Cyanate Assimilation by the Alkaliphilic Cyanide-Degrading Bacterium Pseudomonas pseudoalcaligenes CECT5344: Mutational Analysis of the cyn Gene Cluster

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 can grow with cyanate, cyanide, or cyanide-containing industrial residues as the sole nitrogen source, but the assimilation of cyanide and cyanate takes place through independent pathways. Therefore, cyanide degradation involves a chemical reaction between cyanide and oxaloacetate to form a nitrile that is hydrolyzed to ammonium by the nitrilase NitC, whereas cyanate assimilation requires a cyanase that catalyzes cyanate decomposition to ammonium and carbon dioxide. The P. pseudoalcaligenes CECT5344 cynFABDS gene cluster codes for the putative transcriptional regulator CynF, the ABC-type cyanate transporter CynABD, and the cyanase CynS. In this study, transcriptional analysis revealed that the structural cynABDS genes constitute a single transcriptional unit, which was induced by cyanate and repressed by ammonium. Mutational characterization of the cyn genes indicated that CynF was essential for cynABDS gene expression and that nitrate/nitrite transporters may be involved in cyanate uptake, in addition to the CynABD transport system. Biodegradation of hazardous jewelry wastewater containing high amounts of cyanide and metals was achieved in a batch reactor operating at an alkaline pH after chemical treatment with hydrogen peroxide to oxidize cyanide to cyanate

Changes in cell/tissue organization and peroxidase activity as markers for early detection of graft incompatibility in peach/plum combinations

9 páginas, 3 figuras, 4 tablas -- PAGS nros. 9-17Changes in cell and tissue organization and in peroxidase activity have been analyzed to find early markers to predict graft incompatibility occurrence in peach/plum combinations (Prunus persica/Prunus spp.) at 5 months after grafting in the dormancy period. Different compatible and incompatible peach/plum grafts were grown for 5 months in a nursery. The cellular study of the graft interface revealed structural changes associated with graft incompatibility symptoms. The main structural features were cambium cell disorganization, less differentiation of vascular tissues, degeneration of phloem and xylem cells, and accumulation of phenols at the graft interface after 5 months of graft development. The peroxidase study was performed during dormancy and the vegetative growth period, and revealed a significant increase in peroxidase activity in the incompatible unions, with significant differences between compatible and incompatible grafts. Analysis of gel profiles of nonbudded rootstocks and scions revealed an anodal isoperoxidase band [relative front (Rf) = 0.48] present in scions and compatible rootstocks, and another isoperoxidase band (Rf = 0.53) only present in the incompatible rootstocks. Our results show that the analysis of cell organization to detect early structural events and the evaluation of peroxidase activity at graft unions constituted feasible and convenient methods for early diagnosis of graft incompatibility. Also, it was suggested that the presence of band Rf = 0.48 in plum rootstocks and peach cultivars could be used as a marker to predict graft compatibility for peach scions and plum rootstocksFinancial support was provided by Comisión Interministerial de Ciencia y Tecnología (project nos. AGL2005-05533 and AGL2008-00283/AGR, partially founded by BFU2008-00203 and AGL2008-04255), by the CONSID-DGA A 44, and by a fellowship granted to O.Z. from the Agencia Española de Cooperación Internacional (AECI)Peer reviewe

A hybrid recommendation approach for a tourism system

Many current e-commerce systems provide personalization when their content is shown to users. In this sense, recommender systems make personalized suggestions and provide information of items available in the system. Nowadays, there is a vast amount of methods, including data mining techniques that can be employed for personalization in recommender systems. However, these methods are still quite vulnerable to some limitations and shortcomings related to recommender environment. In order to deal with some of them, in this work we implement a recommendation methodology in a recommender system for tourism, where classification based on association is applied. Classification based on association methods, also named associative classification methods, consist of an alternative data mining technique, which combines concepts from classification and association in order to allow association rules to be employed in a prediction context. The proposed methodology was evaluated in some case studies, where we could verify that it is able to shorten limitations presented in recommender systems and to enhance recommendation quality

A dysregulation in CES1, APOE and other lipid metabolism-related genes is associated to cardiovascular risk factors linked to obesity

