Detecció, identificació de Mycobacterium tuberculosis i caracterització molecular de la resistència als principals antituberculosos en mostra clínica directa mitjançant piroseqüenciació: aplicabilitat en l’estudi de la clonalitat de les soques.

L’augment de soques de Mycobacerium tuberculosis multiresistents i l’aparició de soques extremadament resistents, ha posat de manifest la necessitat de disposar de nous mètodes de detecció precoç de M.tuberculosis i les seves resistències als principals fàrmacs antituberculosos, així com l’estudi eficaç dels brots, per a poder dur a terme un control eficient de la malaltia. L’objectiu de l’estudi va ser determinar la capacitat de la piroseqüenciació per a detectar i identificar micobacteris a partir d’aïllats clínics i mostra directa, determinar el seu patró de resistència a Rifampicina, Isoniacida, Etambutol, Fluoroquinolones, Canamicina i Capreomicina i explorar la seva potencialitat per a ser emprada en el tipatge molecular de les soques. Mitjançant la optimització de la tecnologia de la piroseqüenciació pels gens analitzats, i el disseny de primers específics, hem verificat la utilitat de la piroseqüenciació per al maneig de la tuberculosi. S’ha pogut estudiar la presència de mutacions relacionades amb resistències a fàrmacs de primera línia en el 96.5% de les posicions analitzades en soca clínica i en el 70.4% en mostra directa. Van concordar amb el resultats obtinguts per altres mètodes fenotípics i/o fenotípics el 97.1% i el 98.2% del resultats obtinguts en soca clínica i mostra directa respectivament. La piroseqüenciació ens ha permet analitzar la presència de mutacions relacionades amb resistències a antituberculosos de segona línia, servint com a mètode de confirmació en l’anàlisi d’altres mètodes genotípics. La tècnica ens ha permès també identificar els principals micobacteris no tuberculosos implicats en la infecció humana, sòls o en coinfecció. Resultats preliminars mostren que l’anàlisi amb piroseqüenciació pot ser d’utilitat per l’estudi clonal de les soques de M.tuberculosis. Les nostres observacions mostren la piroseqüenciació com una eina valuosa en l’àmbit clínic, ja que permet una reducció de la demora del diagnòstic, estudi filogenètic i detecció de resistències M.tuberculosis, i per tant una millora de l’aplicació del tractament adequat, ajudant al control de la malaltia.

Estudio de marcadores moleculares en neoplasias mieloproliferativas

Las neoplasias mieloproliferativas (NM) son un grupo de enfermedades clonales de la célula hematopoyética madre. Entre las NM clásicas se encuentran la policitemia vera (PV), la trombocitemia esencial (TE) y la mielofibrosis primaria (MFP). Durante muchos años el diagnóstico de estas patologías se hacía por exclusión utilizando biomarcadores de clonalidad poco específicos. En el año 2005, la descripción de la mutación JAK2V617F supuso un avance importante en el diagnóstico de estas patologías. Posteriormente, se han descrito mutaciones en otros genes como mutaciones en MPL, TET2, ASXL1, IDH1, IDH2, c-CBL, EZH2, IKZF1 y LNK, en distintas neoplasias mieloides y en porcentaje variable. Aun así, ninguno de estos genes son marcadores específicos de ninguna NM y todavía existe un porcentaje elevado de pacientes con TE y MFP sin un marcador de clonalidad conocido. Además, todos estos genes se han descrito como eventos genéticos implicados en la transformación de una NM a leucemia mieloide aguda. El objetivo de este proyecto fue estudiar varios marcadores moleculares en neoplasias mieloproliferativas Philadelphia negativas. En primer lugar, se estudió la modulación de la carga alélica JAK2V617F en pacientes con PV o TE que recibieron tratamiento citoreductor y a su vez se analizó la dinámica natural de la carga alélica en pacientes que no recibieron tratamiento. Posteriormente, se analizaron la presencia de alteraciones en los genes previamente mencionados, en distintos grupos de pacientes. En primer lugar, se analizó la presencia de mutaciones en TET2, ASXL1, IDH1, IDH2 y CBL en un grupo de pacientes JAK2 y MPL negativos, para determinar la frecuencia de alteraciones de estos genes en este grupo de pacientes y determinar su valor en el diagnóstico de estas patologías. En segundo lugar, se estudió la presencia de mutaciones en estos genes incluyendo EZH2, IKZF1 y LNK para estudiar la incidencia y el valor pronóstico de estas alteraciones en las NM que progresan a mielofibrosis.

