Identification and expression analysis of the arbuscular mycorrhiza-inducible Rieske non-heme oxygenase Ptc52 gene from tomato.

Arbuscular mycorrhizal (AM) formation enhances plant growth and fitness through improved uptake of water and mineral nutrients in exchange for carbon compounds to the AM fungus. The fungal structure for the reciprocal exchange of nutrients in the symbiosis is the arbuscule, and defence genes expressed in cells containing arbuscules could play a role in the control of hyphal spread and arbuscule formation in the root. We characterized and analyzed the Ptc52 gene from tomato (SlPtc52), a member of the gene family of non-heme oxygenases, whose function has been related to the lethal leaf spot 1 (Lls1) lesion mimic phenotype in plants which is sometimes associated with enhanced disease resistance. Sequence analysis of the SlPTC52 protein revealed conserved typical motifs from non-heme oxygenases, including a Rieske [2Fe-2S] motif, a mononuclear non-heme iron-binding motif and a C-terminal CxxC motif. The level of transcript accumulation was low in stem, flower and green fruits, and high in leaves. Although SlPtc52 expression was perceptible at low levels in roots, its expression increased concomitantly with AM fungus root colonization. Tomato non-mycorrhizal hairy roots expressing the GUS protein under the control of promoter SlPtc52 exhibited GUS activity in the endodermis, the apical meristem of the root tip and in the lateral root primordium. AM fungal colonization also resulted in intensive GUS activity that clearly corresponds to cortical cells containing arbuscules. SlPtc52 gene silencing led to a delay in root colonization and a decrease in arbuscular abundance, suggesting that SlPTC52 plays a regulatory role during AM symbiosis.

Functional Analysis of Root microRNAs by a Constitutive Overexpression Approach in a Composite Plant System.

Agrobacterium-mediated transformation is a fast and efficient method for genome modification in plants. In this protocol, we apply this technique for the analysis of root microRNA functionality. The induction of hairy roots constitutively overexpressing a given microRNA precursor allows us, in a simple way, to modify the accumulation of specific mature microRNA and analyze the consequence of this alteration on a phenotype of interest. This method generates ready-to-phenotype "composite plants" with untransformed aerial part and microRNA-overexpressing root system, in about 20 days.

Embolismo paradójico como causa de trombosis precoz de fístula arteriovenosa para hemodiálisis / Embolism as a cause of early thrombosis of arteriovenous fistula for hemodialysis

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Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells.

A genome-wide analysis identified the set of small RNAs (sRNAs) from the agronomical important legume Phaseolus vulgaris (common bean), including novel P. vulgaris-specific microRNAs (miRNAs) potentially important for the regulation of the rhizobia-symbiotic process. Generally, novel miRNAs are difficult to identify and study because they are very lowly expressed in a tissue- or cell-specific manner. In this work, we aimed to analyze sRNAs from common bean root hairs (RH), a single-cell model, induced with pure Rhizobium etli nodulation factors (NF), a unique type of signal molecule. The sequence analysis of samples from NF-induced and control libraries led to the identity of 132 mature miRNAs, including 63 novel miRNAs and 1984 phasiRNAs. From these, six miRNAs were significantly differentially expressed during NF induction, including one novel miRNA: miR-RH82. A parallel degradome analysis of the same samples revealed 29 targets potentially cleaved by novel miRNAs specifically in NF-induced RH samples; however, these novel miRNAs were not differentially accumulated in this tissue. This study reveals Phaseolus vulgaris-specific novel miRNA candidates and their corresponding targets that meet all criteria to be involved in the regulation of the early nodulation events, thus setting the basis for exploring miRNA-mediated improvement of the common bean-rhizobia symbiosis.

Selección de temas de actualidad en cardiología clínica, cardiología geriátrica e insuficiencia cardiaca y trasplante 2015 / Clinical cardiology, geriatric cardiology, heart failure, and transplantation 2015: a selection of topical issues

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Emergence of quinolone-resistant, topoisomerase-mutant Brucella after treatment with fluoroquinolones in a macrophage experimental infection model

