulating Rhodococcus jostii RHA1.

The bacterium Rhodococcus jostii RHA1 synthesizes large amounts of triacylglycerols (TAGs) under conditions of nitrogen starvation. To better understand the molecular mechanisms behind this process, we performed proteomic studies in this oleaginous bacterium. Upon nitrogen starvation, we observed a re-routing of the carbon flux towards the formation of TAGs. Under these conditions, the cellular lipid content made up more than half of the cell's dry weight. On the proteome level, this coincided with a shift towards non-glycolytic carbohydrate-metabolizing pathways. These pathways (Entner-Doudoroff and pentose-phosphate shunt) contribute NADPH and precursors of glycerol 3-phosphate and acetyl-CoA to lipogenesis. The expression of proteins involved in the degradation of branched-chain amino acids and the methylmalonyl-CoA pathway probably provided propionyl-CoA for the biosynthesis of odd-numbered fatty acids, which make up almost 30 % of RHA1 fatty acid composition. Additionally, lipolytic and glycerol-degrading enzymes increased in abundance, suggesting a dynamic cycling of cellular lipids. Conversely, abundance of proteins involved in consuming intermediates of lipogenesis decreased. Furthermore, we identified another level of lipogenesis regulation through redox-mediated thiol modification in R. jostii. Enzymes affected included acetyl-CoA carboxylase and a ß-ketoacyl-[acyl-carrier protein] synthase II (FabF). An integrative metabolic model for the oleaginous RHA1 strain is proposed on the basis of our results.

Evaluación de los efectos tóxicos de ftalatos sobre poblaciones naturales de Chironomus riparius (Diptera): implicaciones en estudios de ecotoxicidad / Evaluation of the toxic effects of phthalates on natural populations of Chironomus riparius (Diptera): implications for ecotoxicity studies

Los quironómidos, un género de mosquitos no picadores, son organismos centinela en la evaluación de la calidad de las aguas y un modelo de relevancia ecotoxicológica para el estudio de los efectos de contaminantes ambientales. Son todavía muy escasos los trabajos con poblaciones naturales de este organismo en los que se valore la utilidad de biomarcadores moleculares en el análisis de la salud de individuos expuestos a múltiples variables de estrés. Nuestros resultados muestran que exposiciones de larvas de poblaciones naturales de Chironomus riparius a di (2-etilhexil) ftalato (DEHP) y butil bencil ftalato (BBP), dos compuestos incluidos por la ECHA en la lista de sustancias de muy alta preocupación, provocan alteraciones rápidas en la expresión de diferentes genes relacionados con la respuesta celular de estrés (hsp70 y hsc70), la ruta hormonal (EcR y ERR) y los mecanismos de destoxicación (CYP4G), así como en la actividad enzimática de GST. Ambos compuestos provocan respuestas diferentes en estas dianas, especialmente en las exposiciones más prolongadas, y tienen la capacidad de producir efectos tóxicos retardados. Son de especial relevancia los resultados que muestran la alteración de la ruta hormonal de la ecdisona, confirmando la capacidad disruptora endocrina en insectos de estos ftalatos. Por último, existen diferencias respecto a datos previos obtenidos con larvas de laboratorio, tanto en la toxicidad de DEHP y BBP como en el comportamiento de algunas dianas, lo que acentúa la necesidad de llevar a cabo análisis con diferentes poblaciones para conseguir una aproximación más realista a los efectos de contaminantes (AU)