Assaig d’ aclarida química en raïm de vinificació garnatxa negra

La Garnatxa negra és la varietat negra autòctona i majoritària a la Denominació d’OrigenEmpordà. La facilitat actual per obtenir rendiments elevats (selecció clonal, terrenys fèrtils, pràctiques agrícoles, ...) obliga a regular-ne la producció per obtenir collites de qualitat. L’eliminació d’una part de la collita mitjançant l’aclarida manual ha estat una pràctica efectiva i àmpliament utilitzada per la millora de la qualitat en situacions puntuals de sobreproducció. Però els seuselevats costos de mà d’obra la fan privativa per a la majoria de productors. L’aclarida química amb Etefon es perfila com una bona alternativa a l’hora de regular la producció amb uns costos d’execució acceptables. L’objectiu del treball era provar l’eficàcia de l’Etefon com a aclaridor químic en vinya per a la varietat Garnatxa negra com a alternativa a l’aclarida manual tradicional

Seasonal changes of whole root system conductance by a drought-tolerant grape root system

The role of root systems in drought tolerance is a subject of very limited information compared with above-ground responses. Adjustments to the ability of roots to supply water relative to shoot transpiration demand is proposed as a major means for woody perennial plants to tolerate drought, and is often expressed as changes in the ratios of leaf to root area (AL:AR). Seasonal root proliferation in a directed manner could increase the water supply function of roots independent of total root area (AR) and represents a mechanism whereby water supply to demand could be increased. To address this issue, seasonal root proliferation, stomatal conductance (gs) and whole root system hydraulic conductance (kr) were investigated for a drought-tolerant grape root system (Vitis berlandieri×V. rupestris cv. 1103P) and a non-drought-tolerant root system (Vitis riparia×V. rupestris cv. 101-14Mgt), upon which had been grafted the same drought-sensitive clone of Vitis vinifera cv. Merlot. Leaf water potentials (ψL) for Merlot grafted onto the 1103P root system (–0.91±0.02 MPa) were +0.15 MPa higher than Merlot on 101-14Mgt (–1.06±0.03 MPa) during spring, but dropped by approximately –0.4 MPa from spring to autumn, and were significantly lower by –0.15 MPa (–1.43±0.02 MPa) than for Merlot on 101-14Mgt (at –1.28±0.02 MPa). Surprisingly, gs of Merlot on the drought-tolerant root system (1103P) was less down-regulated and canopies maintained evaporative fluxes ranging from 35–20 mmol vine−1 s−1 during the diurnal peak from spring to autumn, respectively, three times greater than those measured for Merlot on the drought-sensitive rootstock 101-14Mgt. The drought-tolerant root system grew more roots at depth during the warm summer dry period, and the whole root system conductance (kr) increased from 0.004 to 0.009 kg MPa−1 s−1 during that same time period. The changes in kr could not be explained by xylem anatomy or conductivity changes of individual root segments. Thus, the manner in which drought tolerance was conveyed to the drought-sensitive clone appeared to arise from deep root proliferation during the hottest and driest part of the season, rather than through changes in xylem structure, xylem density or stomatal regulation. This information can be useful to growers on a site-specific basis in selecting rootstocks for grape clonal material (scions) grafted to them.

Functional dissection of the ash2 and ash1 transcriptomes provides insights into the transcriptional basis of wing phenotypes and reveals conserved protein interactions

Background: The trithorax group (trxG) genes absent, small or homeotic discs 1 (ash1) and 2 (ash2) were isolated in a screen for mutants with abnormal imaginal discs. Mutations in either gene cause homeotic transformations but Hox genes are not their only targets. Although analysis of double mutants revealed that ash2 and ash1 mutations enhance each other's phenotypes, suggesting they are functionally related, it was shown that these proteins are subunits of distinct complexes.Results: The analysis of wing imaginal disc transcriptomes from ash2 and ash1 mutants showed that they are highly similar. Functional annotation of regulated genes using Gene Ontology allowed identification of severely affected groups of genes that could be correlated to the wing phenotypes observed. Comparison of the differentially expressed genes with those from other genome-wide analyses revealed similarities between ASH2 and Sin3A, suggesting a putative functional relationship. Coimmunoprecipitation studies and immunolocalization on polytene chromosomes demonstrated that ASH2 and Sin3A interact with HCF (host-cell factor). The results of nucleosome western blots and clonal analysis indicated that ASH2 is necessary for trimethylation of the Lys4 on histone 3 (H3K4).Conclusion: The similarity between the transcriptomes of ash2 and ash1 mutants supports a model in which the two genes act together to maintain stable states of transcription. Like in humans, both ASH2 and Sin3A bind HCF. Finally, the reduction of H3K4 trimethylation in ash2 mutants is the first evidence in Drosophila regarding the molecular function of this trxG gene.