AIM: of the study To determine the activity of fluoroquinolones (FQ) and the selection of FQ-resistant mutants in a macrophage experimental infection model (MEIM). MATERIAL AND METHODS: Canine macrophages were inoculated with Brucella melitensis ATCC 23457 (WT), achieving intracellular counts of around 105 CFU/mL. Cell cultures were incubated in the presence of ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MOX), and doxycycline (DOX). After cell lysis, surviving microorganisms were plated for count purposes, and plated onto antibiotics-containing media for mutant selection. Topoisomerases mutations were detected by PCR and sequencing. RESULTS: Bacterial counts after cell lysis were 14.3% (CIP), 65.3% (LEV), and 75% (MOX) lower compared to the control. Quinolone-resistant mutants emerged in cell cultures containing CIP and LEV with a frequency of around 0.5 × 10-3. All mutants showed an Ala87Val change in GyrA. Mutants had FQs MICs around 10 × WT. The ability of these mutants for infecting new macrophages and the intracellular lysis after antibiotic exposure did not change significantly. No 2nd step FQ-resistant mutants were selected from 1st step mutants. CONCLUSIONS: Intracellular activity of FQs is low against WT and gyrA-mutant Brucella. FQs easily select gyrA mutants in MEIM. The ability of mutants for infecting new macrophages remains unchanged. In this MEIM, 2nd step mutants do not emerge

OBJETIVO: del estudio Conocer la actividad de fluoroquinolonas (FQ) y la selección de mutantes resistentes (MR) a FQ en un modelo experimental de infección en macrófagos. MATERIAL Y MÉTODOS: Se inocularon macrófagos de origen canino con la cepa tipo Brucella melitensis ATCC 23457 (CT) hasta alcanzar recuentos intracelulares de aproximadamente 105 UFC/mL. Los cultivos celulares fueron incubados en presencia de ciprofloxacino, levofloxacino, moxifloxacino y doxiciclina. Una vez lisadas las células, los microorganismos supervivientes fueron sembrados en placas sin antibiótico para recuento, y en medios de cultivo conteniendo antimicrobianos para selección de MR. Los MR a topoisomerasas se caracterizaron mediante PCR y secuenciación. RESULTADOS: Los recuentos de microorganismos supervivientes tras el tratamiento con FQ y la lisis celular fueron: 14,3% ciprofloxacino, 65,3% levofloxacino y 75% moxifloxacino con respecto al control. Se seleccionaron MR a FQ en los cultivos celulares que contenían ciprofloxacino y levofloxacino, con una frecuencia en torno a 0,5 × 10-3. Todos los MR seleccionados portaban un cambio Ala87Val en gyrA, y mostraban CIM de FQ en torno a 10x la de la CT. La capacidad de estos MR para infectar nuevos macrófagos y su lisis intracelular tras tratamiento antibiótico no se modificaron de manera significativa con respecto a la CT. Usando la misma metodología, no se seleccionaron MR de segundo nivel a partir de los MR de primer nivel obtenidos. CONCLUSIONES: La actividad intracelular de FQ frente a Brucella es baja, tanto frente a la CT como frente a mutantes con cambios en gyrA. Las FQ seleccionan con facilidad mutantes con cambios en gyrA en este modelo experimental de infección en macrófagos. La capacidad de estos mutantes para infectar nuevos macrófagos permanece intacta. En este modelo experimental de infección en macrófagos no se observó la selección de mutantes de segundo nivel

Emergence of quinolone-resistant, topoisomerase-mutant Brucella after treatment with fluoroquinolones in a macrophage experimental infection model.

AIM OF THE STUDY: To determine the activity of fluoroquinolones (FQ) and the selection of FQ-resistant mutants in a macrophage experimental infection model (MEIM). MATERIAL AND METHODS: Canine macrophages were inoculated with Brucella melitensis ATCC 23457 (WT), achieving intracellular counts of around 105 CFU/mL. Cell cultures were incubated in the presence of ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MOX), and doxycycline (DOX). After cell lysis, surviving microorganisms were plated for count purposes, and plated onto antibiotics-containing media for mutant selection. Topoisomerases mutations were detected by PCR and sequencing. RESULTS: Bacterial counts after cell lysis were 14.3% (CIP), 65.3% (LEV), and 75% (MOX) lower compared to the control. Quinolone-resistant mutants emerged in cell cultures containing CIP and LEV with a frequency of around 0.5×10(-3). All mutants showed an Ala87Val change in GyrA. Mutants had FQs MICs around 10×WT. The ability of these mutants for infecting new macrophages and the intracellular lysis after antibiotic exposure did not change significantly. No 2nd step FQ-resistant mutants were selected from 1st step mutants. CONCLUSIONS: Intracellular activity of FQs is low against WT and gyrA-mutant Brucella. FQs easily select gyrA mutants in MEIM. The ability of mutants for infecting new macrophages remains unchanged. In this MEIM, 2nd step mutants do not emerge.