Chironomids, a genus of non-biting midges, are considered sentinel organisms for the evaluation of water quality and an outstanding model in Ecotoxicology for studying the effects of environmental pollutants. In contrast to its widespread use in studies with laboratory cultures, the use of natural populations (chronically exposed to complex mixtures of pollutants in their environment) is still uncommon to assess the usefulness of molecular biomarkers in studying the health of populations under multiple stress conditions. In Chironomus riparius, our results show that both bis (2-ethylhexyl) phthalate (DEHP) and benzyl butyl phthalate (BBP) cause rapid alterations in the activity of GST enzyme as well as in the expression profile of various genes related to cell stress response (hsp70 and hsc70), to the hormonal route (EcR and ERR) and to detoxification mechanisms (CYP4G). Both compounds cause different responses to these targets, especially in longer exposures, and have the ability to produce delayed toxicity. The alteration of the ecdysone hormone pathway in our experiments has special relevance, since it proves that both compounds are endocrine disruptors in insects. Finally, this study shows differences with previous data obtained with laboratory cultures in both the toxicity of these phthalates and the behavior of some targets, which emphasizes the need of carrying out studies with different populations to get a more realistic approach to the effects of contaminants (AU)

[Psychopathy and suicidal behaviour in a sample of mentally disordered offenders]. / Psicopatía y conducta suicida en una muestra de delincuentes con trastorno mental.

Empirical literature has yielded a positive association between psychopathy levels and suicide attempts. This association is centred around impulsivity and disinhibitory facets of psychopathy, whereas suicide and emotional poverty remain independent. Evidence about the relation between suicide and psychopathy in mentally disordered offenders is not conclusive. The present work explores the relation between several measures of antisocial personality, suicide attempt and deliberate self mutilation in a sample of inmates from a forensic psychiatric hospital. Results support the association between disinhibitory aspects of personality and suicide in this population.

Psicopatía y conducta suicida en una muestra de delincuentes con trastorno mental / Psychopathy and suicidal behaviour in a sample of mentally disordered offenders

La literatura empírica ha constatado una asociación positiva entre el nivel de psicopatía y la presencia de intentos de suicidio. Esta asociación se centra en los aspectos de impulsividad y desinhibición de la psicopatía, mientras que suicidio y pobreza emocional se muestran independientes. Los datos sobre la asociación entre suicidio y psicopatía en personas con enfermedad mental no son concluyentes. Este trabajo explora la relación entre distintas medidas de la personalidad antisocial y la presencia de intentos de suicidio y episodios de autolesión deliberada en una muestra de internos de un Hospital Psiquiátrico Penitenciario. Los resultados apoyan la asociación entre aspectos desinhibidos de personalidad y suicidio también en esta población(AU)

Empirical literature has yielded a positive association between psychopathy levels and suicide attempts. This association is centred around impulsivity and disinhibitory facets of psychopathy, whereas suicide and emotional poverty remain independent. Evidence about the relation between suicide and psychopathy in mentally disordered offenders is not conclusive. The present work explores the relation between several measures of antisocial personality, suicide attempt and deliberate self mutilation in a sample of inmates from a forensic psychiatric hospital. Results support the association between disinhibitory aspects of personality and suicide in this population(AU)

Validation of the PCR-dHPLC method for rapid identification of Candida glabrata phylogenetically related species in different biological matrices.

Since two new species phylogenetically related to Candida glabrata with slightly different phenotypes and antifungal susceptibility profiles have been described, it seems to be necessary from clinical point of view, to develop a rapid and accurate identification system in order to distinguish between these three fungal species. We studied the performance of denaturing high performance liquid chromatography (dHPLC) as a faster (less than 7 min) and alternative novel technique for simultaneous analysis of Candida species in different biological matrices. The analyses show the good low limit of detection (LLOD) in all biological matrices studied (5.16-9.56 ngµL(-1), 4.14-4.70 ng µL(-1) and 3.99-4.66 ng µL(-1) for Candida bracarensis, Candida nivariensis and C. glabrata, respectively). 180 Candida isolates were analyzed in order to demonstrate the method suitability for screening analysis to identify C. glabrata and its cryptic species (C. bracarensis and C. nivariensis) in clinical routine.

Toxicological evaluation of three contaminants of emerging concern by use of the Allium cepa test.