The effect of recombination in Aeromonas

Although several approaches have been attempted, the estimation of recombination frequencies in natural populations ofbacteria remains challenging. Previous studies have demonstrated awide variety of situations among bacterial species, ranging from theclonal diversification of Salmonella or Escherichia coli, which aremainly due to mutation, to the frequent recombination found inNeisseria gonorrhoeae or Helicobacter pylori. Most of the populationstudies done with bacterial species suggest that recombination occursin nature but that it is infrequent compared to mutation. Consequently,bacterial populations consist largely of independent clonal lineages.Our research suggests little or null influence of recombination in thegenetic structure of "Aeromonas hydrophila Species Complex", despite the presence of some strains with recombinant gene fragments.

The effect of recombination in Aeromonas

Although several approaches have been attempted, the estimation of recombination frequencies in natural populations ofbacteria remains challenging. Previous studies have demonstrated awide variety of situations among bacterial species, ranging from theclonal diversification of Salmonella or Escherichia coli, which aremainly due to mutation, to the frequent recombination found inNeisseria gonorrhoeae or Helicobacter pylori. Most of the populationstudies done with bacterial species suggest that recombination occursin nature but that it is infrequent compared to mutation. Consequently,bacterial populations consist largely of independent clonal lineages.Our research suggests little or null influence of recombination in thegenetic structure of "Aeromonas hydrophila Species Complex", despite the presence of some strains with recombinant gene fragments.

Unexpected genomic variability in clinical and environmental strains of the pathogenic yeast Candida parapsilosis

Invasive candidiasis is the most commonly reported invasive fungal infection worldwide. Although Candida albicans remains the main cause, the incidence of emerging Candida species, such as C. parapsilosis is increasing. It has been postulated that C. parapsilosis clinical isolates result from a recent global expansion of a virulent clone. However, the availability of a single genome for this species has so far prevented testing this hypothesis at genomic scales. We present here the sequence of three additional strains from clinical and environmental samples. Our analyses reveal unexpected patterns of genomic variation, shared among distant strains, that argue against the clonal expansion hypothesis. All strains carry independent expansions involving an arsenite transporter homolog, pointing to the existence of directional selection in the environment, and independent origins of the two clinical isolates. Furthermore, we report the first evidence for the existence of recombination in this species. Altogether, our results shed new light onto the dynamics of genome evolution in C. parapsilosis.

Estudi d’adaptació de clons de pollancre (Populus sp.) mitjançant models mitxes en la vall intermèdia de l’Ebre

Durant la dècada dels 80 la DGA (Diputación General de Aragon), va engegar una xarxa d’assaigs experimentals. La major part d’aquests assaigs es troben a la vall de l’Ebre. Padró el 1992 publica les dades de creixement i aporta informació sobre el comportament clonal de cada assaig. Malgrat tot, la enorme desestructura dels assaigs dificultà, en el seu moment, l’anàlisi simultani de les dades. En el treball es proposa un reanàlisi de les dades de la xarxa de la DGA basat en l’ús de models mitxes, amb l’objecte d’interpretar patrons adaptatius i amb especial interès en la descripció de la interacció genotip-ambient. S’utilitzen el models de estabilitat de Shukla, regressions de Finlay-Wilkinson y Eberhart-Russell, AMMI a on el factor ambient és aleatori i el genotip fixe.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

Background: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. Results: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct haracterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. Conclusions: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Allelic diversity and population structure in Vibrio cholerae O139 Bengal based on nucleotide sequence analysis

Comparative analysis of gene fragments of six housekeeping loci, distributed around the two chromosomes of Vibrio cholerae, has been carried out for a collection of 29 V. cholerae O139 Bengal strains isolated from India during the first epidemic period (1992 to 1993). A toxigenic O1 ElTor strain from the seventh pandemic and an environmental non-O1/non-O139 strain were also included in this study. All loci studied were polymorphic, with a small number of polymorphic sites in the sequenced fragments. The genetic diversity determined for our O139 population is concordant with a previous multilocus enzyme electrophoresis study in which we analyzed the same V. cholerae O139 strains. In both studies we have found a higher genetic diversity than reported previously in other molecular studies. The results of the present work showed that O139 strains clustered in several lineages of the dendrogram generated from the matrix of allelic mismatches between the different genotypes, a finding which does not support the hypothesis previously reported that the O139 serogroup is a unique clone. The statistical analysis performed in the V. cholerae O139 isolates suggested a clonal population structure. Moreover, the application of the Sawyer's test and split decomposition to detect intragenic recombination in the sequenced gene fragments did not indicate the existence of recombination in our O139 population.