Role of gibberellins during arbuscular mycorrhizal formation in tomato: new insights revealed by endogenous quantification and genetic analysis of their metabolism in mycorrhizal roots.

Gibberellins (GAs) are key regulators of plant growth and development and recent studies suggest also a role during arbuscular mycorrhizal (AM) formation. Here, complementary approaches have been used to obtain a clearer picture that correlates AM fungal development inside roots with GA metabolism. An extensive analysis of genes associated with GA metabolism as well as a quantification of GA content in roots was made. Application of GA3 and its biosynthesis inhibitor prohexadione calcium (PrCa) combined with a GA-constitutive response mutant (procera) were used to determine whether fungal colonization is altered by the level of these hormones or by changes in the GA-signaling pathway. The increased levels of specific GAs from the 13-hydroxylation pathway in mycorrhizal roots correlate closely with the increased expression of genes coding enzymes from the GA biosynthetic trail. The imbalance of GAs in tomato roots caused by exogenous applications of GA3 or PrCa affects arbuscules in both negative and positive ways, respectively. In addition, procera plants were adversely affected by the mycorrhization process. Our findings demonstrate that an imbalance in favor of an increased amount of GAs negatively affects the frequency of mycorrhization and particularly the arbuscular abundance in tomato mycorrhizal roots and the results point out that AM formation is associated with a change in the 13-hydroxylation pathway of GAs.

Ethylene-dependent/ethylene-independent ABA regulation of tomato plants colonized by arbuscular mycorrhiza fungi.

We investigated the relationship between ABA and ethylene regulating the formation of the arbuscular mycorrhiza (AM) symbiosis in tomato (Solanum lycopersicum) plants and tried to define the specific roles played by each of these phytohormones in the mycorrhization process. We analysed the impact of ABA biosynthesis inhibition on mycorrhization by Glomus intraradices in transgenic tomato plants with an altered ethylene pathway. We also studied the effects on mycorrhization in sitiens plants treated with the aminoethoxyvinyl glycine hydrochloride (AVG) ethylene biosynthesis inhibitor and supplemented with ABA. In addition, the expression of plant and fungal genes involved in the mycorrhization process was studied. ABA biosynthesis inhibition qualitatively altered the parameters of mycorrhization in accordance with the plant's ethylene perception and ethylene biosynthesis abilities. Inhibition of ABA biosynthesis in wild-type plants negatively affected all the mycorrhization parameters studied, while tomato mutants impaired in ethylene synthesis only showed a reduced arbuscular abundance in mycorrhizal roots. Inhibition of ethylene synthesis in ABA-deficient sitiens plants increased the intensity of mycorrhiza development, while ABA application rescued arbuscule abundance in the root's mycorrhizal zones. The results of our study show an antagonistic interaction between ABA and ethylene, and different roles of each of the two hormones during AM formation. This suggests that a dual ethylene-dependent/ethylene-independent mechanism is involved in ABA regulation of AM formation.

Nosocomial infections: new therapeutic alternatives.

This symposium on nosocomial infections, antimicrobial resistance and the benefits of doripenem in this setting was held in Madrid, Spain, on 7 October 2009, and was supported by Janssen-Cilag. The topic was presented under an interdisciplinary approach by different international experts in the field.

Variations in the mycorrhization characteristics in roots of wild-type and ABA-deficient tomato are accompanied by specific transcriptomic alterations.

Abscissic acid (ABA) determines mycorrhiza functionality and arbuscule development. In this study, we performed transcriptome analysis in response to different mycorrhization status according to the ABA content in the root to identify genes that may play a role in arbuscule functionality. Affymetrix Tomato GeneChip (approximately 10,000 probes) allowed us to detect and compare the transcriptional root profiling of tomato (Solanum lycopersicum) wild-type and ABA-deficient sitiens plants colonized by Glomus intraradices. A number of identified genes in tomato belong to a category of genes already described as "mycorrhizal core-set" in other host plants. The impairment in arbuscular mycorrhiza (AM) formation in ABA-deficient mutants was associated with upregulation of genes related to defense and cell wall modification, whereas functional mycorrhization in wild-type plants was associated with activation of genes related to isoprenoid metabolism. The oxylipin pathway was activated in tomato mycorrhizal roots at late stages of interaction, and was related to the control of fungal spread in roots, not with the establishment of the symbiosis. Induction of selected genes, representing a range of biological functions and representative of the three sets of genes specifically upregulated in the different plant phenotype, was confirmed by quantitative reverse-transcription polymerase chain reaction, and their response to phythohormone treatment was tested, showing that ethylene and jasmonic acid are key regulators of gene expression during AM development. Comparative analysis of mycorrhiza upregulated functional categories revealed significant changes in gene expression associated with the different mycorrhization status according to the ABA content in the roots.