OBJECTIVE: The aim of the present study was to investigate the relationship between the differential expression of genes related to lipid metabolism in subcutaneous adipose tissue and metabolic syndrome features in lean and obese subjects with habitual high fat intake. METHODS: Microarray and RT-PCR analysis were used to analyze and validate differential gene expression in subcutaneous abdominal adipose tissue samples from lean and obese phenotype subjects. RESULTS: Several genes and transcripts involved in lipolysis were down-regulated, such as AKAP1, PRKAR2B, Gi and CIDEA, whereas NPY1R and CES1 were up-regulated, when comparing obese to lean subjects. Similarly, transcripts associated with cholesterol and lipoprotein metabolism showed a differential expression, with APOE and ABCA being decreased and VLDLR being increased in obese versus lean subjects. In addition, positive correlations were found between different markers of the metabolic syndrome and CES1 and NPY1R mRNA expressions, while APOE showed an inverse association with some of them. CONCLUSION: Different expression patterns in transcripts encoding for proteins involved in lipolysis and lipoprotein metabolism were found between lean and obese subjects. Moreover, the dysregulation of genes such as CES1 and APOE seems to be associated with some physiopathological markers of insulin resistance and cardiovascular risk factors in obesity

The Social Component of Environmental Enrichment Is a Pro-neurogenic Stimulus in Adult c57BL6 Female Mice

In rodents, the hippocampal dentate gyrus gives rise to newly generated dentate granule cells (DGCs) throughout life. This process, named adult hippocampal neurogenesis (AHN), converges in the functional integration of mature DGCs into the trisynaptic hippocampal circuit. Environmental enrichment (EE) is one of the most potent positive regulators of AHN. This paradigm includes the combination of three major stimulatory components, namely increased physical activity, constant cognitive stimulation, and higher social interaction. In this regard, the pro-neurogenic effects of physical activity and cognitive stimulation have been widely addressed in adult rodents. However, the pro-neurogenic potential of the social aspect of EE has been less explored to date. Here we tackled this question by specifically focusing on the effects of a prolonged period of social enrichment (SE) in adult female C57BL6 mice. To this end, 7-week-old mice were housed in groups of 12 per cage for 8 weeks. These mice were compared with others housed under control housing (2–3 mice per cage) or EE (12 mice per cage plus running wheels and toys) conditions during the same period. We analyzed the number and morphology of Doublecortin-expressing (DCX+) cells. Moreover, using RGB retroviruses that allowed the labeling of three populations of newborn DGCs of different ages in the same mouse, we performed morphometric, immunohistochemical, and behavioral determinations. Both SE and EE increased the number and maturation of DCX+ cells, and caused an increase in dendritic maturation in certain populations of newborn DGCs. Moreover, both manipulations increased exploratory behavior in the Social Interaction test. Unexpectedly, our data revealed the potent neurogenesis-stimulating potential of SE in the absence of any further cognitive stimulation or increase in physical activity. Given that an increase in physical activity is strongly discouraged under certain circumstances, our findings may be relevant in the context of enhancing AHN via physical activity-independent mechanisms

Effects of Trecadrine, a beta3-adrenergic agonist, on leptin secretion, glucose and lipid metabolism in isolated rat adipocytes

Objective: Leptin, a hormone produced in adipocytes, is a key signal in the regulation of food intake and energy expenditure. beta-Adrenergic agonists have been shown to inhibit leptin gene expression and leptin secretion. The mechanisms underlying the inhibitory effects of beta-adrenergic agonists have not been established. In this study, we examined the effects of TrecadrineÒ, a novel beta3-adrenergic agonist, on basal and insulin-stimulated leptin secretion in isolated rat adipocytes. Because insulin-stimulated glucose metabolism is an important regulator of leptin expression and secretion by the adipocytes, the effects of Trecadrine on indices of adipocyte metabolism were also examined. Measurements: Isolated adipocytes were incubated with Trecadrine (10-8-10-4 M) in the absence or presence of insulin (1.6 nM). Leptin secretion, glucose utilization, lactate production, glucose incorporation into CO2 and triglyceride, as well as lipolysis (glycerol release) were determined. Results: Trecadrine induced a concentration-dependent inhibition of basal leptin secretion. Trecadrine also decreased insulin-stimulated leptin secretion; however, the effect was not as pronounced as in the absence of insulin. Treatment of adipocytes with Trecadrine increased basal glucose utilization and produced a further increase in insulin-stimulated glucose utilization. Basal lactate production was also increased by Trecadrine; however, the proportion (percentage) of glucose carbon released as lactate was unaffected. In the presence of insulin, absolute lactate production was unaffected by Trecadrine at 96 h. However, the percentage of glucose carbon released as lactate was significantly decreased by insulin treatment, and was further decreased by the co-treatment with Trecadrine. Trecadrine induced a dose-dependent increase of the absolute amount of glucose incorporated into triglyceride. However, the percentage of glucose utilized that was incorporated into triglyceride was unaffected by Trecadrine. Trecadrine did not modify the proportion of glucose utilized that was oxidized to CO2. Trecadrine increased glycerol release after 96 h of treatment. Glycerol release was negatively correlated with leptin secretion. Conclusions: These results suggest that alterations of glucose metabolism are not directly involved in the effects of beta3-adrenergic agonists to inhibit leptin expression and secretion. The inverse relationship between leptin secretion and the increase of glycerol levels, which is an index of the activation of cAMP-dependent protein kinases, suggests that activation of the cAMP signaling pathway mediates the inhibitory effects of Trecadrine on leptin gene expression and secretion