Di(2-ethylhexyl)phthalate, triclosan and propylparaben are contaminants of emerging concern that have been subjected to extensive toxicological studies, but for which limited information is currently available concerning adverse effects on terrestrial plant systems. The Allium cepa test, which is considered one of the most efficient approaches to assess toxic effects of environmental chemicals, was selected to evaluate the potential risks of these ubiquitous pollutants. Our data demonstrate that all three compounds studied may in some way be considered toxic, but different effects were noted depending on the chemical and the end point analysed. Results derived from the analysis of macroscopic parameters used in testing for general toxicity, revealed that while di(2-ethylhexyl)phthalate had no apparent effects, the other two chemicals inhibited A. cepa root growth in a dose-dependent manner. On the other hand, although all three compounds caused alterations in the mitotic index of root-tip cells, propylparaben was the only one that did not show evidence of genotoxicity in assays for chromosome aberrations and micronuclei. The results of the present study clearly indicate that sensitive plant bioassays are useful and complementary tools to determine environmental impact of contaminants of emerging concern.

An integrated cellular model to evaluate cytotoxic effects in mammalian cell lines.

The ever growing anthropogenic pressure to the environment has lead in 2007 to the revision of the existing legislation and the approval of the new European law regarding the production and importation of chemicals, known as REACH. This new legal framework supports the development of alternative methods to animal experimentation encouraging the improvement and/or design of new methodological strategies for the toxicological evaluation of chemical compounds. Even though cytotoxicity studies are a reductionist approach to acute toxicity in vivo, they offer the best agreement between obtaining relevant information about the mechanism of toxic action and the use of alternative methods. Following this trend, this work presents an integrated cellular strategy in order to know the toxicity and mechanism of action of chemical compounds, using simple and reproducible in vitro systems. The experimental procedures are performed in two steps. The first one involves the systematic analysis of the main cellular targets using proliferation, viability and morphological probes. The second step relies upon the results obtained in the first step, including specific assays that focus on the mechanism of toxic action and the cellular response. The benefits of this strategy are exemplified with two real cases: pentachlorophenol and rotenone.

In vitro assessment of the cytotoxic and mutagenic potential of perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is a perfluorinated compound ubiquitously detected in the environment, including wildlife and humans. Despite the available information, research on the cytotoxicity of PFOA in non-tumoral mammalian cells is relatively limited. In this work, two in vitro toxicity systems were employed to provide further insight into the cytotoxic and mutagenic potential of PFOA. The cytotoxicity of the chemical towards Vero cells was assessed using biochemical and morphological parameters, while mutagenicity was evaluated according to Ames test. High doses of PFOA cause oxidative stress in Vero cells, that was closely linked to cell cycle arrest at the G1 phase and induction of apoptosis. Our results corroborate previous findings in human tumoral cells and suggest that the mode of action of this perfluorinated compound is not a peculiarity among mammalian cell types. On the other hand, the compound was not mutagenic in the Ames test, using four strains of Salmonella typhimurium in the presence or absence of rat S9 metabolic activation system.

Mediación entre iguales y prevención del abuso de benzodiacepinas en un centro penitenciario / Peer mediation and bezodiazepine abuse prevention in a penitentiary centre

El abuso de benzodiacepinas es un problema común en drogodependientes. Se formó a doce internos del Centro Penitenciariode Cáceres (agentes de salud) sobre los riesgos del abuso de benzodiacepinas y su interacción con otros depresoresdel sistema nervioso central. Los agentes se encargaron de difundir la información y de diseñar materiales de apoyo. No seencontraron efectos significativos en la población interna del centro. Se analizan las implicaciones de estos datos (AU)

Benzodiazepine abuse is a common problem in drug addicts. Twelve inmates from the Cáceres Penitentiary Centre (healthagents) were educated on the risks of the benzodiazepine abuse and it’s interaction with other CNS depressors. Agentsdivulged the information and designed support materials. No significant effects were found in the inmate population. Severalimplications are considered (AU)