Profilaxis antifúngica en pacientes oncohematológicos: revisión de la bibliografía médica y recomendaciones / Antifungal prophylaxis in oncohematologic patients: Literature review and recommendations

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Mycorrhization of the notabilis and sitiens tomato mutants in relation to abscisic acid and ethylene contents.

We examined whether the reduced mycorrhization of abscisic acid (ABA)-deficient tomato mutants correlates with their incapacity in ABA biosynthesis and whether this effect is dependent on ethylene production. The mycorrhization of notabilis and sitiens mutants, which have different ABA deficiencies and an excess of ethylene production, was analyzed. Comparative analysis of the ABA-deficient tomato mutants showed both quantitative and qualitative differences in the pattern of arbuscular mycorrhiza (AM) colonization between the two tomato mutant phenotypes. The sitiens mutant showed a great limitation in fungal colonization (mycorrhizal intensity and arbuscule formation) well correlated with their incapacity in ABA biosynthesis. The notabilis plants, which maintained normal ABA levels in roots under our experimental conditions, appeared to be less affected in their capacity for AM formation, and only a decrease in mycorrhizal intensity was noted at the end of the mycorrhization process. Blockage of ABA formation after tungstate application resulted in a reduction in mycorrhization of wild-type tomato plants. The transcript accumulation of the mycorrhiza-responsive LePT4 gene (tomato phosphate transporter) was clearly associated with the ABA content and mycorrhiza development in roots, as the tungstate treatment in wild-type plants and the inherent ABA deficiency in sitiens mutants led to a complete abolishment of their expression. Our results suggest that the decrease in arbuscular abundance in mycorrhizal sitiens roots is directly associated with their ABA biosynthesis deficiency, and the accumulation of ethylene, as a consequence of ABA deficiency in the mutants, primarily affects mycorrhizal intensity.

Multicenter study evaluating the role of enterococci in secondary bacterial peritonitis.

A 1-year prospective multicenter study was performed to explore the significance of the presence of enterococci in cultures of peritoneal fluid from patients with secondary bacterial peritonitis in seven Spanish hospitals. The clinical records of patients with positive peritoneal fluid cultures were reviewed and distributed into cases (patients with cultures yielding enterococci) and controls (patients with cultures not yielding enterococci). Of a total of 158 records, 38 (24.1%) were cases and 120 (75.9%) were controls. The percentages or the scores (cases versus controls) for the variables included in the multivariate analysis were as follows: age of >50 years, 89.5% versus 68.3%; malignancy, 39.5% versus 18.3%; chronic obstructive pulmonary disease (COPD), 15.8% versus 4.2%; postoperative peritonitis, 55.3% versus 30.1%; nosocomial onset, 57.9% versus 34.2%; a higher Charlson comorbidity index, 3.29 +/- 3.38 versus 1.84 +/- 2.31; APACHE II score, 10.71 +/- 4.37 versus 8.76 +/- 5.49; ultimately or rapidly fatal disease, 63.2% versus 34.8%; need for surgical reintervention, 36.1% versus 15.1%; and admission to an intensive care unit, 45.9% versus 30.8%. In the multivariate analysis, enterococci were associated only with postoperative peritonitis (P = 0.009; odds ratio [OR] = 5.0; 95% confidence interval [CI] = 1.49 to 16.80), a higher Charlson comorbidity index (P = 0.002; OR = 1.30; 95% CI = 1.11 to 1.54), and COPD (P = 0.046; OR = 6.50; 95% CI = 1.04 to 40.73). The results of this study showed that enterococci were associated with comorbidity. An association with mortality could not be demonstrated.

Safety and feasibility of returning patients early to their originating centers after transfer for primary percutaneous coronary intervention.