Bone metabolism. Vitamin D and Parathyroid Hormone

La vitamina D es una hormona involucrada en un complejo sistema endocrino que regula la homeostasis mineral, protege la integridad del esqueleto y modula el crecimiento y la diferenciación celular en una amplia variedad de tejidos. El hígado y el riñón son los órganos donde se producen el calcidiol o 25-hidroxivitamina D3 (forma circulante de vitamina D más abundante) y el calcitriol o 1-α,25-dihidroxivitamina D3 (la forma biológicamente más activa), respectivamente. La concentración de calcidiol es el índice más fiable para definir las situaciones de déficit, insuficiencia, hipovitaminosis, suficiencia y toxicidad de vitamina D. Su cuantificación se realiza mayoritariamente por métodos de RIA, aunque se están introduciendo con fuerza en el mercado los de ELISA y quimioluminiscencia; por su parte, los métodos para cuantificar calcitriol son de RIA con inmunoextracción. Hay que destacar que para establecer una correcta definición del umbral exacto de la situación de vitamina D, en una comunidad, se necesita tener un método de cuantificación bien estandarizado y con valores de referencia propios. La paratirina (PTH) es el regulador más importante en la homeostasis del calcio, ya que potencia su reabsorción en el túbulo renal, haciendo que se incremente su concentración sanguínea. En la circulación existen formas moleculares diferentes y de procedencia tisular, vida media, destino y afección en la enfermedad variadas; precisamente el conocimiento de esta heterogeneidad es el que ha permitido poder interpretar resultados procedentes de métodos que cuantifican diferentes regiones de la molécula. Actualmente se usan métodos inmunométricos (sándwich) de segunda generación para cuantificarla, por lo que se puede obviar la presencia de péptidos de cadena larga de la región aminoterminal que se metabolizan en algunos estadios de la insuficiencia renal. La concentración de paratirina tiene un marcado ritmo circadiano, por lo que se aconseja la toma de muestra no antes de las 7 de la mañana y, en sentido académico, una extracción después de las 10 h de la mañana podría discriminar entre población normal e hiperparatiroidismo primario leve. La PTH se ha introducido como prueba protocolizada en campo operatorio abierto en paratiroidectomía y tiroidectomía. En el primer caso, descensos de un 50% respecto a su valor basal representan éxito quirúrgico; en el segundo caso, se pueden seleccionar pacientes con riesgo de hipocalcemia.Vitamin D is a hormone involved in a complex endocrine system that regulates mineral homeostasis, protects the integrity of the skeleton, and modulates growth and cellular differentiation in a wide variety of tissues. Calcidiol, or 25-hydroxivitamin D3 (the more abundant circulating form of vitamin D), is synthesized in the liver, and calcitriol, or 1-α,25-dihydroxivitamin D3 (the biologically more active form), is synthesized in the kidney. The concentration of calcidiol is the most reliable index for defining vitamin D deficiency, insufficiency, hypovitaminosis, sufficiency and toxicity. It is mainly quantified by radioimmunoassay (RIA) methods, although ELISA and chemoluminescence are being strongly introduced in the market. Calcitriol quantification methods usually consist of RIA with immunoextraction. It is important to highlight that to accurately define vitamin D levels in a community, a standardized method of quantification and specific reference values are required. Parathyrin (PTH) is the most important regulator in calcium homeostasis, since it promotes calcium reabsorption in the renal tubules, increasing calcium concentrations in blood. There are several different molecular forms in the circulation with distinct tissular origin, half life, purpose, and involvement in disease. Indeed, knowledge of this heterogeneity is what has enabled results from methods that quantify different regions of the molecule to be interpreted. Currently, second generation immunometric (sandwich) methods are used to quantify PTH, so that the presence of long chain N-terminal peptides appearing in some stages of renal insufficiency can be obviated. PTH concentrations have a marked circadian rhythm and consequently sample extraction after 7 am is advisable, and, theoretically, extraction after 10 am could discriminate between the normal population and that with mild primary hyperparathyroidism. PTH determination has been introduced as a standardized test in open parathyroidectomy and thyroidectomy. In the first case, reductions of 50% with respect to the basal value represent surgical success; in the second case, patients at risk for hypocalcemia can be selected.Ciencias Experimentale

Alternative pathway for 3-cyanoalanine assimilation in pseudomonas pseudoalcaligenes CECT5344 under Noncyanotrophic conditions