Mediación entre iguales y prevención del abuso de benzodiacepinas en un centro penitenciario / Peer Mediation and benzodiazepine abuse prevention in a penitentiary centre

El abuso de benzodiacepinas es un problema común en drogodependientes. Se formó a doce internos del Centro Penitenciario de Cáceres (agentes de salud) sobre los riesgos del abuso de benzodiacepinas y su interacción con otros depresores del sistema nervioso central. Los agentes se encargaron de difundir la información y de diseñar materiales de apoyo. No se encontraron efectos significativos en la población interna del centro. Se analizan las implicaciones de estos datos

Benzodiazepine abuse is a common problem in drug addicts. Twelve inmates from the Cáceres Penitentiary Centre (health agents) were educated on the risks of the benzodiazepine abuse and it’s interaction with other CNS depressors. Agents divulged the information and designed support materials. No significant effects were found in the inmate population. Several implications are considered

Mutations in the third exon of the MYOC gene in spanish patients with primary open angle glaucoma.

Primary open angle glaucoma (POAG) is the second most common cause of blindness in developed countries. It is an optic neuropathy in which a degeneration of the retinal ganglion cells causes a characteristic excavation in the optic disc. Several loci have been identified to be responsible for different types of glaucoma, including the MYOC gene located on chromosome 1. In this work, six mutations have been identified in the third exon of the MYOC gene in patients with POAG. We studied 79 Galician patients with chronic POAG glaucoma and 90 control individuals from the same general population. We identified six mutations, including three novel ones. Two of the six mutations were considered to be polymorphisms, while the other four met the criteria for pathogenicity in this disease as they altered the amino acid sequence and were found in one or more patients with glaucoma and in less of 1% of the control population. These mutations were detected in eight patients suffering from POAG (7.5%) and in two people from the control population (2.2%). POAG can be due to mutations in the myocilin gene (MYOC) on chromosome 1. The glaucoma phenotype associated with this gene may vary from a juvenile severe form to a late-onset chronic open angle glaucoma.

Constructivismo y psicoterapia en la edad avanzada / Constructivism and physiotherapy in the elderly

No disponible

[Diagnosis of anaerobic infection of the pleural fluid using gas-liquid chromatography]. / Diagnóstico de la infección anaerobia del líquido pleural mediante cromatografía de gas-líquido.

BACKGROUND: An assessment of the efficacy of the detection of volatile and non-volatile fatty acids that are produced by microorganisms aimed at the possible diagnosis of pleural empyema. METHODS: 106 pleural fluids were examined by gas-liquid chromatography in contrast to conventional aerobic and anaerobic cultures. The sample previously prepared (1 microliter) was injected in an Hewlett-Packard 5890 chromatograph, that was equipped with a fused silica capillary column and a flame ionization detector. RESULTS: Growth of aerobic or anaerobic microorganisms was obtained in 13 (12.3%) of the samples, and volatile or non-volatile fatty acids were detected in all of them (sensitivity = 1). No growth was obtained in the 93 samples, but volatile or non-volatile fatty acids were detected in 64 (68.8%) of them (specificity = 0.31). A positive predictive value of 0.17 and a negative predictive value of 1 were obtained. Growth of anaerobic microorganisms was obtained in 9 samples (8.5%) and volatile fatty acids were detected in all of them (sensitivity = 1). Anaerobic microorganisms did not grow in 97 samples, however volatile fatty acids were detected in 2 (2.1%) of them. A positive predictive value of 0.82 and a negative of 1 were obtained. CONCLUSIONS: Referring to pleural fluid samples, the detection of volatile and non-volatile fatty acids by gas-liquid chromatography is a highly sensitive method, that may be used to quickly reject negative samples in about 30 mins. Furthermore, when anaerobic microorganisms exist, the high specificity of gas-liquid chromatography will make it possible to confirm their presence in the sample, mainly if there is a clinical suspect of anaerobic infection. Thus, the utilization of this sensitive method may also change the therapeutic behaviour.