INTRODUCTION AND OBJECTIVES: At present, little information is available on returning patients with ST-elevation myocardial infarction (STEMI) to their originating centers after transfer for primary percutaneous coronary intervention (PPCI). The objective of this study was to evaluate the safety and feasibility of the early return of these patients to their originating centers. METHODS: The cohort study involved 200 consecutive STEMI patients (age 62+/-13 years, 83% male) who were returned to their originating centers after PPCI. They were compared with a group of 297 patients with similar characteristics from our healthcare catchment area. The length of stay in the intervention hospital and major adverse cardiovascular events occurring within 30 days were recorded. RESULTS: The median length of stay in the intervention hospital was 8 hours. No adverse events occurred during transport in the group who returned to their originating centers. At 30-day follow-up, no significant difference was observed between patients who returned and the control group in either mortality (1.0% vs. 3.7%; P=.064), readmission (5.0% vs. 4.5%; P=.657), ischemic complications (2.5% vs. 2.0%; P=.721), re-catheterization (5.0% vs. 2.5%; P=.112), stroke (1% vs. 1%; P=.936) or the composite end-point (11% vs. 9.2%; P=.540). Multivariate analysis showed that returning patients after PPCI was not associated with a significantly greater number of major adverse cardiovascular events (odds ratio=1.32; 95% confidence interval, 0.62-2.80). CONCLUSIONS: The early return of patients with low-risk STEMI to their originating centers after PPCI was safe and feasible.

Seguridad y viabilidad del retorno precoz de pacientes transferidos para angioplastia primaria a sus centros de origen / Safety and feasibility of returning patients early to their originating centers after transfer for primary percutaneous coronary intervention

Introducción y objetivos. Hasta la fecha existen pocos datos sobre la posibilidad de retornar a los pacientes con síndrome coronario agudo con elevación del segmento ST (SCACEST) trasladados para angioplastia primaria (AP) a su centro de referencia. El objetivo de este estudio es evaluar la seguridad y viabilidad del retorno precoz de dichos pacientes a sus centros de origen. Métodos. Análisis de cohortes constituido por 200 pacientes consecutivos (edad, 62 ± 13 años; el 83% varones) devueltos a su centro de origen tras la realización de AP, comparándolos con un grupo de 297 pacientes de similares características pertenecientes a nuestra área sanitaria. Se analizó el tiempo de estancia en el hospital intervencionista y los eventos cardiovasculares de más de 30 días. Resultados. La mediana de permanencia en nuestro hospital fue de 8 h. El grupo retornado no presentó ningún evento durante el traslado al hospital de origen. A los 30 días no se observaron diferencias significativas entre los pacientes retornados y los del grupo control respecto a muerte (el 1 frente al 3,7%; p = 0,064), reingreso (el 5 frente al 4,5%; p = 0,657), complicaciones isquémicas (el 2,5 frente al 2%; p = 0,721), realización de nuevo cateterismo (el 5 frente al 2,5%; p = 0,112), accidentes cerebrovasculares (el 1 frente al 1%; p = 0,936) o el evento combinado (el 11 frente al 9,2%; p = 0,540). En un análisis multivariable, el retorno de los pacientes no se asoció con un mayor número de eventos cardiovasculares (odds ratio = 1,32; intervalo de confianza del 95%, 0,62-2,80). Conclusiones. El retorno precoz de pacientes con IAM de bajo riesgo a su centro de origen tras AP es seguro y viable (AU)

Introduction and objectives. At present, little information is available on returning patients with ST-elevation myocardial infarction (STEMI) to their originating centers after transfer for primary percutaneous coronary intervention (PPCI). The objective of this study was to evaluate the safety and feasibility of the early return of these patients to their originating centers. Methods. The cohort study involved 200 consecutive STEMI patients (age 62±13 years, 83% male) who were returned to their originating centers after PPCI. They were compared with a group of 297 patients with similar characteristics from our healthcare catchment area. The length of stay in the intervention hospital and major adverse cardiovascular events occurring within 30 days were recorded. Results. The median length of stay in the intervention hospital was 8 hours. No adverse events occurred during transport in the group who returned to their originating centers. At 30-day follow-up, no significant difference was observed between patients who returned and the control group in either mortality (1.0% vs. 3.7%; P=.064), readmission (5.0% vs. 4.5%; P=.657), ischemic complications (2.5% vs. 2.0%; P=.721), re-catheterization (5.0% vs. 2.5%; P=.112), stroke (1% vs. 1%; P=.936) or the composite end-point (11% vs. 9.2%; P=.540). Multivariate analysis showed that returning patients after PPCI was not associated with a significantly greater number of major adverse cardiovascular events (odds ratio=1.32; 95% confidence interval, 0.62-2.80). Conclusions. The early return of patients with low-risk STEMI to their originating centers after PPCI was safe and feasible (AU)