3-Cyanoalanine and cyanohydrins are intermediate nitriles produced in cyanide degradation pathways in plants and bacteria. 3-Cyanoalanine is generated from cyanide by the 3-cyanoalanine synthase, an enzyme mainly characterized in cyanogenic plants. NIT4-type nitrilases use 3-cyanoalanine as a substrate, forming ammonium and aspartate. In some organisms, this enzyme also generates asparagine through an additional nitrile hydratase activity. The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 assimilates cyanide through an intermediate cyanohydrin, which is further converted into ammonium by the nitrilase NitC. This bacterium also contains three additional nitrilases, including Nit4. In this work, a proteomic analysis of P. pseudoalcaligenes CECT5344 cells grown with 3-cyanoalanine as the sole nitrogen source has revealed the overproduction of different proteins involved in nitrogen metabolism, including the nitrilase NitC. In contrast, the nitrilase Nit4 was not induced by 3-cyanoalanine, and it was only overproduced in cells grown with a cyanide-containing jewelry-manufacturing residue. Phenotypes of single and double mutant strains defective in nit4 or/and nitC revealed the implication of the nitrilase NitC in the assimilation of 3-cyanoalanine and suggest that the 3-cyanoalanine assimilation pathway in P. pseudoalcaligenes CECT5344 depends on the presence or absence of cyanide. When cyanide is present, 3-cyanoalanine is assimilated via Nit4, but in the absence of cyanide, a novel pathway for 3-cyanoalanine assimilation, in which the nitrilase NitC uses the nitrile generated after deamination of the a-amino group from 3-cyanoalanine, is proposed

Epigenetic and post-transcriptional regulation of somatostatin receptor subtype 5 (SST5) in pituitary and pancreatic neuroendocrine tumors

Somatostatin receptor subtype 5 (SST5) is an emerging biomarker and actionable target in pituitary (PitNETs) and pancreatic (PanNETs) neuroendocrine tumors. Transcriptional and epigenetic regulation of SSTR5 gene expression and mRNA biogenesis is poorly understood. Recently, an overlapping natural antisense transcript, SSTR5-AS1, potentially regulating SSTR5 expression, was identified. We aimed to elucidate whether epigenetic processes contribute to the regulation of SSTR5 expression in PitNETs (somatotropinomas) and PanNETs. We analyzed the SSTR5/SSTR5-AS1 human locus in silico to identify CpG islands. SSTR5 and SSTR5-AS1 expression was assessed by quantitative real-time PCR (qPCR) in 27 somatotropinomas, 11 normal pituitaries (NPs), and 15 PanNETs/paired adjacent (control) samples. We evaluated methylation grade in four CpG islands in the SSTR5/SSTR5-AS1 genes. Results revealed that SSTR5 and SSTR5-AS1 were directly correlated in NP, somatotropinoma, and PanNET samples. Interestingly, selected CpG islands were differentially methylated in somatotropinomas compared with NPs. In PanNETs cell lines, SSTR5-AS1 silencing downregulated SSTR5 expression, altered aggressiveness features, and influenced pasireotide response. These results provide evidence that SSTR5 expression in PitNETs and PanNETs can be epigenetically regulated by the SSTR5-AS1 antisense transcript and, indirectly, by DNA methylation, which may thereby impact tumor behavior and treatment response.Junta de AndalucíaMinisterio de EconomíaMinisterio de Ciencia e Innovació

Reuse of Zeolite By-Products Derived from Petroleum Refining for Sustainable Roads

The reduction in consumption of natural resources (fuel, gas, etc.) and contaminant emissions (CO2, CO, NOx, etc.) during the production of asphalt mixtures has become one of the main challenges in road engineering. Warm mix asphalts (WMAs) have been developed in order to achieve this objective while ensuring the mechanical performance and durability of traditional hot mix asphalts (HMAs). However, these materials are commonly manufactured using additives or products whose production could reduce both their environmental benefits and cost effectiveness. /is paper presents a research study that aims to analyse the reuse of zeolite wastes derived from petroleum refining in the production of warm mix asphalts. For this purpose, two different types of zeolite wastes were analysed as additives for the manufacture of two warm mix asphalts, whose mechanical performance was compared with conventional WMA and hot mix asphalt. /e results indicate that zeolite wastes with a lower particles size presented higher capacity to absorb water, while its dosage at 0.3% allows for producing warm mix asphalts at temperatures around 145°C, with comparable workability and densification to conventional HMA at 165°C without reducing its bearing capacity, fatigue life, and resistance to water action and plastic deformation.“Soluciones de Pavimentación Ecológicamente Sostenibles: Ecoasfaltos” funded by the Ministry of Economy and Knowledge of Andalusia and the Ministry of Economy and Competitiveness of Spain in the framework of CTA (Corporación Tecnológica de Andalucía)